Proteins were successfully recovered from all 10 of the dental calculus samples analysed, with total protein identifications ranging from 15 to 128 (following quality filtering) (Table S5). The identified proteins were assigned primarily to the human proteome and to microbial taxa commonly found within the oral microbiome, as well as to common contaminants identified in previous dental calculus proteomic analyses (principally trypsin, human keratins and human collagens) (Hendy et al. 2018a; Mackie et al. 2017; Warinner et al. 2014b) (Fig. S1). The only dietary protein identified within the sample set was BLG. The initial Mascot search strategy only considered peptides which conformed to tryptic cleavage patterns, identifying 34 BLG peptides in 6 individuals (Table S6). These identifications were augmented with spectral data searched using semi-tryptic modifications (Table 2), which identified 29 additional BLG peptides (i.e., not detected in the tryptic dataset) and an additional BLG positive individual (HH3). Using this combined dataset, over half of the dental calculus samples (n = 7) displayed evidence for BLG peptides (Table 2). BLG peptides were identified in all three assemblages (Hambledon Hill (n = 2), Hazleton North (n = 4), Banbury Lane (n = 1)), although with one individual from both Hambledon Hill and Hazelton North represented by only a single BLG peptide. No BLG peptides were identified within blank controls or injection blanks. Using the combined search strategy, 63 spectra (comprising 14 distinct peptides) were assigned to BLG. For each of the samples which tested positive for BLG, a consensus BLG sequence could be assigned to ruminants of the Pecora infraorder of Artiodactyla, with all seven samples containing Bovidae-specific peptides (see Supplementary Information).

Levels of deamidation were examined using MaxQuant to ensure that the recovered BLG peptides and human oral proteins conformed with damage expected from ancient proteins, as opposed to modern contaminants (Mackie et al. 2018). Whilst asparagine (N) tends to deamidate into aspartic acid (D) relatively rapidly (van Duin and Collins 1998; Robinson and Robinson 2001, 2004), deamidation of glutamine (Q) into glutamic acid (E) is markedly slower and therefore can be used to assess protein degradation in archaeological contexts (van Doorn et al. 2012; Wilson et al. 2012). As expected, contaminant proteins (e.g., human keratins, trypsin, etc.) displayed, on average, low-levels of deamidation, with 90.9% undamaged asparagines and 98.5% undamaged glutamines (Fig. S2). The subset of analysed human proteins displayed variable, but on average, more advanced deamidation, with only 17.7% undamaged asparagines and 33.4% undamaged glutamines across the samples (Fig. S2). The BLG peptides displayed the most advanced levels of deamidation. In total, five individuals displayed BLG peptides with at least one deaminating residue: two individuals (HH610 and HN7387) displayed BLG peptides with both Ns and Qs and three individuals (HN11456, BL309.12, HN4786) displayed peptides with either Ns or Qs. For further two individuals (HH3, HN7656), the detected BLG peptides contained neither Ns nor Qs. In the five individuals where at least one deamidating BLG peptide was observed, the deamidation levels were extremely advanced. On average, only 2% of the asparagines and 5% of the glutamines were undamaged across the samples (Fig. 2). These levels of degradation are more advanced than those reported in previous analyses of BLG from ancient dental calculus proteomes (Mays et al. 2018; Mackie et al. 2017), a result consistent with the greater antiquity of these Neolithic individuals compared to previous studies (e.g., Middle Bronze Age, Roman and Medieval individuals).

Fig. 2 Deamidation of all β-lactoglobulin peptides in the samples where a deamidating BLG peptide was found, as measured by relative intensity of peptides with deamidated asparagines (N) and glutamines (Q) versus the total intensity of both the deamidated and unmodified version of the peptides Full size image

Of the two individuals from Hambledon Hill in which BLG peptides were detected here, both indicated BLG deriving from Bovidae (HH3; HH610), a family of ruminants which includes cattle, buffalo, bison, antelopes, sheep, goats and gazelles (Gentry 1992). Therefore, the BLG within the calculus of two individuals at Hambledon Hill may have derived from cattle, sheep or goat milk. However, one tryptic peptide in individual HH610 also suggested the specific presence of goat (Capra sp.) milk. These results are supported by the faunal assemblage from the site, with domesticated cattle (Bos taurus) being the most dominant species present, followed by smaller numbers of ovicaprids (Ovis aries and Capra hircus) (Legge 2008). The age and sex structure of the cattle remains at Hambledon Hill have previously been suggested to be indicative of a dairy herd (Whittle 1992, p. 221; Copley et al. 2003), although others have suggested that the restricted age distribution of the cattle means that the animals were unlikely to have derived from a resident herd (Legge 2008). The high number of cattle skulls has also been interpreted as being representative of an association between the deposition of human and faunal remains (Thomas 1999, p. 28), and parallels between the treatment of human and cattle remains has been suggested to represent ‘respectful consumption’ of beef (Jones 2007, p. 164). The large-scale deposition of cattle remains, however, alongside large amounts of burnt wheat, barley and hazelnuts, has prompted suggestions of feasting at the site (Jones and Legge 2008; Mercer 2008). Additionally, organic residue analysis of pottery from Hambledon Hill has indicated the presence of both porcine and ruminant fats, and ruminant adipose and dairy fats (Copley et al. 2003, 2005a, b, 2008). The presence of dairy fats in > 25% of potsherds analysed has been suggested to indicate that ‘dairying was a very important element of animal husbandry at Hambledon Hill’ (Copley et al. 2008, p. 535).

Of the four individuals from Hazleton North in which BLG peptides were detected, all were also specific to Bovidae, but with peptides distinct from Capra, suggesting that individuals from Hazleton North were likely exploiting milk from cattle and/or sheep, but not goat. Stable isotopic analysis of δ13C and δ15N on human remains from the site has revealed a diet high in animal protein, supplemented by C3 plants (Hedges et al. 2008). Although Hazleton North does not possess a faunal assemblage akin to that at Hambledon Hill, the remains of domesticated cattle were nonetheless recovered within the chambered areas of the tomb at the site (Levitan 1990), and therefore, is consistent with the utilisation of cattle milk by the individuals at Hazleton North.

One individual from Banbury Lane (BL309.12) was also found to have BLG specific to Bovidae, but not Capra, using the tryptic search strategy. Unlike the other two sites, Banbury Lane has very little faunal material or pottery associated with either the monument itself or the human remains (Holmes 2012).

The combined results obtained here from British sites indicate that Neolithic populations were utilising milk and/or dairy products from either cattle, goat or sheep (or potentially combinations of these). This evidence is consistent with the recent discovery of bovine milk consumption in the Middle Neolithic individual recovered from Stonehenge (Mays et al. 2018). The results obtained here also show varying numbers of BLG spectra detected across all individuals, as has been observed in previous studies (Warinner et al. 2014a; Mays et al. 2018). For example, two individuals analysed here (HH3 and HN7656) exhibited only one BLG-specific spectrum each, whereas in contrast, individual HH610 from Hambledon Hill had a total of 36 spectra matching BLG peptides (13 unique peptides). The reasons as to why some individuals have a significantly higher number of BLG spectra are still unclear but is something which certainly warrants further future study. Additional study of the abundance of BLG spectra within different dental calculus samples may reveal if this is linked to the amount of dairy consumed by an individual, or instead if it is the result of preservational biases or environments, or the timings and nature of calculus formation. In order to accurately assess this, however, a much broader scale study, with the likely inclusion of modern dental calculus samples from individuals with known diets, would be needed. Further study should also reveal if individuals with no evidence of BLG peptides within their calculus (as observed in this study, and within other proteomic studies (Warinner et al. 2014a; Hendy et al. 2018a)) were truly not consuming dairy products, or if the absence of milk proteins is instead the result of differential calculus formation processes or taphonomic effects.

It is important to note, however, that due to recent discoveries of LP prevalence in the European Neolithic (Burger et al. 2007; Gamba et al. 2014; Witas et al. 2015; Olalde et al. 2018; Brace et al. 2019), it is unlikely that the British Neolithic individuals studied here would have carried the genetic mutation associated with lactase persistence. Human remains from neither Hambledon Hill nor Hazleton North have been subjected to ancient DNA analysis to date, but none of the individuals (n = 3) genetically analysed thus far from Banbury Lane have a derived lactase persistence allele (Brace et al. 2019). The presence of BLG within the dental calculus however indicates the consumption of dairy products, as supported by other archaeological evidence for dairying from the period, as discussed above. It is important to note however that BLG may reflect either the regular consumption of small quantities of raw milk—based on the observation that individuals without LP can tolerate up to 240 mL of milk per day with negligible symptoms (Suarez et al. 1995; Swallow 2003)—or alternatively, the consumption of processed milk products with reduced lactose content. Distinguishing between these two consumption scenarios is not possible based on the current data and analytical technique. Nevertheless, as organic residue analysis has revealed that the processing of dairy in pottery vessels was widespread from the Early Neolithic onwards in Britain (Copley et al. 2005b), the likely absence of LP, but presence of BLG, is therefore more consistent with the hypothesis that Neolithic populations were processing milk to remove the lactose, but in such a way that at least some BLG was retained.

Lactose can be removed from or decreased in milk products through a range of processing methods. For example, cheese contains little or no lactose, as it is removed during processing with the whey fraction of the milk. Indeed, 98% of lactose is removed in the whey during most cheese production (Izco et al. 2002). The production of cheese within prehistory has however been suggested to have been beneficial for past populations not only due to the reduced lactose content, thereby making it more readily digestible and suitable for non-LP individuals, but also because it allows for the preservation of milk products in a transportable form (Salque et al. 2013). Lactose content is also known to be much decreased in fermented milk products, such as yoghurt, kefir and buttermilk (Alm 1982; O’Brien 1999). As such, fermented milk products have previously been suggested to be suitable for consumption by lactose intolerant individuals (Alm 1982). Although yoghurt does contain small amounts of lactose, it is believed that this lactose is more easily digested than that found within whole milk, due to hydrolysis and autodigestion of lactose by the yoghurt bacteria—thus improving its absorption and creating a ‘lactase activity’ in the gastrointestinal tract (Kolars et al. 1984; Savaiano 2014).

BLG is known to be present in processed milk products, but generally in significantly lower quantities than whole milk, and decreases the more the whole milk is processed. BLG is known to be absent or at very low levels in hard cheeses, for example, due to the removal of the whey fraction of the milk during processing. BLG is however present in yoghurts and other fermented milk products, and it has been noted that it is not markedly subject to hydrolysis or proteolysis during the fermentation process (Bertrand-Harb et al. 2003; Tzvetkova et al. 2007). The heating or fermentation of milk will decrease the levels of BLG within it, but to varying degrees dependent upon the type and intensity of processing utilised (Czerwenka et al. 2007; Bu et al. 2013).

The idea of Neolithic milk processing to reduce lactose content has been proposed previously (e.g., in the form of cheese making (Salque et al. 2013)) and is plausible given the recent genetic evidence for the absence of LP in Neolithic populations. Of the three sites analysed here, only the pottery from Hambledon Hill has previously had organic residue analysis undertaken upon it, indicating the exploitation of dairy fats (Copley et al. 2003). Overall, the evidence for dairy consumption (as evidenced through BLG peptides within calculus detected here and lipid residue analyses on pottery), combined with genetic support for the absence of LP, suggests that either British Neolithic populations were processing raw milk in an attempt to remove or reduce the lactose content, or alternatively consuming relatively small quantities of raw milk. As such, these observations open a range of exciting new research avenues exploring how Neolithic populations may have been consuming and processing raw milk, the required technologies needed to do this, the kinds of products which may have been created, the potential variability which may have existed within these processes in the past, and notions of cuisine. It is conceivable that past populations chose to utilise the milk of different animals purposively, and processed this in different ways, due to cultural reasons or even taste. We can consider the modern regional variability which exists in cheese production in the UK—for example, varying in terms of the types of milk used, the processing methods, if the finished product is a soft or hard cheese, how long the cheese is left to mature for, and often having Protected Designation of Origin or Protected Geographical Indication status (British Cheese Board 2018)—as an indication that similar regional differences may have also existed in the prehistoric past in dairy processing and production. The fact that milk lipids are frequently found in British Neolithic pottery, often with thermally modified compounds, such as long-chain ketones, also suggests that raw milk was processed by heating.