Impact of different crio and lyoprotectans on the viability and reactivation time of Enterococcus faecium LET 301

ARGAÑARAZ MARTINEZ E.; PEREZ CHAIA A.; QUIROGA M.; GARRO M.; BERTANI M.; BABOT J.D.

VIRTUAL

Congreso; XV Congreso Argentino de Microbiología General SAMIGE; 2020

The efficacy and success of probiotic supplements for humans and animals depends mainly on the technology of production and conservation of the cultures, the survival of bacteria in the matrix used as vehicle, and the method of storage of the product. Therefore, the selection of an adequate method for the conservation of a probiotic strain is important to guarantee its viability during processing and storage. Thus, the aim of this work was evaluating the effect of conservation at -20 ºC and lyophilization, using cryo- and lyoprotectants on the viability and reactivation time of the probiotic strain E. faecium LET 301. To this end, E. faecium LET 301 was grown in a 1.5 L fermenter with fixed pH (5.5) at 37ºC in the experimental broth MLQ, which includes by-products of local industries (sugar cane molasses, yeast cream, and cheese whey). The biomass was recovered by centrifugation, ten-fold concentrated in 10% (w/v) milk or MLQ broth, with or without cryoprotectants [10% (w/v) monosodic glutamate, glycerol, or trehalose] or lyoprotectants [10% (w/v) monosodic glutamate or trehalose, or 5% (w/v) sucrose]. For low temperature conservation, samples were frozen and stored at -20 ºC for 120 d. After this time and immediately after freezing, samples were thawed at 37ºC for 5 min and viability (in LAPTg agar) and reactivation time (in MLQ broth and cecal water) were assessed. For conservation by lyophilisation, the powders were stored at 4ºC for 120 d in the dark. After this time and immediately after lyophilisation, samples were resuspended to their original volume in sterile physiological solution or MLQ broth, and viability and reactivation time (Rt) were assessed after 10 min at room temperature. After freezing, E. faecium LET 301 showed a viability percentage higher than 87% in all the conditions tested. After 120 d stored at -20ºC, only samples in milk with monosodic glutamate or trehalose showed viabilities higher than 87%, while samples in milk and MLQ without cryoprotectant evidenced 55 and 65% viabilities, respectively. The Rt of E. faecium LET 301 after 120 d frozen was 2-3 h, depending on the cryoprotectant. On the other hand, the probiotic strain showed viability higher than 86% in presence of lyoprotectectants and 75% in their absence after lyophilisation. Besides, the samples with lyoprotectectants evidenced viabilities higher than 70% after 120 d of storage, while the control without lyoprotectectant retained 50% of viability. The Rt of the lyophilized strain was 3 h in all the conditions tested, except in the control (4 h). These results indicate that E. faecium LET 301, previously isolated from the intestine of poultry, can be conserved by different approaches using cryo- and lyoprotectectants commonly used for non-intestinal strains, such as monosodic glutamate and trehalose. Although better results were obtained by conserving at -20ºC, the lyophilization method could be used for farms in rural areas where a freezer is not available.