Henriet et al., International Journal of Food Microbiology. 2014. 191: 36-44. Full text available here.
Within the world of fermented food, salt is usually regarded as a tool for controlling microbial activity rather than as a source of microbial diversity. But recent studies are beginning to reveal that unrefined salts can carry viable and diverse microbial communities. This paper explores the incidence of members of the domain Archaea in a selection of food-grade salts from around the world.
Introduction: The Archaea
Members of the microbial world fall into two main categories: the prokaryotes (e.g., bacteria) and eukaryotes (e.g., fungi, algae, protozoa). The development of DNA sequencing methods has allowed scientists to classify organisms not simply by how they look or behave, but by how similar their sequences of certain universal genes are to one another. This DNA analysis led scientists to discover that there are actually two types of prokaryotes: Bacteria and Archaea. The Archaea share the cell structure of prokaryotes (no nuclei or organelles), but they are actually more closely related to Eukaryotes. The Archaea consist largely of so-called extremophiles, microbes that are adapted to very salty, acidic, alkaline, or hot or cold environments.
The authors of the paper examined 26 food-grade commercial salts for the presence of Archaea using two complementary approaches:
- Plating samples of the dissolved salts on selective media and then identifying the organisms that grew using DNA sequencing (culture-based method)
- Extracting DNA directly from the salt and identifying the organisms present without a culturing step (culture-independent / metagenomic analysis)
Summary of Findings
Over half (14 of the 26) of the salts grew colonies on at least one of the four selective culture media used in the study. Moreover, the levels found in some salts—such as Sel de Guérande—were quite high (over one million CFU [colony-forming units] per gram). Notably, the three refined salt samples did not contain any viable Archaea.
Most of the Archaea found belonged to a family of extreme halophiles (salt-loving organisms) called Halobacteriaceae, and members of many different genera within that family were identified. Some salts (such as Halen Mon and La Baleine coarse sea salt) showed low diversity, with less than three strains identified; others, such as the Guérande and Camargue salts, contained up to nine isolated strains.
Direct DNA analysis was carried out on nine of the salts, three of which had not yielded any viable colonies when plated out. All of them showed the presence of between 21-27 different genera; the results suggested that the vast majority of the organisms present were similarly members of Halobacteriaceae.
This metagenomic analysis also allowed the scientists to estimate the relative proportion of each genus within each salt sample. Their abundance and proportions varied significantly: in some samples, like the Black Sea salt, one genus dominated the mix; in others such as the Camargue salt there was a more even distribution between many different organisms.
Natural unrefined salt is a haven for large and diverse populations of Archaea, most of which were identified as Halobacteriaceae. These organisms have been shown in other studies to survive in so-called ‘fluid inclusions’: tiny pockets of moisture that form when salt initially crystallises from water. The authors reference a paper where Archaea trapped this way have been shown to be viable over 22,000 years later.
Archaea are not the only group of microbes that can survive within salt. Another group of scientists have also revived a dormant bacterium from a 250-million year slumber in a salt crystal. And some of Dr Benjamin Wolfe’s research in the Dutton lab (which I had a chance to help with last winter) suggested that sea salt used by cheesemakers is host to various Bacillus species and a wide variety of Proteobacteria, some of which thrive on washed and bloomy cheese rinds. (Here’s a presentation of some of that research from the Science of Artisan Cheese Conference last summer.)
The different salts examined in this paper contained different Archaeal genera. Some evidence suggests that the genera isolated from a salt itself is not always directly reflective of the microbial population of the saltern from which it originated. Perhaps certain organisms are more suited to survival in fluid inclusions than others, or the balance of microbes in the saltern changes over time.
The study also compared and contrasted the organisms found in the same salts using the two different methods. For some salts, the results were quite similar; in others the cultured species appeared to be only minor players amongst a large and diverse group of organisms that never grew on the culture plates. This is a great illustration of a principle that applies to all culture-based laboratory analysis: just because something doesn’t grow on a plate doesn’t mean it isn’t there. Conversely, several strains of Archaea that grew successfully on the plates were never identified using the culture-independent method, suggesting that these two complementary methods are still the best way to get a nuanced idea of what organisms are present in a sample.
Archaea can grow and survive within various foods, and have been isolated from fermenting olive brines, salted anchovies, and kimchi, among others. The authors conclude by questioning whether there might be a health implication to their presence in food-grade salt; it seems a pertinent line of inquiry given that viable members of this family have also been isolated from the human intestinal tract. We still have much to learn about the role of these and other microorganisms in salt, and in fermented foods that use natural salt as an ingredient.
Post written by Bronwen Percival