Effects of acid adaptation on the survival of Lactobacillus plantarum NCMIB 8826 in fruit juices

The aims of this work were to study the effect of acid adaptation on the survival of the stationary phase Lactobacillus plantarum NCMIB 8826 cells, a model potential probiotic strain, in several highly acidic fruit juices namely cranberry (pH 2.7), pomegranate (pH 3.5), and lemon & lime juices (pH 2.8) and to investigate the mechanisms involved in cellular response. The results indicated that exposure of the cells in both acidified solutions (buffers and de Man, Rogosa and Sharpe medium; MRS) adjusted to pH 6 to 3 by hydrochloric acid and citric acid for a short period of time significantly improved cell survival in the fruit juices, although the impact on cell viability was less than 107 CFU/ml for 6 weeks which was required for probiotic drinks. Furthermore, the prolonged exposure time (1 to 5 h) and low temperature (10 and 4 ο C) were used to enhance the cell viability of Lactobacillus plantarum NCMIB 8826 but the improvements by these techniques could not make it as a promising strain for probiotic drinks. Analysis of citric and lactic acids as well as γ-aminobutyric acid (GABA) indicated that the citrate fermentation pathway and the glutamate decarboxylase system, which have been implicated in acid response in several lactobacilli, were not involved in this case while the analysis of the cellular fatty acid content showed that the cyclopropane fatty acid, cis-11,12-methylene octadecanoic acid (C19cyclow7c), significantly increased (by ∼1.7 fold) during acid adaptation, which was accompanied by a significant upregulation of the cyclopropane synthase (cfa) gene (by ∼12 fold), as demonstrated by reverse transcription polymerase chain reaction. It was likely that these changes led to a decrease in membrane fluidity and to lower membrane permeability, which prevent the cells from proton influx during storage in fruit juices. Examination of the cell morphology by cryo-scanning electron microscopy revealed that the cell surface of acid adapted cells was rougher and thicker compared to control cells, suggesting that the composition and structure of the peptidoglycan was possibly modified during acid exposure. A significant finding of this study was the observation that alanine, which represented the most abundant intracellular amino acid (> 45%), was significantly reduced in the case of acid adapted cells (∼20%) compared to control cells, which coincided with a significant decrease in the extracellular alanine (~10%). It seems, therefore, likely that upon acid adaptation the cells utilized the available alanine to increase in the D-alanylation of wall teichoic acid, resulting in a positive cell wall with enhanced ability to reduce the influx of protons during storage in fruit juices. Moreover, proteomic analysis was also performed using 2D-gel electrophoresis, which led to the identification of eight proteins exhibiting a difference in % volume of at least 1.4 in expression levels between acid adapted and control cells. Among these, five proteins, molecular chaperone GroEL, aminopeptidase C, 30S ribosomal protein S1, D-alanine-Dligase, and UPF0356 protein Ip_2157 were upregulated, whereas three proteins, 30S ribosomal protein S2, aspartate semialdehyde dehydrogenase, and the hypothetical protein HMPREF0531_11643 were downregulated in acid adapted cells compared to control cells.