Selection of mutants tolerant of oxidative stress from respiratory cultures of Lactobacillus plantarum C17.

AIMS: Lactobacillus plantarum is a lactic acid bacterium involved in the production of many fermented foods. Recently, several studies have demonstrated that aerobic or respiratory metabolism in this species leads to improved technological and stress response properties. METHODS AND RESULTS: We investigated respiratory growth, metabolite production and stress resistance of Lact. plantarum C17 during batch, fed-batch and chemostat cultivations under respiratory conditions. Sixty mutants were selected for their ability to tolerate oxidative stress using H2 O2 and menadione as selective agents and further screened for their capability to growth under anaerobic, respiratory and oxidative stress conditions. Dilution rate clearly affected the physiological state of cells and, generally, slow-growing cultures had improved survival to stresses, catalase production and oxygen uptake. Most mutants were more competitive in terms of biomass production and ROS degradation compared with wild-type strain (wt) C17 and two of these (C17-m19 and C17-m58) were selected for further experiments. CONCLUSIONS: This work confirms that, in Lact. plantarum, respiration and low growth rates confer physiological and metabolic advantages compared with anaerobic cultivation. SIGNIFICANCE AND IMPACT OF THE STUDY: Our strategy of natural selection successfully provides a rapid and inexpensive screening for a large number of strains and represents a food-grade approach of practical relevance in the production of starter and probiotic cultures.

Temperature and respiration affect the growth and stress resistance of Lactobacillus plantarum C17.

AIMS: The aim of the study is to gain further insight on the respiratory behaviour of Lactobacillus plantarum and its consequences on stress tolerance. METHODS AND RESULTS: We investigated the effect of temperature and respiration on the growth and stress (heat, oxidative, freezing, freeze-drying) response of Lact. plantarum C17 during batch cultivations. Temperature as well as respiration clearly affected the physiological state of cells, and generally, cultures grown under respiratory conditions exhibited improved tolerance of some stresses (heat, oxidative, freezing) compared to those obtained in anaerobiosis. Our results revealed that the activities in cell-free extracts of the main enzymes related to aerobic metabolism, POX (pyruvate oxidase) and NPR (NADH peroxidase), were significantly affected by temperature. POX was completely inhibited at 37°C, while the activity of NPR slightly increased at 25°C, indicating that in Lact. plantarum, the temperature of growth may be involved in the activation and modulation of aerobic/respiratory metabolism. CONCLUSIONS: We confirmed that respiration confers robustness to Lact. plantarum cells, allowing a greater stress tolerance and advantages in the production of starter and probiotic cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on respiratory metabolism on a strain other than the model strains WCFS1; novel information on the role of temperature in the modulation of aerobic/respiratory metabolism in Lact. plantarum is presented.

Aerobic metabolism and oxidative stress tolerance in the Lactobacillus plantarum group.

Aerobic metabolism and response to oxidative stress and starvation were studied in 11 Lactobacillus plantarum, L. paraplantarum and L. pentosus strains in order to assess the impact of aerobic metabolism on the growth and on the stress response. The strains were grown in aerobiosis without supplementation (AE), with hemin (AEH) or with hemin and menaquinone (AEHM) supplementation and in anaerobiosis (AN) in a complex buffered substrate. Growth rate, biomass yield, glucose and O2 consumption, production of lactic acid and H2O2, catalase activity, oxidative and starvation stress tolerance were evaluated. Aerobic growth increased biomass yield in late stationary phase. Further increase in yield was obtained with both hemin (H) and menaquinone (M) addition. With few exceptions, the increase in biomass correlated with the decrease of lactic acid which, however, decreased in anaerobic conditions as well in some strains. Addition of H or H + M increased growth rate for some strains but reduced the duration of the lag phase. H2O2 production was found only for aerobic growth with no supplementation due to catalase production when hemin was supplemented. To our knowledge this is the first study in which the advantages of aerobic growth with H or H + M in improving tolerance of oxidative stress and long-term survival is demonstrated on several strains of the L. plantarum group. The results may have significant technological consequences for both starter and probiotic production.

A comparison of fluorescent stains for the assessment of viability and metabolic activity of lactic acid bacteria.

Lactic acid bacteria (LAB) are used as starter or probiotic cultures in the food and pharmaceutical industries and, therefore, rapid and accurate methods for the detection of their viability are of practical relevance. In this study 10 LAB strains, belonging to the genera Enterococcus, Lactococcus, Leuconostoc, Lactobacillus, Streptococcus and Weissella, were subjected to heat and oxidative stresses and cell injury or death was assessed comparing different fluorescent probes (Syto 9; Propidium Iodide, PI; 4,6-diamidino-2-phenylindole, DAPI; 5,(6)-carboxyfluorescein diacetate, cFDA) to identify the stain combination which most reliably allowed the detection of live/metabolically active and dead cells. Protocols for specimen preparation and staining were optimized and a simple procedure for automated cell counts was developed using NIH ImageJ macros. Cysteine and semi-solid agar solution were efficiently used as anti-fading agent and mounting medium, respectively. The double staining cFDA-PI apparently offered the best and most versatile indication of both cell metabolic activity and membrane integrity. An excellent correlation between manual and automated cell counts for the majority of strain/stain combinations was found. This work provides a simple protocol for specimen preparation and staining based on the use of safe, easy to prepare and inexpensive reagents as compared to other methods. Additionally, the automated cell count procedure developed can be applied to several bacterial species and allows an increase in the number of experimental trials and the reproducibility and sensitivity of the analysis.