Bronze Age cheese reveals human and Lactobacillus kefiranofaciens interactions throughout evolutionary history
NEWS - Food fermentation using microorganisms is the oldest food processing practice in human history. Milk fermentation can be traced back to 6000–4000 BC in India and Mediterranean peoples produced and consumed cheese as early as 7000 BC.
However, little is known about the history of microbial use and the associated history of cultural transmission. In particular, the evolutionary trajectories, particularly the functional adaptations of these fermenting microorganisms through long-term interactions with humans, remain unclear.
Recently, Chinese researchers conducted the world’s first metagenomic study of Bronze Age kefir cheese excavated from the Xiaohe cemetery, the oldest dairy remains known to date. Molecular evidence from ancient fermented milk residues serves as a powerful tool for understanding fermentation-microbe interactions.
The researchers have now extracted high-quality genomes of Lactobacillus kefiranofaciens (Lactobacillus kefiranofaciens) from three ancient cheese samples excavated from the Xiaohe cemetery in Xinjiang from around 3,500 years ago. The key was designing probes to enrich Lactobacillus DNA, increasing the target DNA from less 1% to 64-80% and allowing the reconstruction of the entire genome.
“We have been working on this project for more than 11 years. This is an unprecedented study, allowing us to observe how bacteria have evolved over the past 3,000 years,” said Fu Qiaomei, Chinese Academy of Sciences.
“In addition, we have gained a clearer picture of the lives of ancient humans and their interactions with the world. This is just the beginning, and with this technology, we hope to explore other previously unknown artifacts,” Fu said.
The cheese sample excavated from the Xiaohe Cemetery in Xinjiang is the earliest cheese product ever discovered. It was previously identified through paleoproteomics as kefir cheese, a fermented milk product made using kefir grains, which contain fermenting microbes.
The study confirmed that the cheese was made using lactic acid bacteria and yeast. The researchers also found that the milk used to make the cheese came from a group of goats that were widespread in Eurasia during the post-Neolithic period, distinct from contemporary domesticated goats from the interior of East Asia.
These findings suggest that the ancient people of Tarim Basim may have learned kefir production techniques from Eurasian steppe populations. The spread of milk fermentation technology was largely accompanied by human migration and interaction, a process that drove the evolution of lactic acid bacteria.
However, this study also uncovered a new route of dispersal of L. kefiranofaciens by analyzing the phylogenetic relationships of ancient L. kefiranofaciens in Xinjiang. The researchers found that L. kefiranofaciens used in fermentation consisted of two clades.
The first clade mainly consisted of strains from Europe (e.g., modern Germany) and coastal and island regions of Asia (e.g., modern Guangdong, Taiwan Island, Japan, and Singapore). This distribution corresponds to a dispersal route from the Caucasus to Europe on the one hand and to coastal regions of Asia and Southeast Asia on the other. The other clade mainly consisted of strains distributed inland in East Asia (including Tibet).
The ancient strains are located at the base of the clade, suggesting an additional route of dispersal of kefir production technology from Xinjiang to inland East Asia through technological-cultural exchange. The divergence of the two clades is likely the result of the dispersal of the originally domesticated ancestor in different populations, reflecting migrations and interactions that occurred during the use and domestication of microorganisms.
“It is fascinating to see how much information can be extracted from this cheese. Organic residues provide a window into past human behavior and culture that is lost in history and records,” said Yang Yimin from the Chinese Academy of Sciences.
The study also found that human use and domestication played an important role in the evolution of L. kefiranofaciens. Comparative analysis of the genomes of L. kefiranofaciens from the Bronze Age and modern times allows us to characterize the adaptive evolution of L. kefiranofaciens over the past few thousand years.
Extensive horizontal gene transfer is one of the main mechanisms of adaptive evolution in lactic acid bacteria. By comparing the functional profiles of ancient and modern L. kefiranofaciens, the study identified three main evolutionary trajectories of L. kefiranofaciens.
Adaptation to environmental stress with the emergence of drug resistance-related genes in modern strains. Enhanced bacterial genomic defense mechanisms with modern L. kefiranofaciens showing gene clusters such as the R-M system and associated toxin-antitoxin systems that can counteract the entry of exogenous DNA and thereby reduce associated fitness costs.
Adaptations related to the human gut environment, for example, modern strains acquired gene clusters that potentially interact with the host gut, most likely due to long-term interactions with humans.
The study also found that long-term human use and domestication of L. kefiranofaciens likely also influenced evolution. Compared with Bronze Age L. kefiranofaciens, modern strains from Tibet showed two horizontally transferred gene clusters associated with reduced gut inflammatory responses.
Given that ancient populations could easily spread kefir production technology via kefir grains, the presence of these gene clusters is likely related to a preference among contemporaneous populations for kefir grains carrying distinct clades of L. kefiranofaciens.
By using ancient DNA information about fermenting microorganisms that are closely associated with past human activities and cultures, this study offers a new framework for using ancient DNA to study past human-cultural interactions.
Original research
Liu, Yichen et al. (2024). Bronze Age cheese reveals human-Lactobacillus interactions over evolutionary history. Cell, DOI:10.1016/j.cell.2024.08.008
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