Effect of reducing agents on the acidification capacity and the proton motive force of Lactococcus lactis ssp. cremoris resting cells.

International audience; Reducing agents are potential inhibitors of the microbial growth. We have shown recently that dithiothreitol (DTT), NaBH(4) and H(2) can modify the proton motive force of resting cells of Escherichia coli by increasing the membrane protons permeability [Eur. J. Biochem. 262 (1999) 595]. In the present work, the effect of reducing agents on the resting cells of Lactococcus lactis ssp. cremoris, a species widely employed in dairy processes was investigated. DTT did not affect the acidification nor the DeltapH, in contrast to the effect previously reported on E. coli. The DeltaPsi was slightly increased (30 mV) at low pH (pH 4) in the presence of 31 mM DTT or 2.6 mM NaB…

Proton-dependent kinetics of citrate uptake in growing cells ofLactococcus lactissubsp.lactisbv.diacetylactis

The kinetic analysis of citrate uptake in growing cells of Lactococcus lactis subsp. lactis biovar. diacetylactis identified a proton-dependent transport and suggested the divalent anionic species as the form of citrate transported across cell membranes. The reaction followed Michaelis-Menten kinetics for a two-substrate reaction. The limiting steps were the formation of the ternary complex and the rate of transport. Temperature modified the activity of the permease, increasing the uptake rate.

Maitrise de la fermentation butyrique par la microflore lactique réductrice

Clostridium tyrobutyricum, bactérie anaérobie stricte, endosporulée, est à l’origine de l’apparition du défaut appelé «gonflement tardif » lors de l’étape d’affinage de nombreux fromages à pâtes pressées non cuites et cuites. Selon d’Incecco et al. (2018), elle passerait de l’état spore à un état de cellule végétative à la fin de l’étape d’acidification. Par ailleurs dans le projet ANR FoodREDOX, il a été montré qu’un ensemencement dans le lait d’une microflore abaissant le potentiel d’oxydoréduction, ralentirait la consommation de lactates et la production de butyrate par C. tyrobutyricum lors de l’affinage des fromages. Ainsi, connaître et comprendre les mécanismes de germination, sporula…

Effect of chelatants on gellan gel rheological properties and setting temperature for immobilization of living bifidobacteria.

The effect of various concentrations of sequestrants (sodium citrate, sodium metaphosphate, and EDTA) was studied on gellan gel (1.5-2.5% (w/v)) setting temperature and rheological properties. Addition of EDTA between 0 and 0.8% (w/v) led to a progressive decrease of setting temperature. Citrate and metaphosphate decreased this parameter when added up to 0.4 or 0.6%, depending on gellan gum concentration, eventually resulting in the absence of gel formation at room temperature for the 1.5% gellan solution containing 0.4% citrate. This effect was accompanied by a significant decrease of gel strength and stiffness and might be attributed to the binding of the divalent cations required for cha…

Transfers of small analytes in a multiphasic stirred fruit yoghurt model

International audience; The transfer of small analytes in a multiphasic stirred fruit yoghurt model, made of a pectin gel aimed to mimic fruit pieces and of a dairy gel done with milk acidified by glucono-δ-lactone hydrolysis, have been studied. The concentration gradients between the pectin gel and the dairy gel were the driving force for the migration of small analytes (i.e. water, protons and colorants). Water migrated from the dairy to the pectin gel, causing modifications in the water content of both gels and an equilibration of their water activity at 0.938±0.003. Inversely, protons migrated from the pectin to the dairy gel. These changes in composition of both gels being likely to ha…

Addition of reducing agent dithiothreitol improves 4-decanolide synthesis by the genus Sporidiobolus.

Two species of the genus Sporidiobolus, S. johnsonii and S. ruinenii, were used to study the effect of the reducing agent, dithiothreitol (DTT), on 4-decanolide production using ricinoleic acid as the substrate. The results indicate that the addition of DTT into the cultures significantly enhanced 4-decanolide biosynthesis by the two species.

Influence of a reducing environment on the bioconversion of fatty acids to lactones and methyl-ketones by microorganisms : cellular and enzymatic approaches

National audience

Localization of Lactococcus lactis ssp lactis bv diacetylactis in alginate gel beads affects biomass density and synthesis of several enzymes involved in lactose and citrate metabolism

Lactococcus lactis ssp lactis bv diacetylactis, immobilized in calcium alginate beads, was grown in synthetic medium in a continuous flow reactor. Cell distribution inside the gel, as well as the activity of various enzymes, was measured after 30 h of operation. The included biomass tended to concentrate at the periphery of the bead along a section of radius about 100 μm long. ATPase activity was maximal in this zone. The activity of NADH oxidase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase, which are repressed in the presence of citrate, were higher in the deeper zones than at the surface of the beads. This result shows that only the peripheral zone of the bead is respo…

pH Homeostasis and Citric Acid Utilization: Differences Between Leuconostoc mesenteroides and Lactococcus lactis

This study presents the effects of citric acid and extracellular pH (pHe) on the intracellular pH (pHi) of wild-type and citrate negative variants (cit−) Leuconostoc mesenteroides subsp. mesenteroides (Ln. mesenteroides M) and Lactococcus lactis subsp. lactis bv. diacetylactis (L. lactis LD). A recent method using a pH-sensitive fluorescent indicator carboxyfluorescein succinimidyl ester (cFSE) was adapted to measure the pHi of these two lactic acid bacteria in resting cells. Energized cells with 10 mM lactose of Ln. mesenteroides M and L. lactis LD modified their pH gradient (ΔpH) in the same manner; when the pHe was decreased from 7 to 4, the pHi decreased from 7 to about 5. The adjunctio…

Screening of lactic acid bacteria based on their reduction kinetics

Lactic Acid bacteria (LAB) are widespread in the dairy industry for their use as starters and probiotics. They are able to reduce redox potential (E h) of food matrices, influencing oxidation-reduction reactions involved in the organoleptic quality of the product and also preventing the development of undesired microflora. Amongst LAB, some species are able to reduce E h below negative values while others do not [1]. This diversity of reducing activity found in LAB allows the use of selected strains providing optimal reduction values necessary to the overall quality of food dairy products. Currently, there are different ways to compare LAB based on their reducing properties. Cachon et al., …

Screening of lactic acid bacteria for reducing power using a tetrazolium salt reduction method on milk agar.

WOS:000315703100020 ; www.elsevier.com/locate/jbiosc; International audience; Reducing activity is a physiological property of lactic acid bacteria (LAB) of technological importance. We developed a solid medium with tetrazolium dyes enabling weakly and strongly reducing LAB to be discriminated. It was used to quantify populations in a mixed culture (spreading method) and screen strains (spot method).

Chap. 7.8.1. Biotechnological Processes : Culture of cells and microorganisms

International audience; No abstract

Impact of oxidoreduction potential and of gas bubbling on rheological properties of non-fat yoghurt

International audience; The aim of this work was to study the effect of different gaseous conditions on the physico-chemical properties of yoghurt. Four conditions of oxidoreduction potential (Eh7), +433 mV (milk gassed with air), +405 mV (ungassed milk), +283 mV (milk gassed with N2) and −349 mV (milk gassed with N2–H2) were applied to milk. The rheological properties and microstructure of these yoghurts were determined by measuring apparent viscosity, whey separation (WS) and using confocal laser scanning microscopy. Exopolysaccharide (EPS) production by lactic acid bacteria (LAB) was also studied, and production was increased for extreme Eh7 values: the highest values were obtained for a…

Chap. 7.2.2.1. Modified Atmosphere Packaging and Controlled Atmosphere Packaging : Effect of gases on microorganisms

No abstract

Screening of Lactococcus lactis starters with Absorbance Reduction Activity Method (ARAm)

Lactococcus lactis is one of the most used species in the dairy industry. This species has the ability to reduce to very negative values the redox potential (Eh) of food matrices, making it one of the most reducing Lactic Acid Bacteria (LAB) [1]. This reducing activity influences oxidation-reduction reactions involved in the organoleptic quality of the product and prevents the undesired microorganisms development [2–4]. The main mechanism involved are exofacial thiol groups, NoxE NADH Oxidase and the Electron Transport Chain mechanism [5-7]. For Eh measurement, the commonly used method consists of a redox probe placed in a liquid medium or food matrix [8]. With this probe, Cachon et al. (20…

Additional file 6: Table S1. of Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

Strains and plasmids. (PDF 355 kb)

A way to follow the viability of encapsulated Bifidobacterium bifidum subjected to a freeze-drying process in order to target the colon: Interest of flow cytometry

The aim of this work was to apply flow cytometry in order to assess and compare the viability of freeze-dried entrapped bacteria with an usual technique by quantification by plate count techniques. It also aimed at studying the effect of various cryoprotectants on the viability of an entrapped Bifidobacterium bifidum subjected to freeze-drying to check their ability to be delivered all along the gastro-intestinal tract. The alginate-pectinate beads were chosen as the encapsulation matrix added with different protectants. The beads were characterized by scanning electron microscopy and the viability was checked by both methods. The best combination to improve viability of entrapped bacteria …

A descriptive model for citrate utilization by Lactococcus lactis ssp lactis bv diacetylactis

A model for the use of citrate by Lactococcus lactis ssp lactis bv diacetylactis CNRZ 125 is proposed. Citrate metabolism by this strain leads to the production of acetate, CO2 and C4 compounds (diacetyl, acetoin, 2,3-butylene glycol). The model furnishes correct simulations, consistent with published results on the pathways used and on lactose-citrate co-metabolism. Citric acid is incorporated independently of growth. The production of flavoring compounds is a complex process, depending on the rate of citrate utilization, on the proportion of pyruvate arising from citrate and which condenses to form α-acetolactate and CO2, on the rate of transformation of α-acetolactate to diacetyl and ace…

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 pen…

Wine Production by Immobilised Cell Systems

Contribution of exofacial thiol groups in the reducing activity of Lactococcus lactis

Lactococcus lactis can decrease the redox potential at pH 7 (E(h7)) from 200 to -200 mV in oxygen free Man-Rogosa-Sharpe media. Neither the consumption of oxidizing compounds or the release of reducing compounds during lactic acid fermentation were involved in the decrease in E(h7) by the bacteria. Thiol groups located on the bacterial cell surface appear to be the main components that are able to establish a greater exchange current between the Pt electrode and the bacteria. After the final E(h7) (-200 mV) was reached, only thiol-reactive reagents could restore the initial E(h7) value. Inhibition of the proton motive force showed no effect on maintaining the final E(h7) value. These result…

Effect of oxidoreduction potential and of gas bubbling on rheological properties and microstructure of acid skim milk gels acidified with glucono-delta-lactone

Milk oxidoreduction potential was modified using gases during the production of a model dairy product and its effect on gel setting was studied. Acidification by glucono-delta-lactone was used to examine the physicochemistry of gelation and to avoid variations due to microorganisms sensitive to oxidoreduction potential. Four conditions of oxidoreduction potential were applied to milk: milk was gassed with air, nongassed, gassed with N(2), or gassed with N(2)H(2). The rheological properties and microstructure of these gels were determined using viscoelasticimetry, measurement of whey separation, and confocal laser scanning microscopy. It appeared that a reducing environment led to less-aggre…

Additional file 2: Figure S2. of Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

Deletion of llmg_0524 or llmg_0526 decreases modestly operon expression. The plasmid P0524-pTCV-lac is established in mutant ∆llmg_0524 and ∆llmg_0526. Cells were grown in M17Glu0.5 up to OD600= 0.1 for β-galactosidase determination. Results, plus standard deviation, are means of three independent experiments. They are expressed in percentage of values of wild type strain. (PDF 171 kb)

Recombinant hydroperoxide lyase for the production of aroma compounds: Effect of substrate on the yeast Yarrowia lipolytica

International audience; The aim of this study was to investigate the action mechanism of linoleic acid hydroperoxides (HPOD), which are the major substrates of hydroperoxide lyase for the production of flavour compounds, on the yeast Yarrowia lipolytica by evaluating their effect on the oxidative state of the cells. The total antioxidant capacity (TAC) and the activity of the main antioxidant enzymes, such as glutathione reductase, glutathione peroxidase and superoxide dismutase, of cells treated with HPOD were studied. The potential role of intracellular glutathione, including reduced glutathione (GSH) and oxidized glutathione (GSSG), in conferring HPOD resistance was also been examined. T…

Impact of Ageing on Pea Protein Volatile Compounds and Correlation with Odor

Vegetal proteins are of high interest for their many positive aspects, but their ‘beany’ off-flavor is still limiting the consumer’s acceptance. The aim of this work was to investigate the conservation of pea protein isolate (PPI) during time and especially the evolution of their organoleptic quality under two storage conditions. The evolution of the volatile compounds, the odor and the color of a PPI has been investigated during one year of storage. PPI was exposed to two treatments mimicking a lack of control of storage conditions: treatment A with light exposition at ambient temperature (A—Light 20 °C) and treatment B in the dark but with a higher temperature …

Effect of Microbial strains on Hexanal

The positive effects of plant-based protein consumption have been reported, nonetheless, several disadvantages suchgrassy or beany off-flavors are encountered (1) and are limiting the consumers acceptance towards these proteins.In this project, we investigated the potential of selected microorganisms to metabolize compounds responsible forthe leguminous off-flavor. Hexanal, well-known as a main contributor to the beany descriptor (2), was chosen for thisstudy.A selection of 16 strains representative of different fermentative types has been used to treat a liquid model matrixcontaining hexanal at 2 mg/L. The strains were applied on the medium at the end of their growing phase. Theinoculum wa…

Gases in Agro-food Processes

Gases in Agro-food Processes is the ultimate reference covering all applications of gases in agro-Food processes, from farm to fork. Divided into 11 sections, the book covers chemical and physical gas properties, gas monitoring, regulation, heat and mass transfers. Sections are dedicated to agriculture and food processing, wastewater treatment, safety applications and market trends. Users will find this to be a valuable resource for industrial scientists and researchers in technical centers who are developing agro-food products. In addition, the book is ideal for graduate students in agro-food science, chemistry and the biosciences.

Additional file 4: Figure S4. of Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

UV-visible spectra of protein fusions. 20 μM of proteins were used. Analysis was performed in 50 mM Tris–HCl buffer, pH 7.4, at room temperature with a Libra S22 spectrophotometer. (PDF 193 kb)

Additional file 1: Figure S1. of Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

Amino acid sequence of Llmg_0524 and Llmg_0526. Llmg_0524 has 200 amino-acid residues, including four cysteines in the Nter region and two transmembrane domains (TMDs). Llmg_0526 has 421 amino-acid residues, including four cysteines in the Nter region and a transmembrane domain. The cysteine residues cluster in a CX2CX10CX2C motif in both proteins. Cysteine amino-acid residues are in bold red; predicted membrane helices are in bold black. (PDF 304 kb)

Additional file 6: Table S1. of Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

Strains and plasmids. (PDF 355 kb)

Additional file 3: Figure S3. of Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

Determination of PhoA activity of different fusion proteins. Data are the means of results, ± standard deviations, from three independent experiments. PhoA1 contains only the Nter extremity whereas PhoA2 contains the Nter extremity and the predicted transmembrane domain. (PDF 178 kb)

Modeling of growth and lactate fermentation by Lactococcus lactis subsp. lactis biovar. diacetylactis in batch culture

The kinetic behaviour of Lactococcus lactis subsp. lactis biovar. diacetylactis was studied in batch culture under non-limiting conditions that allow high growth and product formation. A model based on laboratory results is proposed for growth and l-lactate fermentation. It shows the necessity for differentiating biomass into three physiological states, two active, Xg (growth + acidification) and Xng (acidification), and one inactive, Xi. The kinetic theory of the model demonstrates the non-competitive nature of fermentation end-product inhibition on growth and acidification, and describes the passage from one physiological state to another. Satisfying simulations were obtained for batch fe…

Kinetics of citrate uptake in growing cells ofLeuconostocspp.

Citrate uptake was studied in growing cells of Leuconostoc mesenteroides subsp. mesenteroides. A Michaelis-Menten pattern with the dianionic form of citrate as the limiting substrate has been proposed. It was validated for different fermentations varying the initial citrate concentrations and the pH medium. This latter did not modify the rate of the process which was clearly confirmed using experiments with resting cells. The model was used to compare the kinetics of citrate consumption between several strains of Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc mesenteroides subsp. cremoris and Leuconostoc lactis.

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…

Additional file 5: Table S2. of Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

Proteins containing the CX2CX10CX2C motif in various bacterial species. (PDF 512 kb)

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…

Hydrodynamic characterization of a new small-scale reactor mixed by a magnetic bar

International audience; The purpose of this study was to evaluate the mixing characteristics of a new milliliter-scale (mL-scale) reactor developed for studying enzymatic activity or physiological cell response. The mL-scale reactor was designed to enable the integration of several sensors to carry out dynamic measurements in a controlled environment. Rapid homogeneity of the entire system is essential to ensure reproducible and reliable results, consequently the reactor was stirred to optimize both mass and heat transfers. A comparative study using three different techniques was undertaken to study mixing performances in the system. Firstly, mixing time (4,) was estimated in the reactor us…

Potential of microorganisms to decrease the "beany" off-flavor

Changes in the proton-motive force in Escherichia coli in response to external oxidoreduction potential.

International audience; The pH homeostasis and proton-motive force (Deltap) of Escherichia coli are dependent on the surrounding oxidoreduction potential (ORP). Only the internal pH value and, thus, the membrane pH gradient (DeltapH) component of the Deltap is modified, while the membrane potential (DeltaPsi) does not change in a significant way. Under reducing conditions (Eh < 50 mV at pH 7.0), E. coli decreases its Deltap especially in acidic media (21% decrease at pH 7.0 and 48% at pH 5.0 for a 850-mV ORP decrease). Measurements of ATPase activity and membrane proton conductance (CH+m) depending on ORP and pH have shown that the internal pH decrease is due to an increase in membrane prot…

Extracellular oxidoreduction potential modifies carbon and electron flow in Escherichia coli.

ABSTRACT 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 enzy…

Immobilised Cell Technology in Winery and Fruit Wine Production

Winemaking is largely concerned with the progress of biotechnology and especially with the use of high cell density reactors. Entrapment is the most widely method to immobilise cells; several matrix can be used (alginate, carrageenan, agar) with different geometry (beads, fibres, plates). Alcoholic fermentation of wine, malolactic fermentation, bottle fermentation known as the "Methode champenoise" and sparkling wines are among the industrial applications. Whereas prospects for this technology appear encouraging, further research is needed to optimise reaction variables, improve the long-term stability of the reactors, and understand more about secondary metabolite production by yeasts unde…

Influence of lactose-citrate co-metabolism on the differences of growth and energetics in Leuconostoc lactis, Leuconostoc mesenteroides ssp. mesenteroides and Leuconostoc mesenteroides ssp. cremoris

International audience; The biodiversity of growth and energetics in Leuconostoc sp. has been studied in MRS lactose medium with and without citrate. On lactose alone, Ln. lactis has a growth rate double that of Ln. cremoris and Ln. mesenteroides. The pH is a more critical parameter for Ln. mesenteroides than for Ln. lactis or Ln. cremoris; without pH control Ln. mesenteroides is unable to acidify the medium under pH 4.5, while with pH control and as a consequence of a high Y(ATP) its growth is greater than Ln. lactis and Ln. cremoris. In general, lactose-citrate co-metabolism increases the growth rate, the biomass synthesis, the lactose utilisation ratio, and the production of lactate and …

Additional file 5: Table S2. of Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

Proteins containing the CX2CX10CX2C motif in various bacterial species. (PDF 512 kb)

Impact of probiotics on risk factors for cardiovascular diseases. A review.

International audience; 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., Crohn's 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 identi…

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), …

Engineering a Saccharomyces cerevisiae Wine Yeast That Exhibits Reduced Ethanol Production during Fermentation under Controlled Microoxygenation Conditions

ABSTRACTWe recently showed that expressing an H2O-NADH oxidase inSaccharomyces cerevisiaedrastically 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 i…

Modeling of continuous Ph-stat stirred tank reactor withLactococcus lactisssp.lactisbv.diacetylactisimmobilized in calcium alginate gel beads

A dynamic diffusion-reaction-growth model is proposed for the study of lactic fermentation, the bioconversion of citric acid, and cell release in an immobilized cell reactor [pH-stat continuous stirred tank-reactor (CSTR)]. The model correctly simulates the onset of fermentation and colonization of the gel, followed by the steady state. External diffusion is nonlimiting and internal diffusion is limited by high cell densities at the periphery of the gel beads. Lactose-citrate cometabolism in the gel is related to the distribution of active included biomass within the gel and to gradients of substrates (lactose, citrate) and products (lactate, pH) in the beads. The utilization of lactose is …

Resistance of Immobilized Lactic Acid Bacteria to the Inhibitory Effect of Quaternary Ammonium Sanitizers

Abstract The inhibitory effect of quaternary ammonium sanitizers (QAS) on the fermentation activity of lactic acid bacteria was investigated. Benzalkonium chloride and didecyl dimethyl ammonium chloride show the same inhibitory effect. In MRS agar medium and MRS liquid medium their minimum inhibitory concentration was below 2 μg/mL. Interfering molecules in milk increased this concentration of inhibitory QAS above 8 μg/mL. Both growth and acidification were decreased but by immobilizing cells in calcium alginate gel beads this inhibitory effect could be reduced. The longer the beads were used for successive acidification and were colonized by lactic acid bacteria, the better was the acidifi…

Criblage des bactéries lactiques en fonction de leur cinétique de réduction : approche méthodologique

Les bactéries lactiques (BL) sont des micro-organismes importants dans l'industrie alimentaire pour la fabrication de produits fermentés ou comme probiotiques. Parmi les BL, certaines espèces sont capables de réduire le potentiel d’oxydoréduction (Eh) jusqu’à des valeurs négatives alors que d'autres non [1]. Cette diversité d'activité réductrice retrouvée chez les BL permet l'utilisation de souches sélectionnées apportant des valeurs de réduction optimales nécessaires à la qualité globale des produits laitiers [2–6]. Les principaux mécanismes impliqués sont les groupes thiol exofaciaux, la NoxE NADH Oxydase et le mécanisme mettant en jeu la chaîne de transport d'électrons [7,8,9].Actuelleme…

Combined action of redox potential and pH on heat resistance and growth recovery of sublethally heat-damaged Escherichia coli

International audience; The combined effect of redox potential (RP) (from -200 to 500 mV) and pH (from 5.0 to 7.0) on the heat resistance and growth recovery after heat treatment of Escherichia coli was tested. The effect of RP on heat resistance was very different depending on the pH. At pH 6.0, there was no significant difference, whereas at pH 5.0 and 7.0 maximum resistance was found in oxidizing conditions while it fell in reducing ones. In sub-lethally heat-damaged cells, low reducing and acid conditions allowed growth ability to be rapidly regained, but a decrease in the redox potential and pH brought about a longer lag phase and a slower exponential growth rate, and even led to growt…

Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity

Background Due to its extraordinary chemical properties, the cysteine amino acid residue is often involved in protein folding, electron driving, sensing stress, and binding metals such as iron or zinc. Lactococcus lactis, a Gram-positive bacterium, houses around one hundred cysteine-rich proteins (with the CX2C motif) in the cytoplasm, but only a few in the membrane. Results In order to understand the role played by this motif we focused our work on two membrane proteins of unknown function: Llmg_0524 and Llmg_0526. Each of these proteins has two CX2C motifs separated by ten amino-acid residues (CX2CX10CX2C). Together with a short intervening gene (llmg_0525), the genes of these two protein…

Eh and pH gradients in Camembert cheese during ripening: Measurements using microelectrodes and correlations with texture

International audience; This study investigated the use of microelectrodes to continuously and simultaneously monitor pH and redox potential (Eh) gradients in Camembert cheeses from the rind to the core, as a function of ripening time (15 and 35 days). Cheeses were shown to be heterogeneous throughout their mass. An Eh gradient existed from the rind to the core. The surface was oxidizing (+330 to +360 mV) and the core was reducing and became more so in the course of ripening (−300 to −360 mV). A relationship between firmness and Eh was shown. pH gradients were in line with those previously described in the literature and related to ammonium and lactate gradients. Gradients evolved from the …

Generalized model of the effect of pH on lactate fermentation and citrate bioconversion in Lactococcus lactis ssp. Lactis biovar. diacetylactis

An aroma-imparting mesophilic lactic starter (Lactococcus lactis ssp. lactis biovar. diacetylactis) was studied in batch culture in medium with 50 g·l−1 lactose and 2 g·l−1 citrate. The effect of pH on the physiology of growth and the production of flavour compounds was investigated with a mathematical model. The specific rates of growth and of lactose fermentation obeyed a law of non-competitive inhibition by lactic acid produced, inhibition increasing as the pH of the medium decreased. The pH thus acted indirectly by increasing the proportion of non-dissociated lactic acid, identified as the inhibiting form of lactic acid. The generalized model, taking into account the effect of pH, was t…

Effects of oxidoreduction potential combined with acetic acid, NaCl and temperature on the growth, acidification, and membrane properties of Lactobacillus plantarum.

International audience; The effects of oxidoreduction potential (Eh) combined with acetic acid, NaCl and temperature on the growth, acidification, and membrane properties of Lactobacillus plantarum were studied. The culture medium was set at pH 5, and two different Eh values were adjusted using nitrogen (Eh = +350 mV) or hydrogen (Eh = -300 mV) gas. In reducing condition, the growth was slowed and the acidification delayed at 37 degrees C, but not at 10 degrees C. A synergistic inhibitory effect of reducing Eh, acetic acid and NaCl was observed, mainly for delaying the lag phase before acidification. These results may be explained by changes in ATPase activity, membrane fluidity and surface…

Modeling hexanal production in oxido-reducing conditions by the yeast Yarrowia lipolytica

International audience; Hexanal produced by cells of a recombinant Yarrowia lipolytica yeast expressing the hydroperoxide lyase (HPL) from green bell pepper fruit was studied under oxido-reducing conditions using the reducing dithiotreitol and oxidizing potassium ferricyanide compounds. The combined effect of pH, linoleic acid 13-hydroperoxides concentration, temperature and oxido-reducing molecules on the hexanal production was studied. Significant positive effects for the hexanal production were found using high concentrations of hydroperoxides (100 mM, 30 g/L). Adding reducing molecules enhanced significantly hexanal production while the oxidizing molecules had an inhibitory effect. Comb…

Impact of oxydoreduction potential on biosynthesis of volatile compounds in acid skim milk gel: identification and quantification

International audience; Oxydoreduction potential (Eh) is an environmental parameter which defines balances between oxidant species (acceptors of electrons) and reduced species (donors of electrons) of a medium. It may modify the growth capacity of microorganisms, the metabolic fluxes, participate to the quality of fermented products (2, 6, 7) and affect the production and/or stability of volatile compounds (3,5). (...)

Kinetics of lactate fermentation and citrate bioconversion by LactococcusIactisssp.Iactisin batch culture

The growth kinetics of Lactococcus lactis ssp. lactis were studied in batch culture in conditions of non-limiting lactose and the presence of citric acid. The control of pH modified growth and citrate metabolism but did not change the yield of acid formation. At controlled pH the growth rate was unaffected by citrate metabolism. Lactose was transformed to L-lactate and assay of the metabolic by-products showed some heterofermentation at the end of the growth of cultures with low growth rates. This heterofermentation was interpreted as a slowing down of glycolysis with activation of both the pyruvate formate lyase (PFL) and the pyruvate dehydrogenase complex (PDHC). Under these conditions th…