Impact of oxidative stress on <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> mitochondrial bioenergetics depends on cell growth stage

Addition of a moderate (1.4 mM) concentration of H₂O₂ to protozoon Acanthamoeba castellanii cell cultures at different growth phases caused a different response to oxidative stress. H₂O₂ treatment of exponentially growing cells significantly delayed their growth; however, in mitochondria isolated from these cells, no damage to their bioenergetic function was observed. In contrast, addition of H₂O₂ to A. castellanii cells approaching the stationary phase did not influence their growth and viability while seriously affecting mitochondrial bioenergetic function. Although mitochondrial integrity was maintained, oxidative damage was revealed in the reduction of cytochrome pathway activity, uncoupling protein activity, and the efficiency of oxidative phosphorylation as well as the membrane potential and the endogenous ubiquinone reduction level of the resting state. An increase in the alternative oxidase protein level and activity as well as an increase in the membranous ubiquinone content were observed in mitochondria isolated from late H₂O₂-treated cells. For the first time, the regulation of ubiquinone content in the inner mitochondrial membrane is shown to play a role in the response to oxidative stress. A physiological role for the higher activity of the alternative oxidase in response to oxidative stress in unicellular organisms, such as amoeba A. castellanii, is discussed.     

Influence of oxygen on sulfate reduction and growth of sulfate-reducing bacteria

The ambivalent relations of sulfate-reducing bacteria to molecular O₂ have been studied with ten freshwater and marine strains. Generally, O₂ was reduced prior to sulfur compounds and suppressed the reduction of sulfate, sulfite or thiosulfate to sulfide. Three strains slowly formed sulfide at O₂ concentrations of below 15 M (6% air saturation). In homogeneously aerated cultures, two out of seven strains tested, Desulfovibrio desulfuricans and Desulfobacterium autotrophicum, revealed weak growth with O₂ as electron acceptor (up to one doubling of protein). However, O₂ was concomitantly toxic. Depending on its concentration cell viability and motility decreased with time. In artificial oxygen-sulfide gradients with sulfide-containing agar medium and also in sulfide-free agar medium under an oxygen-containing gas phase, sulfate reducers grew in bands close to the oxic/anoxic interface. The specific O₂ tolerance and respiration capacity of different strains led to characteristically stratified gradients. The maximum O₂ concentration at the surface of a bacterial band (determined by means of microelectrodes) was 9 M. The specific rates of O₂ uptake per cell were in the same order of magnitude as the sulfate reduction rates in pure cultures. The bacteria stabilized the gradients, which were rapidly oxidized in the absence of cells or after killing the cells by formaldehyde. The motile strain Desulfovibrio desulfuricans CSN slowly migrated in the gradients in response to changing O₂ concentrations in the gas phase.     

The effect of oxygen tension on rat hepatocytes in short-term culture

Summary Cell viability, cytochrome P-450 content, cell respiration, and lipid peroxidation were all investigated as a function of oxygen tension in adult rat hepatocytes in short-term culture (less than 9 h). The various oxygen tensions used in this study were obtained by equilibrating culture medium with air, air + nitrogen, or air + oxygen. Cell viability, as assessed by trypan blue exclusion, was significantly greater at all time points tested when hepatocytes were cultured in Ham's F12 medium containing 132 μM O₂, as compared to medium equilibrated with air (220 μM O₂) or air + oxygen (298 μM O₂). Cells cultured in 220 μM O₂ (air) also exhibited a gradual loss of cytochrome P-450, so that by 9 h of incubation less than 60% of the active material remained. This loss of P-450 was minimized when cells were cultured in 163 μM O₂ and abolished when cells were cultured in 132 μM O₂. The 132 μM O₂ exposure conditions also maintained cell respiration at the 1 h incubation values, whereas there was a continuous loss in cell respiration over time when the cells were cultured in either 220 μM O₂ (air) or 298 μM O₂ (air:O₂). These cytotoxicity findings may be related to oxidative cell damage inasmuch as it was additionally demonstrated that lipid peroxidation (as measured by malondieldehyde equivalents) was consistantly lower in hepatocytes cultured in air:N₂ as compared to air or air:O₂. These results suggest that hepatocyte culture in low oxygen tension improves not only cell viability but also maintains other functional characteristics of the cell. This work was supported by a Biomedical Research Support Grant S-S07-RR 05448 awarded to the University of Minnesota School of Public Health by the Biomedical Research Grant Program, Division of Research and Resources, National Institutes of Health, Bethesda, MD.     

Metabolic stability and inhibitory effect of O-methylated theaflavins on H2O2-induced oxidative damage in human HepG2 cells

Seven new O-methylated theaflavins (TFs) were synthesized by using O-methyltransferase from an edible mushroom. Using TFs and O-methylated TFs, metabolic stability in pooled human liver S9 fractions and inhibitory effect on H₂O₂-induced oxidative damage in human HepG2 cells were investigated. In O-methylation of theaflavin 3′-O-gallate (TF3′G), metabolic stability was potentiated by an increase in the number of introduced methyl groups. O-methylation of TF3,3′G did not affect metabolic stability, which was likely because of a remaining 3-O-galloyl group. The inhibitory effect on oxidative damage was assessed by measuring the viability of H₂O₂-damaged HepG2 cells treated with TFs and O-methylated TFs. TF3,3′G and O-methylated TFs increased cell viabilities significantly compared with DMSO, which was the compound vehicle (p?<?0.05), and improved to approximately 100%. Only TF3′G did not significantly increase cell viability. It was suggested that the inhibitory effect on H₂O₂-induced oxidative damage was potentiated by O-methylation or O-galloylation of TFs.     

Protective effect of augmenter of liver regeneration on hydrogen peroxide-induced apoptosis in SH-SY5Y human neuroblastoma cells

Background: Hydrogen peroxide, as other reactive oxygen species (ROS) produced during redox processes, induces lipid membrane peroxidation and protein degeneration causing cell apoptosis. ROS are recently considered as messengers in cell signalling processes, which, through reversible protein disulphide bridges formation, activate regulatory factors of cell proliferation and apoptosis. Disulphide bridges formation is catalysed by sulphydryl oxidase enzymes.

Aim: The neuroprotective effect of ALR protein (Alrp), a sulphydryl oxidase enzyme, on H₂O₂-induced apoptosis in SH-SY5Y cells has been evaluated.

Methods: Cell viability, flow cytometric evaluation of apoptotic cells, fluorescent changes of nuclear morphology, immunocytochemistry Alrp detection, Western blot evaluation of mitochondrial cyt c release and mitochondrial swelling were determined.

Results: Alrp prevents the H₂O₂-induced cell viability loss, apoptotic cell death and mitochondrial swelling in SH-SY5Y cells in culture.

Conclusions: The data demonstrate that Alrp improves SH-SY5Y cells survival in H₂O₂-induced apoptosis. It is speculated that this effect could be related to the Alrp enzymatic activity.     

Metabolic depression and sodium-potassium ATPase in the aestivating frog, Neobatrachus kunapalari

In aestivation the metabolic rate of the Australian desert frog Neobatrachus kunapalari was 50–67% lower than in the non-aestivating state. The rate of O₂ consumption of isolated muscle, skin and brain was measured in both metabolic states. The average rate of O₂ consumption of muscle was 30% lower and brain 50% lower in aestivating frogs, while the rate of O₂ consumption of skin was the same. The reduction in muscle could account for a large proportion of whole animal metabolic depression. To look for evidence of a reduction in energy demand in the tissues we measured the ouabain-sensitive fraction of tissue rate of O₂ consumption, which is considered to be the proportion of metabolism used for transmembrane Na⁺/K⁺ pumping. Ouabain inhibited the in vitro rate of O₂ consumption of skin by a average of 20% and of brain by an average of 30%. However, in muscle, ouabain stimulated in vitro O₂ consumption. Despite the 50% reduction in the in vitro rate of O₂ consumption of brain during aestivation, neither the ouabain-sensitive nor ouabain-insensitive fractions were found be statistically different, possibly because of the large individual variation in the degree of ouabain inhibition. A reduction in the level of ion pumping during aestivation was therefore not demonstrated in any tissue. Measurement of the level of the enzyme Na⁺K⁺-ATPase in skeletal muscle, ventricle, kidney and brain showed that there was no change in the amount of this enzyme in the aestivating frogs. Measurement of the levels of adenylates in muscle and liver showed that the adenylate energy charge was maintained in aestivation, but that there was a reduction in ATP in liver and a reduction in the level of total adenylates in both tissues, which could be an adaptation of the tissues to a lower energy turnover. Accepted: 22 July 1996     

Light‐dependent oxygen consumption in nitrogen‐fixing cyanobacteria plays a key role in nitrogenase protection1

All colonial diazotrophic cyanobacteria are capable of simultaneously evolving O₂ through oxygenic photosynthesis and fixing nitrogen via nitrogenase. Since nitrogenase is irreversibly inactivated by O₂, accommodation of the two metabolic pathways has led to biochemical and/or structural adaptations that protect the enzyme from O₂. In some species, differentiated cells (heterocysts) are produced within the filaments. PSII is absent in the heterocysts, while PSI activity is maintained. In other, nonheterocystous species, however, a “division of labor” occurs whereby individual cells within a colony appear to ephemerally fix nitrogen while others evolve oxygen. Using membrane inlet mass spectrometry (MIMS) in conjunction with tracer ¹⁸O₂ and inhibitors of photosynthetic and respiratory electron transport, we examined the light dependence of O₂ consumption in Trichodesmium sp. IMS 101, a nonheterocystous, colonial cyanobacterium, and Anabaena flos‐aquae (Lyngb.) Bréb. ex Bornet et Flahault, a heterocystous species. Our results indicate that in both species, intracellular O₂ concentrations are maintained at low levels by the light‐dependent reduction of oxygen via the Mehler reaction. In N₂‐fixing Trichodesmium colonies, Mehler activity can consume ～75% of gross O₂ production, while in Trichodesmium utilizing nitrate, Mehler activity declines and consumes ～10% of gross O₂ production. Moreover, evidence for the coupling between N₂ fixation and Mehler activity was observed in purified heterocysts of Anabaena, where light accelerated O₂ consumption by 3‐fold. Our results suggest that a major role for PSI in N₂‐fixing cyanobacteria is to effectively act as a photon‐catalyzed oxidase, consuming O₂ through pseudocyclic electron transport while simultaneously supplying ATP in both heterocystous and nonheterocystous taxa.     

Role of Reactive Oxygen Species and Glutathione in Inorganic Mercury-Induced Injury in Human Glioma Cells

The present study was undertaken to examine the role of reactive oxygen species (ROS) and glutathione (GSH) in glia cells using human glioma cell line A172 cells. HgCl₂ caused the loss of cell viability in a dose-dependent manner. HgCl₂-induced loss of cell viability was not affected by H₂O₂ scavengers catalase and pyruvate, a superoxide scavenger superoxide dismutase, a peroxynitrite scavenger uric acid, and an inhibitor of nitric oxide N^G-nitro-arginine Methyl ester. HgCl₂ did not cause changes in DCF fluorescence, an H₂O₂-sensitive fluorescent dye. The loss of cell viability was significantly prevented by the hydroxyl radical scavengers dimethylthiourea and thiourea, but it was not affected by antioxidants DPPD and Trlox. HgCl₂-induced loss of cell viability was accompanied by a significant reduction in GSH content. The GSH depletion was almost completely prevented by thiols dithiothreitol and GSH, whereas the loss of viability was partially prevented by these agents. Incubation of cells with 0.2 mM buthionine sulfoximine for 24 hr, a selective inhibitor of -glutamylcysteine synthetase, resulted in 56% reduction in GSH content without any change in cell viability. HgCl² resulted in 34% reduction in GSH content, which was accompanied by 59% loss of cell viability. These results suggest that HgCl₂-induced cell death is not associated with generation of H₂O₂ and ROS-induced lipid peroxidation. In addition, these data suggest that the depletion of endogenous GSH itself may not play a critical role in the HgCl₂-induced cytotoxicity in human glioma cells.     

Low oxygen tension potentiates proliferation and stemness but not multilineage differentiation of caprine male germline stem cells

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O₂) environment, whereas, their in-vitro expansion is typically performed under normoxia (20–21% O₂). The comparative information about the effects of low and normal O₂ levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O₂) and normoxia (21% O₂). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p?<?0.05) improved, thus, yielding a significantly (p?<?0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O₂ conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p?<?0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

     

Cytochrome c oxidase: the mechanistic significance of structural H+ in energy transduction

Changes in the bulk-phase concentration of O₂ and H⁺ associated with the reduction of O₂ to water are simultaneously determined in reactions catalyzed by fully reduced cytochrome c oxidase both isolated and embedded in liposomes. Consistent with the polyphasic kinetics of electron transfer through the oxidase, the time course of O₂ consumption and H⁺ translocation exhibit the following novel characteristics: (1) The uptake of scalar protons (H_m ⁺), the ejection of vectorial protons (H⁺ _v), and the consumption of O₂, all proceed in a kinetically polyphasic process. (2) During the first phase of the reaction the rates of O₂ uptake and H⁺ transfer are extremely fast and compatible with the rates of electron flow through the oxidase. (3) The K_m of the oxidase for O₂ is close to 75 M, the same for O₂ consumption and scalar H⁺ uptake. The V_max of O₂ reduction to water in reactions catalyzed by the isolated enzyme is, at least, 0.5 × 10⁴ s^–1. (4) The extent of vectorial H⁺ ejection by cytochrome c oxidase embedded in liposomes is an exponential function dependent on both enzyme concentration and extent of O₂ consumption. (5) The H⁺/O stoichiometry of H⁺ ejection is a variable that may reach a maximum value of 4.0 only when the enzyme undergoes net oxidation at extremely high enzyme/O₂ molar ratios. It is postulated that the generation of useful energy at the level of cytochrome c oxidase depends not only on the number of molecules of O₂ reduced to water but also on the extent and state of reduction and/or protonation of the enzyme.     

Ellagic acid mitigates arsenic‐trioxide‐induced mitochondrial dysfunction and cytotoxicity in SH‐SY5Y cells

In the current study, neuroprotective significance of ellagic acid (EA, a polyohenol) was explored by primarily studying its antioxidant and antiapoptotic potential against arsenic trioxide (As₂O₃)‐induced toxicity in SH‐SY5Y human neuroblastoma cell lines. The mitigatory effects of EA with particular reference to cell viability and cytotoxicity, the generation of reactive oxygen species, DNA damage, and mitochondrial dynamics were studied. Pretreatment of SH‐SY5Y cells with EA (10 and 20 μM) for 60 min followed by exposure to 2 μM As₂O₃ protected the SH‐SY5Y cells against the harmful effects of the second. Also, EA pre‐treated groups expressed improved viability, repaired DNA, reduced free radical generation, and maintained altered mitochondrial membrane potential than those exposed to As₂O₃ alone. EA supplementation also inhibited As₂O₃‐induced cytochrome c expression that is an important hallmark for determining mitochondrial dynamics. Thus, the current investigations are more convinced for EA as a promising candidate in modulating As₂O₃‐induced mitochondria‐mediated neuronal toxicity under in vitro system.     

Effect of quercetin and its glucuronide metabolite upon 6-hydorxydopamine-induced oxidative damage in Neuro-2a cells

Abstract

Quercetin is ubiquitously distributed in plant foods. This antioxidative polyphenol is mostly converted to conjugated metabolites in the body. Parkinson disease (PD) has been suggested to be related to oxidative stress derived from abnormal dopaminergic activity. We evaluated if dietary quercetin contributes to the antioxidant network in the central nervous system from the viewpoint of PD prevention. A neurotoxin, 6-hydroxydopamine (6-OHDA), was used as a model of PD. 6-OHDA-induced H₂O₂ production and cell death in mouse neuroblastoma, Neuro-2a. Quercetin aglycone suppressed 6-OHDA-induced H₂O₂ production and cell death, although aglycone itself reduced cell viability at higher concentration. Quercetin 3-O-β-d-glucuronide (Q3GA), which is an antioxidative metabolite of dietary quercetin, was little incorporated into the cell resulting in neither suppression of 6-OHDA-induced cell death nor reduction of cell viability. Q3GA was found to be deconjugated to quercetin by microglial MG-6 cells. These results indicate that quercetin metabolites should be converted to their aglycone to exert preventive effect on damage to neuronal cells.     

High-affinity oxygen uptake byBifidobacterium bifidum

Oxygen uptake by washed cell suspensions ofBifidobacterium bifidum DSM 20082 was studied by using spectrophotometric measurements of the degree of oxygenation of added myoglobin as a measure of the concentration of dissolved O₂. The absorbance changes during consumption of O₂ in a closed reaction vessel were analysed by computer to obtain estimates of the changes in dissolved O₂ concentration. The cell were then used to calculate the rate of O₂ uptake as a function of the dissolved O₂ concentration. The cell suspensions showed Michaelis-Menten kinetics with an apparent K_m value of 0.06 M O₂. Cell-free extracts contained a soluble NADH oxidase activity with a stoichiometry corresponding to the reduction of O₂ to H₂O and with a high affinity for O₂.     

Identification and physiological characterization of the nitrogen fixing bacterium Corynebacterium autotrophicum GZ 29

The coryneform hydrogen bacterium strain GZ 29, assigned to Corynebacterium autotrophicum fixed molecular nitrogen under autotrophic (H₂, CO₂) as well as under heterotrophic (sucrose) conditions. Physiological parameters of nitrogen fixation were measured under heterotrophic conditions. The optimal dissolved oxygen concentration for cells grown in a fermenter with N₂ was rather low (0.14 mg O₂/l) compared with cells grown in the presence of NH ₄ ⁺ (4.45 mg O₂/l). C. autotrophicum GZ 29 had a doubling time of 3.7 h at 30°C with N₂ as N-source and sucrose as carbon source and at optimal pO₂. Acetylene reduction reached values of 12 nmoles of ethylene produced/minxmg protein. Although the oxygen concentration in the growing culture was kept constant, the optimal dissolved oxygen tension for the acetylene reduction assay shifted to higher pO₂-values. The overall efficiency of nitrogen fixation amounted to 22 mg N fixed/g sucrose consumed; it reached a maximal value of 65 mg N fixed/g sucrose consumed at the beginning of the exponential growth phase. Intact cells reduced acetylene even under anaerobic test conditions; further anaerobic metabolic activity could not be ascertained so far.     

Protective role of ortho-substituted Mn(III) N-alkylpyridylporphyrins against the oxidative injury induced by tert-butylhydroperoxide

Abstract

The present work addresses the role of two ortho-substituted Mn(III) N-alkylpyridylporphyrins, alkyl being ethyl in MnTE-2-PyP⁵⁺ and n-hexyl in MnTnHex-2-PyP⁵⁺, on the protection against the oxidant tert-butylhydroperoxide (TBHP). Their protective role was studied in V79 cells using endpoints of cell viability (MTT and crystal violet assays), intracellular O₂?– generation (dihydroethidium assay) and glutathione status (DTNB and monochlorobimane assays). MnPs per se did not show cytotoxicity (up to 25 μM, 24 h). The exposure to TBHP resulted in a significant decrease in cell viability and in an increase in the intracellular O₂^?– levels. Also, TBHP depleted total and reduced glutathione and increased GSSG. The two MnPs counteracted remarkably the effects of TBHP. Even at low concentrations, both MnPs were protective in terms of cell viability and abrogated the intracellular O₂^?– increase in a significant way. Also, they augmented markedly the total and reduced glutathione contents in TBHP-treated cells, highlighting the multiple mechanisms of protection of these SOD mimics, which at least in part may be ascribed to their electron-donating ability.     

Effect of fusaric acid on prooxidant and antioxidant properties of the potato cell suspension culture

Cell suspension cultures of potato (Solanum tuberosum, cv. Tamasha) were treated with fusaric acid (FA), a nonspecific fungal toxin produced by Fusarium species to study the effects of FA on H₂O₂ generation, lipid peroxidation, and activities of antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase, and ascorbate peroxidase (APX). The toxicity of various FA doses was evaluated from viability of cultured cells of S. tuberosum. The toxic concentration of FA (10⁻³ M) reduced cell viability by 32% after 48-h incubation and induced alkalinization of the medium; the nontoxic concentration of FA (10⁻⁶ M) had no effect on cell viability and pH of the culturing medium. The treatment of cells with FA caused rapid reversible accumulation of H₂O₂ in cells, promoted lipid peroxidation, and elevated the activity of antioxidant enzymes. The toxic FA concentration elevated the intracellular H₂O₂ content by 51–59% and stimulated lipid peroxidation rate by 35–40%. The nontoxic FA concentration raised the H₂O₂ content by 84–91% and enhanced lipid peroxidation rate by 18–24%. The addition of FA induced transient biphasic induction of the antioxidant enzymes; the action of toxic and nontoxic concentrations differed in terms of the response amplitudes and dynamics. The results confirm the well-known toxic impact of high doses of FA on the cultured cells, which is determined by membrane transport disorders. In addition, the results reveal that toxic and nontoxic concentrations of FA are able to induce pro- and antioxidant systems in the cultured cells of S. tuberosum.     

Changes in redox states of respiratory pigments recorded from the eyes of live blowflies exposed to light stimuli and hypoxia

Time courses of mitochondrial responses to illumination-induced physiological loads and to hypoxia, were recorded optically from eyes of blowflies Calliphora vicina chalky. We isolated changes in redox states of haems a₃, a, c, and b. Two types of responses to light stimulation were observed. Haems b and a₃ responded with transient oxidation and haems a and c with reduction. The same two groups emerged in response to anoxic exposure. The onset of reduction of haems a and c had virtually no latency, while haems a₃ and b exhibited a transient oxidation followed by reduction only after 10–20 s. The dependence of the steady-state reduction level on P_\textO2 P_{{{\text{O}}_{2} }} produced the same groups. Haems a and c were significantly reduced at P_\textO2 P_{{{\text{O}}_{2} }} levels around 10 kPa while with haems b and a₃ load-induced oxidation was only replaced by reduction below 2 kPa. We propose haems respond to physiological loads in accordance with their steady-state reduction, which in turn depends largely on barriers for electron transport imposed by the mitochondrial membrane potential. We also propose it may be possible to assess the values of tissue P_\textO2 P_{{{\text{O}}_{2} }} and O₂ consumption by monitoring haems that are highly oxidized at rest such as haem a.     

Enhanced lactic acid bacteria viability with yeast coincubation under acidic conditions

ABSTRACT

The enhancing effects of yeasts on the viability of lactic acid bacteria (LAB) under acidic conditions were investigated. Meyerozyma guilliermondii, coaggregative with both LAB strains under acidic conditions, significantly enhanced the viability of Lactobacillus pentosus and L. paracasei in pH 3.0 lactic acid (LA) buffer at 10°C (p < 0.05). Non-coaggregative yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Cyberlindnera saturnus) also significantly enhanced the LAB viability (p < 0.05), and physical contact between LAB and yeasts was not essential for the viability-enhancing effect, indicating that the coaggregation had no relation to the enhancing mechanism. Although yeast metabolites and LA assimilation had no enhancing effect, hydrogen peroxide (H₂O₂) decreased after yeast coincubation, and H₂O₂ elimination improved L. pentosus viability. H₂O₂ elimination alone did not sufficiently improve L. paracasei viability, but the addition of antioxidants was effective. These results suggest that the antioxidant activity of yeast increased the LAB viability under acidic conditions.