The history of humankind is marked by the constant adoption of new dietary habits affecting human physiology, metabolism, and even the development of nutrition-related disorders. Despite clear archaeological evidence for the shift from hunter-gatherer lifestyle to agriculture in Neolithic Europe [], very little information exists on the daily dietary habits of our ancestors. By undertaking a complementary -omics approach combined with microscopy, we analyzed the stomach content of the Iceman, a 5,300-year-old European glacier mummy []. He seems to have had a remarkably high proportion of fat in his diet, supplemented with fresh or dried wild meat, cereals, and traces of toxic bracken. Our multipronged approach provides unprecedented analytical depth, deciphering the nutritional habit, meal composition, and food-processing methods of this Copper Age individual.

Results and Discussion

1 Fowler C.

Harding J.

Hofmann D. Defining the ‘Neolithic in Europe’: Diverse and Contemporaneous Communities, c. 6500-2500 BC. 4 Shennan S.

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Thomas M.G. Regional population collapse followed initial agriculture booms in mid-Holocene Europe. 5 Richards M.P. A brief review of the archaeological evidence for Palaeolithic and Neolithic subsistence. 6 Nicklisch N.

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Alt K.W. Holes in teeth - Dental caries in Neolithic and Early Bronze Age populations in Central Germany. 7 Diamond J. Evolution, consequences and future of plant and animal domestication. 13C/12C) and nitrogen (15N/14N) isotope analyses of bone collagen have been widely used to study the dietary adaptations of Neolithic populations [ 8 Richards M.P.

Schulting R.J.

Hedges R.E. Archaeology: sharp shift in diet at onset of Neolithic. 9 Evershed R.P.

Payne S.

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et al. Earliest date for milk use in the Near East and southeastern Europe linked to cattle herding. 10 Fowler C.

Harding J.

Hofman D. Movement of Plants, Animals, Ideas and People. Human evolution is closely linked to dietary changes and the development of new food-processing techniques. This is clearly observed in the transition from hunter-gatherer lifestyle to agriculture, which gave rise to cultivation of crops, animal husbandry, and permanent settlements []. The more stable availability of food boosted the growth of Neolithic European populations []. However, changes in diet had drawbacks for health, such as increased rates of caries []. Added to this, permanent large settlements combined with the adoption of agriculture promoted the spread of density-dependent infectious diseases []. Considering the impact of dietary habits on human evolution and health, the reconstruction of past human subsistence appears to be vital to our understanding of past societies. Stable carbon (C/C) and nitrogen (N/N) isotope analyses of bone collagen have been widely used to study the dietary adaptations of Neolithic populations [], lipid analysis on pottery residues dated the onset of milk use in Neolithic Europe [], and numerous archaeological studies describe the occurrences of wild and domesticated animal and plant remains as indirect measures of past diets []. All these studies provide insights into major dietary changes during the Neolithic period. However, specific details on past diets such as the exact meal composition or food processing are missing. Knowledge of daily dietary habits of our ancestors is pertinent as food type, intake quantity, and food-processing techniques influence human metabolism and health.

2 Gostner P.

Pernter P.

Bonattie G.

Graefen A.

Zink A.R. New radiological insights into the life and death of the Tyrolean Iceman. 11 Maixner F.

Krause-Kyora B.

Turaev D.

Herbig A.

Hoopmann M.R.

Hallows J.L.

Kusebauch U.

Vigl E.E.

Malfertheiner P.

Megraud F.

et al. The 5300-year-old Helicobacter pylori genome of the Iceman. 15N/14N) of the Iceman’s hair suggested first a vegetarian lifestyle [ 12 Macko S.A.

Engel M.H.

Andrusevich V.

Lubec G.

O’Connell T.C.

Hedges R.E. Documenting the diet in ancient human populations through stable isotope analysis of hair. 13 Macko S.A.

Lubec G.

Teschler-Nicola M.

Andrusevich V.

Engel M.H. The Ice Man’s diet as reflected by the stable nitrogen and carbon isotopic composition of his hair. 14 Dickson J.H.

Oeggl K.

Holden T.G.

Handley L.L.

O’Connell T.C.

Preston T. The omnivorous Tyrolean Iceman: colon contents (meat, cereals, pollen, moss and whipworm) and stable isotope analyses. 14 Dickson J.H.

Oeggl K.

Holden T.G.

Handley L.L.

O’Connell T.C.

Preston T. The omnivorous Tyrolean Iceman: colon contents (meat, cereals, pollen, moss and whipworm) and stable isotope analyses. 15 Oeggl K. The Diet of the Iceman. 16 Oeggl K.

Kofler W.

Schmidl A.

Dickson J.H.

Egarter-Vigl E.

Gaber O. The reconstruction of the last itinerary of “Ötzi”, the Neolithic Iceman, by pollen analyses from sequentially sampled gut extracts. 17 Oeggl K.

Kolfer W.

Schmidl A. New aspects on the diet of the Tyrolean Iceman, ‘‘Ötzi”. 18 Rollo F.

Ubaldi M.

Ermini L.

Marota I. Otzi’s last meals: DNA analysis of the intestinal content of the Neolithic glacier mummy from the Alps. 19 Dickson J.H.

Hofbauer W.

Porley R.

Schmidl A.

Kofler W.

Oeggl K. Six mosses from the Tyrolean Iceman’s alimentary tract and their significance for his ethnobotany and the events of his last days. 20 Dickson J.H.

Oeggl K.

Handley L.L. The iceman reconsidered. Figure 1 The Iceman’s Gastrointestinal Tract Show full caption (A) Gastrointestinal (GI) tract preservation and content texture. The radiographic image shows the completely filled stomach (asterisk) and the intestinal loops of the lower GI tract (arrows). Content samples of the stomach (left, asterisk) and of two different sites in the lower GI tract (middle, right) that were re-hydrated in phosphate buffer are shown below the radiographic image. (B) Animal muscle fibers detected in the stomach content using light microscopy. Scale bar, 50 μm. The black box contains a zoomed-in view of one muscle fiber (scale bar, 20 μM). (C) Plant tissue detected in the stomach content using light microscopy. Scale bar, 50 μm. See also Figure S1 for additional plant and animal remains detected in the Iceman intestinal contents. A recent radiological re-examination of the Iceman, a 5,300-year-old European natural ice mummy, identified his completely filled stomach ( Figure 1 A) []. The well-preserved stomach content still contains ancient endogenous biomolecules as demonstrated from the complete reconstruction of the Iceman’s Helicobacter pylori genome []. Previous isotopic analysis (N/N) of the Iceman’s hair suggested first a vegetarian lifestyle [] which was later, after careful re-examination of the data, changed to a omnivorous diet []. Further analyses of lower intestinal tract samples of the Iceman confirmed that he was omnivorous, with a diet consisting of both wild animal and plant material. Among the plant remains, there were cereals, pollen grains of hop-hornbeam, and fragments of bracken and mosses []. The detection of the Iceman’s stomach content with its pristine yet undigested food mix, provides the unique opportunity to fully reconstruct a Copper Age meal.

The study material included 11 gastrointestinal (GI) content biopsies of the stomach and small and large intestines ( Figure 1 A; Data S1 ). Using microscopy and a combined multi-omics approach targeting different biomolecules (ancient DNA, proteins, metabolites, and lipids) (see STAR Methods ), we determined the exact composition of the Iceman’s diet preceding his death and present evidence how people during the Copper Age processed food.

21 Schiaffino S.

Reggiani C. Fiber types in mammalian skeletal muscles. Initial macro- and microscopic analyses showed that, compared to the small and large intestine contents, samples of the stomach content still contain compact pieces of food that display a hydrophobic “fatty-like” character ( Figure 1 A). The most prevalent tissue types in the stomach content samples are animal muscle fibers arranged mostly in bundles ( Figures 1 B and S1 ) and plant fragments identified as bran and glumes belonging to the Triticum/Secale type ( Figures 1 C and S1 ). The striated muscle fiber structures are characteristic for cardiac and skeletal muscle tissue ( Figure S1 B) []. Further microscopic analysis of the plant micro-remains identified cereal pollen from the Triticum-type as well as airborne arboreal pollen and an abundance of spores and sporangia from the bracken fern Pteridium aquilinum ( Figure S1 J).

Metabolite, glycan, stable isotope, and elemental analyses confirm the omnivorous character of the Iceman’s last meal ( Data S2 ). Metabolic compounds indicate the presence of ruminant fat or dairy products (phytanic acid) and support the existence of whole-grain cereals (azealic acid) in the Iceman’s diet. In addition, gamma-terpinene that occurs in coriander oil, lemon oil, and other essential oils suggest the usage of herbs. The most abundant elements found in the stomach content were the nutritional minerals iron, calcium, zinc, magnesium, and sodium, consistent with the consumption of red meat or dairy products. Minor concentrations of trace nutrients of chromium, copper, manganese, selenium, molybdenum, and cobalt were also detected. These data suggest that the Iceman’s last meal was well balanced in terms of essential minerals required for good health with no evidence of toxic heavy metals such as lead, cadmium, or arsenic.

Importantly, the presence of ibex and red deer in the stomach contents was independently confirmed in the Molecular Population Genetics Laboratory, Trinity College, Dublin, by sequencing of a mammalian mitochondrial capture ( Data S1 ). Moreover, PCR-based analysis targeting animal- and plant-DNA barcodes and further assignment of the shotgun data against the DNA Barcode of Life Data dataset ( www.boldsystems.org ) support the first metagenomic results on a phylogenetic higher hierarchical level (genus, family) ( Figure S4 ).

Our analysis of the ancient DNA identified the main components in the Iceman’s meal. However, the highly degraded state of this biomolecule excluded any further quantification of certain foods.

23 Guo G.

Lv D.

Yan X.

Subburaj S.

Ge P.

Li X.

Hu Y.

Yan Y. Proteome characterization of developing grains in bread wheat cultivars (Triticum aestivum L.). 24 Jerkovic A.

Kriegel A.M.

Bradner J.R.

Atwell B.J.

Roberts T.H.

Willows R.D. Strategic distribution of protective proteins within bran layers of wheat protects the nutrient-rich endosperm. 25 Labeit S.

Kolmerer B. Titins: giant proteins in charge of muscle ultrastructure and elasticity. Using multistep solubilization and fractionation proteomics, we identified 167 animal and plant proteins in the stomach metaproteome, of which 13 were taxonomically unambiguously assigned to the Triticinae, 6 to the Caprinae, and 1 protein to the Cervinae subfamily ( Figure 2 C; Data S2 ). The proteomic hits not only confirm the previous metagenomic results but also provide important information on the food source. The detected Triticinae plant proteins are primarily expressed in the endosperm (Globulin 3A, Triticin) [] and pericarp (Oxalate oxidase 2) [] of wheat seeds. The Caprinae Titin-like protein is part of the sarcomere apparatus in muscle tissue []. Thus, we have combined genomic and proteomic evidence that the Iceman’s last meal contained whole seeds of the T. monococcum/T. urartu-like wheat and muscle fibers of ibex. However, due to the lack of diagnostic proteins, we cannot determine which tissues of the red deer were ingested by the Iceman.

27 Cordain L.

Watkins B.A.

Florant G.L.

Kelher M.

Rogers L.

Li Y. Fatty acid analysis of wild ruminant tissues: evolutionary implications for reducing diet-related chronic disease. 28 Mayer B.X.

Reiter C.

Bereuter T.L. Investigation of the triacylglycerol composition of iceman’s mummified tissue by high-temperature gas chromatography. Figure 3 Lipid Analysis of the Iceman’s Stomach Content Show full caption (A) Triacylglycerol (TAG) distribution in the Iceman’s stomach’s content in comparison to the TAG distribution in ibex muscle and fat tissue and goat dairy products. Displayed is the relative abundance of TAGs based on their chain length. (B) Saturation level of the TAGs in the stomach content and the fresh tissue samples. The Iceman’s stomach TAGs contain a much higher saturated bond content than fresh tissue samples. 26 Mirabaud S.

Rolando C.

Regert M. Molecular criteria for discriminating adipose fat and milk from different species by NanoESI MS and MS/MS of their triacylglycerols: application to archaeological remains. (C) The dispersion factor (DF) plotted against the average carbon number (M) of each TAG detected in the ancient and modern samples. The DF measures the spread of the TAG distribution. Together with M, DF can be used for discrimination between archaeological artifacts []. See also Data S2 for further details to the relative abundance of TAG species and to the mass spectrometry results. Approximately 46% ± 19% (w/w) of the stomach content is fat residues. Comparative lipid analysis of the Iceman’s stomach content to contemporary wild ruminant muscle and adipose tissue, and to goat dairy products (milk and cheese), using reverse-phase liquid chromatography separation and quadrupole time-of-flight (QToF) tandem mass spectrometry analysis (RP-LC/MSMS) identified triacylglycerols (TAGs) as a major component of the stomach content, with molecular compositions consistent with ruminant muscle/adipose fat ( Figure 3 A). The TAGs identified in the Iceman’s stomach contain between 44 and 56 total carbon atoms in the fatty acyl chains and resemble the TAG profile of contemporary muscle and adipose tissue. The high level (up to 20%) of odd numbered fatty acyl chains is consistent with TAG profiles from ruminant animals ( Data S2 ) []. Short-chain TAG species characteristic of dairy products are absent in the Iceman TAG profile. However, the high level of saturated bonds in the TAGs of the Iceman’s stomach content compared to the fresh tissues indicates an already ongoing preferential degradation of unsaturated fatty acids in the ancient specimen ( Figure 3 B) []. Therefore, we cannot fully exclude that short-chain dairy-specific lipid species were degraded, although the identification of di- and poly-unsaturated TAG indicates a still high level of preservation. Furthermore, we have histological evidence for the presence of adipose tissue in the Iceman’s stomach content ( Figure S1 C). Further comparison of the dispersion factor with the average carbon number (M) of each detected TAG in the analyzed samples points to the ibex as potential source of the adipose tissue ( Figure 3 C). Thus, the ibex served the Iceman as food source for both meat and fat.

16 Oeggl K.

Kofler W.

Schmidl A.

Dickson J.H.

Egarter-Vigl E.

Gaber O. The reconstruction of the last itinerary of “Ötzi”, the Neolithic Iceman, by pollen analyses from sequentially sampled gut extracts. 29 Böcker U.

Ofstad R.

Wu Z.

Bertram H.C.

Sockalingum G.D.

Manfait M.

Egelandsdal B.

Kohler A. Revealing covariance structures in fourier transform infrared and Raman microspectroscopy spectra: a study on pork muscle fiber tissue subjected to different processing parameters. Figure 4 Atomic Force Microscope Images and Raman Spectra of Muscle Fibers from the Iceman’s Stomach Content Show full caption (A) Fibers obtained from the content of the Iceman’s stomach (I) show structures similar to those of recent fibers (II and VI). The typical Z-line is observed in raw (II) and air-dried lamb muscle fibers (IV). After thermal treatment such as frying or cooking of the meat, the sarcomeres disappear (III). The surface of the fibers becomes blurred, and only faint fibrillary structures can be found. (B) Raman spectra of samples extracted from the Iceman’s stomach content show similarities to untreated (fresh) lamb meat spectra. See Data S2 for details to the modern comparative animal samples. Charcoal particles detected in a previous study in the Iceman’s lower intestine content suggested the involvement of fire in the food processing []. We experimentally tested the effect of heat and drying on contemporary animal muscle tissue and compared their structural and chemical composition with the muscle fibers detected in the Iceman’s stomach by using atomic force microscopy (AFM) and Raman spectroscopy. High magnification AFM images revealed striated fiber structures in both ancient and fresh or dried contemporary muscle fibers while structural changes of the fibers were observed when the samples were thermally treated ( Figure 4 A). After frying or cooking, the regular patterns in the meat fibers disappeared, indicating the denaturation of their protein compounds. Complementary confocal Raman spectroscopy measurements support the AFM results and indicate heat-related conformational changes of the molecular composition of the meat fibers ( Figure 4 B). The Raman spectra of the ancient muscle fibers largely matched the spectra taken of fresh and dried contemporary meat fibers. After heat treatment, the Raman band at about 1,093 cm-1 becomes weaker, and bands at around 1,240 cm-1 appear that are indicative of heat-induced changes in the secondary structure of the myofibrils []. Thus, both the AFM and Raman spectroscopy results suggest that the Iceman consumed either fresh or dried wild meat. Besides, a slow drying or smoking of the meat over the fire would explain the charcoal particles detected previously in the lower intestine content.