Review pubs.acs.org/JAFC
Generation of Acetoin and Its Derivatives in Foods Zijun Xiao*,† and Jian R. Lu*,§ †
Centre for Bioengineering and Biotechnology, China University of Petroleum, Qingdao 266580, China Biological Physics Laboratory, School of Physics and Astronomy, University of Manchester, Manchester M13 9PL, United Kingdom
§
ABSTRACT: Acetoin is a common food flavor additive. This volatile compound widely exists in nature. Some microorganisms, higher plants, insects, and higher animals have the ability to synthesize acetoin using different enzymes and pathways under certain circumstances. As a very active molecule, acetoin acts as a precursor of dozens of compounds. Therefore, acetoin and its derivatives are frequently detected in the component analysis of a variety of foods using gas chromatography−mass spectrometry. Because of the increasing importance of these compounds, this paper reviews the origins and natural existence of these substances, physiological roles, the biological synthesis pathways, nonenzymatic spontaneous reactions, and the common determination methods in foods. This work is the first review on dietary natural acetoin. KEYWORDS: acetoin, derivatives, key enzymes, spontaneous reactions, food additive, flavouring agent
■
INTRODUCTION Acetoin (3-hydroxy-2-butanone) is a pale to yellowish liquid with a pleasant yogurt creamy odor and a fatty butter taste. With FEMA No. 2008, it is a substance generally recognized as safe (GRAS) and mainly used in food industries to enhance the flavor of their products (visit http://ntp.niehs.nih.gov/ for toxicity and related information). As the threshold for taste is affected by various conditions such as temperature, solution, and personal physiological and psychological status, acetoin is perceptible at different levels in different foods and drinks. For example, the sensation threshold of acetoin in different types of beers is quite different, ranging from 6 to 50 ppm by different authors.1 In contrast, its reported levels of use in foods could be hundreds of parts per million (Table 1). Acetoin can be biosynthesized in a variety of dietary materials. For example, in yogurt and cheese, lactic acid bacteria convert lactose and citrate to important metabolites including acetoin and its analogue diacetyl, which add a strong buttery and cheesy flavor to the products. Vinegar is another
example of a bacterially fermented product that is also rich in acetoin. As for yeast-fermented products, acetoin exists in almost all alcoholic beverages and is indispensable in contributing to their flavors.3 Natural acetoin has also been detected in fruits, vegetables, and flours, contributing to their distinct natural flavors. As a very active molecule, acetoin forms various derivatives, which can also be frequently identified during gas chromatography−mass spectrometry (GC-MS) analysis. We have previously discussed acetoin metabolism in bacteria focusing on bacterial physiology and catabolism.4 Very recently, we also reviewed the main approaches for enhancing fermentative acetoin production using bacterial strains with industrial potential.5 However, acetoin-related metabolic mechanisms in eukaryotes including humans are fundamentally different from those in prokaryotes. For example, acetoin esters and glycosides were mainly found in higher plants, but the biological mechanisms remain unclear to date. Given its increasing importance in foods and our daily life, we present here the first review disseminating the knowledge of the dietary natural acetoin. We will outline the natural origins of the dietary acetoin, introduce the key relevant enzymes and nonenzymatic spontaneous reactions of acetoin in food-related higher species, and, where relevant, incorporate a basic discussion to cover the main acetoin derivatives. Improvement in our scientific understanding in this area will help us to better control and use the dietary acetoin compounds in foods and drinks.
Table 1. Acetoin Average Maximum Use Levels in Foods2 food category baked goods beverages, alcoholic beverages, nonalcoholic breakfast cereals cheese chewing gum condiments and relishes confection and frosting fats and oils frozen dairy fruit juice gelatins and puddings
use levels (ppm) 750 3.1 17 0.67 10 0.42 8 100 750 50 0.03 81
food category
use levels (ppm)
gravies hard candy imitation dairy
0.029 84.89 100
meat products milk products other grains reconstituted vegetables seasonings and flavours snack foods soft candy soups sweet sauce
24.27 0.03 400 200
© 2014 American Chemical Society
■
COMMERCIAL PRODUCTION OF ACETOIN Being highly active, acetoin is also very useful in chemical synthesis in addition to its importance in foods and drinks. For example, acetoin acts as the precursor for a range of
90 98 50 0.05 98
Received: Revised: Accepted: Published: 6487
March 22, 2014 June 26, 2014 June 26, 2014 June 26, 2014 dx.doi.org/10.1021/jf5013902 | J. Agric. Food Chem. 2014, 62, 6487−6497
Journal of Agricultural and Food Chemistry
Review
transport and store. It can become easily lost during handling, resulting in environmental pollution and compromising safety. Most of these issues can be resolved by using paraldehyde but require the extra step of paraldehyde depolymerization, increasing the complexity and decreasing the yield.8
compounds and as the chelating agent of alkoxides. As a bioactive compound, it shows great potential in microbiology, botany, and pest control as well.5 Because acetoin has broad usages, it is valuable to study its production. Although there is industrial natural acetoin available from several small fermentation plants, current commercial acetoin is mainly produced by chemical synthesis from fossil-based raw materials and then added into our foods as a flavor enhancer.5 There are currently three main routes that can be used for the synthesis of acetoin: route A, exploiting the transformation processes from structural analogues such as diacetyl and 2,3-butanediol; route B, utilizing the hydrolysis processes from halogenated butanone; and route C, seeking a direct synthesis from aldehyde. Route A. The main disadvantage of using diacetyl as the raw material for the production of acetoin is the high cost as diacetyl or related compounds are fine chemical products. There are also additional issues associated with high equipment requirements and more stringent reaction conditions and controls. If over-reduced, acetoin can be further converted to 2,3-butanediol, decreasing the reaction yield. Similar issues would occur if 2,3-butanediol is used as the raw material. For these reasons there has been no report of industrial uptake of this route.6 Route B. In this route butanone is usually used as the starting material, involving the first step of halogenation to make 3halogenated butanone and the second step of hydrolysis to make acetoin (Figure 1, top). Methyl ethyl ketone is a
■
NATURAL ORIGINS OF ACETOIN In contrast to the organic chemistry routes, however, acetoin can be generated by various natural or “green” routes. Generally speaking, natural acetoin has three origins, microorganisms, plants, and animals, and exists widely in some fermented products, fruits, crops, insects, mammals, and so on. Table 2 gives some examples of the natural origins of acetoin. Table 2. Some Natural Origins of Acetoin material artisanal Spanish cheese9 French blue cheeses10 Chinese vinegars11 European vinegars12 beers13 wines from different countries3 young red wines14 Garnacha Tintorera-based sweet wines15 sherry wines16 palm wine17 pawpaw18 dekopon19 durian20 banana21 lychee22 coconut23 oil palm23 guar bean24 ash gourd25 corn tortillas26 rice cakes27 honeys28 Avicennia marina29 sweet olive30 red clover31 kiwi fruit flowers32 Solomon’s lily33 summer chafer34 rhinoceros beetle35 cockroaches36 liver homogenate and perfused liver of rats37
Figure 1. Industrial chemical synthesis routes of acetoin.
commonly used raw material. Route B thus has much reduced raw material cost. In addition, it has milder reaction conditions, lower equipment specifications, and no special catalysts required either. It is thus not surprising to see its extensive uses in industry. However, the hydrogens on the α-position of butanone tend to have greater reactivity. Apart from the target halogen-substituted ketone, other reactions also occur to produce 1-substituted butanone, 3,3-dihalobutanone, and other derivatives, causing the yield reduction of the 3halogenated product. Furthermore, under the alkali hydrolysis in step 2, byproducts such as 1-hydroxy-2-butanone make separation and purification more difficult.7 Route C. Acetaldehyde is used as the starting material, and the product is produced from condensation reaction under the catalysis of triazolium ylide or thiazolium ylide (Figure 1, bottom). This method also benefits from the lower cost of the starting material, less demanding equipment, fewer byproducts, higher yield, and simpler separation and purification, thereby enjoying more widespread industrial applications than route B. The main disadvantage lies in the complicated process of the catalyst preparation. Furthermore, acetaldehyde has a low boiling point. It is volatile and flammable and is difficult to
blood and urinary from normal male students after ethanol ingestion38 human sweat39
origin of acetoin microbial fermentation microbial fermentation microbial fermentation microbial fermentation microbial fermentation microbial fermentation microbial fermentation microbial fermentation microbial fermentation microbial fermentation fruit, plant synthesis fruit, plant synthesis fruit, plant synthesis fruit, plant synthesis fruit, plant synthesis fruit, plant synthesis fruit, plant synthesis vegetable, plant synthesis vegetable, plant synthesis corn, plant synthesis rice, plant synthesis flower, plant synthesis flower, plant synthesis flower, plant synthesis flower, plant synthesis flower, plant synthesis flower, plant synthesis insect, animal synthesis insect, animal synthesis insect, animal synthesis mammal, animal synthesis mammal, animal synthesis mammal, animal synthesis
■
PHYSIOLOGICAL ROLES Acetoin biogenesis has different physiological roles in different biological species. Current knowledge on the physiological functions of acetoin biogenesis can be summarized into three categories: biological functions in microorganisms, semiochemical functions in higher plants and insects, and biological functions in higher animals. 6488
dx.doi.org/10.1021/jf5013902 | J. Agric. Food Chem. 2014, 62, 6487−6497
Journal of Agricultural and Food Chemistry
Review
be successful in female mate choice. 52 In male−male interactions, the secretion of acetoin plays an important role as well. Microgram levels of acetoin are released by aggressive cockroaches at high rates in strong attacks.53 The dominant males release acetoin strategically to flaunt their dominance.42 Therefore, acetoin is multifunctional in the world of cockroaches. Acetoin is a well-known fermentative product, but it is perhaps less well-known to be the third most abundant volatile compound in fresh human sweat (ethanol, 15.1%; acetic acid, 10.9%; acetoin, 9.5%).39 Acetoin was also detected in blood and urinary samples from normal male students after ethanol ingestion.38 A pathway with acetoin as a key metabolite was proposed to explain the detoxication mechanism of acetaldehyde in mammals.37,38 Under pathological conditions, acetoin has a role in regulating intermediate metabolism in mammalian systems.54 However, the underlying mechanisms of acetoin biogenesis in higher organisms are largely unknown.
The biogenesis of acetoin in bacteria and yeasts has been well investigated partly because acetoin is tightly related to many traditional fermentative dietary products. Furthermore, just like acetic acid, ethanol, and lactic acid, acetoin/2,3-butanediol is another popular primary metabolite in bacteria. The ability to secrete acetoin is a routine classification marker (i.e., the Voges−Proskauer test) used in bacterial identification. In a sugar-rich culture medium, the fast assimilation of carbon source and accumulation of 2,3-butanediol other than organic acids can avoid cellular acidification. When the fermentable sugars are depleted, 2,3-butanediol is transformed back to acetoin, regenerating the reducing force (NADH or NADPH).40 Acetoin is further reutilized as an alternative carbon and energy source.41 In addition, acetoin biosynthesis is closely related to the branched-chain amino acid pathway and the regulation of cell life in certain bacteria.4 In higher organisms, the secretion ability of acetoin/2,3butanediol is substantially weakened or completely abolished. However, due to its moderate volatile ability, characteristic odor, and chemical structure, acetoin has great potential as a semiochemical, especially in some insects (Table 3), flowers,
■
ACETOIN BIOSYNTHESIS As shown in Table 2, acetoin biosynthesis widely exists in various food materials. In contrast, bacteria have very efficient acetoin biosynthesis systems.5 Although the production of acetoin by eukaryotic cells is relatively less efficient, it is important due to its relationships with our foods. Acetoin biogenesis and dissimilation were seldom reported in archaea. Thus, the following discussion will focus on acetoin biosynthesis in eukaryotic systems with emphasis on enzymatic aspects by comparative study. Currently, acetoin’s precursor 2-acetolactate can be synthesized by five enzymes: α-acetolactate synthase (ALS, EC 2.2.1.6), acetohydroxy acid synthase (AHAS, EC 2.2.1.6), pyruvate oxidase (POX, EC 1.2.3.3), pyruvate decarboxylase (PDC, EC 4.1.1.1), and pyruvate dehydrogenase (PDH, EC 1.2.4.1). These enzymes share substantial similarities not only in amino acid sequences (Figure 2) but also in protein structures and catalytic mechanisms. All five enzymes are thiamin pyrophosphate (TPP)-dependent, and they all need a divalent metal ion such as Mg2+ to work properly. Furthermore, all five enzymes except ALS contain flavin adenine dinucleotide (FAD) in their structures. However, ALS replaced the position of FAD with reorientated amino acid side chains, and the overall structure of ALS remained unchanged.55 Another good example of structural and functional similarity among the enzymes is that the D28A and D28N variants of yeast PDC resemble ALSs, which synthesize 2-acetolactate as a major product.56 All five enzymes follow a common mechanism of 2acetolactate synthesis. Presumably, a pyruvate and an enzyme form an initial intermediate, an enzyme-bound lactyl-TPP (lactyl-TPP-E). Then the initial intermediate decarboxylates to form hydroxyethyl-TPP-E (“active acetaldehyde”). The active acetaldehyde reacts with another pyruvate to form 2acetolactate (the so-called carboligase reaction).49 POX, PDC, and PDH can also perform the carboligation of hydroxyethylTPP-E with an acetaldehyde moiety to direct yield acetoin. However, although these five enzymes retain much commonality, evolution has taken place to fit differentiation and refinement functions. ALS exists only in some bacteria. The five genera in the ALS clade in Figure 1 are representatives of the most efficient acetoin producers.5 ALS serves as the first key enzyme of the acetoin/2,3-butanediol biosynthesis pathway in these bacteria. Its sole duty is the condensation of pyruvate to
Table 3. Acetoin as a Semiochemical in Insects species 42
cockroaches Nauphoeta cinerea, Henschoutedenia f lexivitta, and Leucophaea maderae43 summer chafer Amphimallon solstitiale34 white-spotted flower chafer Protaetia brevitarsis44 rhinoceros beetle Scapanes australis35 American palm weevil Rhynchophorus palmarum23 fruit fly Drosophila melanogaster45 fly46 nymphalid butterflies Kaniska canace and Vanessa indica47 African fruit chafer Pachnoda marginata48
function 36
male pheromone female pheromone attractant male pheromone attractant attractant attractant attractant attractant
and fruits (Table 2). Acetoin and some other characteristic fermentative products such as acetic acid and isovaleric acid convey a selective message of fermentation and sugar-rich substrates,48 which is highly attractive to ants, bugs, beetles, flies, wasps, honeybees, and moths. Acetoin synthesis in plant cells was confirmed,49 and together with other odorous compositions, acetoin gives these plants rich fragrances, attracting insects and higher animals to help their pollination, breeding, and propagation. However, on the opposite side, pests are attracted. Mass-trapping techniques have been developed using acetoin as an active component of artificial lures against the corresponding pests (as some references in Table 3). In the model organism fruit fly Drosophila melanogaster, acetoin has been recognized as a natural ligand for an olfactory sensory neuron50 and the strongest known stimulus for the glomerulus VA2, which is associated with the close-range attraction of this fly to vinegar.51 As a male pheromone, acetoin has been well studied in cockroaches. Nauphoeta cinerea, Leucophaea maderae, and Henschoutedenia flexivitta all belong to Oxyhaloinae and have the same mating behavior pattern. However, acetoin is the only compound common to the pheromone blends of the three species.36 Acetoin is attractive to female L. maderae at low dosages (0.1−5 ng).43 In N. cinerea, males must have the ability to secrete acetoin; otherwise, physiologically defective individuals without this ability will not 6489
dx.doi.org/10.1021/jf5013902 | J. Agric. Food Chem. 2014, 62, 6487−6497
Journal of Agricultural and Food Chemistry
Review
Figure 2. Simplified phylogenetic tree based on typical amino acid sequences of putative acetoin and/or 2-acetolactate biosynthesis enzymes. Representative sequences from typical species were extracted from the UniProt database. Accession numbers: AHAS large subunit, P17597 chloroplastic Arabidopsis, Q42768 cotton, Q6K2E8 chloroplastic rice, P0A622 Mycobacterium, P08142 E. coli, P37251 Bacillus, P45261 Haemophilus, P07342 Saccharomyces; ALS, Q04789 Bacillus, G0VU08 Paenibacillus, D4E3C0 Serratia, V5AYP9 Enterobacter, B5XWE0 Klebsiella; POX, Q8X6L4 E. coli, E0U346 Bacillus, P37063 Lactobacillus, Q54970 Streptococcus; PDC, Q8NK64 Rhizopus, P06169 Saccharomyces, M1QLG2 Bacillus, E8Y3L9 E. coli, P06672 Zymomonas, Q8L388 Acetobacter, B0ZS79 plum, Q9FFT4 Arabidopsis, A2XFI3 rice; PDH E1 α subunit, Q8NNF6 Corynebacterium, Q10504 Mycobacterium, P0AFG8 E. coli, P21881 Bacillus, P60089 Staphylococcus, P75390 Mycoplasma, O24457 chloroplastic Arabidopsis, Q7XTJ3 chloroplastic rice, P51267 seaweed, Q9R9N5 Rhizobium, O66112 Zymomonas, Q68XA9 Rickettsia, P08559 mitochondrial human, P35487 mitochondrial mouse, P16387 mitochondrial Saccharomyces, Q6Z5N4 mitochondrial rice, P52901 mitochondrial Arabidopsis, P52903 mitochondrial potato. The tree was constructed with the MEGA5 program using the neighbor-joining cluster algorithm (1000 bootstrap replicates). Scale bar, 0.2 substitutions/site.
The ALS sequences form a close cluster, and they belong to a larger group that includes AHAS and POX in the phylogenetic tree (Figure 1), indicating that ALS came from the same POXlike ancestor as AHAS did.55 However, mutations occurred in ALS; that is, amino acid substitutions took place and ALS became dispensed with the requirement for FAD, after separation from the AHAS evolutionary lineage. By structural evolution, ALS achieved great activity enhancement. For example, the turnover number of the ALS from Klebsiella pneumoniae is 533 s−1. This speed is about 10-fold faster than that of any other AHAS.58 POX, PDC, and PDH have their own main functions in different organisms. Acetoin and 2-acetolactate are only the byproducts of the three enzymes under certain circumstances. This side reaction has been well studied, for example, POX from Escherichia coli and Pediococcus pseudomonas;59 PDC from Zymomonas mobilis,60 yeast,56 mammalian liver,61 and very likely plant cells;49 PDH from Streptococcus mutans,62 animal tissues,63 and mammalian tumor mitochondria.64 Therefore, it is the general consensus from these studies that probably most,
yield 2-acetolactate with highest biological efficiency. Another major enzymatic reason for efficient acetoin biosynthesis in these bacteria is that they apply a specific enzyme, 2acetolactate decarboxylase (ALDC, EC 4.1.1.5), to catalyze the yield of acetoin from 2-acetolactate. Similarly, ALDC has not been found in eukaryotes or archaea. To avoid 2acetolactate accumulation during beer brewing, ALDC genes were often integrated into the chromosomes of brewer’s yeasts. AHASs were mainly found in bacteria, fungi, and plants. It has vital roles in the branched-chain amino acids (BCAAs) biosynthetic pathway. Each AHAS has a larger subunit and a smaller subunit, which perform catalysis and regulation functions, respectively. AHAS catalyzes the same reaction as ALS does but with comparatively much lower efficiency. The product 2-acetolactate is then channelled into the synthesis pathway of leucine and valine. AHAS also turns pyruvate and 2oxobutyrate into 2-aceto-2-hydroxybutyrate, which is an intermediate of the isoleucine biosynthesis pathway. Although AHAS evolved from an FAD-dependent POX-like ancestor,57 FAD works purely for structural integrity in AHAS. 6490
dx.doi.org/10.1021/jf5013902 | J. Agric. Food Chem. 2014, 62, 6487−6497
Journal of Agricultural and Food Chemistry
Review
if not all, TPP-dependent enzymes have the ability to form acetoin by condensing the hydroxyethyl group of the active acetaldehyde intermediate with an acetaldehyde acceptor molecule to give acetoin.59 The rate of acetoin formation by POX of E. coli is much slower than the oxidative reaction (approximately 1/10 of the oxidative rate).59 In yeast, the formation of acetoin and 2acetolactate was estimated to be about 1% of the total reaction catalyzed by PDC.56 Even though this kind of side reaction takes place at very low levels, the apparent widespread presence may account for important roles as discussed under Physiological Roles.
■
INTERCONVERSIONS AND DERIVATIVES As discussed above, acetoin can be generated from 2acetolactate by ALDC or the direct carboligation of pyruvate and acetaldehyde by POX, PDC, or PDH. 2-Acetolactate is not a stable molecule, and it can decarboxylate spontaneously to yield acetoin at acid pH or high temperature. Acetoin can also be generated from diacetyl by reductase. The formation of diacetyl in vivo is generally accepted to be the nonenzymatic oxidative decarboxylation result of 2-acetolactate.4 As a central intermediate, acetoin plays important roles in the production of various types of derivatives: diols, esters, glycosides, heterocyclic compounds, etc. (Figure 3). Most of the derivatives are of importance in foods due to their rich odors and potential dietotherapy functions. Diols, Esters, Glycosides, and Other Possible Enzymatic Derivatives. 2,3-Butanediol, the reduced form of acetion, is another regular product of the acetoin pathway in bacteria.5 The reversible transformation of acetoin and 2,3butanediol is catalyzed by acetoin reductase (2,3-butanediol dehydrogenase) in vivo. Acetoin’s precursor, 2-acetolactate, can be spontaneously decarboxylated to diacetyl. Even though this spontaneous decarboxylation remains in very low levels in different cultures, diacetyl can be easily detected, for example, by scent or odor due to its very low perceptible threshold (0.001 ppm in Emmentaler cheese).9 Therefore, it is not surprising that acetoin, 2,3-butanediol, and diacetyl often occur simultaneously in fermented foods, for example, in cheese9 and wine.17 In acetoin-degrading bacteria, diacetyl could have another choice of transformation other than the production of acetoin by reductase. In this case diacetyl is catalyzed by the acetoin dehydrogenase enzyme system (AoDH ES) to yield 3-hydroxy3-methyl-2,4-pentanedione (common name, acetylacetoin), which could be further reduced to a diol product, 3,4dihydroxy-3-methylpentan-2-one (common name, acetylbutanediol).4 Xiao et al. proposed a new biosynthetic method of acetylbutanediol from acetoin using Bacillus pumilus, a safe industrial species.65 Thereafter, a novel diol, butyrylbutanediol (2,3-dihydroxy-3-methylheptan-4-one), was discovered during a thermophilic fermentation study of acetoin and 2,3-butanediol.66 Butyrylbutanediol was postulated to be the synthetic product of acetoin and butyryl-CoA, following a similar synthetic mechanism as acetylbutanediol. However, the biosynthesis mechanisms of these diols largely remained unclear. These small molecular diols may have multiple usages in chemical synthesis, foods, and cosmetics due to their reactive hydroxyl groups, high water solubility, and hygroscopic properties.65 Dietary fruits usually have pleasant odors, and volatile esters account for a large portion of the aroma compounds. Alcohol
Figure 3. Generation pathways of acetoin and its derivatives. ALS, αacetolactate synthase, which catalyzes the conversion of pyruvate to 2acetolactate, was found only in bacteria. AHAS, acetohydroxy acid synthase, which catalyzes the synthesis of 2-acetolactate and 2-aceto-2hydroxybutyrate, was mainly found in bacteria, fungi, and plants. POX, pyruvate oxidase, which converts pyruvate to acetate and carbon dioxide as its main catalytic function, was reported only in bacteria. PDC, pyruvate decarboxylase, which converts pyruvate to acetaldehyde as its main catalytic function, was found from microorganisms to higher plants. PDH, pyruvate dehydrogenase complex, which catalyzes the decarboxylation of pyruvate to acetyl-CoA as its main catalytic function, exists from lower prokaryotes to higher organisms including human beings. POX, PDC, and PDH can transfer pyruvate to 2acetolactate and/or acetoin as a side reaction. NER, nonenzymatic reaction.
acyltransferases usually link alcohols to acyl moieties in the last step of ester biosynthesis. The hydroxyl groups of acetoin and 2,3-butanediol are also ready for the esterification reaction in some fruits. For example, plentiful acetoin esters and 2,3butanediol esters were detected in pawpaw fruit,18 and 2,3butanediol acetates were also detected in banana extracts.21 The odors and tastes of acetoin and 2,3-butanediol esters18 support the popular characteristics of these fruits. Acetoin acetate and 2,3-butanediol acetate are sometimes present in some particular flowers, vinegars, and wines as well.33 Acetoin was found to be capable of acting as an aglycone and exist in glycosidically bound form in some vegetables and fruits. These glycosides are nonvolatile but may contribute to the 6491
dx.doi.org/10.1021/jf5013902 | J. Agric. Food Chem. 2014, 62, 6487−6497
Journal of Agricultural and Food Chemistry
Review
As early as 1988, Rizzi reviewed the possible formation mechanisms of food-related pyrazines and expressed his preference for the second mechanism.76 In the same period, he observed the spontaneous formation of TTMP from acetoin and ammonium salts in water solution under mild conditions.77 If other kinds of acyloins were supplied, other kinds of alkylpyrazines would be produced. During further studies on the reactions between acetoin and ammonia precursors under mild conditions, another major product, 2-(1-hydroxyethyl)2,4,5-trimethyl-3-oxazoline (HE-TM-oxazoline), was identified.78 The dynamic processes of TTMP and HE-TM-oxazoline were recorded in a 3 week storage study, and the mechanisms were postulated (Figure 3). Mechanistic studies of Fu and Ho supported these postulates, and another two heterocyclic derivatives, 2,4,5-trimethyl-3-oxazoline and 2,4,5-trimethyloxazole, were identified from an acetoin/ammonium acetate aqueous system.79 Le Quéré et al. also hypothesized a chemical synthesis route of 2-alkyl-2,4,5-trimethyl-2,5-dihydrooxazole homologues in cheese from the precursors including acetoin, ammonia, and methyl ketones, which are abundant in cheeses.10 If sulfide is present in acetoin/ammonium reaction systems, similar sulfur-containing heterocyclic compounds will be produced.80 HE-TM-oxazoline was manifested to be relatively unstable, and TTMP would be the main product either under an elevated reaction temperature79 or in a prolonged storage time.78 The dynamics of TTMP and its precursors during the industrial production process of a Chinese vinegar were investigated using GC-MS.73 The results indicated that the main precursor of TTMP, that is, acetoin, was accumulated in the fermentation process, and then it condensed to TTMP during the subsequent storage period. The phenomenon is consistent with the “Biochem-Chem” route and the previous result of the 3 week storage dynamic study by Shu and Lawrence.78 Other kinds of alkylpyrazines and pentaheterocyclic compounds were often detected in acetoin-containing fermentation systems. For example, 2,3-dimethylpyrazine was found in tortillas26 and rice cakes,27 and 2,3,5-trimethylpyrazine was found in rice cakes27 and Chinese vinegars.11 During TTMP formation in a Bacillus fermentation process using glucose as the carbon source (actually the “Biochem-Chem” process), 2,3,5-trimethylpyrazine, 2-ethyl-3,5,6-trimethylpyrazine,72 and a homologous product of 2,4,5-trimethyloxazole, 2,4,5-trimethylimidazole, were detected.81 An extreme example is that a dozen kinds of alkylpyrazines, along with a very complex mixture of other volatile compounds, were also detected in a Bacillus subtilis fermentation process when cooked, roasted soybean cotyledons were applied as the substrate.82 These diverse products might be closely related to the selection and pretreatment of raw materials for fermentation. Soybean cotyledons are rich in complex compounds, and the cooking and roasting processes will increase the complexity significantly. Shu used acetoin to react with amino acids at 250 °C and showed that all reactions between acetoin and α-amino acids generate TTMP and the corresponding Strecker aldehydes, indicating that alkylpyrazines could also be generated during food processing.83 Then, acetoin, sometimes with diacetyl, could be responsible for the generation of many kinds of heterocyclic derivatives, but these derivatives should share a common characteristic: each molecule has at least two adjacent methyl groups, a clear hallmark from the molecular structure of acetoin. Other kinds of alkyl heterocyclic compounds could be the derivatives of
tastes. For example, acetoin was revealed as the major and key odor active aroma constituent (7.6 μg/kg) of ash gourd. Acetoin was also found to be the major constituent (24.00 mg/ kg) existing as glycosidic conjugate in ash gourd.25 In guar beans, the proportion of acetoin in free aroma constituents is even up to 95%, and acetoin is also the major component among the glycosidically bound aglycones in both guar beans (50%) and French beans (54%).24 In lychee fruit acetoin was postulated to be a major characteristic aroma compound, but only a trace amount of glycosidically bound acetoin was detected.22 Even until now, the formation mechanisms of these glycosides remain unknown. However, 2,3-butanediol-glucuronide was postulated to be the conjugation result of 2,3butanediol and uridine diphosphate glucuronide by glucuronyltransferase in rats37 and humans.38 Therefore, the glycosidic bound reaction of acetion might be an enzymatic process in plant cells. Acetoin could be precursors of other biological products. Speranza et al. postulated that the 2-butanol existing in distillates and wines most likely originated from 2,3-butanediol by the action of lactobacilli.67 Yokota and Sasajima studied the enzymatic transformation of acetoin and D-ribose 5-phosphate to a monosaccharide, 1-deoxy-D-altro-heptulose 7-phosphate, by a transketolase mutant of Bacillus pumilus.68 However, these two examples have no further or closely relevant reports. Heterocyclic Compounds. Acetoin-derived heterocyclic derivatives include alkylpyrazines, oxazolines, oxazoles, and their homologues. Pyrazines are extensively used in food, agriculture, and medicine. Their occurrence is nearly ubiquitous in nature, and the alkylated ones are the most abundant among pyrazines.69 According to the nature of the alkyl substituents, alkylpyrazines possess different flavors, but these flavors can be generally described as nutty, roasty, and toasty aroma.70 Oxazolines and oxazoles are responsible for green, sweet, and nutty aromas and have strong sensory quality even at low concentrations. They have wide occurrence in processed foods such as coffee, soy sauce, wheat, and cooked beef.71 2,3,5,6-Tetramethylpyrazine (TTMP) is one of the main pyrazines found in Chinese vinegars,73 tortillas,26 rice cakes,27 cocoa bean- or soybean-based fermented foods,72 etc. In these foods, acetoin, sometimes with 2,3-butanediol and diacetyl, will always appear accompanied by the emergence of TTMP. TTMP is also a GRAS substance with FEMA No. 3237. Furthermore, TTMP is a biologically active ingredient in the medicinal plant Ligusticum wallichii. In addition to the flavoring contribution to foods, TTMP has potential dietotherapy functions. For example, TTMP is held to be good for cardiovascular and cerebrovascular health74 and has a possible cognitive enhancement effect.75 Therefore, research on TTMP has attracted widespread interests. The formation mechanism of alkylpyrazines in vivo has two main controversial viewpoints: (I) The enzymatic condensation biogenic pathway. This viewpoint is supported by only a few authors.69 However, the enzymes or genes responsible for the condensation reaction have never been reported. (II) The “Biochem-Chem” route. The precursors of alkyl pyrazines are prepared by biosynthesis pathways in vivo, but the subsequent condensation is a nonenzymic process. This route has gained convincing experimental support and obtained wider approval. 6492
dx.doi.org/10.1021/jf5013902 | J. Agric. Food Chem. 2014, 62, 6487−6497
Journal of Agricultural and Food Chemistry
Review
Table 4. Electron Ionization (EI) and Chemical Ionization (CI) Mass Spectrometry Data of Acetoin and Its Derivativesa common name or abbrev acetoin acetoin acetylbutanediol butyrylbutanediol diacetyl acetoin acetate acetoin acetate acetoin butanoate acetoin hexanoate acetoin octanoate 2,3-butanediol 2,3-butanediol monoacetate 2,3-butanediol diacetate 2,3-butanediol monobutanoate 2,3-butanediol monohexanoate 2,3-butanediol monooctanoate 2,4,5-trimethyl-3oxazoline 2,4,5trimethyloxazole HE-TM-oxazoline HE-TM-oxazoline
IUPAC systematic name
ionization source
m/z (relative intensity)
3-hydroxybutan-2-one 3-hydroxybutan-2-one 3,4-dihydroxy-3-methylpentan2-one 2,3-dihydroxy-3-methylheptan4-one butane-2,3-dione 3-acetoxy-2-butanone 3-acetoxy-2-butanone 3-oxobutan-2-yl butyrate 3-oxobutan-2-yl hexanoate 3-oxobutan-2-yl octanoate
EI, 70 eVb CI, methane65 CI, methane65
88 (10), 73 (2), 55 (3), 45 (100), 43 (56) 89 (100), 71 (61), 45 (6), 43 (33) 133 (8), 115 (8), 89 (100), 87 (13), 73 (9), 71 (24), 45 (54), 43 (31)
CI, methane66
161 (4), 143 (6), 115 (100), 89 (79), 73 (58)
butane-2,3-diol 3-acetoxy-2-butanol
EI, 70 eVb EI, 70 eV33
86 (19), 43 (100) 130 (
