Rémy Cachon

Extracellular Oxidoreduction Potential Modifies Carbon and Electron Flow in Escherichia coli

Wild-type Escherichia coli K-12 ferments glucose to a mixture of ethanol and acetic, lactic, formic, and succinic acids. In anoxic chemostat culture at four dilution rates and two different oxidoreduction potentials (ORP), this strain generated a spectrum of products which depended on ORP. Whatever the dilution rate tested, in low reducing conditions (-100 mV), the production of formate, acetate, ethanol, and lactate was in molar proportions of approximately 2.5:1:1:0.3, and in high reducing conditions (-320 mV), the production was in molar proportions of 2:0.6:1:2. The modification of metabolic fluxes was due to an ORP effect on the synthesis or stability of some fermentation enzymes; thus, in high reducing conditions, lactate dehydrogenase-specific activity increased by a factor of 3 to 6. Those modifications were concomitant with a threefold decrease in acetyl-coenzyme A (CoA) needed for biomass synthesis and a 0.5- to 5-fold decrease in formate flux. Calculations of carbon and cofactor balances have shown that fermentation was balanced and that extracellular ORP did not modify the oxidoreduction state of cofactors. From this, it was concluded that extracellular ORP could regulate both some specific enzyme activities and the acetyl-CoA needed for biomass synthesis, which modifies metabolic fluxes and ATP yield, leading to variation in biomass synthesis.

Measurement of the intracellular pH in Escherichia coli with the internally conjugated fluorescent probe 5- (and 6-)carboxyfluorescein succinimidyl ester

The 5- (and 6-)carboxyfluorescein succinimidyl ester (cFDASE) is a pH-dependent fluorescent probe which conjugates with the aliphatic amine functions of the cells. Because of this property, the fluorescence efflux rate is low, 10 to 40% after 30 min of fermentable carbon source incubation. Thus, this technique allows a continuous high-time measurement (>30 min) without correction of the signal, but also a dynamic study phenomenon of few seconds. Due to the advantages presented by this technique, the method was adapted for Escherichia coli. The internal pH measurement obtained under various conditions are in accuracy with those from radiolabelled technique and from other fluorescence probe, with the advantages mentioned previously.

Engineering a Saccharomyces cerevisiae wine yeast that exhibits reduced ethanol production during fermentation under controlled microoxygenation conditions

ABSTRACT We recently showed that expressing an H2O-NADH oxidase in Saccharomyces cerevisiae drastically reduces the intracellular NADH concentration and substantially alters the distribution of metabolic fluxes in the cell. Although the engineered strain produces a reduced amount of ethanol, a high level of acetaldehyde accumulates early in the process (1 g/liter), impairing growth and fermentation performance. To overcome these undesirable effects, we carried out a comprehensive analysis of the impact of oxygen on the metabolic network of the same NADH oxidase-expressing strain. While reducing the oxygen transfer rate led to a gradual recovery of the growth and fermentation performance, its impact on the ethanol yield was negligible. In contrast, supplying oxygen only during the stationary phase resulted in a 7% reduction in the ethanol yield, but without affecting growth and fermentation. This approach thus represents an effective strategy for producing wine with reduced levels of alcohol. Importantly, our data also point to a significant role for NAD+ reoxidation in controlling the glycolytic flux, indicating that engineered yeast strains expressing an NADH oxidase can be used as a powerful tool for gaining insight into redox metabolism in yeast.

Theme 4: Immobilized Cell Technology in Wine Production

AbstractIn spite of its traditional nature, wine making is largely concerned with the progress of biotechnology. High cell density reactors have potential for enology: improved performance of alcoholic and malolactic fermentations, smaller scale fermentation facilities, adaptation to continuous processes. Among the immobilization techniques, cell entrapment in alginate beads seems to be an impressive one. Alcoholic fermentation of wine, malolactic fermentation, bottle fermentation known as “Methode champenoise” and sparkling wine are among the industrial applications. Knowledge of kinetics and physiology in microorganisms in heterogeneous media has expanded in the last few years. The use of immobilized yeast cells for the champagne method would greatly simplify “remuage”. The compared metabolism of entrapped and free cells during the bottle fermentation shows differences, but the final product does not reveal significant sensory disparity. New products can be obtained with more thoroughly controlled condi...

Impact of Probiotics on Risk Factors for Cardiovascular Diseases. A Review

Probiotic microorganisms have historically been used to rebalance disturbed intestinal microbiota and to diminish gastrointestinal disorders, such as diarrhea or inflammatory bowel diseases (e.g., Crohns disease and ulcerative colitis). Recent studies explore the potential for expanded uses of probiotics on medical disorders that increase the risk of developing cardiovascular diseases and diabetes, such as obesity, hypercholesterolemia, arterial hypertension, and metabolic disturbances such as hyperhomocysteinemia and oxidative stress. This review aims at summarizing the proposed molecular and cellular mechanisms involved in probiotic–host interactions and to identify the nature of the resulting beneficial effects. Specific probiotic strains can act by modulating immune response, by producing particular molecules or releasing biopeptides, and by modulating nervous system activity. To date, the majority of studies have been conducted in animal models. New investigations on the related mechanisms in humans need to be carried out to better enable targeted and effective use of the broad variety of probiotic strains.

Experimental conditions affect the site of tetrazolium violet reduction in the electron transport chain of Lactococcus lactis

The reduction of tetrazolium salts to coloured formazans is often used as an indicator of cell metabolism during microbiology studies, although the reduction mechanisms have never clearly been established in bacteria. The objective of the present study was to identify the reduction mechanisms of tetrazolium violet (TV) in Lactococcus lactis using a mutagenesis approach, under two experimental conditions generally applied in microbiology: a plate test with growing cells, and a liquid test with non-growing (resting) cells. The results showed that in both tests, TV reduction resulted from electron transfer from an intracellular donor (mainly NADH) to TV via the electron transport chain (ETC), but the reduction sites in the ETC depended on experimental conditions. Using the plate test, menaquinones were essential for TV reduction and membrane NADH dehydrogenases (NoxA and/or NoxB) were partly involved in electron transfer to menaquinones. In this case, TV reduction mainly occurred outside the cells and in the outer part of the plasma membrane. During the liquid test, TV was directly reduced by NoxA and/or NoxB, probably in the inner part of the membrane, where NoxA and NoxB are localized. In this case, reduction was directly related to the intracellular NADH pool. Based on these findings, new applications for TV tests are proposed, such as NADH pool determination with the liquid test and the screening of mutants affected in menaquinone biosynthesis with the plate test. Preliminary results using other tetrazolium salts in the plate test showed that the reduction sites depended on the salt, suggesting that similar studies should be carried out with other tetrazolium salts so that the outcome of each test can be interpreted correctly.

Use of redox potential modification by gas improves microbial quality, color retention, and ascorbic acid stability of pasteurized orange juice

The aim of this paper was to study the effect of both redox potential (Eh) and pasteurization of orange juice on stability of color and ascorbic acid, and growth recovery of microorganisms during storage at 15 degrees C for 7 weeks. Three conditions of Eh, +360 mV (ungassed), +240 mV (gassed with N2), and -180 mV (gassed with N2-H2) were applied to orange juice. Both thermal destruction and recovery of sublethally heat-injured cells of Lactobacillus plantarum and Saccharomyces cerevisiae were investigated. While oxidizing conditions were the most effective for thermal destruction of L. plantarum and S. cerevisiae, reducing conditions decreased recovery of heated cells of S. cerevisiae. In addition, gassing the juice with N2 or N2-H2 increased color retention and ascorbic acid stability. The present study demonstrated that juice must be reduced just after the heat treatment in order, firstly, to maximize microbial destruction during pasteurization, and secondly, to prevent the development of microorganisms and stabilize color and ascorbic acid during storage.

Effect of oxidoreduction potential on aroma biosynthesis by lactic acid bacteria in nonfat yogurt

The aim of this study was to investigate the effect of oxidoreduction potential (Eh) on the biosynthesis of aroma compounds by lactic acid bacteria in non-fat yogurt. The study was done with yogurts fermented by Lactobacillus bulgaricus and Streptococcus thermophilus. The Eh was modified by the application of different gaseous conditions (air, nitrogen, and nitrogen/hydrogen). Acetaldehyde, dimethyl sulfide, diacetyl, and pentane-2,3-dione, as the major endogenous odorant compounds of yogurt, were chosen as tracers for the biosynthesis of aroma compounds by lactic acid bacteria. Oxidative conditions favored the production of acetaldehyde, dimethyl sulfide, and diketones (diacetyl and pentane-2,3-dione). The Eh of the medium influences aroma production in yogurt by modifying the metabolic pathways of Lb. bulgaricus and Strep. thermophilus. The use of Eh as a control parameter during yogurt production could permit the control of aroma formation.

Use of gases to improve survival of Bifidobacterium bifidum by modifying redox potential in fermented milk.

The aim of this work was to study the effect of the oxidoreduction potential, modified using gas, on the growth and survival of a probiotic strain, Bifidobacterium bifidum, and 2 yogurt strains, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. Three fermented milks were manufactured with an initial oxidoreduction potential value adjusted to +440 mV (control milk), +350 mV (milk gassed with N(2)), and -300 mV [milk gassed with N(2) plus 4% (vol/vol) H(2) (N(2)-H(2))]. Acidification profiles, growth during milk fermentation and survival during storage at 4 °C for 28 d were determined. This study showed that fermented probiotic dairy products made from milk gassed with N(2) and, more particularly, those made from milk gassed with N(2)H(2) were characterized by a significant increase in B. bifidum survival during storage without affecting the fermentation kinetics and the survival of Strep. thermophilus and L. delbrueckii ssp. bulgaricus.

Behavior of Lactobacillus plantarum and Saccharomyces cerevisiae in fresh and thermally processed orange juice

Lactobacillus plantarum and Saccharomyces cerevisiae are acid-tolerant microorganisms that are able to spoil citrus juices before and after pasteurization. The growth of these microorganisms in orange juice with and without pasteurization was investigated. Two samples of orange juice were inoculated with ca. 10(5) CFU/ml of each microorganism. Others were inoculated with ca. 10(7) CFU/ml of each microorganism and then thermally treated. L. plantarum populations were reduced by 2.5 and <1 log10 CFU/ml at 60 degrees C for 40 s and at 55 degrees C for 40 s, respectively. For the same treatments, S. cerevisiae populations were reduced by >6 and 2 log10 CFU/ml, respectively. Samples of heated and nonheated juice were incubated at 15 degrees C for 20 days. Injured populations of L. plantarum decreased by ca. 2 log10 CFU/ml during the first 70 h of storage, but those of S. cerevisiae did not decrease. The length of the lag phase after pasteurization increased 6.2-fold for L. plantarum and 1.9-fold for S. cerevisiae, and generation times increased by 41 and 86%, respectively. The results of this study demonstrate the differences in the capabilities of intact and injured cells of spoilage microorganisms to spoil citrus juice and the different thermal resistance levels of cells. While L. plantarum was more resistant to heat treatment than S. cerevisiae was, growth recovery after pasteurization was faster for the latter microorganism.