The best liquor comes from the oldest mud pits.
Over 70% of all distilled beverages produced in China are a flavourful liquor, traditionally made in a process that includes sealing grains, yeast, and other materials into mud pits for months at a time.
Microbes in the mud impart a unique taste to the liquor with compounds like butyric acid, caproic acid, and ethyl caproate.
A new paper in the Canadian Journal of Microbiology investigated how the microbe communities in mud pits changed with age and how they interacted with each other to make high-quality liquor.
Led by Yan Zheng from Zhengzhou University of Light Industry, the team extracted DNA from one-, six-, and ten-year-old mud pits. They compared variations in the 16S ribosomal RNA gene in each sample, allowing them to identify which microbes were present.
One-year-old mud pits had very different microbe communities than six- and ten-year-old pits, and differences in age were responsible for nearly 48% of the microbial variation between pits.
The bacteria Firmicutes dominated young mud pits, representing 72% of all 16S ribosomal RNA sequences detected in one-year-old samples. Firmicutes abundance dropped with age, representing only about 32% of microbes in the six- and ten-year-old mud pits.
A key member of Firmicutes was Lactobacillus, which converts sugars to lactic acid and makes the mud more acidic. Lactobacillius also releases anti-bacterial proteins, making the pit environment less hospitable for sensitive microbes. With the decrease in Firmicutes over time, other bacteria took their place including Aminobacterium.
Aminobacterium breaks down amino acids into acetate that can indirectly fuel the production of caproic acid, one of the key flavour compounds in mud pit liquor.
The archaea, Euryarchaeota, also increased with age, representing from 11% of the microbes in one-year-old pit mud to 37% in the ten-year-old mud. Some key members of Euryarchaeota are methanogens, microbes with an unusual affinity for consuming hydrogen.
Delving deeper into the ecology of young and old mud pits, the researchers investigated how the microbes interacted with each other to produce the liquor’s unique flavor.
To do this, they used network analysis, a mathematical technique that identifies the relationships between members of a community. Network analysis can reveal which community members are the most influential and uncover subtle, indirect relationships.
The network analysis indicated that some organisms tended to occur together, suggesting a synergistic relationship.
For example, the bacterial caproic acid producers, Aminobacterium, tended to co-occur with the methanogenic archaea. Synthesizing caproic acid makes a lot of hydrogen, and high levels of hydrogen can be harmful to microbes. By gobbling up the extra hydrogen, the methanogens protect the Aminobacterium, allowing the pit to produce flavourful, high-quality liquor.
There were also microbes that tended to avoid each other. For example, Lactobacillus showed a strong negative correlation with several types of microbes. Too much Lactobacillus in the pit can lead to a scarcity of hydrogen, which limits the growth of the beneficial methanogens.
Brewing the perfect bottle of liquor is a process that takes years, even decades; in the case of traditional Chinese liquor, it comes down to building a well-balanced community of microbes.
Read the paper: Co-occurrence patterns among prokaryotes across an age gradient in pit mud of Chinese strong-flavor liquor in the Canadian Journal of Microbiology.
