Investigating Biases Associated With Dietary Starch Incorporation and Retention With an Oral Biofilm Model

Abstract

Dental calculus has proven to contain a wealth of information on the dietary habits of past populations. These insights have, to a large extent, been obtained by the extraction and identification of starch granules contained within the mineralised dental plaque from a wide range of regions and time periods. The scope of previous studies have been limited to microfossil extraction and identification to reconstruct dietary preferences from the archaeological record, and few studies have attempted to address the biases of starch retention in dental calculus. Those that have considered this problem have been limited to in vivo studies on modern humans and non-human primates. Here, we present a multispecies oral biofilm model, which allows experimental research on starch incorporation and retention to be conducted on in vitro dental calculus in a controlled laboratory setting. The biofilms were exposed to treatment solutions with known quantities of dietary starches (wheat and potato) during the 25 days growth period. After this, the starch granules were extracted from the mature biofilm (by dissolution in EDTA), and counted. We show that the granule counts extracted from the model dental calculus represented a low proportion (ranging from 0.06% to 0.16%) of the total number of granules exposed to the biofilms throughout the experiment. Additionally, we found that the ratios of granule sizes from the extracted starch granules differed from the original treatment solutions, with large granules (>20 μm) consistently being under-represented. We also found a positive correlation between the absolute granule counts and dry-weight of the biofilm (r = 0.659, 90%CI[0.463, 0.794]), meaning the absolute quantity of starch granules will increase as the size of the calculus deposit increases. A similar, but weaker correlation was found between the concentration (count per mg) of granules and dry-weight (r = 0.3, 90%CI[0.0618, 0.506]). Our results complement and reinforce previous in vivo studies suggesting that dental calculus presents a very small, and partly biased picture of the original dietary intake of starches, with an over-representation of plants producing granules smaller than 20 μm in size. The experimental model presented here is well-suited to address the need for further validation of methods and biases associated with dietary research on dental calculus.

Ecological flexibility and adaptation to past climate change in the Middle Nile Valley: A multiproxy investigation of dietary shifts between the Neolithic and Kerma periods at Kadruka 1 and Kadruka 21

“…However, there is increasing recognition of the interpretive constraints that limit the utility of these analyses as a proxy for past diet [84][85][86]. Studies continue to highlight the variability of recovered microparticle assemblages that present a partial record of dietary intake and fail to capture dietary breadth at the individual level [87,88]. As such, dental calculus studies are increasingly applying larger population approaches embedded in multiproxy frameworks [57,58,[89][90][91][92].…”

Assessing the validity of a calcifying oral biofilm model as a suitable proxy for dental calculus

“…Raw potato and wheat starch solutions were added during the biofilm growth to explore the biases involved in their incorporation and extraction from dental calculus. These results are presented in a separate article (Bartholdy & Henry, 2022).…”

“…An 𝛼-amylase assay conducted on samples from days 3, 6, 8, 9, 10, 12, and 14 also showed that there was no host salivary 𝛼-amylase activity in the system. The results of the starch incorporation and 𝛼-amylase activity assay have been reported in a separate article (Bartholdy & Henry, 2022).…”

“…Our goals for additional validation measures involve functional profiles of bacteria, to see if metabolic behaviour of bacteria is consistent with in vivo conditions, and whether this is affected by the presence/absence of amylase and starch treatments. The absence of host salivary 𝛼-amylase activity in our model (as shown in Bartholdy & Henry (2022)) provides an opportunity to explore the effect of various amylase levels on biofilm growth and composition, as well as the incorporation of dietary compounds in dental calculus.…”