Expression Of Cold Shock Proteins Of L.lactis And Their Effects In Cryoprotection

Lactic acid bacteria (LAB) play an important role in the food industry because of their wideapplication as starter cultures in many fermentation processes. L.lactis, which is one of the mostimportant LAB involved in the dairy industry, have a fermentative metabolism and was widelyadopted for manufacturing dairy products like buttermilk, yogurt and cheese. During processingand ripening of fermented dairy products, Cold stress might be important for the survival of startercultures after frozen storage and for fermentations taking place at low temperatures.Many bacteria, including lactic acid bacteria, are able to adapt to temperatures far below theiroptimum growth temperatures, and a set of 7-kDa proteins is strongly induced in response to arapid decrease in growth temperature, named cold shock proteins.They are 65-70 amino acidresidues long and all of the structures contain five antiparallelβstrands that form aβbarrel. CSPsmay function as RNA chaperones, as they possess binding messenger RNA. In this way they couldminimize the secondary folding of mRNA, thereby facilitating the translation process.In this study, there is great interest on the effect of dealing with different cold shock stresseson Cold Shock Proteins and freeze-survival capacity of L.lactis and the effects of overproductionsCspC,CspD in cryoprotection. Several experiments were carried out:1. In order to set cold-shock temperatures, an Arrhenius plot of the growth of KLDS4.013 wasestablished. Cold shock subrange are from 28℃to 13℃and below 13℃.2. An approximately 200-fold increased survival after freezing was observed when cells wereshocked to 10℃for 4h compared to mid-exponential-phase cells grown at 30℃, Upon addition ofchloramphenicolthis culture is not able to recover from a cold shock,indicating an active protectionagainst freezing.3. Using SDS-PAGE 7.3,6.1KDa cold-induced proteins (CSPs) was identified after a coldshock treatment.4. CspC and CspD genes of L.lactic KLDS4.013 were PCR amplified with Dynazyme DNApolymerase.5. CspC and CspD was overexpressed using the nisin-controlled expression system. Analysisof the SDS-PAGE revealed that 7kDa cold-shock protein CspD and 6.2kDa cold-shock proteinCspC were identified respectively after induced with nisin.6. In order to study the function played by CspC and CspD, We overproduced each of them inturn using the nisin controlled expression system, three experiments were carried out: We firstobtained the growth curves include control strain and CSP-overproducing strains. And then Exponentially growing cultures (OD₆₀₀=0.8) were transferred from 27 to 7℃and growth wasmonitored for 30h. At last, The number of CFU was determined just before freezing and after fourconsecutive freeze-thaw challenges. The result shows that overproducing CspC improves growthresumption and overproducing CspD enhance the survival capacity after freezing (30-40 fold).In this study we provide evidence for an active adaptation response of L.lactis KLDS4.013 toa repetitive freezing challenge after exposure to low temperature and protein synthesis is found inthe class of 7kDa CSPs.which are the implication of CSPs in cryotolerance; In addition,overproduction of CSP in relation to freeze survivalwas studied. This revealed that L. lactis cellsoverproducing CspC improves growth resumption and overproducing CspD enhances cryotolerancebut that this protein is probably not the only factor determining cryoprotection.