Effect of the ratio of (+) and (−) mating type of Blakeslea trispora on carotene production from cheese whey in submerged fermentation

The effect of the ratio of (+) and (?) mating type of Blakeslea trispora on carotene production from deproteinized hydrolysed whey in shake flask culture was investigated. Also, the inoculum ratio of the two mating types on the morphology of the microorganism and the relationship between morphological changes of the fungus and product formation were studied. The concentration of carotenes was significantly affected by the ratio of (+) and (?) mating type of B. trispora. A ratio of 1:10 up to 1:100 of (+) and (?) type was found to achieve the highest carotene yields. The optimum ratio of the (+) and (?) mating types for the maximum pigment production (175.0 mg/g dry biomass at 8 days of fermentation) was found to be 1:10. The carotene content in B. trispora consisted of β-carotene, γ-carotene, and lycopene. At the maximum concentration of carotenes the proportion of β-carotene, γ-carotene, and lycopene (as percent of total carotenes) was 80, 12, and 8%, respectively. B.trispora growing in submerged fermentation is able to develop complex morphologies which have been classified into three major groups: freely dispersed hyphae, clumps and pellets. These parameters are strongly influence the production of carotenes.     

Carotene production from waste cooking oil by Blakeslea trispora in a bubble column reactor: The role of oxidative stress

The oxidative stress induced by hydroperoxides and reactive oxygen species (ROS) during carotene production from waste cooking oil (WCO) and corn steep liquor (CSL) by the fungus Blakeslea trispora in a bubble column reactor was investigated. The specific activities of the intracellular enzymes superoxide dismutase (SOD) and catalase (CAT) as well as the micromorphology of the fungus were measured in order to study the response of the fungus to oxidative stress. The changes of the morphology of microorganism leaded to pellets formation and documented using a computerized image analysis system. As a consequence of the mild oxidative stress induced by hydroperoxides of WCO and ROS a significant increase in carotene production was obtained. The highest carotene concentration (980.0 mg/l or 51.5 mg/g dry biomass) was achieved in a medium consisted of CSL (80.0 g/L) and WCO (50.0 g/L) at an aeration rate of 5 vvm after 6 days of fermentation. In this case the carotenes produced consisted of β‐carotene (71%), γ‐carotene (26%), and lycopene (3%). The strong oxidative stress in the fungus caused a significant increase of γ‐carotene concentration. Bubble column reactor is a useful fermentation system for carotene production in industrial scale.     

Role of hydrolytic enzymes and oxidative stress in autolysis and morphology of <Emphasis Type="Italic">Blakeslea trispora</Emphasis> during β-carotene production in submerged fermentation

The role of hydrolytic enzymes (proteases and chitinase) and oxidative stress in the autolysis and morphology of Blakeslea trispora during β-carotene production from a chemically defined medium in shake flask culture was investigated. The process of cellular autolysis was studied by measuring the changes in biomass dry weight, pH, concentration of β-carotene, specific activity of the hydrolytic enzymes and micromorphology of the fungus using a computerized image analysis system. In addition, the phenomenon of autolysis was associated with high concentrations of reactive oxygen species (ROS). The accumulation of ROS produced during fermentation causes oxidative stress in B. trispora. Oxidative stress was examined in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). The profile of the specific activities of the above enzymes appeared to correlate with the oxidative stress of the fungus. The high activities of CAT and SOD showed that B. trispora is found under oxidative stress during β-carotene production. The culture began to show signs of autolysis nearly in the growth phase and autolysis increased significantly during the production phase. The morphological differentiation of the fungus was a result of the degradation of the cell membrane by hydrolytic enzymes and oxidative stress. Increased β-carotene production is correlated with intense autolysis of clumps, which has as a consequence the increase of the freely dispersed mycelia.     

Improved β-carotene production by oxidative stress in Blakeslea trispora induced by liquid paraffin

When 3 % (v/v) liquid paraffin was added to the medium, β-carotene production increased from 397 to 715 mg l^?1 in mated cultures of Blakeslea trispora. Liquid paraffin also enhanced the oxygen concentration and induce high oxidative stress, as observed by the increase in activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). After 84 h of cultivation in the presence of liquid paraffin, the activities of SOD, CAT and POD in B. trispora increased 77, 52.5 and 76.6 %, respectively.     

The role of oxidative stress on carotene production by Blakeslea trispora in submerged fermentation

In aerobic metabolism, reactive oxygen species (ROS) are formed during the fermentation that can cause oxidative stress in microorganisms. Microbial cells possess both enzymatic and non-enzymatic defensive systems that may protect cells from oxidative damage. The antioxidant enzymes superoxide dismutase and catalase are the two key defensive enzymes to oxidative stress. The factors that induce oxidative stress in microorganisms include butylated hydroxytoluene (BHT), hydrogen peroxide, metal ions, dissolved oxygen tension, elevated temperature, menadione, junglone, paraquat, liquid paraffin, introduction to bioreactors of shake flask inocula and synthetic medium sterilized at initial pH 11.0. Carotenes are highly unsaturated isoprene derivatives. They are used as antioxidants and as coloring agents for food products. In fungi, carotenes are derived via the mevalonate biosynthesis pathway. The key genes in carotene biosynthesis are hmgR, ipi, isoA, carG, carRA and carB. Among microorganisms, Βlakeslea trispora is the main microorganism used for the production of carotenes on the industrial scale. Currently, the synthetic medium is considered the superior substrate for the production of carotenes in a pilot plant scale. The fermentation systems used for the production of carotenes include shake flasks, stirred tank fermentor, bubble column reactor and flat panel photobioreactor. This review summarizes the oxidative stresses in microorganisms and it is focused on the current status of carotene production by B. trispora including oxidative stress induced by BHT, enhanced dissolved oxygen levels, iron ions, liquid paraffin and synthetic medium sterilized at an initial pH 11.0. The oxidative stress induced by the above factors increases significantly the production of carotenes. However, to further reduce the cost of carotene production, new biotechnological methods with higher productivity still need to be explored.     

A pH control strategy for increased β-carotene production during batch fermentation by recombinant industrial wine yeast

The effects of different pH values, ranging from 4.0 to 7.0, on cell growth and β-carotene production by recombinant industrial wine yeast Saccharomyces cerevisiae T73-63 in a synthetic grape juice medium was investigated. Based on the kinetic analysis of the batch fermentation process, a two-stage pH control strategy was developed in which the pH was maintained at 7.0 for the first 24 h and then shifted to 5.0 after 24 h. Using this strategy, the highest β-carotene production (50.39 mg/l) and the formation rate (1.40 mg/l/h) were increased by 19.1% and 18.6%, respectively, compared to the maximum values of constant pH fermentation. The oxidative stress during β-carotene production was also determined in terms of the catalase (CAT) and superoxide dismutase (SOD) activities. Oxidative stress appears to be induced by the lowering of pH as indicated by the increase in activities of CAT and SOD due to pH shift from pH 7.0 to pH 5.0. Pre-treating cells with ascorbic acid (an antioxidative agent) reversed the improvement of β-carotene production while addition of H₂O₂ enhanced it. Considering that induction of oxidative stress is associated with increased β-carotene production, it was concluded that the enhancement of β-carotene production by the low-pH strategy involved the induction of oxidative stress.     

Antioxidant defence system during exponential and stationary growth phases of Phycomyces blakesleeanus: Response to oxidative stress by hydrogen peroxide

An analysis of the components of the antioxidant defence system in exponential and stationary growth phases of filamentous fungus Phycomyces blakesleeanus and the response to the oxidative stress hydrogen peroxide were performed. There is a strong positive correlation between mycelial antioxidant capacity and the contents of gallic acid, d-erythroascorbate (d-EAA) or d-erythroascorbate monoglucoside (d-EAAG). These secondary metabolites are specifically synthesized by this fungus and reach maximal values in the stationary growth phase, suggesting that they can play some role in the antioxidant defence system of this fungus. There is a differential expression of the two more notable antioxidant activities, catalase (CAT) and superoxide dismutase (SOD), depending of the growth stage of P. blakesleeanus, CAT being expressed in the exponential and SOD in the stationary phase. Phycomyces blakesleeanus showed a high resistance to the oxidative stress caused by H₂O₂ (50 and 200 mM) which was higher in exponential phase. This higher resistance can be explained by the presence of CAT, glutathione peroxidase (GPx), and the probable contribution of glutathione-S-transferase (GST) and high levels of reduced form of glutathione (GSH). The transition to stationary phase was accompanied with a higher physiological oxidative damage illustrated by the higher protein carbonylation. In this growth stage the resistance of the fungus to the oxidative stress caused by H₂O₂ could be explained by the presence of SOD, GPx, and the probable contribution of GST as well as of secondary metabolites, mainly d-EAA and d-EAAG. These results highlight a specific response to oxidative stress by H₂O₂ depending on the growth phase of P. blakesleeanus.     

Oxidative stress response of Blakeslea trispora induced by H2O2 during β-carotene biosynthesis

The cellular response of Blakeslea trispora to oxidative stress induced by H₂O₂ in shake flask culture was investigated in this study. A mild oxidative stress was created by adding 40 μm of H₂O₂ into the medium after 3 days of the fermentation. The production of β-carotene increased nearly 38 % after a 6-day culture. Under the oxidative stress induced by H₂O₂, the expressions of hmgr, ipi, carG, carRA, and carB involving the β-carotene biosynthetic pathway all increased in 3 h. The aerobic metabolism of glucose remarkably accelerated within 24 h. In addition, the specific activities of superoxide dismutase and catalase were significantly increased. These changes of B. trispora were responses for reducing cell injury, and the reasons for increasing β-carotene production caused by H₂O₂.     

Enhancement of β-Carotene Synthesis by Citrus Products

β-Ionone, a stimulatory compound in the microbiological production of β-carotene by mated cultures of Blakeslea trispora, could be replaced with low-cost agricultural by-products (citrus oils, citrus pulp, or citrus molasses) with as good or better carotene yields. Peak yields (81 to 129 mg of carotene per g of dry solids) were achieved in 5 days. The various citrus products tested did not change the pigments produced; all trans-β-carotene remained the pre-dominant pigment. The acid-hydrolyzed soybean meal and corn used in previous production media could be replaced with unhydrolyzed cottonseed embryo meal and corn in a medium that also contained a natural lipid, deodorized kerosene, nonionic detergent, and a precursor.     

The isolation, purification, and characterization of cis-zeta-carotene and the demonstration of its conversion to acyclic, monocyclic and dicyclic carotenes by a soluble enzyme system obtained from the plastids of tangerine tomato fruits

Cis-ζ-carotene was isolated, purified in several chromatographic systems, and then identified as an intermediate in the biosynthesis of poly-cis-carotenes. The structure of cis-ζ-carotene was tentatively established from its visible light-absorption spectrum, and by a comparison of the infrared spectrum with that of trans-ζ-carotene. Confirmation of the identity of this compound was obtained by high resolution mass spectroscopy. The presence of the cis-configuration was indicated by a bathochromic shift of 6–10 nm in the visible spectrum when the compound was subjected to iodinecatalyzed photoisomerization. The infrared spectrum also showed characteristic peaks for the cis-configuration. Proof of the conversion of cis-ζ-[¹⁴C]carotene to trans-ζ-carotene, proneurosporene, prolycopene, neurosporene, lycopene, β- and γ carotenes was obtained on incubation with soluble enzyme systems obtained from plastids of fruits of two different tangerine varieties of tomato. Proof for the formation of each of the carotenes was provided by column and thin-layer chromatography A close correspondence of radioactivity and optical density was observed for each carotene. Additional proof was obtained by gas-liquid chromatography of each hydrogenated carotene. A coincidence of mass and radioactivity was observed for each carotene.     

β-Carotene biosynthesis by extracts of the C115 mutant of Phycomyces blakesleeanus

A cell extract of the yellow C115 car-42 mad-107(?) mutant of Phycomyces blakesleeanus, capable of converting MVA-[2-¹⁴C] into isoprenoids, was used to investigate the formation of β-carotene. The incorporation of radioactivity into β-carotene was reduced by the addition of unlabelled carotenes, solubilised using detergent, to the incubation mixtures. On reisolation of these carotenes after anaerobic incubations, they were found to carry radioactivity. The relative efficiencies of these carotenes as trapping agents are discussed in relation to the pathways of carotene cyclisation and to the apparent operation of a system for the negative feedback control of carotene biosynthesis.     

Microbiological Production of Carotenoids: VIII. Influence of Hydrocarbon on Carotenogenesis by Mated Cultures of Blakeslea trispora

Synthesis of β-carotene by mated strains of Blakeslea trispora in shaken-flask culture was considerably enhanced by adding either 5% kerosene after 2 days of fermentation or acid-refined kerosene at the start of fermentation to a grain-based medium that also contained a natural lipid, nonionic detergent, and β-ionone; average yields of 17,500 μg per g of dry fermentation solids (86,000 μg per 100 ml of medium) were attained when refined kerosene was used. Almost all of the carotene was retained within the mycelium. Peak yields were achieved in 5 days.     

The enzymatic conversion of cis-(14C)phytofluene, trans-(14C)phytofluene, and trans-zeta-(14C)carotene to more unsaturated acyclic, monocyclic, and dicyclic carotenes by a cell-free preparation of red tomato fruits

This paper reports the conversion of cis-[¹⁴C]phytofluene to trans-[¹⁴C|phytofluene and the conversion of the latter compound to trans-ζ-[¹⁴C]carotene by a soluble enzyme system obtained from the plastids of red tomato fruits. Each of these radioactive compounds was also converted to labeled neurosporene, lycopenc, α-carotene, and β-carotene by the same enzyme system. The incorporation of each substrate into more unsaturated carotenes was carried out under nitrogen at pH 7.5–8.2 (borate buffer), at 25 °C in the dark.Proof of the formation of the above carotenes from each of the three radioactive substrates was demonstrated by cochromatography with authentic nonradioactive carotenes on an alumina chromatographic column. A close correspondence between radioactivity and light absorbance for each carotene was observed. Confirmation of these conversions was achieved by cochromatography with authentic samples on thinlayer plates. Final proof for the formation of the acyclic and cyclic carotenes from the above radioactive substrates was obtained by gas-liquid chromatography of the hydrogenated products. Coincidence between mass and radioactivity was observed.Maximum conversion of cis- and trans-phytofluenes to more unsaturated carotenes by the red tomato fruit enzyme system appears to be dependent upon the presence of NADP⁺, FAD, and Tween 80. The formation of the carotenes is also increased in the presence of Mg²⁺ or Mn²⁺ ions.     

Activation of the biosynthesis of carotenoids by Blakeslea trispora

The research carried out by several scientists has made possible the industrial preparation of β-carotene by fermentation. A fungus, Blakeslea trispora, abundantly synthesizes carotenoids when its two opposite forms are cultivated together in a special fatty medium. When ionones or other natural substances are introduced into the culture, a very obvious increase in the biosynthesis of carotenoids, more specifically of β-carotene, is obtained. Our own work has shown that; (1) several synthetic products chemically related to β-ionone, such as 2,6,6-trimethyl-l-acetyleyelohexene, can advantageously replace either partially or totally the ionones as inductors of the biosysnthesis of β-carotene; (2) various nitrogen-containing substances when added to the culture medium can considerably enhance the biosysnthesis of carotenoids while sometimes very specically orienting it. Their action comes on top of that of the ionones or their substitutes; actually this action is unexplained. Thus certain amides, imides, lactams, hydrazides, or substituted pyradines, and in particular succinimide and isonicotinoylhydrazine, have produced a two or threefold increase in the quantity of β-carotene present in the culture media of Blackeslea trispora. Conversely some heterocyclic substances such as pyridine itself or imidazole totally inhibit the biosysnthesis of β-carotene but induce the production of very important quantities of lycopene.     

Carotene biosynthesis by cell extracts of mutants of Phycomyces blakesleeanus

Cell extracts capable of converting MVA-[2-¹⁴C] into isoprenoids were obtained from the yellow C115-mad-107(−) and red C9-carR21(−) mutants of Phycomyces blakesleeanus. Neither air nor light was essential for carotene biosynthesis. The specific activities of the terpenoid-synthesizing enzymes varied with the age of the cultures although the formation of lycopene (ψ,ψ-carotene) in the C9 and of β-carotene (β,β-carotene) in the C115 'mutants. respectively, followed the increase in the dry weight yield of the cultures. The significance of these results to the biosynthesis of carotenes and to the classification of these compounds as secondary metabolites is discussed.     

Zygote Formation in Blakeslea trispora: Morphological Peculiarities and Relationship with Carotenoid Synthesis

Changes associated with zygospore formation in the mucorous fungus Blakeslea trispora were studied. Zygospores are dormant cells with thickened cell walls and large central lipid vacuoles containing large amounts of lycopene. We established for the first time that B. trispora gametangia of different sexes differ in their carotenoid content and revealed that zygote formation involves a novel structure that consists of densely intertwined hyphae. Using inhibitory analysis (blocking -carotene synthesis with diphenylamine and 2-amino-6-methylpyridine), we showed that suppression of carotene producion results in the inhibition of zygote formation. Hence, we established a manifest dependence of zygote formation on -carotene synthesis.     

Effects of anisosmotic medium on cell volume,transmembrane potential and intracellular K+ activity in mouse hepatocytes

Summary Mouse hepatocytes in primary monolayer culture (4 hr) were exposed for 10 min at 37°C to anisosmotic medium of altered NaCl concentration. Hepatocytes maintained constant relative cell volume (experimental volume/control volume) as a function of external medium relative osmolality (control mOsm/experimental mOsm), ranging from 0.8 to 1.5. In contrast, the relative cell volume fit a predicted Boyle-Van't Hoff plot when the experiment was done at 4°C. Mouse liver slices were used for electrophysiologic studies, in which hepatocyte transmembrane potential (V _m ) and intracellular K⁺ activity (a _K ⁱ ) were recorded continuously by open-tip and liquid ion-exchanger ion-sensitive glass microelectrodes, respectively. Liver slices were superfused with control and then with anisosmotic medium of altered NaCl concentration.V _m increased (hyperpolarized) with hypoosmotic medium and decreased (depolarized) with hyperosmotic medium, and ln [10(experimentalV _m /controlV _m )] was a linear function of relative osmolality (control mOsm/experimental mOsm) in the range 0.8–1.5. Thea _K ⁱ did not change when medium osmolality was decreased 40–70 mOsm from control of 280 mOsm. Similar hypoosmotic stress in the presence of either 60mm K⁺ or 1mm quinine HCl or at 27°C resulted in no change inV _m compared with a 20-mV increase inV _m without the added agents or at 37°C. We conclude that mouse hepatocytes maintain their volume anda _K ⁱ in response to anisosmotic medium; however,V _m behaves as an osmometer under these conditions. Also, increases inV _m by hypoosmotic stress were abolished by conditions or agents that inhibit K⁺ conductance.