Malt properties
The main chemical changes that grains undergo during malting are the reduction in assayable starch and hemicellulose contents. During this physiological process, the cell bound components weaken due to the synthesis of degrading enzymes like cellulases and arabinoxylanases. The hydrolyzed cell walls allow the entry of other relevant enzymes that will degrade protein bodies and matrix and starch granules (Boulton and Quain 2001). There are many investigations which clearly conclude that malted sorghum has significantly lower diastatic activity compared to barley malt especially in terms of maltose-producing β-amylase. These deficiencies have been counteracted by the addition of exogenous enzymes like α and β-amylases, glucoamylase or amyloglucosidase and even proteolytic enzymes that improve the exposure of the starch granules to amylolytic enzymes (Serna-Saldivar and Rubio-Flores 2016). The idea of inoculating A. oryzae during the malting process of sorghum is based in the fact that this mold synthesizes relevant enzymes like amylases and proteases that enhance the conversion of starch and proteins into fermentable carbohydrates and simpler and soluble nitrogenous compounds, respectively. Heredia-Olea et al. (2017) observed that α-amylase activity of sorghum malt was positively affected by addition of A. oryzae but it did not affect β-amylase activity. The conversion of starch into linear and branched dextrins explains the significant reduction of starch content during germination. In addition, the proteases generated are distributed to the entire caryopsis and hydrolyze conjugated proteins associated with amylases in order to activate starch-degrading enzymes. The proteases also degrade germ and endosperm proteins, solubilizing approximately 30% of the total protein. The sorghum malt contained higher amounts of readily assimilable amino acids (FAN) compared to the barley malt and other cereals such as wheat (Hill and Stewart 2019) likely enhancing yeast nutrition during beer fermentation and production of relevant fusel alcohols.
Wort properties
The regular sorghum malt generated less 15° P wort yield compared to the barley malt (Table 2). Similar difference was reported by Espinosa-Ramírez et al. (2013a) who observed that barley malt yielded was up to 24% more 12 °P wort and similar FAN and pH values compared with their counterparts produced with white or red sorghum malts. According to Espinosa-Ramírez et al. (2014) and Heredia-Olea et al. (2017), the use of regular sorghum malt usually yields worts with lesser amounts of fermentable carbohydrates and FAN contents and their fermented beers with lower alcohol contents. These differences, especially in terms of generation of maltose and glucose, may be attributed to the higher β-amylase and amyloglucosidase activities of the barley and sorghum–A. oryzae malts. It is well known that malt and adjunct starches are hydrolyzed to dextrins and sugars during mashing (Serna-Saldivar and Espinosa-Ramirez 2018). In terms of the preferred carbon source, Carlsen and Nielsen (2001) studied the influence of maltose and maltodextrins differing in chain length, glucose, fructose, galactose, sucrose, glycerol, mannitol and acetate on α-amylase production by A. oryzae. Productivity was found to be higher during growth on maltose and maltodextrins, which are present in relatively high amounts in typical worts. Maltose was the most abundant carbohydrate in the control and experimental worts (Table 2). This is due to the concerted enzymatic action of α- and β-amylases. The sorghum worts contained more glucose and less maltose than the barley wort. This is related to the higher β-amylase activity reported in the barley malt. Even though the worts were adjusted to 15° Plato, the total amount of fermentable sugars did not total 15% due to the presence of dextrins. Dextrins commonly account for 90% of the residual carbohydrate of beer because regular yeast is not capable of fermenting these carbohydrates. Approximately, 40–50% of the dextrins are oligosaccharides containing 4–9 glucose units, and the remaining 50–60% are higher dextrins with 10 or more glucose units (Boulton and Quain 2001). Espinosa-Ramírez et al. (2013a) enhanced the amounts of fermentable sugars in the sorghum malt worts with the addition of β-amylase. When amyloglucosidase was added, the total sugar content increased 20% and consequently the glucose content was five times higher compared with worts without exogenous enzymes. It is well known that A. oryzae is a mold that synthesizes large amounts of amylolytic, proteolytic and lipolytic enzymes. In fact, commercial mold cultures are used to produce and isolate enzymes widely used by the food industries. The main disadvantage of sorghum malt is its relatively low production of β-amylase, key enzyme in brewing operations because it complements the activity of α-amylase. Furthermore, the color of the beer is influenced by Maillard reactions that occur between sugars and amino-compounds (including the amino acids) during the hop-boil step, which gave rise to colored and flavored substances. The proportions of the flavored fermentation products made by yeast are dependent on the nitrogenous substances that are present. Nitrogenous components are present in the form of amino acids, small peptides, and proteins. Recommended FAN concentrations of wort range from 150 to 200 mg/L (Boulton and Quain 2001). Table 1 clearly depicts that the barley wort contained slightly lower FAN values compared to the sorghum with and without A. oryzae counterparts. In fact, the sorghum wort with A. oryzae contained 27% more FAN compared to the control barley counterpart. The significant difference is attributed to the synthesis and action of proteases (mainly carboxypeptidases) produced by the Aspergillus. Malt carboxypeptidases have optimum activity at temperatures between 40 and 60 °C and are inactivated at 70 °C. Likely the double mashing process gave these enzymes the opportunity to hydrolyze proteins into FAN components (Boulton and Quain 2001). The proteases generated by the mold facilitated the entrance of amylases associated to the sorghum malt and A. oryzae. This mold is known to express high amounts of amyloglucosidases that convert linear and branched dextrins into glucose (Heredia-Olea et al., 2017). The hydrolyzed peptides are converted to higher alcohols during fermentation (Barredo-Moguel et al. 2001). Briggs et al. (2004) indicate that 100 to 140 mg/L is regarded as the minimum level of FAN needed for trouble-free fermentations. The soluble proteins and polypeptides that remain in the fermented wort contribute to the `body’ and `mouth-feel’ of the beer, its foaming properties, and its susceptibility to haze formation.
Fermentation and beer parameters
Glucose and fructose consumption profiles during fermentation followed the expected trend. According to Cason et al. (1987), glucose and fructose are taken up by the same membrane transport system which explains the similar consumption profiles. The maltose consumption profiles were the same for all treatments. Cason et al. (1987) observed that utilization rates of maltose were identical independently of the adjunct concentration. The rapid consumption of glucose and fructose exerted catabolite repression on the maltose membrane transport system or any of the subsequent metabolic steps of maltose catabolism to glucose. Therefore, maltose started to be utilized when glucose or fructose levels fall to a cut-off point below which catabolite repression did not occur (Cason et al. 1987). The metabolism of maltose and maltotriose is highly interconnected. Both sugars are α-glucosides transported by the activated α-glucoside-Hc symporter encoded by gen AGT1. This permease, which is maltose inducible, has the same affinity for maltose and maltotriose (Zastrow et al. 2000). These authors observed a single exponential growth phase of maltotriose fermented by Saccharomyces cereviseae grown in medium containing glucose, maltose or maltotriose as carbon and energy sources indicating that the metabolism of this particular fermentable sugar was oxidative. In terms of sugar consumption, fermented beers for the three malt treatments did not show any significant differences.
Final specific gravity value in beer is closely related to the final ethanol content (Esslinger 2009) and this relationship was observed in all beers. In case of attenuation, the values obtained herein were lower compared to the ones reported by Esslinger (2009) for regular barley and sorghum beers where a final attenuation of 82.1% and 79.7% was observed, respectively. In terms of beer specific gravity, pH, ethanol, and FAN contents, the use of sorghum–A. oryzae improved values compared to the use of only sorghum malt and similar to the barley malt beer. Espinosa-Ramírez et al. (2014) obtained similar ethanol contents when barley malt beers were compared with sorghum malt beers produced with exogenous amyloglucosidase. The superior alcohols’ yeast metabolism was not affected by the malt treatment despite dissimilarities in FAN concentrations. Esters are the products of the enzymatic catalysis of organic acids, ethanol and higher alcohols. Their formation is closely related to yeast propagation and lipid metabolism (Pires et al. 2014). Beer contains more than 50 different esters, from which three are of higher relevance because they greatly affect beer flavor: ethyl acetate, iso-amyl acetate, iso-butyl acetate. Since the sorghum–A. oryzae treatment had sixfold and fourfold less hydrogen sulfide than barley beer and sorghum beer, respectively, the difference can be attributed to its higher rate of catabolism mediated by the mold. In terms of hydrogen sulfide production, all beer treatments were statistically different, where only the sorghum–A. oryzae beer was under the threshold level of 5 ppb. It arises through yeast autolysis at the end of fermentation or during maturation. The best understood carbonyl flavor compounds associated with yeast fermentation are the VKD, which include diacetyl and 2,3 pentanedione. This led to high levels of VKD, during fermentation owing to effects on the regulation of valine synthesis by the yeast. According to Esslinger (2009) the beer taste thresholds of diacetyl and 2,3 pentanedione range from 0.08 to 0.2 ppm and from 0.5 to 0.6 ppm. Beer from barley contained significantly higher levels of both butanedione and 2,3 pentanedione whereas beer manufactured with the sorghum–A. oryzae malt contained about 96% lower content of VDK. This important difference in volatile compounds needs to be further researched especially in terms of beer stability and sensory analysis. According to the EBC scale, the sorghum beers are classified as pale whereas the slightly darker yellowish barley beer as Pilsner. The final beer color is influenced by the type of malt, color of brewing adjuncts, concentration and type of hops and pH (Esslinger 2009). The almost twice as high color score observed in the barley beer is attributed to the utilization of a malt rich in glumes that contain phenolic compounds that lixiviated into the wort and the production of Maillard type of compounds during boiling (Granato et al. 2011). It is worth mentioning that both sorghum beers were produced from naked caryopses of white sorghum that do not contain tannins and were low in phenolic compounds (Serna-Saldivar and Espinosa-Ramirez, 2018). Differences of ethyl hexanoate, hydrogen sulfide, methanethiol and VKD compounds could have also affected beer color. According to Dack et al. (2017), Maillard reaction products inhibit the synthesis of esters due to possible suppression of enzymes and/or gene expression linked to ester synthesis. The impact of FAN on formation of flavor and aroma compounds during fermentation has been previously studied. Initial wort FAN content and the amino acid and ammonium ion equilibrium in the medium impact the formation of esters, aldehydes, VKD, superior alcohols and acids, as well as sulfur compounds. Even small differences in wort composition can exert significant effect on the flavor of the resulting beer (Hill and Stewart 2019). According to Taylor et al. (2013), and Kobayashi et al. (2008), there is some indication that the differences in the free amino acid profile of sorghum malt worts compared with barley malt worts could influence beer flavor by affecting yeast metabolism. Sorghum malt worts were found to contain low levels of branched chain valine. The amino acid profiles of barley and sorghum differed because the first commonly contained 2.2 g of methionine and 1.8 g of cysteine per 100 g of protein whereas the second 1 g of methionine and 1.6 g of cysteine per 100 g of protein (Serna-Saldivar 2010).