Exploring the diversity of extremely halophilic archaea in food-grade salts

(eng) Introduction Salting is one of the oldest means of food preservation: adding salt decreases water activity and inhibits microbial development. Worldwide production of salt is led by China (24 %), Europe (21 %) and USA (17 %). Applications for food and feed represent 3 % of the European consumption. Salted and fermented food products often contain halophilic archaea. The presence of Halorubrum and Halosarcina species has been reported in brines during fermentation of table olives [1]. Histamine-degrading archaea belonging to genera Halobacterium and Natrinema were reported in salted-fermented fishery products and Haloarcula marismortui was isolated from salted anchovies [2] [3]. Kimchi, a Korean fermented food, was shown to maintain archaeal population of the Natronococcus, Natrialba, Halosimplex, Halobiforma and Halococcus genera [4]. The salt itself has proven to contain viable microbial cells [5]. However, to our knowledge, no studies have investigated the occurrence and diversity of archaea in food-grade salts. Material and methods The archaeal diversity of twenty-six commercial food-grade salts from worldwide origin was assessed by culture on solid media (MGM, Hv-YPC, DBCM2 and CDM with pyruvate). Colony forming units (CFU) were counted and phenotypically distinct isolates were identified by 16S rRNA gene sequencing. Additionally, high-throughput sequencing was performed on nine of these salts. Results Viable archaea were observed in 14 salts and colony counts reached more than 105 CFU per gram in three salts. All archaeal isolates identified by 16S rRNA gene sequencing belonged to the Halobacteriaceae family and were related to 16 distinct genera among which Haloarcula, Halobacterium, Halarchaeum and Halorubrum were the most represented. High-throughput sequencing generated extremely different profiles for each salt. Four of them contained a single major genus (Halorubrum, Halonotius or Haloarcula) while the others had three or more genera of similar occurrence. The number of distinct genera per salt ranged from 21 to 27. Discussion Halorubrum had a significant contribution to the archaeal diversity in seven salts; this correlates with its frequent occurrence in crystallization ponds. On the contrary, Haloquadratum walsbyi, the halophilic archaea most commonly found in solar salterns, was a minor actor of the food-grade salt diversity. This supports the hypothesis that the crystallization process modifies the archaeal diversity, possibly due to distinct survival capabilities in extremely low water activity environments. Although not primarily sought, halophilic bacteria were also recovered from four food-grade salts. They were isolated from the richest substrate media MGM and Hv-YPC. The latter not only supported the growth of halophilic bacteria, but also impeded archaeal development. The surprisingly high content of viable archaea in popular food-grade salts frequently used in food preparation raises the question of their fate after ingestion. Therefore, their survival and potential activity in human intestinal tract should now be considered and further investigated. References 1. Abriouel H. et al. (2011). Int J Food Microbiol 144(3): 487-496. 2. Moschetti G. et al. (2006). Ann Microbiol 56(2): 119-127. 3. Tapingkae W. et al. (2010). Enzyme Microb Tech 46(2): 92-99. 4. Chang H-W. et al. (2008). Int J Food Microbiol 126(1-2): 159-166. 5. Minegishi H. et al. (2010). Int J Syst Evol Microbiol 60(11): 2513-2516.