3.1. Analysis of Non-volatile Parameters
Results for the non-volatile analyses of beers are shown in Table 4
. We can see significant differences (p
< 0.05) among all samples on every parameter analysed (ABV, IBU, TAC and TPC).
The content of alcohol (ABV) was significantly higher in beers that contained barley malt than in those made only with corn malt. This might be explained as corn has shown a low diastatic power compared to barley [5
], which leads to wort contained less fermentable sugars and thus, less alcohol content. As the brewing process remained under the same conditions for all beers, it was surprising to find that the bitterness unit (IBU) in beers were significantly different only for the blended beer made of red corn and barley malt (15.72 IBU) and the blue corn beer (14.57 IBU). These beers showed lowest IBU than the rest of beers (ranged between 15.7 to 19.6). The IBU is a measurement of how much iso-α-acids (1 IBU = 1 ppm iso-humulone) is in the final product, but it does not always really tell if a beer is bitter or not [24
]. The amount of iso-α-acids in the beer depends on the time and temperature the hops spend in the boiling step [25
]. Thus, minor changes in temperature or time the hops are added to the wort could change the amount of iso- α-acids in beer. Additionally, some authors have reported the susceptibility of this method to the interference from other compounds present in beer, such as polyphenols, that absorb light at the wavelength of measurement (275 nm). Therefore, minor contributions from compounds unrelated to bitterness can be detected (oxidised fatty acids), whereas others contributing to bitterness are not detected [36
]. Moreover, coloured beers absorb light which directly decrease the emission intensity and result in lower IBU values [37
]. Despite limitations, the IBU method is widely used as an indicator of bitterness in quality control [24
Beers containing only corn malt showed a higher content of anthocyanins (TAC) than those blended beer made of barley and corn malt. The anthocyanins value for beers made of blue corn and red corn malt varied from 14.6 to 8.84 mg C3G/L respectively. These results are in agreement with Flores-Calderón et al. [5
] who assessed different styles of blue corn beer and reported values that ranged from 13.2 to 18.7 mg C3G/L. A significantly higher difference between beers made of blue corn malt than the one made of red corn malt is expected as a greater amount of anthocyanins has been reported in varieties of blue corn than in the red corn variety [38
]. Also, as was expected, the beer made of 100% barley malt did not show presence of anthocyanins. Red and blue corn contain anthocyanins, such as pelargonidin-3-glucoside and cyanidin-3-glucoside, which are responsible of the colour of the grains. Additionally, these anthocyanins have been reported to have various biological activities, such as antioxidant, antimicrobial, antimutagenic and anticancer effects [3
]. Regarding sensory profile, presence of anthocyanins in beer not only has an effect on colour (ranging from amber-red-cooper) but also on taste and mouthfeel as these compounds could contribute with bitterness and astringency attributes. Thus, the presence of anthocyanins in pigmented corn beers could improve the quality of these beverages.
Finally, all the beers showed considerable amounts of total phenolic content (TPC). The main polyphenols present in a typical barley beer are hydroxybenzoic, cinnamic and ferulic acids. Malt is the main source of polyphenol compounds, providing 70 to 80% of them. Also, a small proportion is originated from hops (20–30%), such as α- and β- acids and their isomeric forms [36
]. In beers made of pigmented corn malt, the presence of polyphenols is also expected. Blue and red corn also have shown the presence of phenolic compounds such as cyanidin-3-glucoside and pelargonidin-3-glucoside, respectively. In addition, ferulic acid and p
-coumaric acid could be found in these varieties of corn [5
]. The results showed significant difference between beers. Higher quantities of TPC were found in those beers made of blue corn malt (BC) and the blended beers made of red and blue corn and barley (RCBa, BCBa). The value of polyphenols ranged between 398.5 to 750 mg GAE/L. Other studies have shown similar results for beers made of blue corn (342 to 560 mg GAE/L) [5
] and traditional beers made of barley malt (152.0 to 339.12 mg GAE/L) [40
]. The differences of the total content of polyphenols may be explained by the variation in the quantity and quality of raw material, the brewing process and the storage conditions during ageing. Polyphenols provide beer with bitterness and astringency but also improve its functionality in terms of foamability, oxidative stability and heat stability which help to preserve the beverage during storage and ageing [39
3.2. Volatile Composition
One hundred and twenty-one volatile compounds were identified in beer samples by HS-SPME/GC-MS. The chromatographic data of the volatile compounds of each beer is summarised in Table 2
. Compounds were classified into 12 groups of which, the most abundant include esters, representing ~29% of the volatiles, followed by alcohols (~20%), terpenes (~15%) and phenols (~6%). These compounds, particularly alcohols and esters have been the most reported volatiles in barley beers [42
]. The major volatiles detected in this study were consistent with those of previously published studies [11
As mentioned before, esters were the largest group found in all beer samples. Esters are the most common compounds in the majority of beers and these volatiles are considered desirable as they act in synergy with other compounds and contribute with most of the pleasant fruity-floral aromas in beer [44
]. According to our results, it seems that beers made with barley malt contain higher number of esters than the beers made with corn malt (Table 2
). For instance, ethyl propanoate, ethyl isobutanoate, ethyl pentanoate, ethyl isohexanoate, ethyl benzoate and isopropyl palmitate were only found in beers made with barley (Ba, BCBa, RCBa). It is well known that the presence of alcohols leads the production of esters [44
]. Thus, the presence of a greater number of esters in barley beers could be attributed to their content of alcohols, which are precursors of these compounds.
Esters such as ethyl acetate, 3-methylbutyl acetate, ethyl hexanoate, phenethyl acetate, ethyl 9-decenoate and ethyl decanoate were found in all samples in higher abundance than the rest of the esters.
Ethyl octanoate, a product of fermentation by Saccharomyces
yeast, was detected in all five beers that contain corn malt except in the one made of 100% barley malt. Conversely, octanoic acid was more abundant in the barley beer than in the corn beers, which is consistent with Saerens et al. [45
] who found that higher levels of unsaturated fatty acids in beers, like in the corn beer samples, result in a decrease in ethyl ester production. The contrary effect can be seen for ethyl hexanoate and hexanoic acid, where in samples that exhibited a higher peak area of the ester, the presence of the acid seems to be reduced (BCBa and RCBa).
Alcohols were the second largest group of volatiles found in beers. We identified 24 alcohols and some of them were found in all six beer samples such as ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2-ethyl-1-hexanol, phenylethyl alcohol and citronellol. These alcohols come mainly from alcoholic fermentation while others such as citronellol and phenylethyl alcohol come from the essential oils of hops. According to Lyu et al. [12
] and Dong et al. [29
] aromas like sweet alcohol, rough, whiskey, fruity and rose could be attributed to these compounds.
In addition, some alcohols such as 2-furanmethanol, 4-methyl-1-pentanol, 3-methyl-1-hexanol and iso-geraniol were only found in those beers that contain barley malt (Ba, BCBa and RCBa). Of them, 2-furanmethanol is a product of Maillard reactions that occur during the roasting process of malt, especially in the production of ‘dark’ and ‘caramel’ malts; hence the caramel malt used in Ba, RCBa and BCBa beers could be the source of this volatile [30
]. Interestingly, to our knowledge, there are no reports of iso-geraniol in beers. This compound is the result of the partial oxidation of geraniol. It was previously identified in some flowers, fruits (grapes) and the essential oil of lemon, imparting a pleasant rose odour [46
In beers, terpenic compounds are generally derived from the hop essential oils, which are added to the wort during the boiling process. These compounds have been related to pleasant aromas like citrus, floweryand lilac [29
]. We identified 18 terpenes in the beer samples, most of them have previously been reported in barley beers [30
]. Only linalool, geraniol and humulene, associated with flower, geranium and wood aromas respectively, were detected in all six samples of beer. In turn, limonene and β-myrcene were found in beers made 100% with corn malt (RC, BC). In addition, these beers (RC, BC) showed more abundance of limonene and linalool than the other samples of beer. Interestingly, δ-cadinol and α-cadinol were found in those beers made with blue corn malt but (BC and BCBa) and 3-methoxy-2-naphthalenol was found only in those that contain barley malt (Ba, BCBa, RCBa). Among these terpenes, limonene have been previously reported in corn starch and corn products [48
Seven phenol volatile compounds were identified among the beer samples. These compounds contribute to clove and spice aromas in beers, which are desirable in some Belgian styles (amber and Trappist beers) and wheat beers [50
]. For instance, 4-ethyl-2-methoxy-phenol was detected in all beers containing red and/or blue corn malt, but not in barley beer. Buttery and Ling [49
] reported that 4-ethyl-2-methoxy-phenol is one of the major components in products like corn tortillas and tortilla chips. Furthermore, 2-methoxy-phenol was found only in beers containing blue corn malt. Even though 4-ethyl-phenol and 2-methoxy-4-vinylphenol were found in all beers, these compounds exhibited a higher peak area in those beers that contain both red and blue corn malt (RBC) than in the other beers. Of those, 4-ethyl-phenol is usually found in beers made of wheat malt. This molecule is formed from the biodegradation of hydroxycinnamic acids, such as ferulic and coumaric acid, during wort boiling. In high concentrations it imparts unpleasant aromas like medicinal, phenolic, clove-like, or smoky. However, in some beer styles such Belgian wheat and German Weizen these aromas are appreciated [51
]. 2-methoxy-4-vinylphenol and 4-vinylphenol (the precursor of 4-ethyl-phenol) have been reported as major components of sweet corn products such as tortillas [53
Styrene was the most abundant hydrocarbon found in all beer samples. This compound usually comes from the malt and it derives from the metabolism of cinnamic acid in barley malt by top-fermenting yeast [18
]. Its presence in the corn beers is explained as its formation occurs in parallel to the formation of 2-methoxy-4-vinylphenol and 4-vinylphenol. Styrene has been described as a “sweet-smelling colourless fluid” [54
Interestingly, dimethyl sulfide (DMS) exhibited a higher peak area in the beer made 100% with barley malt (Ba), followed by those made with corn malt (BC, RC, RBC). DMS is usually lost during the kilning of malt and the boiling of the wort, however its presence in the beer depends on the type of malt used. This sulphur compound has been reported in barley beers. Its presence is desirable in some styles of beers, like some lagers, while in others is not desirable as it adds sweet corn aroma to the beer [10
]. In addition, DMS has been identified as an important contributor to the aroma of corn products [48
Additionally, β-ionone was only found in beers made with blue and red corn malt (BC, RC, BCBa, RCBa) with the exception of RBC. This ketone has been previously reported as potential contributor of hop aroma. It has been identified in tortillas and corn dough [49
], in late-hopped and dry-hopped beers [55
] and in samples of whiskey made with corn [56
3.3. Descriptive Sensory Analysis
The sensory panel developed a list of 30 attributes to describe the appearance, odour, taste, aroma and mouthfeel characteristics perceived in all beer samples (Table 3
). The panel was asked to be as specific as possible in identifying attributes. Some terms and references were similar to those defined in the “beer flavour wheel”, developed by Meilgaard [33
], but others were unique attributes related to the presence of pigmented corn and chili.
The mean scores of the attributes were plotted in a radial diagram (except for the colour attribute) (Figure 1
). Significant differences (p
< 0.05) were found in 17 out the 30 attributes across the samples (Supplementary Table S1
). In order to have a complete description of all sensory characteristics of the beers, all attributes were kept and used in the subsequent analysis. We can see that the non-significant attributes were mainly those pertaining to the odour category. These odour characteristics are common to most of the commercial beers and some of them are the result of the volatile compounds developed during the fermentation process (e.g., banana, apple, floral, fruity). Thus, as all steps in the brewing process remained the same, we can expect some similarities between beers.
All beers in this study exhibited a range of sensory characteristics commonly found in most of the commercial beer samples, however some characteristics such as ‘dried fruits-O’, ‘dried-chili-O’, ‘brown sugar-O’, ‘tortillas-A’ and ‘spicy-M’ are not in the common lexicon of beers [33
]. Thus, the pigmented corn malt and the chili used in these beers appear to contribute to the development of these attributes. Despite the fact that cooked vegetable-A and cooked corn-O are usually associated with off-aromas in barley beers, we could expect that the pigmented corn beers develop these characteristics as they are sensory attributes found in the ‘Sendechó’ beverage [4
] and in many corn-derived products [48
The beer made 100% with barley malt (Ba) had a significantly higher intensity of brown sugar and caramel attributes than the other beers, which was expected as the caramel malt used in this beer contributes with the development of these aromas. Furthermore, alcohol aroma was higher in barley beer (Ba) than in the others, this is reasonable as barley malt contributes more to the formation of fermentable sugars than corn and therefore barley beers had higher alcohol content than the beers made with corn malt (see Table 4
RC and RCBa, both containing red corn malt, were rated higher in bitter taste, as compared to the other beers. In general, those beers containing red corn malt (RC and RCBa) were characterised by higher intensity of aroma attributes such as cooked vegetables and tortillas, related to the type of corn used. In addition, sour taste, oxidised and metallic sensations were scored high in the RC beer. The latter attributes are usually associated to an ageing effect [32
Despite the fact that Guajillo chili was added to all the beers in the same proportion and conditions during the brewing process, the perception of spicy attribute was different in all the beers. For instance, the beer made of blue corn and barley (BCBa) was rated significantly higher in spicy mouthfeel than the rest of beers, followed by blue corn beer (BC). The perception of the ‘spicy’ or ‘pungent’ sensation elicited by the capsaicin (the active ingredient of the Guajillo chili) may be influenced by factors such as the temperature, acidity and carbonatation of the beverage [57
]. In addition, phenolic compounds that evoke an oral irritation [39
] might increase the perception of this sensation. Thus, the content of polyphenols in BC and BCBa might contribute to the increase perception of the attribute spicy. Beers made with barley (Ba, BCBA, RCBa) had a higher carbonatation sensation than those beers made with pigmented corn malt (BC, RC, RBC). The perception of the fullness, which is associated with the body of the beer, was higher in the beers that contain blue corn and/or barley malts (BC, Ba, RCBa and BCBa) than in the ones made with red corn malt (RC and RCBa). The fullness palate sensation is related to the unfermentable sugars namely dextrins, developed during the mashing process. These compounds contribute to the body of the beer without imparting sweetness [10
The assessment of a beer’s appearance includes its colour, which according to the SMR colour chart it can range from straw to black. All beer samples analysed are in the range of the colours that goes from 10 SMR to 15 SMR units. Significant difference can be observed (Supplementary Table S1
) between the RC beer with a ‘medium amber’ colour (9 SMR), the BC beer with a ‘light brown-reddish’ colour (15 SMR) and the rest of the beers with a ‘cooper-red’ colour (12–13 SMR). It is well known that malt has the greatest impact on beer colour because of its content of melanoidins and Maillard compounds, which add colours that range from yellow, orange to red and brown [58
]. In this case, the anthocyanins in the pigmented corn beers contribute to develop of these ‘amber–red-cooper’ colours, especially in those beers made 100% with red and blue corn malt. In acidic solutions such as beer, anthocyanins are chemically stable and turns their colours to reddish tones [3
With the aim of illustrating the differences among beers produced by different types of malt (red corn, blue corn and barley), a PCA was applied on the total data set of 30 attributes. The biplot obtained is shown in Figure 2
. The first two components (PC) explained 72.58% of the total variation in the samples with contributions of 40.39% by PC1 and 32.19% by PC2, where most of the attributes contributed considerably to samples discrimination.
PCA permitted a clear-cut separation of the samples based on the type of malt used.
PC1 opposed the beers made with barley malt like Ba, RCBa and BCBa (on the left) to the RC and RBC beer (on the right). On the other hand, PC2 opposed beers made of red corn malt (positive side) to beers made of blue corn malt (negative side). The RC beer was characterised by attributes such as fermented fruits-O, olive-O, tortillas-A, cooked vegetables-A, metallic-M and oxidised-M. On the contrary, BC and BCBa were characterised by spicy-M, sweet-T, Turbidity-Ap.
The beer made of 100% barley malt (Ba) was discriminated along PC1 (at the negative side) and was characterised by brown sugar-O, apple-O, alcohol-A, carbonatation-M and fullness-M.
Blended beer made of both type of corn malt (RBC) was placed in between red corn beer (RC) and blue corn beer (BC), sharing attributes of both malts used such as bread-O, cooked corn-O and dried chili-O and dried fruits-O. This behaviour was also shown in blended beer made of red corn and barley malt (RCBa), preserving the sensory characteristics of both 100% barley (Ba) and 100% red corn (RC) beers such as apple-A, fruity-A, banana-A, malty-A and floral-A, attributes that are more common in typical barley beers.
These sensory data showed that by adding corn malt to the beer formulation, the sensory profile of the typical barley beer can be reached easily, while preserving at the same time odours and aromas of corn products, especially those of the Sendechó beverage such as corn and spicy and dried chili [4
3.4. MFA of Sensory Attributes and Chemical Data
In this study, MFA was used to explore the differences and similarities between beers due to the type of malt used in brewing. In addition, MFA helped to identify associations between sensory and chemical datasets that brought us to know those components (sensory and chemical) that can be used as markers of beers made with pigmented corn malt.
The first two dimensions (Dim 1 and Dim 2) in Figure 3
accounted for 56.31% of the total variation with contributions of 31.19% by Dim 1 and 25.12% by Dim 2.
First, the variable plot (Figure 3
b) shows that Dim 1 separates samples based on the sensory ‘odour-aroma’ attributes (in green; 34.05% of the variance) and the ‘non-volatile’ components (in pink; 34.53% of the variance). For Dim 2, the groups of variables ‘volatiles’ (in orange) and ‘taste-mouthfeel’ (in blue) are those that contribute the most to the dimension with 22.41% and 44.91% of variance respectively. The plot of the individuals (Figure 3
a) allows us to visualise the global resemblance between beers by considering the information of all variables (sensory and chemical). It clearly showed that Dim 2 separated the samples based on the type of malt used, with beers made with pigmented corn (red and blue) on the top of the plot, and the beers that contain barley malt plotted on the bottom (Figure 3
Second, the RV coefficients (Table 5
) show the relationship between the data matrices, the closer the RV coefficient to 1, the more similar the matrices [21
]. According to the RV, a good correlation can be observed between the ‘odour-aroma’ and ‘non-volatile’ variables (0.740). Moreover, a better correlation between ‘volatiles’ and ‘taste-mouthfeel’ variables (0.649) than for ‘odour-aroma’ and ‘volatiles’ data matrices (0.509).
A deeper analysis of Figure 3
allows detailing these relations between the different types of variables that strengthen the characterisation of the beers. On the negative side of Dim 1 of the variable plot (Figure 3
b), it can be observed that the sensory attributes floral-O, hoppy-O and pineapple-O are positively correlated mainly with esters (i.e., ethyl butanoate (48), phenylethyl acetate (61), ethyl (E)-4-decenoate (65), ethyl decanoate (67), isoamyl octanoate (68), terpenes (i.e., geraniol (108), δ-cadinene (112), humulene oxide (115), δ-cadinol (120), and alcohols (i.e., phenylethyl alcohol (15), citronellol (19) and 1-decanol (22)). Numbers correspond to those on Table 2
. Esters and alcohols are well known for their floral and fruity contribution to the beers, and terpenes are more likely associated with herb and green odours-aromas, which are consistent with the description of the hoppy odour. These correlations between the sensory attributes and the volatiles compounds strengthen the aromatic profile of the barley beer (Ba). Also compounds such as 2-nonanone (88), heptanoic acid (37), 2-ethylhexanoic acid (38) and acetaldehyde (25) were also correlated with the sensory attributes mentioned before. The positive correlation of these fruity and floral sensory attributes with carboxylic acid compounds could suggest that the presence of esters, even in low levels, might reduce the perception of off-aromas like sweat and rancid, caused by octanoic acid [59
On the positive side of Dim 1, RBC (Figure 3
a) can be separated from the other beers mainly by the presence of phenol volatile compounds. Among them, phenol (94), 2-methoxyphenol (95), 4-ethylphenol (96) and 4-ethyl-methoxy-phenol (97) showed association with the sensory attributes related to the presence of pigmented corn malt such as cooked vegetables-A, cooked corn-A, olive-O and fermented fruits-O (Figure 3
b). These compounds and the sensory attributes allow us to differentiate between the beers made 100% with corn malt, suggesting that these phenol compounds could be use as indicators of the use of pigmented corn in the brewing process.
On the negative side of Dim 2, we found positive correlations between attributes such as malty-A banana-O, brown sugar-O, tortillas-A and fruity-O and the compounds 2-furanmethanol (8), ethyl propanoate (44), propyl acetate (45), ethyl pentanoate (51), ethyl isohexanoate (52), ethyl hexanoate (54), iso-geraniol (20), acetophenone (87), 2-acetylpyrrol (101) and tetramethyl-pyrazine (102). The presence of these compounds, characterised by fruity, bready, brown sugar and caramel aromas [45
], is consistent with the use of roasted malts (caramel malt) in the beers associated to these compounds (RCBa and Ba). Furthermore, on Dim2 (negative side), a weak correlation was also found for benzeneacetaldehyde (26) with astringent, which is consistent with the results obtained by Owusu et al. [61
], where the presence of this compound has been associated with the astringent mouthfeel in products as cocoa and dark chocolates.
The positive side of Dim 2 is positively correlated with beers made from red corn malt (RC) and blue corn malt (BC) (Figure 3
a, top side). These beers are well characterised by compounds such as linalool (106), limonene (105), β-ionone (90) and 4-ethyl-2-methoxy-phenol (90). These volatile compounds have been found in other corn products such as tortillas and pop-corn [49
] and especially limonene and β-ionone have also been reported in samples of whiskey made with corn [56
]. Thus, these compounds could also be used as markers of the presence of corn in beers.
In addition, the spicy attribute was strongly correlated with 2-methyl-5-(1-methylethyl)-phenol (98) well known as carvacrol -a key aroma compound in oregano spice- that is concordant with the pungent mouthfeel associated with this compound [60
]. Unexpectedly, dimethyl sulfide (103) which usually imparts cooked vegetable off-aroma also showed a positive correlation with the spicy attribute. This behaviour could be attributed to the high abundance of phenylethyl alcohol (15) that could suppressed the perception of this compound [8
Correlations between the non-volatiles variables (ABV, IBU, TPC, TAC) and the sensory and volatile data were also studied. For instance, a positive correlation was found between alcohol sensory attribute and alcohol content (ABV). Regarding the total polyphenol content (TPC), a negative correlation was observed between TPC and metallic and oxidised sensory attributes, confirming that polyphenols help to retard the development of these attributes in beer [54
]. Moreover, TPC showed a positive correlation with carvacrol volatile (98). According to Lee et al. [62
] carvacrol is a volatile compound that has exhibited potent antioxidant activity.
It is well known that anthocyanins do not impart aromas, but sometimes these compounds have been related to an astringent or bitter taste [41
]. Even though, no obvious correlations were found between TAC and bitter or astringent attributes. The results showed a positive correlation between TAC and phenol compounds such as phenol (94), 2-methoxy-phenol (95), 4-ethyl-phenol (96) and 4-ethyl-2-methoxy-phenol. This could suggest an interaction between the anthocyanins that comes from corn malt and those phenol volatile compounds. According to Dufour and Sauvaitre [63
] and Ruta and Farcasanu [64
], interactions between anthocyanins and some aroma compounds such as phenol and 2-methoxy-phenol, lead the formation of copigments, which improve the stability of the anthocyanins and hence the colour stability of the beverage.
Apparently, no positive correlation was found between IBU parameter and bitter sensory attribute. However, there are other components that could contribute to the perception of bitterness such as the Maillard products formed during the kilning and roasting process of caramel and dark malts [57
]. In addition, bitterness can be masked by sweetness due to sugars (residual sugar) that remain after the fermentation process. As has been mentioned before, IBU measures a beer’s bitterness due to the α-acids of the hops, which gives an approximate idea of beer bitterness but there are other compounds that could impart or mask the bitter taste. Thus, it is not possible to directly correlate IBU to the perceived sensory bitterness [41
Finally, the different groups of variables (sensory and chemical) had different influences in each beer. The major difference was found for the BCBa and RCB which were mainly described based on taste-mouthfeel attributes and non-volatile parameters respectively. Beers Ba and RCBa were mainly described based on the odour-aroma attributes and volatile compounds. For beers made 100% with pigmented corn (RC and BC) the group of volatiles had more influence in their characterisation. Overall, the volatile composition also separates beers depending on the presence of corn, supporting the fact that the use of corn as an ingredient clearly alters the sensory profile of beers.