Melanoidins

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Melanoidins, which consist of tan and brown compounds, are formed via Maillard reactions during kilning, mashing and wort boiling and have been detected in beer.8 Melanoidins play an important role in the formation of color, aroma and flavor, foam stability and can influence the oxidation–reduction processes of beer due to their strong antioxidant properties and other biological effects.

Melanoidins exhibit both antioxidant and pro-oxidant properties, which contribute to the stabilization of color, aroma, flavor and foam.

Melanoidins formed as a result of heat treatment of the malt made from reductive sugars and amino acids or proteins contribute to the forming of the antioxidative activity of worts and the final beers [16–18]. Colored macromolecules formed in the final stage of the Maillard reaction are subjected to a number of studies consequential to their interesting biological properties and unknown chemical structure [19]. Melanoidins positively affect antioxidative abilities of food products, as well as storage stability. The mechanism of their antioxidative activity relies on the ability to break chain reactions of radicals, chelation of metals, H 2 O 2 reduction and scavenging of free radicals [16,17]. Melanoidins, apart from their antioxidative properties, also demonstrate antiviral and antimutagenic activity, and the ability to reduce cholesterol levels and stimulate growth of intestinal bacteria. However, some of the compounds, formed during early stages of the Maillard reaction process, are considered carcinogenic and mutagenic. Therefore, it is difficult to explicitly decide which of the activities are dominant [20,21]. One of the compounds formed during the heating of the malt is 5-hydroxymethylfurfural (HMF), which is also formed during thermal treatment of food, e.g., malt, dried fruit, fruit juices, coffee, bread or vinegar. Toxic effects of HMF consumption have been proved after receiving a dose of 75 mg/kg of body weight [22]. Compared to other food products, the HMF content in beer is relatively low.

During roasting, the main chemical reactions are the Maillard reactions. These are a series of changes initiated by the reaction between reducing sugars and amino groups, leading to the formation of compounds responsible for the color and flavor of heat-treated products. The non-enzymatic browning reactions can be divided into several stages. In the first of them, the early phase reaction products are created. They are mainly products of the Amadori rearrangement. The second step involves the formation of intermediates such as: HMF, Strecker aldehydes and pyrazines. The last, third stage is the formation of the melanoidins, which are reaction end products [39]. Melanoidins are a group of compounds with a very diverse structure. They differ in chemical properties depending on the origin. Melanoidins give beer its color and provide it with antioxidant activity [40].

An increase of melanoidins fosters the Strecker degradation of amino acids and the oxidation of alcohols.

Melanoidins are macromolecular, nitrogenous and brown colored products of Maillard reactions, which are formed during the malting and brewing process. The Maillard Reaction (MR) is a complex network of reactions summarized in Figure 5. The initial stage involves the condensation of a carbonyl group, mostly form a reducing sugar, with a free amino group within peptides or proteins. This results in an Amadori rearrangement product, which can react further to give colored, low molecular mass products and melanoidins.

Maillard reaction products change depending on the time and temperature applied during the brewing process. The formation of high-molecular-weight (HMW) compounds occurs during the final stages of the Maillard reaction by polymerization of highly reactive intermediates [86,87]. Several works have demonstrated that malt roasting induces the polymerization of early-formed low-molecular-weight compounds (LMW) (< 10 kDa) into HMW brown compounds (> 300 kDa), reason why the content of LMW in roasted malts is lower than in pale malts [87,88]. Therefore, pale and caramel malts are characterized by light brown LMW colorants while roasted malts are characterized by intense brown HMW [88,89,90].

The most important free MRPs in beer are fructosyllysine (6.8–27.0 mg/L) and maltulosyllysine (3.7–21.8 mg/L). In addition, the analyzed beers contained comparatively high amounts of late-stage free MRPs such as pyrraline (0.2–1.6 mg/L) and MG-H1 (0.3–2.5 mg/L) and minor amounts of formyline (4–230 μg/L), maltosine (6–56 μg/L), and argpyrimidine (0.1–4.1 μg/L) [97]. Perlolyrine (which is a Maillard reaction product from tryptophan), has also been identified in various kinds of beer.

Although melanoidins are consumed regularly as part of the daily human diet, they are generally considered poorly absorbable and poorly bio-available compounds (and therefore are not a significant source of dietary antioxidants like phenolic compounds).

There are various mechanisms by which Maillard reaction products may act as antioxidants: oxygen scavengers, reactive oxygen scavengers, reducing agents and metal chelating agents [92]. Traditionally, the use of colored malt is known to improve the stability of the finished beer, and it has also been shown that more-highly colored beers retain a greater reducing power during storage [87]. Several works carried out showed the existence of positive correlations between antioxidant activity and malt color, which have been ascribed to the presence of Maillard components [87,95]. More recently, Zhao et al. (2013) [94] also found positive correlations between melanoidin content in beers and antioxidant capacity.

During malting, the development of MRPs during thermal processing has been associated with a pro-oxidative effect and with a negative effect on the oxidative stability of malt and beer. The mechanisms behind anti/prooxidant effects of MRPs are still unclear since their structures are still unknown. The mechanisms are assumed to be based on their ability to trap positively charged electrophilic species, to scavenge oxygen radicals, to have reducing power, and to chelate metals to form inactive complexes.

The Maillard reaction is a non-enzymatic chemical reaction. Mela­noidins are final products of the Maillard reaction, have been widely regarded as the most important antioxidants in Maillard products, and are important sources of antioxidants in food. In the process of beer production, malt withering (86 ◦ C), kilning (250 ◦C), mashing and wort boiling processes can produce melanoidins, ketones and other antioxidant substances through the Maillard reaction. the AOX of barley increase with the intensity of heating, in parallel with color for­ mation. The content of melanoidins with HMW in wort and beer prepared from black malt are significantly higher, and could effectively inhibit the isomerization of xanthohumol, thus improving the AOX of beer.

The free radical scavenging ability of melanoidins has been confirmed through the study of electron spin resonance (ESR) of dark beer. Effective chelation of melanoidins to iron and copper ions has also been found. However, experiments in a model system using sulfites (the main antioxidants in beer) found melanoidins showed a pro-oxidation reac­ tion and may also increase the degree of oxidation and free radical content of dark beer (Cortés, Suárez, & Methner, 2010; Nøddekær & Andersen, 2007). For example, the Maillard reaction is also accompa­ nied by the formation of advanced glycation end products (AGEs), greatly increasing the free radicals in the reaction system. (Poulsen, Andersen, Nielsen, Skibsted, & Dragsted, 2013). Therefore, the anti­ oxidation and pro-oxidation properties of Maillard products are worthy of further study.

melanoidin content has been found to increase by 25% after the wort is boiled, indicating that a violent Maillard reaction takes place during the boiling process, which is the main stage of melanoidin for­mation (Zheng, 2017). Melanoidins carry xanthohumol; the combina­ tion of the two can prevent the isomerization of xanthohumol in the boiling process, and combination with xanthohumol does not signifi­cantly affect the content of melanoidins in wort. The ABTS radical scavenging activity of boiled wort (with no hops??) has been found to be 51% higher than that at the end of the mashing stage (Zheng, 2017), indi­cating the melanoidins produced in the process of boiling exert strong scavenging of ABTS free radicals.

Thermal treatment of malt may result in nonenzymatic browning also known as Maillard reaction (Coghe and others 2004, 2005, 2006; Yahya and others 2014). As illustrated in Figure 3, MRPs can result from the reaction of reducing sugars with amino acids and amino groups of peptides or proteins, involving a cascade of consecutive and parallel reactions, resulting in a complex mixture of compounds (Morales and others 2005; Wang and others 2011). Formation of MRPs largely depends on time and temperature applied during kilning and roasting. It has been shown that conditions of intermediate moisture content and moderate temperatures favored aqueous-phase Maillard reactions, while lower moisture contents (less than 2%) and high temperatures (200 °C) led to extensive pyrolysis reactions and generations of compounds such as maltol and methylpyrazine (Yahya and others 2014). --see article for a flow chart of melanoidin formation

HMW brown compounds formed in the late stages of the Maillard reaction are often referred to as melanoidins (MLDs). MLDs can be defined as polymeric nitrogenous compounds of HMW with high reducing potential and an intense brown color, responsible for the brown color development in roasted malts (Morales and others 2005; Echavarr´ıa and others 2012). However, it has also been reported that the occurrence of sugar–sugar caramelization, involving aldolization/dehydration products of sugars, may be linked to proteins or other sources of amino nitrogen (Rizzi 1997). Cammerer and others (2002) have found that intact carbohydrate structure can be part of the MLD skeleton in model systems obtained under water-free reaction conditions, similar to roasting conditions. It seems that the Maillard reaction under water-free conditions can induce the incorporation of a relevant amount of dimer- and oligomer carbohydrates with intact glycosidic bond into the MLD skeleton with consequent side chains formation (Cammerer and others 2002). MLD with a carbohydrate-based skeleton (Figure 4A) can also be formed in water-free conditions due to aldol condensations of α-dicarbonyl compounds and from transglycosylation reactions of saccharides (Cammerer and others 2002; Wang and others 2011). Amino acids may well react with the unsaturated carbonyl structure to form MLDs with amino compounds (Figure 4B). The sudden formation of HMW MLDs also coincides with the abrupt decrease of the level of vicinal diketones and aldehydes, indicating a possible involvement of these compounds in the polymerization reactions and formation of advanced HMW MLDs (Coghe and others 2006). Additionally, phenolic compounds, ascorbic acid, and other carbonyl compounds may also take part in the Maillard reaction itself, a reason why their contents can decrease during heat treatments (Rizzi 1997).

Malt obtained by using the hottest kilning regimen possessed higher antioxidant activity due to higher levels of MRPs (Inns and others 2011). Also, antioxidant contribution of MRPs was higher for malts kilned using a rapid regimen (Woffenden and others 2002). Even though the development of radical-scavenging and reducing activities coincided with color formation in the early caramelization phase, higher roasting temperatures did not continuously produce MRPs with antioxidant activity (Coghe and others 2006). Herein, the existence of at least 2 types of Maillard reactionrelated antioxidants in malt has been proposed: redox indicatorreducing antioxidants and radical-scavenging antioxidants (Coghe and others 2003). In fact, MRPs can contribute to the antioxidant capacity of malt due to their metal-chelating properties, reducing power, and radical-scavenging properties (Sovrano and others 2006; Wang and others 2011; Echavarr´ıa and others 2012). Accordingly, the antioxidant properties of kilned and roasted malts have been demonstrated, in several cases, as having the capacity to quench radicals or to reduce redox indicators (Woffenden and others 2002; Coghe and others 2003; Samaras and others 2005). Coghe and others (2003) have suggested that the initial steps of the Maillard reaction led to the production of antioxidants with quenching properties, while redox-reducing antioxidants are formed during malt color development and the late stages of the Maillard reaction. Heat-induced advanced HMW MLDs have shown a 4-fold higher reducing power and a 3-fold higher antioxidant capacity, as tested by the metmyoglobin assay, compared to LMW compounds. As demonstrated, they can act as antioxidants by scavenging radical species or by having reducing properties (Carvalho and others 2014). However, during mild and intermediate roasting only reductive capacity increased while intensive roasting led to an increase of DPPH radial-scavenging capacity. During continuous roasting at high temperatures (above 157 °C) the redox indicator-reducing capacity stagnated while scavenging properties decreased (Coghe and others 2006). The soluble HMW fraction (>10 kDa) isolated from Maillard reaction model systems and beer by ultrafiltration are able to scavenge hydroxyl radicals, but no correlation between browning and scavenging efficiency was found, meaning that chromophore residues linked to MLD are not responsible for the observed effect (Morales 2005). Moreover, Morales (2005) has shown that beer-isolated MLD displayed the same properties of model MLD obtained from the combination of sugar (glucose or lactose) with amino acids. However, another study has shown that chromophores linked to the beer MLD skeleton largely contribute to their peroxyl radical-scavenging properties (Morales and Jimenez-P ´ erez 2004). Malts roasted using temperatures above ´ 150 °C exibith lower antiradical activity comparing to malts roasted at lower temperatures for longer periods (Coghe and others 2006). This indicates that structural groups responsible for the antiradical properties are involved in the advanced stages of nonenzymatic browning reactions that occur at high temperatures. So, according to Coghe and others (2006), the maximum antiradical activity appears to be more related to a specific endtemperature than to a specific malt color. The higher reducing power observed for roasted malts could also come from the reaction between phenolic compounds with MRPs (Samaras and others 2005; Bekedam and others 2008). In coffee, the levels of caffeic accids correlated with melanoidin levels, indicating that they are incorporated in melanoidins not linked via its carboxyl group (Bekedam and others 2008) . Samaras and others (2005) have also found that ferulic acid can react with Maillard reaction intermediates, which are formed from glucose and proline (the most abundant free amino acid in malt) at kilning temperatures, leading to higher antioxidant activity. However, LMW compounds bound to MDL have exhibited higher antioxidant activity than pure MLD to which they are linked. Nevertheless, no correlation between color and antioxidant activity was found, except for ABTS (2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay, supporting the idea that MLD chromophores are not responsible for these actions (Rufian-Henares and Morales 2007).

MLD has been reported to exert both antioxidant and pro-oxidant activity (Woffenden and others 2002; Carvalho and others 2014). In fact, some studies suggest that MRPs exhibit antioxidant properties with a positive influence on the oxidative stability of wort due to its reducing properties (Coghe and others 2003, 2006), while other studies suggest a negative influence of these compounds on malt and beer stability (Nøddekær and Andersen 2007; Cortes and others 2010; Hoff and others 2012; Kunz and others 2013; Wunderlich and others 2013)

higher the amount of caramel malt, the higher the antioxidant activity.

Melanoidins contribute to the antioxidative activity of malt. However, there is a debate about whether intermediates of the Maillard reaction and Maillard reaction products such as melanoidins have pro- or antioxidative character. Reductones and melanoidins have high antioxidative capacities. On the other hand, intermediates in the Maillard reaction can markedly enhance the formation of specific aldehydes during beer storage, and blockage of α-dicarbonyls yielded a significantly lower accumulation of these aldehydes. There is a correlation between the content of Maillard reaction products in special malt, caramel beers, stout and an increase of radicals in the Fenton reaction assay resulting in an acceleration of prooxidative action. In this context there is a reaction mechanism of specific intermediate Maillard reaction products with reductone/enediol structure on prooxidative processes caused by their reduction potential against oxidised metal ions, especially Fe3+, resulting in an acceleration of oxygen activation and the generation of highly reactive oxygen species (ROS) such as •OH radicals.

Dark malts kilned at high temperatures are responsible for higher concentrations of radicals in wort and beer, causing lower oxidative stability in dark beers compared to pale beers (Cortes and others 2010). Dark worts are less stable with high radical intensities and high iron content, contrary to light worts that were less reactive toward oxidation with low radical intensity and low iron content. Recent studies demonstrated a direct correlation between the content of MRPs and higher reducing power of roasted malts, as well as higher levels of radicals measured by ESR spectroscopy. Furukawa Suarez and others (2011) showed that specialty malt ´ leads to a decrease of endogenous anti-oxidative potential of beer, related to an increase of the reducing power and the reductone/endio structure of MRPs. However, MRPs rapidly reduce oxidized metallic ions, such as Fe3+ to Fe2+, leading to oxygen activation and intensification of the Fenton–Haber–Weiss reaction system. Consequently, the oxidative processes are accelerated and the formation of reactive radicals is increased (Kunz and others 2013). Wunderlich and others (2013) have also shown that the development of radical formation and reducing power are linked during roasting. More recently, it was also demonstrated that MLD can induce a pro-oxidant effect in a Fenton system, leading to a decrease of the oxidative stability of malt worts, due to the catalytic formation of hydroxyl radicals in the presence of ferric ions in a Fenton reaction-based system (Carvalho and others 2014). Transition metals have a significant effect on the oxidative stability of malt and beer since they can act as catalysts in radical generation and oxidation reactions. The decreased lag phase for radical formation and reduction of the oxidative stability of beer after the addition of MRPs can be caused by reactions that are able to induce the formation of radicals by means other than iron-catalyzed reactions. Other study suggested a mechanism of auto-oxidation of MRPs. MLD are able to quench hydroxyl radicals, but are not able to reduce Fe3+, proving there is no effect in the reducing of iron in Fenton-type reactions (Morales 2005). The pro-oxidative effect of MRPs probably involves other mechanism than the Fenton catalysis, since stout beer was able to decrease the levels of radicals and the lag phase for formation of radicals in a beer model system based in a Fenton chemistry and measured by spin trapping and ESR spectroscopy, but not as much as lager beer (Nøddekær and Andersen 2007). It was also proposed that, since polymerization process can involve different groups with radical-scavenging properties, it can lead to a decrease in the overall radical-scavenging capacity due to the involvement of antiradical compounds in the formation of MLD (Coghe and others 2006).

Melanoidins are not only capable of scavenging superoxide to a greater extent than is ascorbate, but they are also capable of interaction with peroxide and hydroxyl (38). Again, the melanoidin radicals may serve only the purpose of carrying oxidative potential onto a further molecule. Compare this with Hashimoto's proposals that whereas melanoidins suppress the oxidative degradation of isohumulones and unsaturated fatty acids, they promote the oxidation of amino acids and higher alcohols (37). The concept that whole chains of electron carriers may carry oxidizing potential through to potentiate staling in low-oxygen final beer was discussed many years ago (22,23). More recently, chemiluminescence has been used to detect free radical formation in beer without any firm indication of the chemical nature of such radicals (49).

Higher alcohols are not oxidized to aldehydes unless tnelanoidins are present (37). Indeed, melanoidins can oxidize higher alcohols even in the absence of oxygen. It seems that melanoidin radicals, formed by the oxidation of ^–C=O groups to O—C^–, are responsible. Such melanoidin radicals also participate in the Strecker degradation of amino acids, with the formation of the corresponding aldehyde. By contrast, Hashimoto claims that the electron donor activity of melanoidins suppresses the oxidative degradation of iso-a-acids and the autoxidation of unsaturated fatty acids. Conversely, catechin has been shown to suppress the melanoidin-promoted oxidation of ethanol (24). The situation is complex. It is, however, difficult to conceive of beers produced without the presence of the very substances that can (at least potentially) break down to give staling aldehydes. Elimination of higher alcohols and iso-alpha-acids is not a practical alternative. Attention to the minimization of oxygen levels and, probably more importantly, the minimization of oxygen activation potential, is the only realistic course of action. By preventing oxidation of polyphenols and, perhaps through them, the reducing power of melanoidins is maintained.

Regarding Maillard reaction products, Andersen et al. (2000) found them to be pro-oxidants, whereas Bright et al. (1999) and Coghe et al. (2003) described the benefit to flavor stability of these materials, especially from more roasted malts. Melanoidins, like phenolic compounds, can absorb radicals to form relatively stable products, essentially halting the oxidation chain reaction.

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