Yeast Candida Utilis Research Articles

Export of an invertase by yeast Candida utilis cells

Export and accumulation of various forms of invertase (EC 3.2.1.26) in the cell wall and culture medium of the yeast Candida utilis was investigated. It was found that there is the high-molecular-weight invertase in the cell wall (CW-form). This form is not exported into the culture medium, and it is by a third more glycosylated than the previously described exported S-form. It was shown that one of the two forms of invertase exported into the culture medium—the glycosylated S-form—is retained in the cell wall, while the other one-the nonglycosylated F-form—was not detected in the cell wall. Based on these results, as well as data on the distribution dynamics of the enzyme in the culture medium and in the cell wall during different growth stages of a yeast culture, we suggested that the nonglycosylated form was exported into the culture medium via the zone of abnormal cell wall permeability and the glycosylated forms of this enzyme (both exported and nonexported) did not use this pathway and the degree of N-glycosylation is an important factor determining the final localization of the enzyme.

Antimicrobial Activity of Coleus For skohlii (Wild) Briq and Costus Igneus N.E.Br

Medicinal plants are the wealthy source of antibacterial agents and curatives. The use of plants in treatment of burns, dermatophytes and infectious diseases is common in traditional medicine. The development of new antimicrobial agents against resistant pathogens is increasing interest. It was found that most plant extracts studied had antibacterial and antifungal activities. In the present investigation, the effect of different extracts namely petroleum ether, methanol, ethyl acetate, acetone and chloroform were tested on Gram positive bacteria like Bacillus subtilis, Staphylococcus aureus and Gram negative bacteria E coli, Pseudomonas aeruginosa and the yeast Candida utilis by disc diffusion and agar well diffusion method. All the extracts showed significant activity against all pathogens, but the methanolic extract showed maximum zone of inhibition against all the microorganisms. Both the gram positive bacteria tested are found to be more sensitive compared to gram negative bacteria which recorded minimum inhibition zone. The plant extracts were found to be more inhibitory towards fungus tested. This study scientifically supports the usage of whole plant as a remedy for Costus igneus, common name Fiery Costus or Spiral Flag, is a species of herbaceous plant in the Costaceae family. The plant grows very quickly and the propagation is by stem cutting. It needs sunshine but it also grows in slightly shady areas. This plant has large fleshy looking leaves. The undersides of these large, smooth, dark green leaves have light purple shade. The leaves are spirally arranged around the stem, forming attractive, arching clumps arising from underground rootstocks. The maximum height of these plants is about two feet. The flowers are orange in color and are beautiful, 1.5-inch diameter. Flowering occurs during the

Export of an invertase by yeast cells (Candida utilis)

Export and accumulation of various forms of invertase (EC 3.2.1.26) in the cell wall and culture liquid of the yeast Candida utilis was investigated. It was found that the high-molecular-weight CW-form of invertase is present in the cell wall. This form is not exported into the culture liquid, and it is by a third more glycosylated than the previously described exported S-form. It was shown that one of the two liquid forms of invertase exported into the culture-the glycosylated S-form--is retained in the cell wall, while the other one--the nonglycosylated F-form--was not detected in the cell wall. Based on these results, as well as data on the distribution dynamics of the enzyme in the culture liquid and in the cell wall during different growth stages of a yeast culture, we suggested that the nonglycosylated form was exported into the culture liquid via the zone of abnormal cell wall permeability and the glycosylated forms of this enzyme (both exported and nonexported) did not use this pathway (the degree of N-glycosylation is an important factor determining the final localization of the enzyme).

Candida utilis and Chlorella vulgaris Counteract Intestinal Inflammation in Atlantic Salmon (Salmo salar L.)

Intestinal inflammation, caused by impaired intestinal homeostasis, is a serious condition in both animals and humans. The use of conventional extracted soybean meal (SBM) in diets for Atlantic salmon and several other fish species is known to induce enteropathy in the distal intestine, a condition often referred to as SBM induced enteropathy (SBMIE). In the present study, we investigated the potential of different microbial ingredients to alleviate SBMIE in Atlantic salmon, as a model of feed-induced inflammation. The dietary treatments consisted of a negative control based on fish meal (FM), a positive control based on 20% SBM, and four experimental diets combining 20% SBM with either one of the three yeasts Candida utilis (CU), Kluyveromyces marxianus (KM), Saccharomyces cerevisiae (SC) or the microalgae Chlorella vulgaris (CV). Histopathological examination of the distal intestine showed that all fish fed the SC or SBM diets developed characteristic signs of SBMIE, while those fed the FM, CV or CU diets showed a healthy intestine. Fish fed the KM diet showed intermediate signs of SBMIE. Corroborating results were obtained when measuring the relative length of PCNA positive cells in the crypts of the distal intestine. Gene set enrichment analysis revealed decreased expression of amino acid, fat and drug metabolism pathways as well as increased expression of the pathways for NOD-like receptor signalling and chemokine signalling in both the SC and SBM groups while CV and CU were similar to FM and KM was intermediate. Gene expression of antimicrobial peptides was reduced in the groups showing SBMIE. The characterisation of microbial communities using PCR-DGGE showed a relative increased abundance of Firmicutes bacteria in fish fed the SC or SBM diets. Overall, our results show that both CU and CV were highly effective to counteract SBMIE, while KM had less effect and SC had no functional effects.

Research of Producing Single Cell Protein with Molasses Wastewater

This paper is to study the optimum conditions for extracting the protein feed by using the mixed microbial fermentation. And using the single factor and orthogonal methods, the microbial content as the main indicators, investigated the effects of the combination of strain, strain ratio, the amount of bacteria, fermentation temperature, the concentration of fermentation, fermentation pH, nitrogen sources and inorganic salts and other factors on the fermentation of protein yield. The test results showed that the best fermentation process is sterilization process, the best combination of strains of Geotrichum candidum, Candida tropicalis and Candida utilis yeast, the cast of bacteria was 10%, the best molasses fermentation concentration 20g / 100 mL, and temperature 30 ° C, initial pH 5.0, appropriate amount of Yeast extract, magnesium sulfate, potassium dihydrogen phosphate and super phosphate. The Test provides a theoretical and experimental basis for Molasses wastewater utilization

The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 is functional in Agrobacterium tumefaciens

The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 was cloned to create a novel integrative vector for Agrobacterium tumefaciens-mediated transformation. The new binary vector harbors β-glucuronidase activity as reporter and kanamicin/geneticin resistance as selection marker. Recombinant clones of A. tumefaciens show kanamycin resistance and high β-glucuronidase activity under the control of the C. utilis promoter. This finding can be explained by the presence of a prokaryotic core in the yeast promoter, predicted by in silico analysis of the sequence. This is the first report about functionality of a yeast promoter in A. tumefaciens.

Application of yeast Candida utilis to ferment Eisenia bicyclis for enhanced antibacterial effect.

In this study, fermentation broth of Eisenia bicyclis with Candia utilis YM-1 exhibited enhanced antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) and food-borne pathogenic bacteria. To perform a more detailed investigation on the antibacterial activity, the fermented broth of E. bicyclis was extracted with methanol and further fractionated with organic solvents. After 1-day fermentation, the ethyl acetate (EtOAc)-soluble extract exhibited the highest anti-MRSA activity with minimum inhibitory concentration values ranging from 128 to 512 μg/mL, suggesting that the fermentation of E. bicyclis with C. utilis YM-1 may enhance antibacterial activity against MRSA. This effect was correlated to the result obtained by an increase in total phenolic contents in EtOAc-soluble extract. In addition, high-performance liquid chromatography analysis revealed that eckol, dieckol, dioxinodehydroeckol, and phlorofucofuroeckol-A contents in the EtOAc-soluble extract increased significantly. Thus, these results show that anti-MRSA activity of E. bicyclis fermented with C. utilis most likely originated from phlorotannins and allow the possible application of a variety of seaweed functional foods.

Metabolic engineering of Candida utilis for isopropanol production

A genetically-engineered strain of the yeast Candida utilis harboring genes encoding (1) an acetoacetyl-CoA transferase from Clostridium acetobutylicum ATCC 824, (2) an acetoacetate decarboxylase, and (3) a primary-secondary alcohol dehydrogenase derived from Clostridium beijerinckii NRRL B593 produced up to 0.21g/L of isopropanol. Because the engineered strain accumulated acetate, isopropanol titer was improved to 1.2g/L under neutralized fermentation conditions. Optimization of isopropanol production was attempted by the overexpression and disruption of several endogenous genes. Simultaneous overexpression of two genes encoding acetyl-CoA synthetase and acetyl-CoA acetyltransferase increased isopropanol titer to 9.5g/L. Moreover, in fed-batch cultivation, the resultant recombinant strain produced 27.2g/L of isopropanol from glucose with a yield of 41.5% (mol/mol). This is the first demonstration of the production of isopropanol by genetically engineered yeast.

Heterologous protein secretion by Candida utilis

The yeast Candida utilis (also referred to as Torula) is used as a whole-cell food additive and as a recombinant host for production of intracellular molecules. Here, we report recombinant C. utilis strains secreting significant amounts of Candida antarctica lipase B (CalB). Native and heterologous secretion signals led to secretion of CalB into the growth medium; CalB was enzymatically active and it carried a short N-glycosyl chain lacking extensive mannosylation. Furthermore, CalB fusions to the C. utilis Gas1 cell wall protein led to effective surface display of enzymatically active CalB and of β-galactosidase. Secretory production in C. utilis was achieved using a novel set of expression vectors containing sat1 conferring nourseothricin resistance, which could be transformed into C. utilis, Pichia jadinii, Candida albicans, and Saccharomyces cerevisiae; C. utilis promoters including the constitutive TDH3 and the highly xylose-inducible GXS1 promoters allowed efficient gene expression. These results establish C. utilis as a promising host for the secretory production of proteins.

Evaluation of Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae yeasts as protein sources in diets for Atlantic salmon (Salmo salar)

The use of the yeasts Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae as protein sources in diets for Atlantic salmon (Salmo salar) pre-smolts was investigated. An 89day study was conducted using triplicate groups of Atlantic salmon with 28g initial weight kept in fresh water. The dietary treatments consisted of a control diet based on high-quality fish meal (FM diet) and three experimental diets with C. utilis (CU diet), K. marxianus (KM diet) or S. cerevisiae (SC diet) yeast, substituting 40% of the crude protein (CP) from FM. There was no difference in final weight or specific growth rate (SGR) of fish fed the CU diet and the FM diet. The SC diet resulted in lower final weight and SGR, and higher average daily feed intake (ADFI) and feed conversion ratio (FCR) than the FM diet, and higher ADFI and FCR than the KM and CU diets. Fish fed the SC diet had decreased retention of nitrogen and energy, while fish fed the CU diet had increased nitrogen retention compared with those fed the FM diet. In general, feeding the SC diets gave lower digestibility of CP, amino acids, and energy compared with the FM, KM and CU diets, whereas there was no difference in nutrient or amino acid digestibility between the KM and CU diet and the FM diet. Feeding KM and SC gave higher total gut weight and higher distal intestinal weight compared with the FM diet. Plasma cholesterol level was lower for the KM and SC diets while plasma glutathione peroxidase levels were higher for the KM diet compared with the FM diet. In conclusion, feeding moderate levels of S. cerevisiae reduced growth performance and nutrient utilization, while C. utilis and K. marxianus were shown to be promising protein sources in diets for Atlantic salmon, capable of replacing 40% of the protein from high-quality fishmeal without adversely affecting growth performance, digestibility or nutrient retention.

Ethanol Production from Xylose by a RecombinantCandida utilisStrain Expressing Protein-Engineered Xylose Reductase and Xylitol Dehydrogenase

The industrial yeast Candida utilis can grow on media containing xylose as sole carbon source, but cannot ferment it to ethanol. The deficiency might be due to the low activity of NADPH-preferring xylose reductase (XR) and NAD(+)-dependent xylitol dehydogenase (XDH), which convert xylose to xylulose, because C. utilis can ferment xylulose. We introduced multiple site-directed mutations in the coenzyme binding sites of XR and XDH derived from the xylose-fermenting yeast Candida shehatae to alter their coenzyme specificities. Several combinations of recombinant and native XRs and XDHs were tested. Highest productivity was observed in a strain expressing CsheXR K275R/N277D (NADH-preferring) and native CsheXDH (NAD(+)-dependent), which produced 17.4 g/L of ethanol from 50 g/L of xylose in 20 h. Analysis of the genes responsible for ethanol production from the xylose capacity of C. utilis indicated that the introduction of CsheXDH was essential, while overexpression of CsheXR K275R/N277D improved efficiency of ethanol production.

Value-added fermentation of Ecklonia cava processing by-product and its antioxidant effect

The interest in the extraction of polyphenolic compounds from plant materials is focused on upgrading of the large amount of by-products coming from food or cosmetics industries from which the press residues have particularly high contents of phenolics. In this study, for value-added use of the brown seaweed Ecklonia cava processing by-product (ECPB), which can be obtained after polyphenolic extraction of E. cava, it was fermented by the yeast Candida utilis and its antioxidant activities were evaluated by 1,1-diphenyl-2-picrylhydrazyl, hydroxyl, and alkyl radical scavenging using electron spin resonance spectrometer. ECPB was fermented for 1~4 days prior to being extracted with 80% ethanol, and significant differences were observed in extraction yields, total phenolic contents (TPC), and radical scavenging activities with the fermentation time. Extract from the ECPB fermented for 1 day exhibited the highest TPC and also found to be the strongest antioxidant. The 1-day fermented ECPB strongly enhanced cell viability against H2O2-induced oxidative damage in Vero cell line. This sample also exhibited good protective properties against H2O2-induced cell apoptosis as was demonstrated by a decreased quantity of sub-G1 hypodiploid cells and decreased apoptotic body formation in the flow cytometry analysis. This study demonstrated that the fermentation elevated functionally important polyphenolic contents of ECPB and resultant antioxidant activities were enhanced. Therefore, the fermentation could offer a tool to further increase the bioactive potential of ECPB.

Biocalorimetry as a process analytical technology process analyser; robust in-line monitoring and control of aerobic fed-batch cultures of crabtree-negative yeast cells

Control of bioprocesses requires reliable and robust on- or in-line monitoring tools providing real-time information on process dynamics. Heat generation related to metabolic activity of living systems is currently gaining importance in bioprocess industry due to its non-invasive and essentially instantaneous characteristics. This study deals with monitoring and control of pure aerobic fed-batch cultures of three Crabtree-negative yeast strains, Kluyveromyces marxianus, Candida utilis and Pichia pastoris, based on in-line measured, metabolic heat flow signals. A high resolution biocalorimeter (BioRC1) was developed from a standard bench-scale heat flow calorimeter (RC1). The BioRC1 was equipped with in-line (dielectric spectroscopy, pH probe and dissolved oxygen probe) and at-line (exit gas analyser) sensors to characterise the growth behaviour of the yeast cells. Both metabolic heat flow and biomass profiles exhibited similar behaviour proving the significance of employing heat flow signal as a key-parameter for the system under investigation. A simple estimator for biomass concentration and specific growth rate was formulated based on heat flow values. In order to evaluate the potential of calorimetry as a reliable and powerful process monitoring tool, the robustness, reliability as well as the broad applicability of the developed estimators was assessed through comparison with off-line measurement techniques and showed promising results for general applicability with a wide range of bioprocesses.

EFFECT OF COMBINATION OF YEAST (Saccharomyces cerevisae + Candida utilis) AND HERBS SUPPLEMENTATION IN FINISHING DIET ON CARCASS CHARACTERISTICS OF BEEF CATTLE

A 75 days feeding trial was completed to study the effect of inclusion of a combined probioticyeast+herbs in finishing diet on carcass characteristics of beef cattle. Thirty bull of Ongole crossbred,age 2.5-3 years old with average body weight (BW) of 320 kg were used in this experiment. They weredivided into 2 groups, each of 15 animals and were allotted to control and treatment. They wereslaughtered at around 450 kg BW. The treated animals were given a supplementation containingcombined yeast (Saccharomyces cerevisae and Candida utilis) and herbs. All animals were fed a diet ofammoniated rice straw and commercial concentrate with a ratio of 10: 90. Addition of combinedprobiotics + herbs in the diet increased percentage of carcass, dressing and meat, but there were noeffect on live weight gain (LWG), % bone, meat:bone ratio, % body fat, back fat thickness and eyemuscle area. There was a reduction in the percentage of offal due to a decrease in intestinal weight in thetreated animal.

EFFECT OF INCREASED EPIPHYTIC POPULATION ON REDUCTION OF FIRE BLIGHT (ERWINIA AMYLOVORA) INFECTION ON APPLE TREES

Fire blight is a limiting factor for organic apple production. The use of antibiotics is not allowed in organic systems. The use of an innocuous microorganism consortium including bacteria like Pseudomonas fluorescens and Lactobacillus acidophilus, combined with yeast Saccharomyces boullardii and Candida utilis, is discussed as an alternative management for the disease. The spray application of the living consortium on trees, especially on flowers or pruning wounds, reduced infection. The enhancing action of the consortium to increase the competition for space and nutrients is demonstrated. Experiments in vitro and in vivo demonstrated good disease control.

Oil phytoremediation potential of hypersaline coasts of the Arabian Gulf using rhizosphere technology

The rhizosphere and phyllosphere of the halophyte Halonemum strobilaceum naturally inhabiting hypersaline coastal areas of the Arabian Gulf harbor up to 8.1×104g−1 and 3×102g−1, respectively, of extremely halophilic oil-utilizing microorganisms. Such organisms were 14- to 38-fold more frequent in the rhizosphere than in the plant-free soil. Frequent genera in the rhizosphere were affiliated to the archaea Halobacterium sp. and Halococcus sp., the firmicute Brevibacillus borstenlensis, and the proteobacteria Pseudoalteromonas ruthenica and Halomonas sinaensis. The phyllospheric microflora consisted of the dimorphic yeast Candida utilis and the two proteobacteria Ochrobactrum sp. and Desulfovibrio sp. Individual strains grew on a range of pure aliphatic and aromatic hydrocarbons, as sole sources of carbon and energy. All the strains, except C. utilis which could not tolerate salinities >2M NaCl, grew also in media with salinities ranging between 1 and 4M NaCl, with optimum growth between 1 and 2M NaCl. With the exception of the two archaeal genera, all isolates could grow in a nitrogen-free medium. The total rhizospheric and phyllospheric microbial consortia could attenuate crude oil in complete (nitrogen-containing) medium, but also equally well in a nitrogen-free medium. It was concluded that H. strobilaceum could be a valuable halophyte for phytoremediation of oil-polluted hypersaline environments via rhizosphere technology.

Use of Pseudomonas stutzeri and Candida utilis in the improvement of the conditions of Artemia culture and protection against pathogens

To evaluate the effect of two bacterial strains isolated from Artemia cysts and yeast (Candida utilis) on the survival, growth and total biomass production of its larvae, challenge tests were performed with Candida utilis, Pseudomonas stutzeri and Pasteurella haemolityca. In addition, a pathogenic strain of Vibrio alginolyticus was tested for comparative purposes. Pseudomonas stutzeri and Candida utilis have no impact on survival, but enhance growth and total biomass production of the larvae. However, we noted that Pasteurella haemolityca affect negatively Artemia larvae. The adhesion and antagonism assay demonstrates that Candida utilis and Pseudomonas stutzeri are fairly adherent and play an important role in the enhancement of the protection of Artemia culture against pathogens. On the basis of these results, it's suggested that it's possible to use Candida utilis and Pseudomonas stutzeri, potential candidates, as probiotic for the culture of Artemia larvae.

USE of pseudomonas stutzeri and candida utilis in the improvement of the conditions of artemia culture and protection against pathogens

To evaluate the effect of two bacterial strains isolated from Artemia cysts and yeast (Candida utilis) on the survival, growth and total biomass production of its larvae, challenge tests were performed with Candida utilis, Pseudomonas stutzeri and Pasteurella haemolityca. In addition, a pathogenic strain of Vibrio alginolyticus was tested for comparative purposes.Pseudomonas stutzeri and Candida utilis have no impact on survival, but enhance growth and total biomass production of the larvae. However, we noted that Pasteurella haemolityca affect negatively Artemia larvae. The adhesion and antagonism assay demonstrates that Candida utilis and Pseudomonas stutzeri are fairly adherent and play an important role in the enhancement of the protection of Artemia culture against pathogens. On the basis of these results, it’s suggested that it’s possible to use Candida utilis and Pseudomonas stutzeri, potential candidates, as probiotic for the culture of Artemia larvae.

Producing Single Cell Protein from Poultry Manure and Evaluation for Broiler Chickens Diets

An experiment was run to study the possibility of elimination of uric acid from poultry manure and increasing its nutritive value by using it as a medium for single cell protein (SCP, yeast) production. Dried Poultry Manure (DPM) was collected freshly from battery brooders of broiler chicken house, dried in an oven at 80 C for 48 h, crushed with mill and stored in plastic bags. Seven strains of yeast (Candida utilis, Candida o tropicalis, 3 strains of Saccharomyces cerevisae, S. uvarum and Rhodotorula rubra) were tested to hydrolyzed uric acid and produce protein when grown on DPM containing medium. In addition, forty five one- day old commercial broiler chicks were used to determine Total Protein Efficiency (TPE) of raw and treated DPM. The chickens were randomly divided among 3 equal groups of approximately similar initial body weight of three replicates each containing 5 chicks. Another, one hundred and sixty two broiler chickens were allotted to 9 dietary treatments of 3 replicates of 6 chickens each in a randomized complete block design. This experiment aimed to evaluate the nutritive value of the treated DPM. The experimental diets were corn- soybean meal diet in which 3, 6, 9 and 12% treated DPM and untreated DPM were added on the expense of soybean meal protein. Candida utilis offered the highest protein yield (12.7%) and the highest efficiency to hydrolyze uric acid; therefore, it was selected for further study. Shorter growth periods ( 5 days) were concomitant to higher uric acid hydrolysis. The optimum amount for protein production and uric acid hydrolysis under the specified fermentation condition were 2 g /50 ml medium and pH range between 6-7. Inoculum size from 2-8% and medium should be consists only 40 g DPM /l without any salt increased the protein yield and high utilized of uric acid. The chemical analysis of the DPM showed 19.1% Crude Protein (CP), 18.2% ash, 7.9% Crude Fiber (CF), 1.7% Ether Extract (EE) and relatively high uric acid content, 7.2%. Fermentation of DPM increased the CP content from 19.1 to 24.9%, the NPN content decreased from 9.6 to 8.7%, uric acid content decreased from 7.2 to 0.3% and the EE increased from 1.7 to 2.4%. Amino acids of fermented DPM were greatly increased than those of the DPM except for glycine, histidine and tyrosine. Results of TPE assay indicated that chickens fed diet containing treated DPM gained significantly more weight than those given diets containing untreated DPM. Chickens fed treated DPM gained significantly more weight than those given the untreated DPM, throughout the experimental period. In conclusion, yeast treatment for DPM improved its nutritive value, thus it could be included up to 9% in broiler diets without adverse effect on growth performance of broiler chickens up to 4 wks of age.

Acriflavine‐Mediated Apoptosis and Necrosis in Yeast Candida utilis

Acriflavine is an antiseptic, fungicide, and effective agent against parasitic infections, inducing petite mutation in the yeast Saccharomyces cerevisiae and kinetoplast loss in Trypanosomidae. Here we showed that acriflavine caused both apoptosis and necrosis in the yeast Candida utilis. Cells were cultured in minimal medium, with 1.5% ethanol as substrate, in the presence of 30-180 micromol/L acriflavine. Fluorescence measurements showed a linear concentration-dependence flux of the drug into the cells. Acriflavine induced a decrease in cell number, an increase in trypan blue-positive cells, and a decrease in cell viability. Cells cultured in the presence of acriflavine showed an alteration in their respiratory control ratio and a decrease in their cytochrome content. Fluorescence microscopy, after acridine orange staining, revealed the presence of apoptotic cells in cultures conducted in the presence of acriflavine. Electron microscopy of cells grown in the presence of acriflavine showed apoptotic cells exhibiting chromatin condensation, cytoplasmic lysis, but reasonably well-preserved mitochondria, whereas necrotic cells showed no distinctive intracellular organelles. Data showed that acriflavine caused both apoptosis and necrosis. Moreover, acriflavine induced oxidative phosphorylation uncoupling. Generally, apoptosis is considered to be mediated either by a change in mitochondrial permeability and cytochrome c release or by plasma membrane death receptor activation. The outer mitochondrial membrane permeability to cytochrome c, with efflux of protons to the cytosol and cytoplasmic acidification, produced a collapse in the electrochemical proton gradient, a decrease in ATP synthesis, and subsequent cytolysis leading to apoptosis and necrosis.