Research On The Heat-Adaptive Kinetics Of Thermo-Tolerance And HSP Gene Expression Of Lactobacillus Plantarum

Probiotics are becoming commercially valuable for their probiotic properties that draw public attention. When consumed in adequate dose of at least 107 CFU ml?1 of live bacteria, they are confirmed to reduce blood cholesterol, counter oxidation or inflammation, modulate immunity, and inhibit pathogenic infection. The higher bacterial viability the probiotic products contain, the more benefits they will confer. However, the adverse conditions during the processing and storing of the products (e.g. extremes in temperature, pH, and osmotic pressure) may lead to substantial loss of viability, which is a technological bottleneck for the probiotic industry. Approaches to enhance the stability of probiotic products, like stress response of heat, acid, and salt, are well on their way to success. Especially heat stress response offers the convenience to be applied to large- and food-scale production. The topic of this research is all about the heat stress response of a commercialized probiotic strain called Lactobacillus plantarum LpOnlly and its thermo-tolerant derivative LpOnlly-Hr.This work compared the relationship between the kinetics of cellular thermo-tolerance and gene expression of heat shock proteins (HSPs) when LpOnlly was heat-adapted under various conditions. The aim is to disclose the factors and the mechanisms that influence the heat stress response of Lactobacillus plantarum. Heat adaptation was performed at 45℃with a combination of such conditions as three bacterial growth phases (mid-exponential, pre-stationary, and stationary phases), two heat-adaptive matrices (natural culture and fresh media), and two strain phenotypes (prototrophic and thermo-tolerant strains). Twelve HSP genes were selected by four groups including dnaK operon, groESL operon, small HSP genes, and genes of proteases belonging to the CtsR regulon. To reveal the impact of the time for heat adaptation on bacterial physiology, LpOnlly mid-exponential-phase cells heat-adapted for 30 min and 90 min, respectively, were recovered under normal growth temperature of 37℃. Kinetics of cellular thermo-tolerance and HSP gene expression were also characterized. Determination in 60-min duration with 6-min interval and heat challenge at 58℃for 1.5 min were adopted for both heat adaptation and recovery.Firstly, the thermo-tolerance kinetics of all samples, except stationary-phase cells in natural culture, was presented in elevating sigmoid under the experimental conditions for heat adaptation. Thermo-tolerance enteres plateau after heat adaptation for 30 min, a node called critical time in this study. In addition, intrinsically increased thermo-tolerance of cells or using fresh media as heat-adaptive matrix for cells generally decreases the inducibility of the thermo-tolerance during heat adaptation. Secondly, heat-adaptive kinetics of HSP gene expression goes into three patterns. Pattern 1 is hump-shaped, namely an expression peak that mainly appears at 24-36 min which corresponds to the critical time of the thermo-tolerance kinetics. Some vital transcriptional regulation may happen at the critical time. Pattern 1 represents a major modality of the HSP gene expression kinetics. As the heat-adaptive time runs through the critical time, the HSP gene expression of Pattern 2 and Pattern 3 do not drop as immediately as that of Pattern 1, suggesting extra modes of transcriptional regulation. When the tested heat-adaptive condition alters, these two rare patterns sometimes transform to Pattern 1. In addition, intrinsically increased HSP gene expression of cells or using fresh media as heat-adaptive matrix for cells generally decreases the inducibility of the HSP gene expression during heat adaptation. Heat adaptation in natural culture of LpOnlly and LpOnlly-Hr cells in mid-exponential phase else shows the repressive effect of hrcA and ctsR from the gene expression of hrcA operon and CtsR regulon. Lastly, after heat stress is canceled, thermo-tolerance lasts longer for LpOnlly cells heat-adapted for 90 min (long term) than for 30 min (short term). Meanwhile, expression of the HSP genes in the recovering cells after short- and long-term heat adaptation all decays exponentially with the former declines in a faster rate, which is tightly related to the thermo-tolerance kinetics. In the late period of the decay, long heat-adapted cells exceed short ones in the expression of an overwhelming majority of the HSP genes. Moreover, it was verified by way of parenthesis in this study that the heat-adapted seed culture of LpOnlly almost lost its induced thermo-tolerance on harvest after the normal batch fermentation.Judging comprehensively from the thermo-tolerance kinetics and HSP gene expression kinetics during heat adaptation and recovery, critical time can be determined before picking a proper heat-adaptive time to meet the experimental need. If maximal thermo-tolerance is required under a specific heat-adaptive condition, adapting till the critical time suffices. Once the aim is durable thermo-tolerance even in case of the removal of heat stress, adapting longer than the critical time will be more effective. If transcription level of the heat stress response is certainly to be studied, the heat-adaptive time has to be decided by the special moment in the expression kinetics of the target gene. On the other hand, high induction of thermo-tolerance is not necessarily accompanied by high expression of the HSP genes during heat adaptation. It seems that induction of the HSP genes is a preliminary resort to combat heat stress, and bacteria will switch to other defensive pathways as the stressed effect continues, e.g. recruiting more HSPs to cure damage, reducing fluidity of the plasmic membrane, changing the internal pH of the cell, etc.