Metagenomic Study Reveals the Most Ancient Dairy Remains Known to Date

The oldest method of producing food with microorganisms in human history is food fermentation. Cheese production and consumption dates back to 7000 before the present (BP) among Mediterranean populations, and milk fermentation, for instance, dates back to 6000–4000 BC in India.

Though humans have consumed fermented foods for quite a while, little is known about the history of the use of fermentative microorganisms and the transmission of related cultures. Specifically, it is unclear how these fermented microorganisms have evolved through long-term interactions with humans, particularly in terms of functional adaptation.

However, by completing the first metagenomic study of Bronze Age kefir cheese discovered in Xiaohe cemetery—the oldest known dairy remains—a group of Chinese scientists have recently advanced our understanding of the spread of early dairy fermentation practices. Ancient fermented dairy residues provide molecular evidence that is a valuable tool for reconstructing past human-fermentative microbial interactions.

In this study, three old cheese samples from the 3,500-year-old Xiaohe Cemetery in Xinjiang were used to extract the high-quality genomes of Lactobacillus kefiranofaciens (L. kefiranofaciens). On September 25^th, 2024, the team’s research was made available online in Cell under the title "Bronze Age cheese reveals human-Lactobacillus interactions over evolutionary history." It was featured as a Cell Highlighted Paper.

We have been working on this project for over 11 years.”

Qiaomei Fu, Study Corresponding Author and Director, Molecular Paleontology Laboratory, Institute of Vertebrate Paleontology and Paleoanthropology (IVPP), Chinese Academy of Sciences

The secret is that she created probes to enrich Lactobacillus DNA, which raises the target DNA from less than 1% to 64–80% and permits the reconstruction of Lactobacillus’ entire genome.

Fu added, “This is an unprecedented study, allowing us to observe how a bacterium evolved over the past 3,000 years. Moreover, by examining dairy products, we’ve gained a clearer picture of ancient human life and their interactions with the world. This is just the beginning, and with this technology, we hope to explore other previously unknown artifacts.”

History of the Use and Spread of Kefir Yogurt Among Ancient Xinjiang Populations

The oldest known cheese product is a sample that was unearthed from Xiaohe Cemetery in Xinjiang. Paleoproteomics previously identified it as kefir cheese, a fermented milk product made from kefir grains that are home to fermentative microbes.

The study verifies that lactic acid bacteria and yeasts were used to make the cheese by reconstructing the fermentation microbial community. In addition, the researchers found that, unlike modern domesticated goats from inland East Asia, the milk used to make the cheese originated from a clade of goats that were widely dispersed throughout Eurasia during the post-Neolithic era.

This discovery implies that Eurasian steppe populations are likely where the ancient Tarim Basim peoples learned how to produce kefir.

The evolution of lactic acid bacteria was largely driven by human migration and interaction, which coincided with the spread of dairy fermentation technology. However, by examining the phylogenetic relationships of ancient L. kefiranofaciens in Xinjiang, this study also reveals a new route of L. kefiranofaciens spread.

The researchers discovered that the L. kefiranofaciens used in fermentation belonged to two clades. The first was primarily composed of strains from Asia’s coasts and islands (such as those occupied by modern Guangdong, Taiwan Island, Japan, and Singapore) and Europe (such as the region that is now Germany).

This distribution followed the path taken by the species to disperse from the Caucasus to Europe and, conversely, to the coastal regions of Asia and Southeast Asia. The strains in the other clade were mostly found in inland East Asia, including Tibet.

The base of the clade contains the reconstructed ancient strains, indicating another possible route for the diffusion of kefir production technology from Xinjiang to inland East Asia via cross-cultural interactions.

The spread of their common ancestor, which was first domesticated in different populations, is likely what caused the two clades of L. kefiranofaciens to diverge. This migration and interaction happened during the consumption and domestication of fermentative microorganisms by various ancient populations.

It is exciting to see how much information can be retrieved from these cheeses. Organic residues open a window into past human behaviors and culture that were lost in history and records.”

Yimin Yang, Professor, University of Chinese Academy of Sciences

Domestication and Evolution of Lactobacillus by the Xiaohe Population

The use and domestication of L. kefiranofaciens by humans was also found to have been a significant factor in its evolution. Lactobacillus kefiranofaciens’ adaptive evolution over the previous few thousand years can be characterized by comparing the genomes of the species from the Bronze Age and the present.

One of the primary mechanisms of adaptive evolution in lactic acid bacteria is extensive horizontal gene transfer. The study identified three main trajectories of L. kefiranofaciens evolution by comparing the functional profiles of ancient and modern strains:

1) adaptation to environmental stress, as evidenced by the emergence of drug-resistance-related genes in modern strains; 2) enhancement of bacterial genome defense mechanisms, as evidenced by the presence of gene clusters such as the R-M system and the associated toxin-antitoxin system in modern strains, which can counteract the introduction of exogenous DNA and thereby reduce related fitness costs; and 3) adaptations related to the human intestinal environment, as evidenced by the acquisition of gene clusters by modern strains that may interact with the host gut, probably as a result of long-term interactions with humans.

This study also discovered that domesticating L. kefiranofaciens and using it for a long time by humans probably had an impact on its evolution. Modern strains of Lactobacillus kefiranofaciens from Tibet exhibit two horizontally transferred gene clusters linked to the reduction of the intestinal inflammatory response, in contrast to strains from the Bronze Age. This supports intestinal function in addition to helping L. kefiranofaciens survive in the human gut.

The presence of these gene clusters is probably related to the preference of ancient populations for kefir grains carrying different clades of L. kefiranofaciens, given that ancient populations could easily spread kefir production technology through kefir grains.

Human-microbial interaction is always fascinating. Fermentative microbes played such an important role in the daily life of these ancient humans, and they propagated these microbes for thousands of years without knowing the existence of them for most of the time.”

Yichen Liu, Study Authors, Associate Professor, Associate Professor, Institute of Vertebrate Paleontology and Paleoanthropology (IVPP), Chinese Academy of Sciences

This study presents a novel framework for using ancient DNA to study past human cultural interactions, based on information about fermentative microorganisms closely related to past human activities and culture.