Adaptive response of Lactobacillus curvatus CRL705 during growth in the presence of curing additivies analyzed through a physiologic and proteomic perspective

Abstract

During the processing, drying and ripening of fermented meat products, a hostile environment develops that selectively limits the microbiota present and to which lactic acid bacteria manage to adapt competitively. The curing additives (CA) are included in meat products to guarantee their best preservation and color development. The most commonly CA used are: sodium chloride, which favors drying and hinders microbial growth; sodium nitrite, which acts as preservative and intervenes in the color; ascorbic acid, an antioxidant; and sucrose, which is used as energy source to favor starter culture dominance. Lactobacillus (L.) curvatus CRL705 was isolated from an Argentinean fermented sausage, and is of great interest as starter and bioprotective culture due to the production of two bacteriocins (Antilisteria and Lactocin 705). In this context, the objective of this work was to study the role of CA into adaptation mechanisms of L. curvatus CRL705 using physiologic and proteomic approaches. L. curvatus CRL705 growth and pH decrease, acid and bacteriocin production were evaluated in a chemically defined medium (CDM) without (control) and with CA (3% NaCl, 200 ppm Sodium Nitrite, 0.1 mg/ml ascorbic acid and 0.75% sucrose) at 25 ºC during 24 h. Differential protein expression was evaluated in both growing conditions by bidimensional electrophoresis (2DE). The selected proteins were identified by Maldi ToF MS/MS. A functional analysis was conducted for identified proteins which were assigned to different COG categories. In addition, interaction protein networks were built using STRING v10.05 and Cytoscape v3.6.1 softwares. Results showed that CRL705 strain presented similar growth and pH decrease when grew in both media but presenting a longer lag phase in the medium containing CA. Also, it could be noticed through HPLC that the consumption of glucose was higher in the CDM control as well as the production of lactic acid. When bacteriocins production was analyzed, no differences in Lactocin CRL705 titers were detected. Nevertheless, AL705 production was higher in the CDM control. 36 proteins presented significantly lower expression level (1.2 - 2.8 times) in the presence of the CA, most of them related with the carbohydrates metabolism and to oxide-reduction processes, among others. In conclusion, L. curvatus CRL705 presented a general repressed metabolism during its growth in the presence of CA although was capable of keeping its growing and acidifying ability under these stressful conditions, which represents encouraging results for the exploitation of CRL705 strain as a starter and/or bioprotective culture for cured meat products.