Tween 80 and respiratory growth affect metabolite production and membrane fatty acids in Lactobacillus casei N87.

AIMS: To evaluate the effect of cultivation (anaerobiosis vs respiration) and Tween 80 supplementation on the production of metabolites and on the composition of membrane fatty acids (FAs) in Lactobacillus casei N87. METHODS AND RESULTS: Anaerobic and respiratory growth, with or without Tween 80 supplementation, was carried out in a chemically defined medium. Production of biomass, organic acids, volatile organic compounds (VOCs), consumption of amino acids and changes in membrane FAs were investigated. Respiration altered the central metabolism rerouting pyruvate away from lactate accumulation, while Tween 80 had a minor effect on metabolic pathways. VOCs were mainly affected by growth conditions and significant amounts of diacetyl were produced by respiratory cultures. Respiration increased desaturation of membrane lipids and Tween 80 improved the production of essential polyunsaturated FAs. Palmitic acid decreased in Tween-supplemented aerated cultures. CONCLUSIONS: Combination of Tween 80 and respiratory growth promoted production of biomass and aroma compounds and affected the composition of membrane FAs in Lact. casei N87. SIGNIFICANCE AND IMPACT OF THE STUDY: Respiration might be exploited in Lact. casei as a natural strategy for the enhanced production of aroma compounds.

Modified chemically defined medium for enhanced respiratory growth of Lactobacillus casei and Lactobacillus plantarum groups.

AIMS: Members of the Lactobacillus casei and Lactobacillus plantarum groups are capable of aerobic and respiratory growth. However, they grow poorly in aerobiosis in the currently available chemically defined media, suggesting that aerobic and respiratory growth require further supplementation. METHODS AND RESULTS: The effect of Tween 80, L-alanine, L-asparagine, L-aspartate, L-proline and L-serine on anaerobic and respiratory growth of Lact. casei N87 was investigated using a 2(5) factorial design. The effectiveness of modified CDM (mCDM) was validated on 21 strains of Lact. casei and Lact. plantarum groups. Tween 80 supplementation did not affect anaerobic growth, but improved respiratory growth. L-asparagine, L-proline and L-serine were stimulatory for respiring cells, while the presence of L-aspartate, generally, impaired biomass production. mCDM promoted the growth of Lact. casei and Lact. plantarum, with best results for strains showing a respiratory phenotype. CONCLUSIONS: The nutritional requirements of anaerobic and respiratory cultures of members of the Lact. casei and Lact. plantarum groups differ. Tween 80 and selected amino acids derived from pathways related to TCA cycle, pyruvate conversion and NADH recycling are required for respiration. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of mCDM will facilitate the study of aerobic metabolism of lactobacilli under controlled conditions.

Biochemical analysis of respiratory metabolism in the heterofermentative Lactobacillus spicheri and Lactobacillus reuteri.

AIMS: This study evaluated the aerobic and respiratory metabolism in Lactobacillus reuteri and Lactobacillus spicheri, two heterofermentative species used in sourdough fermentation. METHODS AND RESULTS: In silico genome analysis, production of metabolites and gene expression of pyruvate oxidase, pyruvate dehydrogenase and cytochrome oxidase were assessed in anaerobic and aerobic cultures of Lact. reuteri and Lact. spicheri. Respiring homofermentative Lactobacillus casei N87 and Lact. rhamnosus N132 were used for comparison. Aerobiosis and respiration increased the biomass production of heterofermentative strains compared to anaerobic cultivation. Respiration led to acetoin production by Lact. rhamnosus and Lact. casei, but not in heterofermentative strains, in which lactate and acetate were the major end-products. Lactobacillus spicheri LP38 showed the highest oxygen uptake. Pyruvate oxidase, respiratory cytochromes, NADH oxidase and NADH peroxidase were present in the genome of Lact. spicheri LP38. Both Lact. spicheri LP38 and Lact. rhamnosus N132 overexpressed pox in aerobic cultures, while cydA was up-regulated only when haeme was supplied; pdh was repressed during aerobic growth. CONCLUSIONS: Aerobic and respiratory growth provided physiological and metabolic advantages also in heterofermentative lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: The exploitation of oxygen-tolerant phenotypes of Lact. spicheri may be useful for the development of improved starter cultures.

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.