Study of freezing properties of aqueous leaf homogenates from Hippophae rhamnoides

Hippophae rhamnoides is a cosmopolitan shrub that has attracted the attention of many scientists worldwide, not only for its therapeutic properties and metabolite richness but also for its ability to sustain freezing down to -40 °C. Endowed with the capacity to withstand harsh conditions, this plant controls freezing processes to cope with seasonal exposure to sub-zero temperatures. Intriguingly, H. rhamnoides is reported to harbour intrinsic ice-nucleating agents (INAs) active at temperatures above -5 °C. Moreover, its fruits provide a habitat for diverse populations of bacteria represented by genera that might exogenously initiate ice formation on vegetative and reproductive parts. Inspired by the multifaceted cold adaptation features H. rhamnoides, in this work, we have examined freezing patterns of its aqueous crude leaf homogenates (CLHs) in admixture with cryoprotective agents (CPAs) known to promote supercooling: antifreeze protein (AFP) type III, sucrose, trehalose, and dimethyl sulfoxide (Me₂SO). For this, we employed infrared thermography, cryomicroscopy, and differential scanning calorimetry (DSC). In order to identify the possible microbial contributions to H. rhamnoides INAs, bulk and filtered CLHs underwent microbiological studies. Analogous to Snomax™ (SM), the addition of CLH to disaccharides increased crystallization duration and reduced both the degree of supercooling and freezing time compared to the respective controls. Findings from experiments involving filtered and heat-treated CLHs suggest the presence of soluble ice nucleators that may originate from the plants themselves, as well as from ice-nucleating bacteria that are active at warmer sub-zero temperatures. Using conventional aerobic cultivation on chromogenic solid media, we obtained three bacterial isolates from bulk H. rhamnoides homogenates. Using 16S rRNA sequencing, the isolates were identified as representatives of the Erwiniaceae family. The sequence of one isolate clustered with those of Pantoea agglomerans (formerly known as Enterobacter agglomerans or Erwinia herbicola), a well-known bacterial ice-nucleating species. Interestingly, two isolates were clustered together with the recently delineated species Duffyella gerundensis. In summary, the investigation into the freezing characteristics of leaves derived from H. rhamnoides is of potential environmental and cryobiological utility.