Lactobacillus paracasei A13 and High-Pressure Homogenization Stress Response

Abstract

Sub-lethal high-pressure homogenization treatments applied to Lactobacillus paracasei A13 demonstrated to be a useful strategy to enhance technological and functional properties without detrimental effects on the viability of this strain. Modification of membrane fatty acid composition is reported to be the main regulatory mechanisms adopted by probiotic lactobacilli to counteract high-pressure stress. This work is aimed to clarify and understand the relationship between the modification of membrane fatty acid composition and the expression of genes involved in fatty acid biosynthesis in Lactobacillus paracasei A13, before and after the application of different sub-lethal hyperbaric treatments. Our results showed that Lactobacillus paracasei A13 activated a series of reactions aimed to control and stabilize membrane fluidity in response to high-pressure homogenization treatments. In fact, the production of cyclic fatty acids was counterbalanced by the unsaturation and elongation of fatty acids. The gene expression data indicate an up-regulation of the genes accA, accC, fabD, fabH and fabZ after high-pressure homogenization treatment at 150 and 200 MPa, and of fabK and fabZ after a treatment at 200 MPa suggesting this regulation of the genes involved in fatty acids biosynthesis as an immediate response mechanism adopted by Lactobacillus paracasei A13 to high-pressure homogenization treatments to balance the membrane fluidity. Although further studies should be performed to clarify the modulation of phospholipids and glycoproteins biosynthesis since they play a crucial role in the functional properties of the probiotic strains, this study represents an important step towards understanding the response mechanisms of Lactobacillus paracasei A13 to sub-lethal high-pressure homogenization treatments.

Keywords: Lactobacillus paracasei; high-pressure homogenization; membrane fatty acid composition; stress response.

Figure 1

Schematic representation of the conserved fatty acids biosynthesis pathway based on the Lb. paracasei ATCC334 fatty acids synthesis model. In red are highlighted the genes considered in this study. Acc genes (accA, accB, accC, accD) are involved in the malonyl-CoA synthesis. Malonyl-ACP Malonyl-CoA:ACP transacylase (fabD) catalyze the coenzyme A substitution with an acyl carrier protein (ACP). Malonyl-ACP is condensed by 3-oxoacyl-ACP synthase (fabH) to acetyl-CoA, producing a β-ketoacyl-ACP. Ketoacyl-ACP reductase (fabG) starts with a reduction and is followed by a dehydration executed by 3-hydroxyacyl-ACP- dehydratase (fabZ). A second reduction by enoyl-ACP-reductase (fabK) produces acyl-ACP. In order to continue the elongation reaction, acyl-ACPs are dehydrated by 3-oxoacyl-ACP synthase (fabF) and the elongation chain cycle starts again. Once sufficiently elongated, acyl-ACPs are processed by different hydrolytic enzymes depending on the chain length. Medium-chain fatty acids (i.e., C:12) are released by the esterase activity of the Medium-chain acyl-ACP hydrolase (mch).

Figure 2

Relative gene expression (RGE) index of Lb. paracasei fatty acids biosynthesis genes accA, accB, accD, fabD, fabH, fabG, fabZ, fabK and fabF in relation to homogenization pressure. RGE index was calculated according to the model proposed by Pfaffl [24] and reviewed by Muller et al. [25] using ileS (Isoleucine tRNA ligase) as reference gene and homogenization at 0.1 MPa as the control condition. The results are the average of three replicates and for the same gene, column followed by different superscript letters are significantly different p < 0.05. Error bars indicate ± SD.