Identification of major carotenoids from green alga Tetraspora sp. CU2551: partial purification and characterization of lutein, canthaxanthin, neochrome, and ß-carotene.

The green algae Tetraspora sp. CU2551 was previously identified as a strain with high potential for biohydrogen production; however, its algal biomass characteristics changed from green to reddish orange within 43 days of biohydrogen production. The crude pigments were extracted, partially purified, and characterized by chemical determination. The present study focused on elucidating the carotenoid composition of the selected green alga Tetraspora sp. CU2551. The pigment extract was partially purified and fractionated using thin layer chromatography, and yielded two major and two minor carotenoid bands. The fractions were confirmed by high-performance liquid chromatography with a diode array detector (HPLC-DAD) before being identified and confirmed using Liquid Chromatograph-Quadrupole Time of Flight-Mass Spectrometry (LC-QTOF-MS). The spectral data of these fractions revealed four sub-fractions of interest that were lutein, canthaxanthin, neochrome, and ß-carotene, which had percentages in the crude extracts of 30.57%, 25.47%, 7.89%, and 0.71%, respectively. Lutein and canthaxanthin were found to be the major carotenoid pigments present. Our findings in this present study are the first reporting of Tetraspora sp. CU2551 as a potential alternate source for carotenoid pigment production.

Identifying Codium fragile extract components and their effects on muscle weight and exercise endurance.

Codium fragile (CF) is a type of green algae consumed as kimchi in Asia. UPLC-QTOF-MS/MS analysis showed that CF contain lysophosphatidyl choline, canthaxanthin, retinoic acid, α-tocopherol, and unsaturated fatty acids, which reportedly improve skeletal muscle health. However, the effect of CF on skeletal muscle mass and function remains to be elucidated. In mice fed with CF extracts, exercise endurance and muscle weight increased. CF extracts enhanced protein synthesis and myogenic differentiation through the mTORC1 pathway. CF extracts also promoted oxidative muscle fiber formation and mitochondrial biogenesis through the PGC-1α-related signaling pathway. Upregulation of PGC-1α by CF extracts was abolished by EX527 SIRT1 inhibitor treatment. Changed signaling molecules in the CF extracts were partially regulated by canthaxanthin, a new compound in CF extracts, suggesting that canthaxanthin contribute synergistically to the effect of CF extracts. Therefore, CF is a potential food source for sport nutrition or prevention of sarcopenia.

Identification of ß-carotene oxidation products produced by bleaching clay using UPLC-ESI-MS/MS.

The removal of plant pigments such as ß-carotene is an aspect of vegetable oil processing often desired by the food and pharmaceutical industries. Adsorption of ß-carotene to acid-activated clay (AAC) is a well-established method for purification. Despite this, the removal mechanism of ß-carotene is not well understood. UPLC-MS/MS analysis of surface compounds extracted from ß-carotene-AAC (BC-AAC) complexes show that AAC acts as an oxidiser. Oxidation products detected included canthaxanthin and 3',4'-didehydro-ß-caroten-4-one. AAC had surface water exchanged with an 18O labelled water and was then exposed to ß-carotene. Carotenoids labelled with 18O were produced from this reaction, suggesting surface water is necessary for ß-carotene removal.

Evaluation of canthaxanthin in eggs and its subsequent dietary risks to Chinese consumers.

Canthaxanthin is a colourant widely used in medicine, food and cosmetics, which is closely related to human eye dysfunction. The EU determines that the daily acceptable intake of canthaxanthin (ADI) is 0.03 mg canthaxanthin per kg body weight. In order to protect people's diet health and avoid the occurrence of food safety accidents, this paper measured the content of canthaxanthin in 114 egg samples sourced from different regions of China using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The dietary exposure assessment method was used to evaluate the dietary risk to Chinese consumers. Results showed that the content of canthaxanthin varies significantly across regions of origin, with eggs produced in Beijing having a lower amount than those from other regions. The average dietary exposure of children (2-10 years old) was 0.242 ~ 0.653 µg/(kg bw∙d), and the dietary exposure of 95/97.5/99 percentile was 0.246 ~ 1.779 µg/(kg bw∙d). The maximum exposure was 3.089 µg/(kg bw∙d), and its risk quotient was 0.103 (<1), which was an acceptable risk. For the teenager group (11-18 years old), the average dietary exposure was 0.141 ~ 0.247 µg/(kg bw∙d), and the dietary exposure at the 95/97.5/99 percentile was 0.143 ~ 0.671 µg/(kg bw∙d), presenting an acceptable risk. For adults, the dietary risk of eating eggs with canthaxanthin is the lowest among the three age groups.

Pigment-depletion in Atlantic salmon (Salmo salar) post-smolt starved at elevated temperature is not influenced by dietary carotenoid type and increasing α-tocopherol level.

Pigment-depletion in the fillets of farmed Atlantic salmon (Salmo salar) arises after periods of elevated water temperatures with voluntary starving. This study tested the effects of dietary pre-loading with different pigment carotenoids (astaxanthin and/or canthaxanthin) combined with two α-tocopherol levels (normal and high: 500 and 1000 mg/kg, respectively) on pigment-depletion in vivo in Atlantic salmon after four weeks of challenge. We also tested whether oxidative stress manifested as an underlying depletion mechanism. Carotenoid levels in whole fillet homogenates were not decreased significantly post-challenge but fillet α-tocopherol concentrations were increased significantly in contrast to decreased oxidative stress indices. However, image analysis revealed localised fillet pigment-depletion following all dietary treatments. These data imply that localised pigment-depletion was not prevented by pre-loading of the fillet with different carotenoid-types/mixtures and increased of α-tocopherol levels from normal to high, respectively. Further, we suggest that oxidative stress might not facilitate pigment-depletion in vivo.

Determination of Tartrazine, Lutein, Capsanthin, Canthaxanthin and ß-Carotene in Animal-Derived Foods and Feeds by HPLC Method.

Pigments are still widely used in food and feed industry and their resides in food might be harmful to human health due to their side effects. A high performance liquid chromatography (HPLC) method for simultaneous determination of pigments including tartrazine, lutein, capsanthin, canthaxanthin and ß-carotene in animal-derived foods (including the muscle and liver of swine, the muscle, liver and skin of chicken and duck, and the muscle of fish) and feeds (swine, chicken and duck) was developed. Lutein, capsanthin, canthaxanthin and ß-carotene were extracted with acetonitrile-ethyl acetate by ultrasonication, and tartrazine was extracted with water, followed by defatting with n-hexane and clean-up by solid phase extraction on weak anion exchange cartridges. The quantitation of the five pigments was performed by HPLC with ultraviolet and visible spectrophotometer detection. Chromatographic separations were performed on a C8 column with gradient elution. The mean recoveries of analytes ranged from 80.4 to 92.5%. The intra- and the inter-day variabilities were below 15.0%. This HPLC method was suitable for the routine determination of pigment residues in animal-derived foods and feeds.

Effects of Potassium Chloride-Induced Stress on the Carotenoids Canthaxanthin, Astaxanthin, and Lipid Accumulations in the Green Chlorococcal Microalga Strain TISTR 9500.

Microalgae are a diverse group of photosynthetic eukaryotic organisms that are widely distributed globally. They are prolific sources of highly valuable compounds with fascinating chemical structures. Due to their balanced nutritional compositions and health benefits, they are increasingly being used as functional food ingredients. Carotenoid-based pigments and polyunsaturated fatty acids (PUFAs) are examples of high-value nutrients that can be accumulated abundantly in microalgae. Here, the effects of potassium chloride-induced stress on the productions of lipids and carotenoids in the green microalga of the Chlorococcaceae family were investigated. Under normal BG11 medium, this green microalga strain TISTR 9500 accumulated high levels of PUFA and primary carotenoid lutein. Stress tests revealed that KCl enhanced and modulated lipid and carotenoid accumulation levels. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that secondary carotenoids astaxanthin and canthaxanthin were robustly produced under KCl stress with the similar content of lutein. Further, this stress led to a significant increase in the total FA amount with the higher proportion of unsaturated FA than saturated FA. Thus, this green microalga could be an attractive and alternative natural biosource for canthaxanthin and astaxanthin, as well as for functional lipids.

HPLC Quantification of astaxanthin and canthaxanthin in Salmonidae eggs.

Astaxanthin and canthaxanthin are naturally occurring antioxidants referred to as xanthophylls. They are used as food additives in fish farms to improve the organoleptic qualities of salmonid products and to prevent reproductive diseases. This study reports the development and single-laboratory validation of a rapid method for quantification of astaxanthin and canthaxanthin in eggs of rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis М.). An advantage of the proposed method is the perfect combination of selective extraction of the xanthophylls and analysis of the extract by high-performance liquid chromatography and photodiode array detection. The method validation was carried out in terms of linearity, accuracy, precision, recovery and limits of detection and quantification. The method was applied for simultaneous quantification of the two xanthophylls in eggs of rainbow trout and brook trout after their selective extraction. The results show that astaxanthin accumulations in salmonid fish eggs are larger than those of canthaxanthin. As the levels of these two xanthophylls affect fish fertility, this method can be used to improve the nutritional quality and to minimize the occurrence of the M74 syndrome in fish populations.

Determination of Other Related Carotenoids Substances in Astaxanthin Crystals Extracted from Adonis amurensis.

Astaxanthin is a kind of important carotenoids with powerful antioxidation capacity and other health functions. Extracting from Adonis amurensis is a promising way to obtain natural astaxanthin. However, how to ensure the high purity and to investigate related substances in astaxanthin crystals are necessary issues. In this study, to identify possible impurities, astaxanthin crystal was first extracted from Adonis amurensis, then purified by saponification and separation. The concentration of total carotenoids in purified astaxanthin crystals was as high as 97% by weight when analyzed by UV-visible absorption spectra. After identified with TLC, HPLC and MS, besides free astaxanthin as main ingredient in the crystals, there existed four other unknown related substances, which were further investigated by HPLC/ESI/MS with the positive ion mode combining with other auxiliary reference data obtained in stress tests, at last it was confirmed that four related carotenoids substances were three structural isomers of semi-astacene and adonirubin.

[Rapid screening and confirmation of non-target pigment in Chinese softshell turtle by liquid chromatography coupled to time of flight mass spectrometry].

A method of non-target pigment screening in Chinese softshell turtle has been established by using liquid chromatography coupled to time of flight mass spectrometry (LC-Q-TOF MS). After being purified by a simple acetonitrile extraction work, the non-target pigment in 20 Chinese softshell turtle samples was detected by liquid chromatography-photodiode array detection (LC-DAD). The S7 sample, which has a strong spectral response, was chosen to extract the mass spectrometry information of the non-target pigment on different gradient elution conditions. In order to get the characteristic molecular mass ion (564.397 73 Da and 564.395 61 Da) of the non-target pigment, qualitative spectral full scan with negative sample was used. The molecular formula generation data and the literature information prompted speculation that the non-target pigment was canthaxanthin with the formula of C40H52O2. By comparing the canthaxanthin standard material MS/MS information, the result was confirmed accurate. A strategy of LC-Q-TOF MS method for the qualitative analysis of unknown compounds is discussed, and the results indicated that the described method can be effectively applied to qualitative analysis for non-target pigment in Chinese softshell turtle.

Astaxanthin and canthaxanthin (xanthophyll) as supplements in rainbow trout diet: in vivo assessment of residual levels and contributions to human health.

Many studies have demonstrated that xanthophylls, such as astaxanthin, have beneficial effects in human health, and their use in food supplements is thus encouraged. Moreover, such nutrients are frequently used in aquaculture to meet consumer demand for salmonoid flesh pigmentation. In this study different xanthophyll administration protocols were tested to verify pigmentation properties and safety of such mixtures of additives in trout diet. Residues of xanthophylls in muscle samples were determined by HPLC-MS/MS, reaching levels of 3.70 ± 0.04 mg/kg (astaxanthin) and 1.21 ± 0.06 mg/kg (canthaxanthin) during a 56 day administration period. On the basis of the average fish consumption in the human diet, the highest astaxanthin and canthaxanthin concentrations detected in trout fillets could result in weekly intakes of 1.63 and 0.53 mg, respectively, in humans; these values are not sufficient to achieve the positive effects described by many authors, but their residues could still represent an important source of carotenoids, alternative to the use of synthetic dietary supplements.

Efeito do armazenamento e da cantaxantina dietética sobre a qualidade do ovo fértil e o desenvolvimento embrionário / The effect of storage and dietary canthaxanthin on fertile egg quality and embryonic development

O presente experimento foi conduzido em lote de matrizes Cobb desde 46 até 60 semanas de idade. Os tratamentos foram definidos pelas dietas (com e sem adição de 6ppm de cantaxantina na dieta das matrizes) e pelos períodos de armazenamento dos ovos (três e sete dias), em delineamento inteiramente ao acaso, em arranjo fatorial 2x2 (duas dietas x dois períodos de armazenamento). A cantaxantina dietética elevou o escore de cor da gema de sete para 14. O armazenamento dos ovos por sete dias prejudicou a qualidade dos ovos, promoveu oxidação de ácidos graxos da gema e retardou o desenvolvimento embrionário.

This trial was done with Cobb broiler breeders. Treatments were defined by the diets (with and without 6ppm canthaxanthin added to broiler breeder diets) and periods of egg storage (three and seven days) in a completely randomized factorial 2 x 2 design (two diets x two periods of egg storage). The dietary canthaxanthin increased the yolk color score from 7 to 14. Egg storage for seven days reduced egg quality, promoted oxidation of yolk fatty acids and delayed embryonic development.

Effect of domestic cooking methods on egg yolk xanthophylls.

Xanthophylls are a class of bioactive compounds known to play an important role in preventing age-related macular degeneration. Egg yolk is a rich source of highly bioavailable xanthophylls including lutein and zeaxanthin. The effects of domestic cooking methods (boiling, frying, microwaving) on egg yolk xanthophyll content were investigated. A LC-(APCI)-MS/MS method was used to identify and quantify all-E- and Z-isomers of lutein, zeaxanthin, canthaxanthin, and ß-apo-8'-carotenoic acid ethyl ester in fresh and cooked egg yolks. Both fresh and cooked yolks showed similar xanthophyll profiles but with higher contents of Z-isomers in cooked samples. All-E-lutein was the most affected, with 22.5%, 16.7%, and 19.3% reductions in boiled, microwaved, and fried yolk extracts, respectively. Total xanthophyll losses ranged from 6% to 18%. The results presented here could be useful in calculating the dietary intake of xanthophylls and also in assessing the xanthophyll profiles and contents of egg-containing products.

Determination of supplemental feeding needs for astaxanthin and canthaxanthin in salmonids by supramolecular solvent-based microextraction and liquid chromatography-UV/VIS spectroscopy.

Development of simple and rapid analytical methods for predicting supplemental feeding requirements in aquaculture is a need to reduce production costs. In this article, a supramolecular solvent (SUPRAS) made up of decanoic acid (DeA) assemblies was proposed to simplify sample treatment in the total and individual determination of carotenoids (red-pink pigments) in farmed salmonids. The analytes were quantitatively extracted in a single step that spends a few minutes using a small volume of SUPRAS (i.e. 800 µL) and directly determined in extracts without the interference from fats or other matrix components. The methods based on the combination of microextraction with SUPRAS and photometry or HPLC-UV/VIS spectroscopy were developed for the determination of total and individual carotenoids, respectively. The applicability of the methods was demonstrated by analysing non-fortified and fortified samples of farmed Atlantic salmons and rainbow trouts. Recoveries obtained by photometry and HPLC-UV/VIS spectroscopy were within the intervals 98-104% and 94-106%, respectively.

Analysis of canthaxanthin production by Gordonia jacobaea.

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., ß-carotene, astaxanthin, and canthaxanthin) have been the focus of much attention. The bacterium Gordonia jacobaea readily synthesizes and accumulates large amounts of canthaxanthin (ß-ß'-carotene-4,4'-dione), which is widely used in the food and cosmetics industries. In the present work, the integral process of canthaxanthin production by G. jacobaea is analyzed together with its application as natural sources for the industry. A great influence of culture media is observed on canthaxanthin levels. Also, the ability is found of extract the pigments with ethanol from bacteria. The concentration of the samples is a crucial point of the process, being mandatory to discard any process of heating the samples, because this provoked the pigment degradation. Despite this, the described method allows to consider G. jacobaea as a potential canthaxanthin producer for the industry.

[Determination of canthaxanthin and astaxanthin in egg yolks by reversed phase high performance liquid chromatography with diode array detection].

OBJECTIVE: A method using reversed phase high performance liquid chromatography (RP-HPLC) coupled with diode array detector (DAD) was developed for the simultaneous determination of canthaxanthin and astaxanthin in egg yolks. METHODS: Samples were extracted with acetonitrile in ultrasonic bath for 20 minutes and then purified by freezing-lipid filtration and solid phase extraction (SPE). After being vaporized to dryness by nitrogen blowing and made up to volume with methanol, the extract solution was chromatographically separated in C18 column with a unitary mobile phase consisting of acetonitrile. The proposed method was validated in terms of linearity, precision, accuracy, and limit of detection (LOD). RESULT: Regression analysis revealed a good linearity between peak area of each analyte and its concentration (r > or = 0.998). The intra- and inter-day relative standard deviations (RSDs) were less than 3.6% and 5.2%, respectively. LODs of canthaxanthin and astaxanthin were 0.035 and 0.027 microg/mL (S/N = 3). The average recoveries of canthaxanthin and astaxanthin were 91.5% and 88.7%. CONCLUSION: The proposed method is simple, fast and easy to apply.

Impact of dietary carotenoid and packaging during frozen storage on the quality of rainbow trout (Oncorhynchus mykiss) fed carotenoids.

BACKGROUND: The storage life of frozen salmonids is often limited primarily by oxidation and flesh discolouration due to carotenoid degradation. The objective of this research was to determine the carotenoid changes and therefore the muscle colour modifications during 6 months of frozen storage (-18 °C) of whole rainbow trout fed astaxanthin (100 ppm) or canthaxanthin (80 ppm), kept under two different packagings: plastic film and cardboard box. RESULTS: After 6 months of frozen storage, the carotenoid type effect was seen for dry matter while there was no packaging material effect on carotenoid, total lipids, and TBARS contents of trout fillets. The time under frozen storage had an effect on carotenoid and TBARS fillet concentration. The carotenoid-type effect was noted for the four colour parameters (lightness difference, chroma difference, hue angle difference, and total colour difference) of rainbow trout fillet, while the packaging material effect was observed only for chroma. Frozen storage time had an effect on the four colour parameters of rainbow trout fillet. CONCLUSION: Carotenoid and packaging material effects were more marked for colour parameters than for biochemical parameters. In this study, as fish were frozen and stored as whole fish, fish skin provided good protection against oxidation.

Determination of para red, Sudan dyes, canthaxanthin, and astaxanthin in animal feeds using UPLC.

A simple high-performance liquid chromatography method was developed for quantitative determination of para red, Sudan I, Sudan II, Sudan III, Sudan IV, canthaxanthin, and astaxanthin in feedstuff. The sample was extracted using acetonitrile and cleaned up on a C(18) SPE column. The residues were analyzed using ultra-performance liquid chromatography coupled to a diode array detector at 500 nm. The mobile phase was acetonitrile-formic acid-water with a gradient elution condition. The external standard curves were calibrated. The mean recoveries of the seven colorants were 62.7-91.0% with relative standard deviation 2.6-10.4% (intra-day) and 4.0-13.2% (inter-day). The detection limits were in the range of 0.006-0.02 mg/kg.

Unique carotenoids in the terrestrial cyanobacterium Nostoc commune NIES-24: 2-hydroxymyxol 2'-fucoside, nostoxanthin and canthaxanthin.

Cyanobacteria produce some carotenoids. We identified the molecular structures, including the stereochemistry, of all the carotenoids in the terrestrial cyanobacterium, Nostoc commune NIES-24 (IAM M-13). The major carotenoid was beta-carotene. Its hydroxyl derivatives were (3R)-beta-cryptoxanthin, (3R,3'R)-zeaxanthin, (2R,3R,3'R)-caloxanthin, and (2R,3R,2'R,3'R)-nostoxanthin, and its keto derivatives were echinenone and canthaxanthin. The unique myxol glycosides were (3R,2'S)-myxol 2'-fucoside and (2R,3R,2'S)-2-hydroxymyxol 2'-fucoside. This is only the second species found to contain 2-hydroxymyxol. We propose possible carotenogenesis pathways based on our identification of the carotenoids: the hydroxyl pathway produced nostoxanthin via zeaxanthin from beta-carotene, the keto pathway produced canthaxanthin from beta-carotene, and the myxol pathway produced 2-hydroxymyxol 2'-fucoside via myxol 2'-fucoside. This cyanobacterium was found to contain many kinds of carotenoids and also displayed many carotenogenesis pathways, while other cyanobacteria lack some carotenoids and a part of carotenogenesis pathways compared with this cyanobacterium.

[Simultaneous determination of canthaxanthin and astaxanthin in feedstuffs using solid phase extraction-reversed-phase high performance liquid chromatography].

A method was developed for the simultaneous determination of canthaxanthin and astaxanthin in feedstuffs using reversed-phase high performance liquid chromatography (RP-HPLC). The sample was extracted by acetonitrile, and cleaned up by an LC-NH2 column. An Agilent ZORBAX Eclipse XDB-C18 analytical column (150 mm x 4.6 mm, 5 microm) was used and kept at 25 degrees C. Acetonitrile-methanol (95 : 5, v/v) was used as the mobile phase at a flow rate of 1.0 mL/min. The detection was performed by a diode array detector at 474 nm. The quantitive analysis of external standard calibration curves was used. The linear ranges of the method for canthaxanthin and astaxanthin were 1.0 - 30.0 mg/L (r = 0.999 0) and 1.0 - 20.0 mg/L (r = 0.999 1), respectively. The average recoveries were 90% - 101% with the relative standard deviations of 0.62% - 3.68%. The detection limits were 0.84 and 0.60 mg/L for canthaxanthin and astaxanthin, respectively. The method is simple, precise, sensitive and reproductive. It can be used to determine the contents of canthaxanthin and astaxanthin in feedstuffs.