Freeze-drying, a biotechnological process employed to dehydrate products at low temperatures, involves freezing the product, such as probiotic strains, under low pressure. However, one of the main disadvantages of this technique is the occurrence of osmotic shock and cell membrane damage, which significantly reduce cell viability. Therefore, the addition of protective substances during freeze-drying may help maintain the viability and survival of these microorganisms. The development of a protocol that guarantees high viability is essential for creating a product based on probiotic bacteria for aquaculture. Therefore, the objective of this study was to investigate the viability and survival of bacteria strains Enterococcus faecium and E. gallinarum isolated from the intestinal microbiota of Arapaima gigas following freeze-dried using different cryoprotectants.
Initially, the E. faecium and E. gallinarum strains were isolated from the intestine of healthy A. gigas juveniles, cultivated in tryptone soy agar (TSA) medium and incubated for 48 h (35 ºC). After confirming purity through Gram staining, the bacteria were inoculated in tryptone soy broth (35 ºC/24 h). Subsequently, the culture media containing E. faecium and E. gallinarum were centrifuged individually, and the bacterial cells harvested by centrifugation were washed twice, homogenized, and resuspended in sterilized phosphate-buffered saline (PBS) to obtain a standard cell suspension (SCS). The SCS was fractionated into equal volumes, and the cryoprotectants dextrose (D), fructose (F), skimmed milk (SK), maltodextrin (M), sucrose (S), and trehalose (T) were added individually. PBS was used as the control. The samples were frozen and then dried by freeze-drying (40 h/-48 ºC). Cell viability (log10 CFU/mL) of both strains before and after lyophilization was determined by serial dilution in PBS (10-1-10-6) and subsequent plating in TSA.
After freeze-drying, E. faecium and E. gallinarum coated with skimmed milk and trehalose, or skimmed milk, trehalose, and dextrose, respectively, did not show any significant reduction in cell viability (p > 0.05). The survival rates of E. faecium and E. gallinarum varied widely (37.26–105.90%) among the cryoprotectants. Probiotic strains freeze-dried with skimmed milk (90.45–97.32%), trehalose (95.72–105.90%), and dextrose (93% for E. gallinarum) showed high survival rates compared to other cryoprotectants and the control treatment (p < 0.05). Therefore, our study highlighted for the first time that all tested cryoprotectants provided high viability for the lactic acid strains E. faecium (≥ 8.6 log CFU/g) and E. gallinarum (≥ 9.4 log CFU/g) after freeze-drying, but only the incorporation of skimmed milk and trehalose promoted very high survival rates (≥90%) for both probiotic strains (Figure 1). This indicates the potential of both cryoprotectants for the protection and stability of bacterial strains of biotechnological interest post-lyophilization. Furthermore, we emphasize that understanding how these probiotics can be preserved effectively for potential applications in aquaculture and health management is essential to strengthening a more sustainable aquaculture.