Isolation and Identification of Canthaxanthin from Micrococcus roseus

Cooney, J. J. (University of Dayton, Dayton, Ohio), H. W. Marks, Jr., and Anne M. Smith. Isolation and identification of canthaxanthin from Micrococcus roseus. J. Bacteriol. 92:342-345. 1966.-The principal colored carotenoid of Micrococcus roseus was purified by solvent partitioning followed by column and thin-layer chromatography. Absorption spectra, partition coefficients, and infrared spectra suggested that the pigment was a diketo derivative of beta-carotene. The pigment was subjected to reduction, and the reduced pigment was subsequently dehydrated. Spectral data and partition coefficients of these derivatives indicated that the original pigment was canthaxanthin (4',4'-diketo-beta-carotene). The pigment was an all-trans isomer; it does not exist as an ester in M. roseus. Canthaxanthin has not previously been identified as a bacterial pigment.     

Influence of culture conditions of Gordonia jacobaea MV-26 on canthaxanthin production.

Commercial interest in the use of natural pigments isolated from microorganisms has increased in recent years; hence, molecules belonging to the polyisoprenoid group (i.e. beta-carotene, astaxanthin, and canthaxanthin) have been the focus of much attention. The bacterium Gordonia jacobaea readily synthesizes and accumulates large amounts of canthaxanthin (beta-beta'-carotene-4,4'-dione), which is widely used in the food and cosmetics industries. In the present work, the effects of different low-cost raw materials on fermentation and canthaxanthin accumulation by a hyperpigmented strain of G. jacobaea were studied. Canthaxanthin production and peak levels of accumulation varied according to the different media used.     

The major carotenoid pigment of a psychrotrophic Micrococcus roseus strain: purification, structure, and interaction with synthetic membranes.

The major carotenoid pigment of a psychrotrophic Micrococcus roseus strain was purified to homogeneity from methanol extracts of dried cells by reverse-phase liquid chromatography and was designated P-3. On the basis of the UV-visible, infrared, mass, and 1H nuclear magnetic resonance spectra of P-3, it was identified as bisdehydro-beta-carotene-2-carboxylic acid. The pigment interacted with synthetic membranes of phosphatidylcholine and dimyristoyl phosphatidylcholine and stabilized the membranes. These results also indicate that P-3 is different from canthaxanthin, the major carotenoid pigment from a mesophilic M. roseus strain.     

Carotenoids in bird plumage: the complement of red pigments in the plumage of wild and captive bullfinch (Pyrrhula pyrrhula)

We have studied the carotenoid pigments in the red plumage of male bullfinch (Pyrrhula pyrrhula) immediately following capture and after the completion of the moult in captivity under dietary control. Astaxanthin, adonirubin, and alpha-doradexanthin, as well as papilioeritrinone and canthaxanthin (in lower amounts) are in every case the dominant carotenoids in the plumage pigment of wild individuals. alpha-Doradexanthin is responsible for the reddish-rose colour, which captive individuals adopt after a diet consisting mainly of lutein as disposable carotenoid. The red pigmentation biogenesis of captive bullfinch is compared with those of other red pigmented Carduelinae in which male individuals usually lose the red colour in captivity, namely Carpodacus roseus, Carpodacus rubricilloides, Uragus sibiricus, Carduelis cannabina, Carduelis flammea, Loxia curvirostra and Pinicola enucleator.     

Nature and Distribution of Carotenoid Pigments in Astasia ocellata

SYNOPSIS. The pigments synthesized by Astasia ocellata include α- and ε-carotene, 4-keto-β-carotene (echinenone), and 4,4'-diketo-β-carotene (canthaxanthin); 4-keto-α-carotene, accounting for about half the pigment in the cells, was tentatively identified; a strongly adsorbed keto-carotenoid, accounting for 25% of the pigments and bearing some similarities to astacin, polytomaxanthin and phoenicoxanthin, was also found.     

玫瑰色微球菌A-04的红色素鉴定及稳定性分析

从石油污染的土壤和水样中筛选出一株玫瑰色微球菌A-04 Micrococcus roseus,对其所产红色色素进行了分离,并初步鉴定了色素种类,基于对影响A-04色素稳定性的单因素分析基础之上,采用3因素3水平响应面分析法,进一步对影响色素稳定性的主要因素进行了优化分析。结果表明A-04所产红色色素为类胡萝卜素,对该菌株的色素稳定性的单因素条件分析,色素对环境条件的耐受性较好,在80℃、pH5. 0～8. 0等条件下依然能长时间保持鲜红而不褪色。经响应面优化分析表明:温度、pH和溶剂是影响该色素稳定性的主要因素,温度与溶剂的交互作用对色素稳定性的影响也较为明显。pH5. 0～8. 0之间时,在80℃范围内,温度越低,同时溶剂的极性越大,越有利于维持色素的稳定性。本研究结果为该色素的实际开发和应用奠定了基础。     

Exceptional molecular organization of canthaxanthin in lipid membranes

Canthaxanthin (β,β-carotene 4,4' dione) used widely as a drug or as a food and cosmetic colorant may have some undesirable effects on human health, caused mainly by the formation of crystals in the macula lutea membranes of the retina of an eye. Experiments show the exceptional molecular organization of canthaxanthin and a strong effect of this pigment on the physical properties of lipid membranes. The most striking difference between canthaxanthin and other macular pigments is that the effects of canthaxanthin at a molecular level are observed at much lower concentration of this pigment with respect to lipid (as low as 0.05 mol%). An analysis of the molecular interactions of canthaxanthin showed molecular mechanisms such as: strong van der Waals interactions between the canthaxanthin molecule and the acyl chains of lipids, restrictions to the segmental molecular motion of lipid molecules, modifications of the surface of the lipid membranes, effect on the membrane thermotropic properties and finally interactions based on the formation of the hydrogen bonds. Such interactions can lead to a destabilization of the membrane and loss of membrane compactness. In the case of the retinal vasculature, it can lead to an increase in the permeability of the retinal capillary walls and the development of retinopathy.     

Canthaxanthin pigment does not maintain color in carmine bee-eaters

Carmine bee-eaters (Merops nubicus) in captivity lost feather color when fed diets supplemented with canthaxanthin (23 mg/kg dry matter), a pigment and concentration known to maintain adequate color in numerous other bird species. Supplementation of whole insects with natural mixed carotenoids including α- and β-carotene, zeaxanthin, cryptoxanthin, and lutein resulted in a quantifiable change in feather color in this species. Positive identification of feather pigments and elucidation of metabolic pathways of color production in bee-eaters remain to be completed; initial data suggest α-carotene or derivatives as primary pigments or precursors in this species. © 1996 Wiley-Liss, Inc.     

Bilirubin conjugates of human bile. Isolation of phenylazo derivatives of bile bilirubin

A method is presented that allows the isolation of eight different phenylazo derivatives of bile bilirubin. In step I of the isolation procedure, three bilirubin fractions (bilirubin fractions 1, 2 and 3) from human hepatic bile are separated by reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from butan-1-ol and 5mm-phosphate buffer, pH6.0. Azo coupling is then performed with diazotized aniline. The three azo pigment mixtures are subjected to step II, in which the above chromatography system is used again. With each azo pigment mixture this step brings about the separation of a non-polar and a polar azo pigment fraction (azo 1A and azo 1B, azo 2A and azo 2B, and azo 3A and azo 3B from bilirubin fractions 1, 2 and 3 respectively). Approximately equal amounts of non-polar and polar pigments are obtained from bilirubin fractions 1 and 2, whereas bilirubin fraction 3 yields azo 3B almost exclusively. In step IIIA the non-polar azo pigment fractions are fractionated further by adsorption chromatography on anhydrous sodium sulphate with the use of chloroform followed by a gradient of ethyl acetate in chloroform. Three azo pigments are thus obtained from both azo 2A (azo 2A(1), azo 2A(2) and azo 2A(3)) and azo 3A (azo 3A(1), azo 3A(2) and azo 3A(3)). The 2A pigments occur in approximately the following proportions: azo 2A(1), 90%; azo 2A(2), 10%; azo 2A(3), traces. The pigments are purified by crystallization, except for the A(3) pigments, which are probably degradation products arising from the corresponding A(2) pigments. In step IIIB the polar azo pigment fractions are subjected to reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from octan-1-ol-di-isopropyl ether-ethyl acetate-methanol-0.2m-acetic acid (1:2:2:3:4, by vol.). Azo pigment fractions 2B and 3B each yield six azo pigments (azo 2B(1) to azo 2B(6) and azo 3B(1) to azo 3B(6) respectively) together with small amounts of products of hydrolysis (azo 2A(B) and azo 3A(B)). Only one azo B pigment is obtained from bilirubin fraction 1, and this azo pigment is probably of the B(2) type. The yields of the azo 3B pigments suggest that these pigments are present in approximately the following proportions: azo 3B(1), 0-0.4%; azo 3B(2), traces; azo 3B(3), traces; azo 3B(4), 10%; azo 3B(5), 50%; azo 3B(6), 40%. Azo pigments 2B(1) to 2B(6) are estimated to occur in similar proportions. Since pairs of correspondingly numbered azo pigments from bilirubin fractions 1, 2 and 3 do not separate on rechromatography together (e.g. azo 2A(1) co-chromatographs with azo 3A(1), and azo 2B(6) co-chromatographs with azo 3B(6)), it is concluded that such pigments are chemically identical. The structures of the isolated phenylazo derivatives are discussed in an accompanying paper (Kuenzle 1970c).     

Isolation and Identification of Echinenone from Micrococcus roseus

An orange carotenoid from Micrococcus roseus was purified by solvent partitioning followed by column and thin-layer chromatography. Absorption spectra, chromatographic mobility, and partition coefficient suggested that the pigment was echinenone (4-keto-beta-carotene). Reduction yielded a pigment with the spectral and polar properties of isocryptoxanthin (4-hydroxy-beta-carotene), the expected product. The orange pigment and its reduction product co-chromatographed with the respective authentic pigments, confirming the original pigment as echinenone. To our knowledge echinenone has not been identified previously as a bacterial pigment.     

A reduction in temperature induces bioactive red pigment production in a psychrotolerant Penicillium sp. GEU_37 isolated from Himalayan soil

Filamentous fungi are being globally explored for the production of industrially important bioactive compounds including pigments. In the present study, a cold and pH tolerant fungus strain Penicillium sp (GEU_37), isolated from the soil of Indian Himalaya, is characterized for the production of natural pigments as influenced by varying temperature conditions. The fungal strain produces a higher sporulation, exudation, and red diffusible pigment in Potato Dextrose (PD) at 15 °C as compared to 25 °C. In PD broth, a yellow pigment was observed at 25 °C. While measuring the effect of temperature and pH on red pigment production by GEU_37, 15 °C and pH 5, respectively, were observed to be the optimum conditions. Similarly, the effect of exogenous carbon and nitrogen sources and mineral salts on pigment production by GEU_37 was assessed in PD broth. However, no significant enhancement in pigmentation was observed. Chloroform extracted pigment was separated using thin layer chromatography (TLC) and column chromatography. The two separated fractions i.e., fractions I and II with Rf values 0.82 and 0.73, exhibited maximum light absorption, λ_max, at 360 nm and 510 nm, respectively. Characterization of pigments using GC–MS showed the presence of the compounds such as phenol, 2,4-bis (1,1-dimethylethyl) and eicosene from fraction I and derivatives of coumarine, friedooleanan, and stigmasterole in fraction II. However, LC-MS analysis detected the presence of derivatives of compound carotenoids from fraction II as well as derivative of chromenone and hydroxyquinoline as major compounds from both the fractions along with other numerous important bioactive compounds. The production of such bioactive pigments under low temperature conditions suggest their strategic role in ecological resilience by the fungal strain and may have biotechnological applications.     

Isolation and identification of carotenoids produced by a green alga (Dictyococcus cinnabarinus) in submerged culture

1. Six carotenoid pigments were produced by and isolated from the green alga Dictyococcus cinnabarinus grown in submerged culture in the presence of glucose. 2. The first, second and fourth pigments were identified respectively as β-carotene, echinenone and canthaxanthin; the physicochemical properties of the other three are described and their similarity to other oxo-carotenoids is shown. 3. Culture techniques, isolation and identification procedures are described.     

Grazing experiments with two freshwater zooplankters:fate of chlorophyll and carotenoid pigments

In order to evaluate the validity of the gut pigment methodto assess grazing and diet in two freshwater zooplankters, experimentswere carried out to check chlorophyll a and xanthophyll conservationduring feeding. For both animals, two sets of experiments wereconducted by incubating animals in the laboratory, either isolatedfrom a reservoir (the calanoid copepod, Eudiaptomus gracilis)or cultured under high-food conditions (the cladoceran, Daphniagaleata). For both animals, gut pigments and clearance rateson different types of algae were determined from the same incubations.Chlorophyll a and derivatives, as well as major algal carotenoids,were analysed by High Performance Liquid Chromatography (HPLC).In copepods, the pigment profiles from the gut extracts reflectedthe diet of the animals poorly. The animal extracts containedalmost exclusively alloxanthin (or an alloxanthin-like pigment)in large amounts, whereas the other pigments were lost in highproportions (>70% for lutein and fucoxanthin; 57 and 78%for a-phorbins). The cladocerans fed on the main types of algaeabundant in the suspensions, with a preference, however, forsmall cells. Although the main xanthophylls from these algaewere detected in the Daphnia extracts, some destruction of luteinand fucoxanthin may have occurred (18.7 and 30%). The loss ratefor alloxanthin seemed more variable (0 and 68%), possibly dependingon food concentration. As for the transformation of a-phorbins,E.gracilis and D.galeata behaved quite differently. The HPLCprofiles of copepod extracts always showed a very small chlorophylla peak, along with phaeophytin a and pyrophaeophytin a. Thosefrom the cladoceran exhibited a large phaeophorbide a peak,along with some chlorophyll a and phaeophytin a. In fact, D.galeatadid not destroy a-phorbins under our experimental conditionsbut converted chlorophyll a mainly into phaeophorbide. Froma comparison of our results with data from other studies, itseems that in these two zooplankters, use of gut pigment datafor quantitative grazing assessment should be considered withcaution.     

The nature of the lamprey visual pigment

From the retina of the land-locked population of the sea lamprey, Petromyzon marinus, a photolabile pigment was extracted which was identified spectrophotometrically as a member of the rhodopsin group of pigments. Using the absorption spectrum of a relatively pure solution and analysis by means of difference spectra, the peak of this pigment was placed at about 497 mµ. The method of selective bleaching by light of different wave lengths revealed no significant amounts of any other pigment in the extracts. A similar pigment was also detected in retinal extracts of the Pacific Coast lamprey, Entospenus tridentatus. These results are significant for two reasons: (a) the lamprey is shown to be an example of an animal which spawns in fresh water but which is characterized by the presence of rhodopsin, rather than porphyropsin, in the retina; (b) the primitive phylogenetic position of the lamprey suggests that rhodopsin was the visual pigment of the original vertebrates.     

Pigments of Staphylococcus aureus, a series of triterpenoid carotenoids.

The pigments of Staphylococcus aureus were isolated and purified, and their chemical structures were determined. All of the 17 compounds identified were triterpenoid carotenoids possessing a C30 chain instead of the C40 carotenoid structure found in most other organisms. The main pigment, staphyloxanthin, was shown to be alpha-D-glucopyranosyl 1-O-(4,4'-diaponeurosporen-4-oate) 6-O-(12-methyltetradecanoate), in which glucose is esterified with both a triterpenoid carotenoid carboxylic acid and a C15 fatty acid. It is accompanied by isomers containing other hexoses and homologs containing C17 fatty acids. The carotenes 4,4'-diapophytoene, 4,4'-diapophytofluene, 4-4'-diapophytofluene, 4-4'-diapo-zeta-carotene, 4,4'-diapo-7,8,11,12-tetrahydrolycopene, and 4,4'-diaponeurosporene and the xanthophylls 4,4'-diaponeurosporenal, 4,4'-diaponeurosporenoic acid, and glucosyl diaponeurosporenoate were also identified, together with some of their isomers or breakdown products. The symmetrical 4,4'-diapo- structure was adopted for these triterpenoid carotenoids, but an alternative unsymmetrical 8'-apo-structure could not be excluded.     

Biosynthesis of Carotenoids in Brevibacterium sp. KY-4313

The biosynthesis of 4-keto and 4,4′-diketo carotenoids in Brevibacterium sp. KY-4313 was studied. Echinenone and canthaxanthin were isolated from the cultures grown on a medium containing several n-alkanes. When glutathione was added to the bacterial cultures, the formation of canthaxanthin was inhibited while β-carotene and its hydroxy derivatives accumulated. It is suggested that these 4-hydroxy compounds, isocryptoxanthin, isozeaxanthin, and 4-hydroxy-4′-keto-β-carotene, are intermediates in the biosynthesis of canthaxanthin. In the presence of 2-(4-chlorophenylthio)-triethylamine hydrochloride or nicotine, lycopene and neurosporene accumulated. The β-carotene level decreased slightly but β-zeacarotene remained unchanged. β-carotene and its derivatives were resynthesized upon removal of the inhibitors. It was concluded that cyclization can take place at either the neurosporene or lycopene level in Brevibacterium sp. KY-4313.     

Reinvestigation of Brevibacterium sp. Strain KY-4313 as a Source of Canthaxanthin

The hydrocarbon-utilizing Brevibacterium sp. strain KY-4313 was reevaluated for its potential to produce canthaxanthin, a carotenoid pigment of strong commercial interest. Three approaches were used to optimize the canthaxanthin yield from this organism, i.e., the preparation of mutants, the addition of supposedly carotenogenic chemicals to the growth medium, and growth promotion. Following treatment of the parent strain with N-nitrosomethylurea, a presumed mutant was isolated which showed a 32% increase in cellular canthaxanthin content. No effective carotenogenic chemicals were found in connection with hydrocarbon fermentations, in which mainly growth promotion through periodic medium renewal proved conducive to enhanced pigment production. Carotenogenesis could be stimulated in brain heart infusion broth by adding alcohols or retinol. Improved growth in this medium was generally not associated with higher canthaxanthin yields. Both superior growth and pigment levels were obtained in a newly designed medium based on fumaric acid-molasses. The maximum yields of canthaxanthin in shake flasks were (in milligrams per liter) 4.2 (brain heart infusion broth plus propanol-zinc sulfate), 3.6 (hydrocarbon medium), and 9.3 (fumaric acid-molasses), which represent a significant improvement over the originally reported optimal result (1 mg/liter). The corresponding yields of echinenone, the direct precursor of canthaxanthin, were 1.2, 1.6, and 2.3 mg/liter, respectively. Two-liter hydrocarbon batch fermentations involving medium renewal maximally produced 7.2 mg of canthaxanthin and 3.7 mg of echinenone per liter.     

Effect of pH and nitrogen source on pigment production by Monascus purpureus

The effect of pH and nitrogen source on pigment production by Monascus purpureus 192F using glucose as the carbon and energy source, was studied in pH-controlled, batch fermentor cultures using HPLC analysis to determine individual pigment concentrations. A maximum of four pigments were detected in fungal extracts. These were the yellow pigments monascin and ankaflavin, the orange rubropunctatin and the red pigment monascorubramine. Monascorubramine was present as the major product in all instances. Fungal growth and ankaflavin synthesis were favoured at low pH (pH 4.0), whereas production of the other pigments was relatively independent of pH. The nature of the nitrogen source affected fungal growth and pigment production, independent of pH. Ammonium and peptone as nitrogen sources gave superior growth and pigment concentrations compared to nitrate. Ankaflavin was not detected in nitrate cultures. The highest red pigment production was obtained using a glucose-peptone medium at pH 6.5, due to the secretion of red pigments into the medium under these conditions. Correspondence to: M. R. Johns     

Chlorophylle und Carotinoide der Chaetophorineae (Chlorophyceae; Ulotrichales)

In air-supplied inorganic liquid cultures, the highly differentiated green alga Fritschiella tuberosa forms only branched filaments with long slender cells. In nitrogen-deficient medium and with ageing of the cultures these cells become much shorter in length by subsequent formation of transverse walls. The chloroplasts of the slender cells contain the typical pigments of green algae. Together with the morphogenetic change to “short cells” secondary carotenoids are synthesized and stored in lipid droplets. Besides traces of lutein, violaxanthin and neoxanthin and reduced amounts of β-carotene, the following pigments have been demonstrated: Esters of astaxanthin (main pigment) and adonixanthin, canthaxanthin, echinenone and a Keto-α-carotinoid, which presumably is identical with α-doradexanthin, (3,3′-Dihydroxy-4-keto-α-Carotene) a pigment not known in plants until now. In nitrogen deficient cultures the chlorophylls are totally decomposed, the total-lipids increase by about 500%. By supplying nitrogen-deficient cultures with nitrogen and subsequent illumination regreening of the cells starts already 16 hrs later.     

Primary and Secondary Carotenoids of Spongiochloris typica

A qualitative and quantitative investigation was made of the pigments of Spongiochloris typica over an 8 week period. The pigments were chromatographed on thin layers of sucrose and measured spectrophotometrically. Pigments present after 1 week of growth were identified as chlorophylls a and b, β-carotene, lutein, zeaxanthin, violaxanthin, trollein, and neoxanthin. In cultures 2 weeks or more old, secondary carotenoids appeared. These were echinenone, canthaxanthin, astacene, and an unidentified ketocarotenoid. Carotenoids comprised nearly 100 percent of the total pigment composition on the 8th week. About 75 percent of the carotenoid fraction on the 8th week consisted of secondary carotenoids.