GC-MS and IR analysis of primary metabolites in the beer residue

45 Compounds belonging to 11 chemical classes of metabolites were identified in the beer residue. Most abundant were inorganic (31.7%), acids (27.8%), and amino acids (27.4%) (Fig. 2A,B, Table 1). Other chemical classes comprised nitrogenous compounds, fatty acids, aromatics, alcohols, ethers, sugars, aldehydes, and mono glyceryl ethers, although these were present at much lower levels. Phosphoric acid, one of the detected inorganics, was found at high levels (26.7%) in comparison with other identified metabolites in all samples. This was confirmed using infrared (IR) analysis that showed bands indicative of phosphoric acid (Fig. 2). These bands corresponded to the following functional groups and their wave numbers: P-H (PO 4 −3), 2360–2368 cm−1; OH (PO 4 −3), 1625–1627 cm−1; P = O (PO 4 −3), 1106 cm−1; P-O (PO 4 −3), 1035–1037 cm−1; P-C (PO 4 −3), 779 cm−1; and PO 4 −3, 530–541 cm−1 (Fig. 3). Today phosphoric acid is a common additive to alcoholic and non-alcoholic beverages and is employed to prolong their shelf life and enhance flavor6,7,8. This is the first time such a high abundance of phosphoric acid has been detected in ancient Egyptian beer9. The source of phosphoric acid is yet to be determined conclusively. However, it is likely that barley grains, which are rich in readily soluble phosphoric acid, are the source for this acid in the beer residue10,11. It is possible that the ancient Egyptians utilized the phosphoric acid in barley as a food preservation, much in the same as hops is currently used to impart a flavor and for its preservative effect during beer fermentation12. Previously, the earliest evidence of phosphoric acid was from two tripod cooking pots from Crete dating to around 1700 BCE where it was found together with dimethyl oxalate, a basic constituent of modern beer13.

Figure 2 Metabolites analysis of the beer resides from the vats. (A) Representative overlaid chromatograms of non-volatile primary metabolites in vats analyzed as silylated products; (B) Relative percentage of chemical classes of compounds in beer residue samples as analyzed using GC-MS for non-volatiles analysis post silylation; (C) Representative GC-MS chromatogram of headspace volatiles collected from vats; (D) Relative percentage of chemical classes of volatile compounds in beer residue samples as analyzed using GC-MS. Full size image

Table 1 Relative percentage of silylated compounds detected in beer residues from vats using GC-MS for primary metabolites analysis (n = 4). Full size table

Figure 3 IR spectra from beer residue vat 1 (A) and vat 2 (B) showing IR absorption bands indicative of organic and phosphoric acid metabolites. Full size image

Twenty-two Organic acids were detected accounting for 27.8% of the primary metabolites pool in the beer residue. IR analysis confirmed bands indicative of organic acids from their respective wave numbers: OH (acid), 3403–3423 cm−1; C=O (acid), 1706–1708 cm−1; and C-O (acid), 1319–1321 cm−1 and in accordance with GC-MS results (Fig. 3). Succinic acid was the predominant organic acid detected at 7.3%, which is in agreement with what is described in modern beer post-fermentation14. Succinic acid is usually found at low levels in barley, but it accumulates with yeast fermentation, as does lactic acid, which was detected at 3.1%15. Oxalic acid, which is commonly found in modern beer16 and is used as a marker for identifying ancient barely-beer fermentation17,18, was detected at 4.7%. Our analysis confirms its usage by the ancient Egyptians in their beer making19 as reported by Maksoud et al. (1994) for residues recovered from another predynastic installation site, HK24A, at Hierakonpolis9. Other minor detected organic acids were malonic, acetic, malic, and hexanoic acid, which are also found in modern beer20,21. In addition, two fatty acids also known in modern brews, viz., palmitic and stearic acids22, were also detected.

Amino acids accounted for 27.4%, with proline as the only major amino acid found at 25.3%, making it the second most abundant acid after phosphoric acid. Ancient Egyptians are believed to have used different fruits, such as dates or figs, in their brewing process to enhance beer flavor and quality2. Maksoud et al. (1994) identified dates and grapes in the Predynastic beer residues9, although further macrobotanical analysis has not confirmed this23. Nevertheless, the high abundance of proline may suggest that dates were added, as proline is enriched in this fruit24,25. Hydroxylamine was the main nitrogenous compound found in the beer residue at 3.1%; it is a by-product of ammonia oxidation and S-nitrosoglutathione detoxification, part of nitrosative stress response26,27.

GC-MS analysis of volatiles in the beer residue

Volatile analysis using solid phase microextraction (SPME) coupled to GC-MS techniques was adopted in parallel for flavor profiling and resulted in the detection of 49 volatiles belonging to 9 chemical classes, with acids (56.3%), ketones/lactones (18.5%), and esters (12.1%) being the major ones alongside terpenes, nitrogenous compounds, aromatics, and aldehydes (Fig. 2C,D, Table 2). 12 Acids were detected, including nonoic, caproic, 2-methyl-butanoic, caprylic, pentanoic, heptanoic, benzoic, α-methylbutyric, and capric acids. Interestingly, caprylic acid, capric acid, heptanoic, and nonoic acids are part of the volatile acid mixture found in modern beer28,29. Caproic acid, a medium-chain carboxylic acids, is a product of fermentation by yeast via chain elongation30. These fatty acids are fermentation-products and signs of beer aging and yielding a rancid or goaty flavor29. γ-Nonalactone was the major lactone found at 7.6% in the beer residue specimens. It exhibits a coconut-like odor and has been detected as the most odiferous compound in American bourbon whisky and other modern beers31,32,33. Nine esters were identified with laurate and 2-methylpropanoate esters being the major ones. Laurate and 2-methylpropanoate esters have been reported in modern beer20,33. The only major terpene that was found in the beer residues was geranyl acetone at 2%, and it is also found in modern beer34.

Table 2 Relative percentage of compounds detected in beer residues from vats using SPME-GC-MS for volatiles analysis (n = 3). Full size table

Archaeo-botanical analyses of remains from various locations at predynastic Hierakonpolis indicate the cultivation of barley and emmer wheat, with emmer being the predominant crop35. Macrobotanical examination of the beer residues also suggested that emmer wheat was the major component in the beer produced at the site5,9. From the point of view of metabolites composition, barley and emmer wheat differ in the relative abundance of their constituents, such as their phenolics and acids, but they do not have any unique constituents that distinguish them36. Thus, it is hard to define what ratio of these grains was used in the beer. Numerous constituents of both were detected in the residue sample; however, suberic acid found at 1%, glycerol at 1.6%, and pyroglutamic acid < 1%, have a higher abundance in wheat than barley, lending support to the macrobotanical observations.