A microfluidic impedance cytometry device for robust identification of H. pluvialis.

H. pluvialis contains rich oleic acid and astaxanthin, which have important applications in the fields of biodiesel and biomedicine. Detection of live H. pluvialis is the prerequisite to obtaining oleic acid and astaxanthin. For this purpose, we successfully developed a reliable microfluidic impedance cytometry for the identification of live H. pluvialis. Firstly, we established a simulation model for detecting H. pluvialis based on their morphology and studied the effect of medium conductivity on the impedance of H. pluvialis at different frequencies. From the simulations, we determined that the optimal solution conductivity for the detection of H. pluvialis was 1500 µS cm-1 and studied the frequency responses of the impedance of H. pluvialis. Secondly, we fabricated the microchannels and stainless-steel detection electrodes and assembled them into microfluidic impedance cytometry. The frequency dependence of live and dead H. pluvialis was explored under different frequencies, and live and dead H. pluvialis were distinguished at a frequency of 1 MHz. The impedance of live H. pluvialis at the frequency of 1 MHz ranges from 33.73 to 52.23 Ω, while that of dead ones ranges from 13.05 to 19.59 Ω. Based on these findings, we accomplished the identification and counting of live H. pluvialis in the live and dead sample solutions. Furthermore, we accomplished the identification and counting of live H. pluvialis in the mixed samples containing Euglena and H. pluvialis. This approach possesses the promising capacity to serve as a robust tool in the identification of target microalgae, addressing a challenge in the fields of biodiesel and biomedicine.

Determination of xanthophylls in egg yolk using a combination of spectrophotometric and diol phase high-performance liquid chromatography methods.

Normal-phase (NP) liquid chromatography is one of the most effective methods for separating isomers with sensitive structural features, including xanthophyll isomers. In this work, reverse-phase (RP) and NP liquid chromatography (LC), with silica gel and diol phase, respectively, were evaluated for the separation of xanthophyll isomers. The results showed that RP LC with monomeric C18 phase not only poorly separate all xanthophyll isomers in egg yolk but also requires additional sample preparation to eliminate triacylglycerols in egg yolk. The diol phase of NP-LC provided the highest efficiency for separating lutein, zeaxanthin, and their cis-isomers with isocratic separation using mobile phases consisting of n-hexane and polar modifiers (such as acetone, methyl tert-butyl ether, or ethyl acetate). To determine the xanthophyll content, peak areas from LC and total absorbance from spectrophotometry measurements were used. The approach was applied to analyze the xanthophylls of nine commercial egg samples. The results revealed that five out of nine analyzed samples contained a high level of canthaxanthin, which contributes to color enhancement but not to prevent age-related macular degeneration. Together, it shows that NP LC with diol phase combined with spectrophotometry is a powerful tool to monitor xanthophylls in eggs.

Applications of high-intensity ultrasound on shrimp: Potential, constraints, and prospects in the extraction and retrieval of bioactive compounds, safety, and quality.

The global shrimp market holds substantial prominence within the food industry, registering a significant USD 24.7 billion in worldwide exportation in 2020. However, the production of a safe and high-quality product requires consideration of various factors, including the potential for allergenic reactions, occurrences of foodborne outbreaks, and risks of spoilage. Additionally, the exploration of the recovery of bioactive compounds (e.g., astaxanthin [AX], polyunsaturated fatty acids, and polysaccharides) from shrimp waste demands focused attention. Within this framework, this review seeks to comprehend and assess the utilization of high-intensity ultrasound (HIUS), both as a standalone method and combined with other technologies, within the shrimp industry. The objective is to evaluate its applications, limitations, and prospects, with a specific emphasis on delineating the impact of sonication parameters (e.g., power, time, and temperature) on various applications. This includes an examination of undesirable effects and identifying areas of interest for current and prospective research. HIUS has demonstrated promise in enhancing the extraction of bioactive compounds, such as AX, lipids, and chitin, while concurrently addressing concerns such as allergen reduction (e.g., tropomyosin), inactivation of pathogens (e.g., Vibrio parahaemolyticus), and quality improvement, manifesting in reduced melanosis scores and improved peelability. Nonetheless, potential impediments, particularly related to oxidation processes, especially those associated with lipids, pose a hindrance to its widespread implementation, potentially impacting texture properties. Consequently, further optimization studies remain imperative. Moreover, novel applications of sonication in shrimp processing, including brining, thawing, and drying, represent a promising avenue for expanding the utilization of HIUS in the shrimp industry.

Kinetic Extraction of Fucoxanthin from Undaria pinnatifida Using Ethanol as a Solvent.

Fucoxanthin (Fx) has been proven to exert numerous biological properties, which makes it an interesting molecule with diverse industrial applications. In this study, the kinetic behavior of Fx was studied to optimize three variables: time (t-3 min to 7 days), temperature (T-5 to 85 °C), and concentration of ethanol in water (S-50 to 100%, v/v), in order to obtain the best Fx yield from Undaria pinnatifida using conventional heat extraction. The Fx content (Y1) was found through HPLC-DAD and expressed in µg Fx/g of algae sample dry weight (AS dw). Furthermore, extraction yield (Y2) was also found through dry weight analysis and was expressed in mg extract (E)/g AS dw. The purity of the extracts (Y3) was found and expressed in mg Fx/g E dw. The optimal conditions selected for Y1 were T = 45 °C, S = 70%, and t = 66 min, obtaining ~5.24 mg Fx/g AS; for Y2 were T = 65 °C, S = 60%, and t = ~10 min, obtaining ~450 mg E/g AS; and for Y3 were T = 45 °C, S = 70%, and t = 45 min, obtaining ~12.3 mg Fx/g E. In addition, for the selected optimums, a full screening of pigments was performed by HPLC-DAD, while phenolics and flavonoids were quantified by spectrophotometric techniques and several biological properties were evaluated (namely, antioxidant, antimicrobial, and cholinesterase inhibitory activity). These results could be of interest for future applications in the food, cosmetic, or pharmaceutical industries, as they show the Fx kinetic behavior and could help reduce costs associated with energy and solvent consumption while maximizing the extraction yields.

Physiological Significance of the Heterogeneous Distribution of Zeaxanthin and Lutein in the Retina of the Human Eye.

Zeaxanthin and lutein are xanthophyll pigments present in the human retina and particularly concentrated in its center referred to as the yellow spot (macula lutea). The fact that zeaxanthin, including its isomer meso-zeaxanthin, is concentrated in the central part of the retina, in contrast to lutein also present in the peripheral regions, raises questions about the possible physiological significance of such a heterogeneous distribution of macular xanthophylls. Here, we attempt to address this problem using resonance Raman spectroscopy and confocal imaging, with different laser lines selected to effectively distinguish the spectral contribution of lutein and zeaxanthin. Additionally, fluorescence lifetime imaging microscopy (FLIM) is used to solve the problem of xanthophyll localization in the axon membranes. The obtained results allow us to conclude that one of the key advantages of a particularly high concentration of zeaxanthin in the central part of the retina is the high efficiency of this pigment in the dynamic filtration of light with excessive intensity, potentially harmful for the photoreceptors.

Encapsulated fucoxanthin improves the structure and functional properties of fermented yogurt during cold storage.

Fucoxanthin (FX) extracted from Undaria pinnatifida by an ultrasonic-assisted extraction (UAE) procedure was successfully added to the fermented yogurt through a stably nanoencapsulation. The physicochemical characteristics, texture analysis, rheological testing, sensory evaluation, simulated digestion analysis, and 16SrDNA sequencing analysis were used to evaluate the effect of encapsulated-FX on the function, structure and stability of the fermented yogurt during 7 days cold storage. Encapsulated-FX with a highly water dispersion, changed the microstructure of yogurt, making it more uniform and denser, enhanced the antioxidant activity, increased the stability of milk protein in simulated gastric environment in vitro and promoted the absorption of protein small molecule fragments in the intestine, and inhibited the growth of harmful bacteria during cold storage. This study provided a simple strategy for the production of FX-fortified yogurt by using an effective nanoencapsulation technology, and promoted the extraction and application of active ingredients of edible brown algae.

The Identification and Quantitative Analysis of Unusual Keto-Carotenoids in Ripe Fruits of Maclura tricuspidate and Its Potential as a Valuable Source of Cryptocapsin.

Ripe fruits of Maclura tricuspidata (MT) are used as food material and a natural colorant in Korea. Although MT fruits have a deep red color due to carotenoid-like pigments, their chemical nature has not been explored in detail so far. The present study aimed at elucidating the chemical structures and composition of carotenoids in MT fruits and changes at different maturity stages. Two carotenoids from saponified MT fruit extract were isolated using repeated silica gel column chromatography. Based on interpretations of spectroscopic data, these compounds were determined as keto-carotenoids, i.e., capsanthin (3,3'-dihydroxy-ß,κ-caroten-6'-one) and cryptocapsin (3'-hydroxy-ß,κ-caroten-6'-one), and the contents of individual carotenoids were quantified with HPLC based on calibration curves obtained from authentic standards. The contents of capsanthin and cryptocapsin in the sample of saponified MT fruits were 57.65 ± 1.97 µg/g and 171.66 ± 4.85 µg/g as dry weight base (dw). The majority of these keto-carotenoids in the MT fruits were present in esterified forms with lauric, myristic or palmitic acid rather than in their free forms. The results also showed that esterification of these compounds occurred starting from early stage (yellow-brownish stage) of maturation. Considering the high cryptocapsin content, MT fruits can be applied as a potentially valuable source of cryptocapsin for food and medicinal application as well as a source of provitamin A.

High-Purity Fucoxanthin Can Be Efficiently Prepared from Isochrysis zhangjiangensis by Ethanol-Based Green Method Coupled with Octadecylsilyl (ODS) Column Chromatography.

The exploitation of new economically valuable microalgae as a sustainable source of minor high-value products can effectively promote the full utilization of microalgae. The efficient preparation of minor products from microalgae remains the challenge, owing to the coexistence of various components with a similar polarity in the microalgae biomass. In this study, a novel approach based on the sustainable-oriented strategy for fucoxanthin (FX) production was proposed, which consisted of four steps, including the culture of microalga, ethanol extraction, ODS column chromatography, and ethanol precipitation. The high-purity FX (around 95%) was efficiently obtained in a total recovery efficiency of 84.28 ± 2.56%. This study reveals that I. zhangjiangensis is a potentially promising feedstock for FX production and firstly provides a potentially eco-friendly method for the scale-up preparation of FX from the microalga I. zhangjiangensis.

The breeder's tool-box for enhancing the content of esterified carotenoids in wheat: From extraction and profiling of carotenoids to marker-assisted selection of candidate genes.

Carotenoid esterification is a new target for cereal biofortification since esterification increases both accumulation and stability of carotenoids. A xanthophyll acyl transferase is responsible for carotenoid esterification in the endosperm of wheat and related cereals. In this chapter we describe the procedures for transferring the carotenoid esterification attribute into wheat using the wild barley Hordeum chilense as donor of the esterification trait, the outline of the breeding program and the protocols for marker assisted selection and the analysis of carotenoids in grain. Biofortified cereals with increased lutein ester content will help to reduce the risk of developing age-related macular degeneration in human populations with limited access to other dietary sources.

Astaxanthin improves the shelf-life of tilapia fillets stored under refrigeration.

BACKGROUND: Astaxanthin, classified as a xanthophyll, has antioxidant properties about 500 times greater than α-tocopherols and ten times greater than ß-carotenes. Based on the antioxidant activity of this carotenoid, this study aimed to evaluate the shelf-life of tilapia fillets (Oreochromis niloticus) fed with astaxanthin, by determining the microbiological quality (colimetry, counts of mesophilic and psychrotrophic microorganisms), physicochemical analyses (colorimetry, pH, thiobarbituric acid reactive substances (TBARS)) and sensory analysis. RESULTS: Tilapia supplemented with astaxanthin presented a reduction in the counts of microorganisms (mesophiles and psychrotrophics) and lower lipid oxidation index (TBARS), when compared to fillets of control fish. Colorimetric changes of fillet degradation were observed, associated with increased pH during storage, as well as loss of brightness and texture in addition to worsening of appearance and odor. These deteriorating changes were minimized using astaxanthin. CONCLUSION: Our results demonstrate the beneficial performance of astaxanthin in the shelf-life of tilapia fillets stored under refrigeration. Therefore, dietary supplementation with astaxanthin (100 and 200 mg kg-1 of feed) improves the microbiological and physicochemical quality of tilapia fillets during 50 days of shelf-life. © 2022 Society of Chemical Industry.

High cell density culture of Paracoccus sp. LL1 in membrane bioreactor for enhanced co-production of polyhydroxyalkanoates and astaxanthin.

A cell retention culture of Paracoccus sp. LL1 was performed in a membrane bioreactor equipped with an internal ceramic filter module to reach high cell density and thus enhance the co-production of polyhydroxyalkanoates (PHA) and astaxanthin as growth-associated products. Cell retention culture results showed that PHA accumulation increased with increasing dry cell weight (DCW), giving rise to a maximum of 113 ± 0.92 g/L of DCW with 43.9 ± 0.91 g/L of PHA (38.8% of DCW) at 48 h. A significant increase in both intracellular and extracellular astaxanthin concentrations was also recorded during fermentation process achieving a maximum of 8.51 ± 0.20 and 10.2 ± 0.24 mg/L, respectively. Amounts of PHA and total astaxanthin produced by cell retention culture were 6.29 and 19.7-folds higher, respectively, than those recorded under batch cultivation. PHA and total astaxanthin productivities by cell retention culture also increased up to 0.914 g/L/h and 0.781 mg/L/h, respectively, which were 3.54 and 11.1-folds higher than those of batch culture. Based on gas chromatography, Fourier transform infrared spectroscopy, and 1H nuclear magnetic resonance spectroscopy, the extracted PHA was identified as a copolymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with a 3-hydroxyvalerate content of 3.78 mol%.

Kinetic and hybrid modeling for yeast astaxanthin production under uncertainty.

Astaxanthin is a high-value compound commercially synthesized through Xanthophyllomyces dendrorhous fermentation. Using mixed sugars decomposed from biowastes for yeast fermentation provides a promising option to improve process sustainability. However, little effort has been made to investigate the effects of multiple sugars on X. dendrorhous biomass growth and astaxanthin production. Furthermore, the construction of a high-fidelity model is challenging due to the system's variability, also known as batch-to-batch variation. Two innovations are proposed in this study to address these challenges. First, a kinetic model was developed to compare process kinetics between the single sugar (glucose) based and the mixed sugar (glucose and sucrose) based fermentation methods. Then, the kinetic model parameters were modeled themselves as Gaussian processes, a probabilistic machine learning technique, to improve the accuracy and robustness of model predictions. We conclude that although the presence of sucrose does not affect the biomass growth kinetics, it introduces a competitive inhibitory mechanism that enhances astaxanthin accumulation by inducing adverse environmental conditions such as osmotic gradients. Moreover, the hybrid model was able to greatly reduce model simulation error and was particularly robust to uncertainty propagation. This study suggests the advantage of mixed sugar-based fermentation and provides a novel approach for bioprocess dynamic modeling.

In-silico HMG-CoA reductase-inhibitory and in-vivo anti-lipidaemic/anticancer effects of carotenoids from Spondias mombin.

OBJECTIVES: Inhibition of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase, the rate rate-determining enzyme for the biogenesis of cholesterol is known to show antineoplastic effects. Therefore, this study investigates the in-silico HMG-CoA reductase (HMGCR)-inhibitory and in-vivo anti-lipidaemic/anticancer effects of carotenoids from Spondias mombin. METHODS: Carotenoids from S. mombin leaves were characterized with the aid of liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The characterized phytochemicals were obtained from PubChem. They were docked into the orthosteric site of human HMGCR (Protein Data Bank code 1HW8) using AutoDock 4.0 suites. DMBA (7,12-dimethylbenz[a]anthracene) model of breast cancer was treated with the carotenoids extract from S. mombin (100 mg/kg and 200 mg/kg doses) to assess its anti-lipidaemic cum anticancer effects. KEY FINDINGS: Carotenoids from S. mombin; beta-carotene-15,15'-epoxide, astaxanthin and 7,7',8,8'-tetrahydro-ß-ß-carotene demonstrate HMGCR inhibition. They form hydrophobic interactions with key residues within the catalytic domain of HMGCR. The carotenoids extract exhibits anti-lipidaemic/anticancer effects, lowering serum triglyceride, LDL and cholesterol concentration. It increases HDL concentration and downregulates the expression of HMGR, AFP, CEACAM-3, BRCA-1 and HIF-1 mRNAs. CONCLUSION: Carotenoids from S. mombin demonstrate HMG-CoA reductase (HMGCR) inhibition, anti-lipidaemic, and anticancer effects. The inhibition of HMGCR by the carotenoids extract further poses it as a potential anti-hypercholesterolaemia compounds.

Valorization of Fermented Shrimp Waste with Supercritical CO2 Conditions: Extraction of Astaxanthin and Effect of Simulated Gastrointestinal Digestion on Its Antioxidant Capacity.

Lactic acid fermentation increases the bioactive properties of shrimp waste. Astaxanthin is the principal carotenoid present in shrimp waste, which can be found esterified in the liquid fraction (liquor) after its lactic acid fermentation. Supercritical CO2 technology has been proposed as a green alternative to obtain astaxanthin from fermented shrimp waste. This study aimed to optimize astaxanthin extraction by supercritical CO2 technology from fermented liquor of shrimp waste and study bioaccessibility using simulated gastrointestinal digestion (GD) of the optimized extract. A Box-Behnken design with three variables (pressure, temperature, and flow rate) was used to optimize the supercritical CO2 extraction. The optimized CO2 extract was obtained at 300 bar, 60 °C, and 6 mL/min, and the estimated characteristics showed a predictive extraction yield of 11.17%, antioxidant capacity of 1.965 mmol of Trolox equivalent (TE)/g, and astaxanthin concentration of 0.6353 µg/g. The experiment with optimal conditions performed to validate the predicted values showed an extraction yield of 12.62%, an antioxidant capacity of 1.784 mmol TE/g, and an astaxanthin concentration of 0.52 µg/g. The astaxanthin concentration decreased, and the antioxidant capacity of the optimized extract increased during gastrointestinal digestion. In conclusion, our optimized supercritical CO2 process is suitable for obtaining astaxanthin from shrimp by-products after lactic acid fermentation.

Carotenoid Nostoxanthin Production by Sphingomonas sp. SG73 Isolated from Deep Sea Sediment.

Carotenoids are used commercially for dietary supplements, cosmetics, and pharmaceuticals because of their antioxidant activity. In this study, colored microorganisms were isolated from deep sea sediment that had been collected from Suruga Bay, Shizuoka, Japan. One strain was found to be a pure yellow carotenoid producer, and the strain was identified as Sphingomonas sp. (Proteobacteria) by 16S rRNA gene sequence analysis; members of this genus are commonly isolated from air, the human body, and marine environments. The carotenoid was identified as nostoxanthin ((2,3,2',3')-ß,ß-carotene-2,3,2',3'-tetrol) by mass spectrometry (MS), MS/MS, and ultraviolet-visible absorption spectroscopy (UV-Vis). Nostoxanthin is a poly-hydroxy yellow carotenoid isolated from some photosynthetic bacteria, including some species of Cyanobacteria. The strain Sphingomonas sp. SG73 produced highly pure nostoxanthin of approximately 97% (area%) of the total carotenoid production, and the strain was halophilic and tolerant to 1.5-fold higher salt concentration as compared with seawater. When grown in 1.8% artificial sea salt, nostoxanthin production increased by 2.5-fold as compared with production without artificial sea salt. These results indicate that Sphingomonas sp. SG73 is an efficient producer of nostoxanthin, and the strain is ideal for carotenoid production using marine water because of its compatibility with sea salt.

Astaxanthin for the Food Industry.

Xanthophyll astaxanthin, which is commonly used in aquaculture, is one of the most expensive and important industrial pigments. It is responsible for the pink and red color of salmonid meat and shrimp. Due to having the strongest anti-oxidative properties among carotenoids and other health benefits, natural astaxanthin is used in nutraceuticals and cosmetics, and in some countries, occasionally, to fortify foods and beverages. Its use in food technology is limited due to the unknown effects of long-term consumption of synthetic astaxanthin on human health as well as few sources and the high cost of natural astaxanthin. The article characterizes the structure, health-promoting properties, commercial sources and industrial use of astaxanthin. It presents the possibilities and limitations of the use of astaxanthin in food technology, considering its costs and food safety. It also presents the possibilities of stabilizing astaxanthin and improving its bioavailability by means of micro- and nanoencapsulation.

Distinguishing between isomeric neoxanthin and violaxanthin esters in yellow flower petals using liquid chromatography/photodiode array atmospheric pressure chemical ionization mass spectrometry and tandem mass spectrometry.

RATIONALE: Liquid chromatography/photodiode array atmospheric pressure chemical ionization mass spectrometry (LC/PDA-APCI-MS) is used for the analysis of various carotenoid pigments in plants. Among them, it is difficult to distinguish between the isomeric violaxanthin/neoxanthin esters. METHODS: The yellow pigments of tomato petals were extracted with acetone, and the extracts were kept at -30°C to allow the contaminating triacylglycerols to settle out physically. The supernatants were analyzed using LC/PDA-APCI-MS with a high-resolution orbitrap mass spectrometer for their exact masses. The expected carotenoid esters were calculated with the combination of carotenoids and fatty acids, and they were matched with the experimental exact masses. The fatty acid structures in the carotenoid esters were also identified using collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). The isomeric violaxanthin/neoxanthin esters were distinguished using CID MS/MS from their in-source dehydrated product ions as pseudoprecursor ions. RESULTS: The in-source dehydrated ions [M - H2 O + H]+ of neoxanthin diesters predominated over their protonated molecules [M + H]+ in LC/MS. By contrast, the protonated molecules of violaxanthin diesters predominated. The 92 u loss product ions [M - H2 O - C7 H8 + H]+ were observed from the dehydrated violaxanthin diesters, but they were not generated from the dehydrated neoxanthin diesters in the CID MS/MS of their dehydrated pseudoprecursor ion [M - H2 O + H]+ . CONCLUSIONS: The allene allyl carbocation in neoxanthin diesters was generated from dehydration after preferential protonation at the hydroxy group. The epoxide group of violaxanthin diesters opens easily after protonation; however, the dehydration did not proceed at this stage. The 92 u loss of C7 H8 was explained by an intramolecular [2 + 2] cycloaddition, which proceeded preferentially in dehydrated violaxanthin diesters because the carbocations in the dehydrated species were conjugated to the polyene and those double bonds were depolarized during CID MS/MS. Therefore, the isomeric neoxanthin/violaxanthin diesters were distinguished using LC/PDA-APCI-MS and MS/MS. This method was a practical and useful method of profiling the carotenoid esters of the yellow petals.

Characterization of nutritionally important lipophilic constituents from brown kelp Ecklonia radiata (C. Ag.) J. Agardh.

This research study presents information for the first time on the nutritionally relevant lipophilic compounds obtained from Ecklonia radiata, a poorly studied brown kelp. The major lipophilic compounds were analyzed utilizing liquid chromatography (LC)-tandem mass spectrometry (MS/MS) and gas chromatography (GC)-mass spectrometry (MS). The LC-MS/MS results revealed the presence of eight major lipophilic compounds, including sterols, carotenoids, vitamin E, and phylloquinone (vitamin K1). Quantitative analysis showed that fucosterol was the most predominant phytosterol in the fronds and stipes of E. radiata. The carotenoids (all-E)-fucoxanthin and (all-E)-ß-carotene were present in higher yield. In terms of vitamin E, α-tocopherol was identified as the main tocol. The coenzyme, phylloquinone, important for protein synthesis, was also identified in E. radiata. GC-MS identified 13 fatty acids with palmitic (C16:0) and oleic acid (C18:1n9c) present in the highest quantities. To our knowledge, this is the first report on E. radiata, and the valuable data presented herein can be used as a baseline for developing novel nutraceuticals.

Profiling fragments for carotenoid esters in Penaeus monodon by ultra-high-performance liquid chromatography/quadrupole-Orbitrap high-resolution mass spectrometry.

RATIONALE: The precise identification of carotenoid esters of Penaeus monodon, especially those in the carotenoid skeleton, needs to occur during mass spectrometry analysis. Detailed structural information about carotenoid esters is significant not only for the assessment of nutritional quality, but also for tracing biosynthetic precursors. METHODS: The profiling of carotenoid esters in P. monodon was elucidated using ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC/Q-Orbitrap-HRMS). The raw LC/MS data were analyzed using Exact Finder™ software. RESULTS: The structurally relevant ions, *l and *m, were considered markers of the astaxanthin monoester. Moreover, the carotenoid skeleton was unequivocally identified using the diagnostic ions *i, *j/*j' and *g/*g' generated by the carbon-carbon bond cleavage between ß-ionone ketones and conjugated polyene moieties. In total, 24 carotenoid esters were identified in P. monodon based on the fragmentation patterns discussed above. The identified carotenoid skeleton includes astaxanthin, astacene, oxidized astaxanthin and adonixanthin, which have been described for the first time. CONCLUSIONS: Characterization of the unknown carotenoid esters demonstrates the capabilities of this methodology, which is significant for enriching the carotenoid species in P. monodon.

Bioaccessibility and cellular uptake by Caco-2 cells of carotenoids and chlorophylls from orange peels: A comparison between conventional and ionic liquid mediated extractions.

Native extracts from orange peels were obtained by a conventional method using acetone and, an alternative method using ionic liquid (1-butyl-3-methylimidazolium chloride ([C4mim]Cl)). The bioaccessibilities and cellular uptakes of carotenoids, esters and chlorophylls were evaluated, since the influence of esterification on bioaccessibility and bioavailability is not well established. For this, the extracts were emulsified, submitted to in vitro simulated digestion model according to the INFOGEST protocol, followed by uptake by Caco-2 cells. Compounds were separated, identified and quantified by HPLC-PDA-MS/MS. After digestion, 22.0% and 26.2% of the total carotenoids and 45.9% and 68.7% of the chlorophylls were bioaccessible from the acetone and [C4mim]Cl extracts, respectively. The bioaccessibilities of xanthophylls and carotenes were significantly higher than those of the mono- and diesters. The uptake by Caco-2 cells varied from 130.2 to 131.9 ng/mg cell protein for total carotenoids and from 243.8 to 234.2 ng/mg cell protein for chlorophylls in the acetone and [C4mim]Cl extracts, respectively. In general, xanthophylls and esters were better absorbed than carotenes.