Identification of Lyoprotectants Released from Gradient-freezing Pretreated Entocyte in Lactococcus Lactis Subsp. Lactis IL1403

Performance of Lactococcus lactis as a starter culture in food production largely depends on the use of lyoprotectants during lyophilization. Gradient-freezing of bacterial cultures was conducted at 4°C, −20°C, and −80°C by storing the samples at each temperature for 2 h, successively. The entocytes extracted from the frozen cells were used as a lyoprotectant in the follow-up freeze-drying process of Lactococcus lactis subsp. lactis IL1403. The cell survival rate of gradient-freezing group increased 6.4-fold by bacterial plate count method. Furthermore, a proteomics and bioinformatics method was applied to elucidate the protein changes of Lactococcus lactis subsp. lactis IL1403 in response to gradient-freezing by gel-free proteomics using tandem mass tags (TMTs). The results showed that 121 stress-related proteins were significantly influenced by gradient-freezing. These proteins were involved in several metabolism pathways including ribosome metabolism, amino acid metabolism, quorum sensing, phosphotransferase system (PTS), pentose phosphate pathway, microbial metabolism in diverse environments, and nitrogen metabolism, etc. Some of these proteins especially the up-regulated proteins are potential lyoprotectants in vitro and they still need to be further investigated.