Preservation of Lacticaseibacillus paracasei 90 by freeze-drying: viability, culturability, cell architecture and metabolic potential

GUILLERMO PERALTA; BERET MV; BURGI M; ALE, E.C.; MARTÍNEZ L.; ALBARRACÍN V.; ERICA HYNES; BERGAMINI CARINA

Conferencia; CYTO Virtual 2021; 2021

Freeze-drying is the main process used to preserve lactic cultures at industrial level, making it possible to have viable cells for a long period of time. However, their viability and metabolic activity could be affected not only by the process per se, but also by the storage conditions. In this sense, the storage temperature is one of the most important factors, as it will have a great impact on the final cell viability. On the other hand, the culture medium might significantly affect the amount of biomass produced and the activity of the cells, having an influential effect on the resistance of microorganisms to freeze-drying and storage. The objective of this work was to evaluate the resistance of Lacticaseibacillus paracasei 90 (L90) to the freeze-drying process and storage after its growth in two economic culture media formulated with industrial residues (M1 and M3), in comparison with a commercial medium (MRS). Briefly, L90 was inoculated (2% v/v) in 1 L of each culture media (M1, M3 and MRS), and was subsequently incubated at 34 °C for 20 h. The cells were harvested and washed twice with 50 mM potassium phosphate buffer (pH 7) and resuspended in 300 mL of a 10% w/v lactose as cryoprotectant. The cell suspension in lactose was fractionated in sterile glass vials and frozen at -80 °C. Finally, these suspensions were freeze-dried in a 24 h cycle (Christ Alpha 1?4 LD Plus). The vials were immediately vacuum sealed once the cycle was completed. The obtained freeze-dried cultures were labelled as FDM1, FDM3 and FDMRS, depending on the medium used to grow the cells. The vacuum-sealed vials were stored for a period of 14 months at two temperatures: RT-room temperature and LT-low temperature (4 °C). Cell counts and scanning electron microscopy (SEM) were performed to evaluate the impact of freeze-drying on culturability and cell architecture, while flow cytometry-FC (at 3, 6 and 14 months) and cell counts (at 1, 3, 6 and 14 months) were performed to assess the effect of storage temperature on viability and culturability, respectively. For FC studies, the cells were stained with thiazole orange and propidium iodide and analysed using a Guava® EasyCyteTM cytometer. Finally, in order to study several parameters related to metabolic activity at the end of storage, ultra-high temperature milk was inoculated (2% v/v) with each treatment and incubated for 24 h at 37 °C. Regardless of the growth medium, the freeze-drying process did not have a negative impact on culturability, which was >9 log cfu/mL before and after freeze drying. The SEM micrographs of the freeze-dried cultures (before being rehydrated) showed that most lactobacilli were trapped within the amorphous matrix that lactose produces when dehydrated. In particular, for FDM1, a greater number of cells outside the lactose matrix was observed in comparison with FDM3 and FDMRS. The percentages of dead, injured, and live cells obtained by FC showed that FDM3 and FDMRS storage at LT presented the highest levels of viability (>96%) at the end of storage. In contrast, storage at RT had a negative effect, especially for FDM1, showing the highest levels of injured and dead cells. The result obtained from microbiological counts showed similar trends. The negative impact of RT on viability and culturability also affected their ability to acidify milk. Overall, these results demonstrate the robustness of L90 to survive freeze-drying and storage for 14 months at LT, highlighting its potential to be applied as an adjunct culture in the food industry.