5
Amines, Amino Acids, and Proteins

5.1 Introduction

Characterized by the presence of a nitrogen atom with a lone pair of electrons, amines are a class of compounds derived from ammonia (NH₃) by replacement of one, two, or three hydrogens to yield primary, secondary, and tertiary amines, respectively. Additional substitution on the amino nitrogen with a hydrocarbyl group yields a quaternary ammonium cation whereas protonation of the amino nitrogen yields an aminium cation. Amines can also be present in molecules containing additional functional groups (e.g. carboxyl, carbonyl, thiol, etc.), and in grapes, the major amine‐group‐containing species are amino acids and their polymers (e.g., proteins).

5.2 Chemistry of amines

The presence of the lone electron pair results in amines and other amine‐containing compounds behaving as weak bases. Analogous to acids (Chapter 3), the predilection of bases to be protonated can be described by an equilibrium constant. It is common to report the pK_a value of the conjugate acid, where a higher pK_a value indicates a weaker conjugate acid and a stronger amine base. Values of pK_a for different amine classes are summarized in Table 5.1.

Table 5.1 Representative structures and acidity constants for weakly basic nitrogenous compounds

Compound class	Amine	Imine	Arylamine	Heteroaromatic amine
Typical pKa of corresponding weak acid	9–11	5–7	2–6	Pyridine (left): 5 Pyrazine (right): 0.6
Examples in wine	Amine group of α‐amino acids	2‐Acetyl‐3,4,5,6‐tetrahydropyridine	Methyl anthranilate	3‐Isobutyl‐2‐methoxypyrazine

The nitrogen lone pair of amino acids and many other amines is protonated at wine pH, and thus many well‐known food chemistry reactions that involve amine groups acting as nucleophiles are expected to occur very slowly, for example, the reaction of amino acids with sugars as occurs in Maillard reactions. Amines with low pK_a values will also have minimal volatility and thus no odor in wine.

Amino acids and polypeptides have multiple ionizable groups at wine and juice pH. As an example, simple amino acids with neutral hydrocarbon side chains (e.g., leucine, glycine) have two ionizable groups: the carboxylic acid (pK_a1 = ~2) and the amine (pK_a2 = 9–10 for the –NH₃⁺ conjugate acid). Application of the Henderson–Hasselbalch equation (Chapter 3) reveals that the majority of amino acids and proteins will exist as zwitterions (both positive and negative charges on the same molecule) in juice or wine.

The charge of a zwitterionic species at a given pH can be determined from its isoelectric point (pI), giving the following possibilities:

• pH < pI – the protein (or amino acid) has a net positive charge.
• pH = pI – the protein has no net charge.
• pH > pI – the protein has a net negative charge.

The importance of pI to wine and other food systems is that it defines the pH at which a protein will be at minimum solubility.¹ Most wine proteins are reported to have pI values just above wine pH, in the range of 4–6 [1], and will thus have a net positive charge in wine or juice. As a result, proteins can non‐covalently bond to species that have formal negative charges or are H‐bond acceptors, such as bentonite and polyphenols. These properties will be discussed more in Chapter 26.2.

5.3 Amino acids and related major nitrogenous compounds in wines

The major nitrogenous constituents of grapes and wines are summarized in Table 5.2, and include [2]:

• Ammonium (NH₄⁺), which represents a major source of usable nitrogen during fermentation.
• Amino acids, characterized by the presence of both a carboxylic acid group (R–COOH) and an amine group (R–NH₂ or R–NH–R′).
• Proteins, which are polymers of amino acids (i.e., large polypeptides) linked by amide groups (R–CONH–R′), and usually containing >100 amino acid monomers.
• Oligopeptides, typically defined as polymers containing 2 to 20 amino acid monomers, again joined through amide bonds.

Table 5.2 Concentrations of major nitrogenous species in must and wine

Nitrogenous compounds	Concentration in grape must [3–5]			Concentration in wine (mg/L)
	mg/L	mg/L as N	Contribute to yeast assimilable nitrogen (YAN)?
Ammonium	100 ± 45	79 ± 35	Yes	Lower than in must
α‐Amino acids (non‐proline)	843 ± 51	135 ± 51	Yes
Proline	Up to 4000	Up to 500	No	Similar to must concentrations
Glutathione (major oligopeptide)	15–100	3–15	Yes	ND – 27 [5]
Proteins	20–250	3–15	No	30‐275 [4,6]

5.3.1 Amino acids and ammonia

In must, the major soluble nitrogen forms exist as ammonium and free amino acids. Yeast require nitrogen for several roles – primarily as a component of proteins, cell walls, and nucleic acids² – and ammonium and free amino acids serve as the main nitrogen source during alcoholic fermentation. The structures of several representative amino acids in grapes are shown in Figure 5.1. The majority of amino acids in grapes and in wines are primary α‐amino acids; that is, the amine group is bonded to only one carbon (R–NH₂), and the acid and amine groups form bonds to the same carbon. Also, most of the amino acids in grapes and wines are proteinogenic, that is, they have an associated codon that utilizes them in the synthesis of proteins during transcription. A few non‐proteinogenic amino acids can also be found in must, especially γ‐aminobutyric acid (GABA), which can exist at concentrations up to 580 mg/L [7].³

Figure 5.1 Structures of major nitrogenous species in must: proline, primary α‐amino acids, and glutathione (tripeptide)

The predominant amino acid in musts is proline, which can exist at concentrations up to 4000 mg/L, followed by arginine, valine, and alanine [2, 8]. Not all forms of amino acids are equally useful to yeast as a nitrogen source, and in particular the secondary amino acids (proline and hydroxyproline) are not well used under anaerobic conditions. The “yeast assimilable nitrogen” (YAN) fraction is defined as [3]

(5.1)

The importance of YAN to nitrogen metabolism is described in more detail later (Chapter ). The major factors affecting must ammonium, amino acid, and YAN concentrations have been reviewed [2] and include cultivar and growing conditions, as well as pre‐fermentation additions of diammonium hydrogen phosphate (DAP) or other supplements.

Concentrations of ammonium and α‐amino acids in finished wines are typically lower than in must due to utilization by yeast [9]. A survey of 128 commercial wines found YAN concentrations ranging from 11–586 mg/L as N. Higher concentrations of YAN in finished wines may result from excessive must nitrogen supplementation [10] and are generally discouraged since they can promote microbial instability [11]. In wine, most amino acids exist at concentrations 1–2 orders of magnitude below their taste thresholds [12]. The only amino acids that approach their thresholds in model wine are proline (“sweet”) and glutamate (“umami”), but reconstitution studies with model wines showed that omitting all amino acids at usual wine concentrations had no effect on wine flavor [12]. Interestingly, proline in wines is reported to correlate with perception of “body” in dry white wines [13], although this may be because it serves as a maturity marker.

5.3.2 Oligopeptides

The best‐studied oligopeptide in grapes and wines is glutathione (GSH, Figure 5.1). GSH is a tripeptide formed from glycine, cysteine, and glutamine. GSH is produced by a wide range of microorganisms, plants, and animals, where it has important roles in preventing oxidative damage and in the metabolism of toxic compounds.

Factors affecting GSH in grapes and wines have been reviewed thoroughly [14]. GSH present in grapes (Table 5.2) can be utilized as a nitrogen source by yeast during fermentation. However, yeast will also synthesize considerable amounts of GSH during fermentation – the intracellular GSH content of S. cerevisiae can be up to 1% dry weight [15] – and some will be excreted. Although it has minor importance as a nitrogen source, GSH has a key role as a nucleophile in reaction with o‐quinones to inhibit browning and other oxidative reactions (Chapters 13 and 24), and with (E)‐2‐hexenal or other unsaturated aldehydes to form S‐glutathione conjugates (Chapters 10 and 23.2).

Yeast autolysis is known to release other oligopeptides into wine, but, historically, their contribution has not been well studied. Recently, an oligopeptide (MW < 3 kDa) formed by degradation of a heat‐shock protein during yeast autolysis was shown to have a sweet taste [16]. This observation may help explain why wines aged on lees are often described as being less astringent or acidic (“softer”) even though they have subthreshold concentrations of sugars.

5.3.3 Proteins

The protein concentration of white musts and wines is reportedly 20–250 mg/L [4] and 30–275 mg/L [4, 6], respectively, although these values are presented cautiously because common methods for protein analysis in wine can suffer from interferences [1]. Wine peptides with MW > 3 kDa, which includes all proteins, are reported to have no flavor at concentrations found in wine [16]. However, the major soluble proteins are heat‐unstable and can denature to cause haziness. The two major classes of grape proteins with molecular weights in the range of 21–32 kDa have been implicated in white wine haze [17], and are discussed in more detail elsewhere (Chapter 26.2).

• Chitinases, glucanases, and other pathogenesis‐related (PR) proteins, which are increased in response to disease pressure.
• Thaumatin‐like proteins, which are associated with grape ripening and berry softening.

S. cerevisiae lacks strong proteolytic activity and thus grape proteins are not used as a nitrogen source. However, most proteins are poorly extracted during fermentation, and likely are bound to the pomace and lees. Protein concentrations in white wines will be further decreased by the use of fining agents, particularly bentonite (Chapter ). Yeast‐derived proteins are not major contributors to the protein concentration of white wines [4]. However, mannoproteins (typically at concentrations of 100–150 mg/L, 30% protein) may be minor contributors to protein content, especially when added exogenously to inhibit potassium bitartrate crystal formation (Chapter 26.1).

Grape and yeast‐derived proteins in red wines are not as well studied as in white wines, in part because of the greater economic importance of protein haze in white wine and in part because common colorimetric methods for protein determination require modification for use in red wines. The protein concentrations of red wines are reported to range from 50 to 100 mg/L [18]. Proteins are well known to bind to grape or wine tannins (Chapter 14), a property exploited by winemakers to decrease tannins during cellar operations (Chapter ). The lower concentration of protein in red wines likely arises from binding to tannin during maceration. Conversely, very high concentrations of proteins in grapes have been correlated with lower concentrations of tannins in finished wines [19].

5.4 Nitrogenous compounds with health effects

5.4.1 Biogenic amines

In fermented products, biogenic amines describe those compounds produced by decarboxylation of amino acids. Commercial and wild yeasts have low ability to form biogenic amines, and their primary source in wines appears to be lactic acid bacteria [20], particularly some strains of the spoilage species Pedioccocus (Chapter 22.5). The major biogenic amines (histamine, tyramine, putrescine) in red wines are shown in Table 5.3. Total concentrations in red wines are usually <50 mg/L, about an order of magnitude less than concentrations observed in sauerkraut and many other lactic fermented foods. Because malolactic fermentation is less common in white wines, concentrations of biogenic amines in whites are typically lower than in reds (<4 mg/L total) [21]. The primary strategies to prevent biogenic amine formation have been reviewed elsewhere [22], and include:

• Using starter bacterial cultures without amino acid decarboxylases
• Preventing the growth of spoilage bacteria
• Decreasing the concentrations of amino acid precursors, that is, by avoiding excess nitrogen additions during fermentation (Chapter 22.3).

Table 5.3 Major biogenic amines in wines

Biogenic aminea (corresponding amino acid)	Structure	Representative concentrations [24, 26] Mean (range), mg/L
Putrescine (Ornithine)		19.4 (2.9–122)
Tyramine (Tyrosine)		3.5 (1.1–10.7)
Histamine (Histidine)		7.2 (0.5–26.9)
Indoleb (Tryptophan)		Sound wine: 1–10 μg/L Faulty wine: up to 350 μg/L

^a Common name.

^b Indole is not a classic biogenic amine in that it is not formed by decarboxylation of an amino acid. However, it does appear to be produced from tryptophan by microorganisms during fermentation through an unknown pathway.

Biogenic amines, particularly histamine, have well‐known adverse health effects and can lead to headaches, heart palpitations, diarrhea, and other ill‐effects at high concentrations [21]. They have been implicated as a potential cause of the “Red Wine Headache” phenomenon, and the European Union has recommended histamine limits of 10 mg/L, although the correlation between histamine and headaches is still not well established [23].

Finally, indole is not a classic biogenic amine, but does appear to be formed as a result of microbial metabolism of an amino acid (tryptophan). Unlike other biogenic amines, indole is a heterocyclic amine and will be predominantly in its volatile (and odorous) form at wine pH. Indole is detectable in most wines at concentrations of 1–10 μg/L [24]. Higher concentrations, up to 350 μg/L [24], appear to arise during sluggish fermentations, resulting in an off‐flavor described as “plastic” at concentrations in excess of 30 μg/L [25].

5.4.2 Ethyl carbamate

Ethyl carbamate (EC) is a known carcinogen and is present in detectable amounts in many fermented products [27]. In Canada, EC must be below 30 μg/L in table wines and 100 μg/L in dessert wines, while in the US a voluntary industry target of 15 μg/L has been established. Two key pathways have been identified for EC formation in wine: (i) a major pathway involving yeast catabolism of arginine to release urea and (ii) a minor amount formed from lactic acid bacteria degradation of arginine to citrulline [2]. Both urea and citrulline can undergo non‐enzymatic ethanolysis to yield EC (Figure 5.2). This conversion of EC from these precursors is non‐quantitative (<10%) and highly temperature dependent (Chapter 25). In a survey of 27 wines, increasing storage temperature from 20 °C to 40 °C resulted in a 20–40‐fold increase in the EC formation rate [28]. Additionally, factors that increase urea, citrulline, or their precursor (arginine) such as excessive vineyard fertilization will potentially increase EC in wine [2]. Several strategies for decreasing EC have been proposed, including enzymatic degradation of EC or urea, and the use of sorbents [29].

Figure 5.2 Reaction of urea with ethanol (left) to yield ethyl carbamate and ammonia (right)

5.5 Odor‐active amines

With the exception of indole, the amines listed above contribute negligibly to wine aroma because their nitrogen lone pair will be protonated at wine pH, rendering the amine non‐volatile. Two classes of grape‐derived amines have pK_a values < 3 either due to inductive effects (methoxypyrazines) or resonance (aniline derivatives), and are thus volatile at wine pH. Both of these compound classes are primary odorants (see Introduction chapter), and will contribute to varietal aromas. Another compound class, the microbially produced cyclic imines, are not volatile at wine pH (pK_a = 5–7), but can deprotonate at mouth pH to become odor‐active.

5.5.1 Varietal amines – methoxypyrazines and “foxy” aniline derivatives

The 3‐alkyl‐2‐methoxypyrazines, also called methoxypyrazines, pyrazines, or MPs, possess odors generally described as “vegetal” or “earthy” (Table 5.4). These compounds have some of the lowest odor thresholds (~1 ng/L) of any compound found in wine. The MPs most often reported in grapes above their sensory thresholds are 3‐isobutyl‐2‐methoxypyrazine (IBMP, “bell pepper”) and 3‐isopropyl‐2‐methoxypyrazine (IPMP, “peas”). Other MPs, including 3‐sec‐butyl‐2‐methoxypyrazine (sBMP), 3‐ethyl‐2‐methoxypyrazine (EMP), and 2,5‐dimethyl‐3‐methoxypyrazine (DMMP) are also reported in grapes and wines, but usually at concentrations below their respective odor thresholds [30,31]. In addition to grapes, IPMP and several other MPs in wines can also derive from contamination of ferments by insects like the multicolored Asian ladybeetle (MALB, Harmonia axyridis) or the 7‐spot ladybeetle (Coccinella septempunctata) [32]. Finally, 2‐methoxy‐3,5‐dimethylpyrazine (“musty, fungal”), has been identified in tainted corks, and will be discussed in more detail in Chapter 18.

Table 5.4 Summary of properties for key varietal amine odorants in wines

Name	Structure	Typical range in Bordeaux‐grape varietals [33–36]	Threshold in wine [33, 37, 38]	Odor descriptor
3‐Isopropyl‐2‐methoxypyrazine (IPMP)		<0.5–5.6 ng/L	0.3–2 ng/L	Asparagus, earth, peas
3‐Isobutyl‐2‐methoxypyrazine (IBMP)		4–30 ng/L	2 ng/L (detection) 8–16 ng/L (recognition)	Bell pepper, vegetal
Methyl anthranilate (MA)		600–3000 μg/L (labruscana) 0.06–0.6 μg/L (vinifera)	300 μg/L	Artificial grape
ortho‐Aminoacetophenone (o‐AAP)		8–12 μg/L (labruscana) <0.5 μg/L (vinifera) 0.8–13 μg/L (ATA)	0.5 μg/L	Corn tortilla, mothball, acacia

While MPs may add complexity or typicity to some wine styles, for example, Sauvignon Blanc, winemakers are often interested in avoiding excess MP concentrations to avoid masking fruity aromas, especially in red wines [39].

The concentration of MPs in grape berries is well‐known to be dependent on genotype, with suprathreshold MP concentrations most often observed in the so‐called “Bordeaux” varieties like Cabernet Sauvignon and Sauvignon Blanc. MPs reach a maximum concentration about 1–2 weeks prior to veraison and degrade during maturation. Generally, lower MP concentrations at harvest are correlated with warmer, drier, and longer growing seasons, well‐exposed clusters pre‐veraison, and less vigorous sites [40]. Within grapes, MPs are located mostly in the skins and stems, and are readily extracted during skin fermentation [41]. MPs appear to be stable post‐fermentation, as wine age is not correlated with MP concentration [42], and widely used fining agents cannot selectively remove MPs [43]. MPs are expected to be less reactive than the major aromatic ring‐containing compounds in wines – the polyphenols – because the ring nitrogens of MPs are more electronegative and will make the ring less susceptible to electrophilic addition (Chapter 11).

Two aniline derivatives, methyl anthranilate (MA, “artificial grape”) and o‐aminoacetophenone (o‐AAP, “acacia, mothball”) are found in supra‐threshold concentrations in large‐berried American grape species and their offspring, for example, V. labruscana cultivars like Concord and Niagara (Table 5.4). MA and o‐AAP are critical compounds for the so‐called “foxy” aroma of labruscana and related American grape species,⁴ MA can be below the sensory threshold in certain foxy‐smelling grapes (e.g., Catawba, V. rotundifolia) [47], with more recent work implicating o‐AAP as the key foxy‐smelling compound [45].⁵ Other non‐nitrogenous compounds such as furaneol and methylfuraneol (“strawberry, caramel”) may contribute to the native character of American grapes [49], although these compounds are more often observed as sugar degradation products of toasted oak (Chapter 25). MA and o‐AAP are detectable in vinifera wines but usually at subthreshold concentrations. One exception to this statement is wines afflicted by so‐called atypical aging (ATA), referred to as UTA in German (Untypische Alterungsnote) [50]. Early work on ATA/UTA suggested that drought stress and nitrogen deficiency induced formation of indole‐3‐acetic acid (IAA) in grapes, and studies on model solutions suggested an oxidative pathway to form o‐AAP from IAA during storage, although more recent investigations have been more equivocal about these hypotheses [50].

MA is highly stable – supratheshold concentrations (2–3 mg/L) can be observed in 5 year old Concord and Niagara wines [47], comparable to concentrations observed in Concord grapes and young Concord wines. Based on thermodynamic predictions, methyl anthranilate should be lost and anthranilic acid and ethyl anthranilate formed during storage via acid‐catalyzed hydrolysis and ethanolysis (Chapter 25). The slow kinetics of MA solvolysis as compared to aliphatic esters may be due either to resonance stabilization of the carbonyl group (so the carbon has less partial positive charge) or because of steric interference (from the ortho‐substituent blocking access to the carboxyl group) [51].

5.5.2 “Mousy” imines

Several cyclic imines, shown in Table 5.5, have been detected in wines: 2‐ethyl‐3,4,5,6‐tetrahydropyridine (ETHP), 2‐acetyl‐3,4,5,6‐tetrahydropyridine (ATHP), and 2‐acetylpyrroline (APY). Although non‐volatile in wine, these compounds can deprotonate and become volatile in the mouth, resulting in “mousy,” “cardboard,” or “cracker” retronasal aromas. These compounds had been previously identified in baked and roasted products like bread crust, where they can contribute positively. They are formed from polynitrogenous amino acids (e.g., ornithine, lysine) by spoilage organisms, and will be discussed in more detail later (Chapter 22.5).

Table 5.5 “Mousy” cyclic imines in wine

Mousy‐smelling compound	Structure	Retronasal threshold (μg/L) [52]	Maximum reported concentration (μg/L) [52]
2‐Ethyl‐3,4,5,6‐tetrahydropyridine (ETHP)		150	162
2‐Acetyl‐3,4,5,6‐tetrahydropyridine (ATHP)		16	108
2‐Acetylpyrroline (APY)		0.1	7.8

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Notes

1 The first step of most cheesemaking processes is to form a casein protein precipitate (“curds”) in milk either by decreasing the milk pH through acidification or by increasing the pI of the casein by the action of a protease. Both approaches bring the pI of casein closer to the pH of the milk.

2 Inspection of the nutritional data on a bread yeast packet reveals that yeast is about 50% protein by dry weight. Approximately one-sixth of the weight of protein is due to nitrogen.

3 GABA can be metabolized to γ-butyrolactone (GBL), discussed later in a Chapter 7 footnote for its role as a “date-rape” drug. GABA concentrations naturally found in wine are typically much less than concentrations used to adulterate drinks (2000 mg/L or more).

4 The “foxy” descriptor was applied to large-berried wild American grapes like V. labrusca and V. rotundifolia (Muscadine) as early as the 1620s by European settlers [44]. The most probable explanation is that Europeans associated the aroma of these grape species with the musky aromas of the European foxes. This hypothesis is given more credibility by the fact that o-AAP is present in the anal sac of carnivores like the Japanese weasel [45], and MA and o-AAP are both bird deterrents [46]. Several other characteristics (large berries, thick skins, downward growth, thick pulp, dull color) support the hypothesis that large-berried grapes were not selected for by birds, but instead by small mammals like raccoons.

5 In 1964, the discovery of o-AAP as a key off-flavor in stale dry milk was sufficiently exciting to justify publication in the prestigious journal Nature [48].