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Background Caseins and whey proteins have great potential for interacting with carotenoids due to their binding sites with hydrophobic domains. Even if a neglected amount of carotenoids is naturally bound to milk proteins, the complexation of these molecules has been proven to be an efficient approach for increasing carotenoid solubility and protection against oxidation, for instance. Scope and approach This review compiled the updated research progress in this field, pointing out all the benefits associated with the interaction between carotenoids and milk proteins and the challenges of characterizing the mechanisms involved in the binding. The impact of temperature, pH, and ionic strength, and the intrinsic characteristics of milk proteins and carotenoids on their binding affinity were reviewed. The main analytical methods applied for the characterization of the binding mechanism were comparatively discussed. Key findings and conclusions The binding of carotenoids to milk proteins is a promising strategy for the development of functional fat-free foods enriched with these bioactive molecules. However, the practical challenges to investigate the interaction between milk proteins and this low-water soluble group of molecules must be considered. Despite being affected by many factors, a binding constant of about 104 M−1 and moderate affinity between carotenoids and milk proteins were frequently reported. Finally, promising techniques and future challenges for this scientific field were addressed. Besides research groups focused on elucidating the mechanism behind the binding between carotenoids and proteins, the scope covered by this review may also interest those involved in the development of healthier formulated food products.

Interest in pigment composition of microalgae species is growing as new natural pigments sources are being sought. However, we still have a limited number of species of microalgae exploited to obtain these compounds. Considering these facts, the detailed composition of carotenoids and chlorophylls of two species of green microalgae (Chlorella sorokiniana and Scenedesmus bijuga) were determined for the first time by high-performance liquid chromatography coupled to diode array and mass spectrometry detectors (HPLC-PDA-MS/MS). A total of 17 different carotenoids were separated in all the extracts. Most of the carotenoids present in the two microalgae species are xanthophylls. C. sorokiniana presented 11 carotenoids (1408.46 μg g−1), and S. bijuga showed 16 carotenoids (1195.75 μg g−1). The main carotenoids detected in the two microalgae were all-trans-lutein and all-trans-β-carotene. All-trans-lutein was substantially higher in C. sorokiniana (59.01%), whereas all-trans-β-carotene was detected in higher quantitative values in S. bijuga (13.88%). Seven chlorophyll compounds were identified in both strains with different proportions in each species. Concentrations of chlorophyll representing 7.6% and 10.2% of the composition of the compounds present in the biomass of C. sorokiniana and S. bijuga, respectively. Relevant chlorophyll compounds are reported for the first time in these strains. The data obtained provide significant insights for microalgae pigment composition databases., Graphical abstract Image 1, Highlights • The carotenoids and chlorophylls profile by HPLC-PDA-MS of microalgae is reported. • Microalgae showed species-specific pigments profiles. • 17 carotenoids and 7 chlorophylls were identified and quantified in details. • The quantitative profile presented a prevalence of chlorophylls over carotenoids. • Green microalgae are proposed as an interesting natural source of food pigments.

Phenolic compounds and carotenoids profile of Amazonian Eryngium foetidum leaves and the quenching ability of their hydrophilic extract against singlet oxygen (1O2) were determined. Chlorogenic acid (4327 μg/g, dry basis, d.b.) was the major phenolic compound in the leaves at very high concentrations, while lutein (205 μg/g, d.b.) and β-carotene (161 μg/g, d.b.) were the major carotenoids. The extract of E. foetidum leaves was able to scavenge DPPH● (91.6% at 5 mg/mL), ABTS●+ (15.77 μM Trolox equivalent/g extract) and it exhibited high efficiency to protect tryptophan against 1O2, with IC50 at 343 μg/mL and 78% of protection at the highest tested concentration (625 μg/mL). Therefore, E. foetidum leaves can be exploited as an accessible natural source of bioactive compounds with antioxidant properties to be used by the food or pharmaceutical industries.

Antrocaryon amazonicum fruits are unexploited sources of bioactive compounds found in the Amazonia region of Brazil. In this study, for the first time, the carotenoid and phenolic compound profiles of the pulp and peel of A. amazonicum fruits, from two varieties at two harvest periods, were determined by LC-MS. Additionally, the potential of the peel and pulp extracts to scavenge physiologically relevant reactive oxygen species (ROS) and reactive nitrogen species (RNS) was assessed. The major carotenoids in both parts of the fruits were lutein, accounting for ≈42% of the identified carotenoids in the peel and ≈25% in the pulp, whereas catechin and hydroxybenzoic acid derivatives were the major phenolics in both parts. The peel extract, which presented the highest bioactive compound contents, was more efficient to scavenge ROS than the pulp. The peel extract showed high scavenging efficiency (IC50 ) for singlet oxygen (1 O2 ; 16 µg/ml), hypochlorous acid (HOCl; 20 µg/ml), peroxynitrite (ONOO- ; 38 µg/ml), and superoxide radical (O2•- ; 47 µg/ml), whereas the pulp extract exhibited high efficiency for ONOO- (13 µg/ml), followed by HOCl (30 µg/ml), ¹O2 (76 µg/ml), and less efficient for O2•- (44 µg/ml). Therefore, A. amazonicum fruits can be seen as an expressive source of bioactive compounds with high antioxidant potential to be further investigated to inhibit or delay oxidative processes both in food and physiological systems triggered by ROS and RNS. PRACTICAL APPLICATION: Bioactive compound extracts of Antrocaryon amazonicum fruits have high potential to be exploited for inhibiting or delaying oxidative processes and increase food stability.

Made available in DSpace on 2021-06-25T10:30:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-09-01 Lignin from pre-treated sugarcane bagasse was sulfomethylated to overcome its high hydrophobicity (Lig-S) and tested at different concentrations as an emulsifier for stabilizing oil-in-water nanoemulsions. The average diameter of the oil droplets was higher in the nanoemulsion prepared with 0.1 % (w/w) Lig-S (∼380 nm) than those prepared with 0.5 and 1.0 % (w/w) (∼180 nm and ∼170 nm, respectively). Zeta potential measurements predicted the long-term stability of Lig-S nanoemulsion. GC–MS analysis of the volatile carbon compounds derived from the oxidation of soybean oil indicated the highest oxidation rates were in preparations with the smallest droplet size. However, all the Lig-S nanoemulsions showed oxidation rates below the threshold values described in the literature. Microscopy analysis confirmed that all the preparations nanosized, dispersed spherical droplets. Collectively, this study has demonstrated that modified lignin isolated from sugarcane bagasse is an excellent emulsifier for the production of oil-in-water nanoemulsions that have both high physical and oxidative stability, providing prospects for the development of nanosystems, based on sustainable strategies, that can be explored for applications such as entrapment and delivery of hydrophobic or bioactive molecules. Brazilian Biorenewables National Laboratory (LNBR) Brazilian Center for Research in Energy and Materials (CNPEM) Faculty of Food Engineering University of Campinas (UNICAMP) Brazilian Nanotechnology National Laboratory (LNNano) Brazilian Center for Research in Energy and Materials (CNPEM) Interdisciplinary Center of Energy Planning University of Campinas (UNICAMP) School of Chemical Engineering University of Campinas (UNICAMP) Department of Bioprocess and Biotechnology College of Agricultural Sciences São Paulo State University (UNESP) Programa de Processos Tecnológicos e Ambientais University of Sorocaba Department of Bioprocess and Biotechnology College of Agricultural Sciences São Paulo State University (UNESP)

Phenotypic characteristics, as well as the relation between carotenoid accumulation and gene expression during ripening were determined in fruits of five new hybrids between tangor cv. ‘Murcott’ and sweet orange cv. ‘Pera’. The genotypes were classified into the orange-like group, showing mainly epoxycarotenoids, oval fruit shape and yellowish color, or in the mandarin-like group, showing mainly β-cryptoxanthin, flattened shape and deep-orange coloration; although some hybrids presented intermediate characteristics. The diversity in carotenoid composition of hybrids and genitors were mostly explained by patterns of gene expression. High carotenoid (250–426 μg/g dry weight [dw]) and β-cryptoxanthin (81–125 μg/g dw) contents, observed in the mandarin-like group, were generally associated with high expression of upstream genes (GGPPS1, PSY, PDS). On the other hand, low expression/repression of these genes and high expression of downstream genes (BCHX and ZEP) were associated with low carotenoid (~158 μg/g dw) and β-cryptoxanthin (5–22 μg/g dw) contents and epoxycarotenoid accumulation, as occurred in the orange-like group. Breeding experiments resulted in hybrids with outstanding higher carotenoid contents than both genitors (up to 426 μg/g dw versus 158–250 μg/g dw in genitors), which was attributed to transgressive segregation. Differences among genotypes have great impact on commercial fruit quality and potential health benefits, such as the provitamin A content.

This study investigated the biocompounds present in the Scenedesmus obliquus biomass and evaluated the effect of the supplementation in the serum lipid profile and tissues antioxidant status of mice. Male mice were divided into control and microalgae biomass treated groups (T1 = 400 and T2 = 800 mg·kg−1 bw·d−1) for four weeks. Serum and tissues (heart, liver, kidneys, and spleen) were analyzed. The microalgae biomass presented a valuable content of biocompounds. In serum, the T2 treatment promoted an increase in high-density lipoprotein but also triglycerides levels. The T1 increased glutathione peroxidase (kidney) and antioxidant potential of all tissues, while T2 treatment was able to raise the glutathione reductase (liver) and glutathione peroxidase (kidneys) activities, as well as the heart antioxidant potential. The two doses reduced lipid peroxidation in all tissues. Thus, we observed that S. obliquus biomass (T1) exerted a protective influence against tissue lipid peroxidation.

Since carotenoids are bioactive compounds that present low oral bioavailability, the knowledge of the steps limiting their bioavailability is mandatory for the design of functional foods/ingredients. Recent findings related to the main food components influencing carotenoids’ bioaccessibility/availability are discussed. A wise choice of food components and processes, as well as carotenoid delivery systems, can successfully drive to an enhanced carotenoid bioavailability in the final product. There are great opportunities not only for the industry to design new formulations/ingredients and foods or beverages with enhanced carotenoid bioavailability, but also for the academy to unveil issues, from the fate of the carotenoids lost through the digestive process to the mechanisms behind the actions of the bioactive compound in a functional food.

Passiflora tenuifila is a Brazilian native passion fruit consumed by the local population and is a dietary source of bioactive compounds with potential biological activity. The aim of this study is to evaluate the nutritional value of P. tenuifila fruit and its bioactive compounds at two ripening stages. Three batches of fruit were collected at mature-green and ripe stages, and phenolic compounds, carotenoids, and polyamines were analyzed by HPLC-DAD and LC-MS/MS. The fruit is a good source of dietary fiber. Proanthocyanidin dimers are the major phenolic compounds (up to 84%) at both stages, followed by the C-glycosylated luteolin. Lutein and β-carotene are the major carotenoids, contributing up to 50% of total carotenoids. The OPLS-DA segregates the mature-green and ripe fruits, as carotenoids are responsible for this separation. In conclusion, passion fruit can be consumed at both stages of maturation without losses of bioactive compound contents or nutritional value.

Using the INFOGEST in vitro digestion protocol adapted to carotenoids, the impact of additional rabbit gastric lipase (RGL) on the hydrolysis extent of β-cryptoxanthin esters was evaluated for the first time, and compared with the addition of porcine cholesterol esterase (CEL). Both the modifications increased the hydrolysis of (all-E)-β-cryptoxanthin esters from mandarin and peach pulps, although the outcomes were different. Addition of RGL consistently increased the average hydrolysis extent from 55.2% to 59.5% in mandarin pulp and from 22.7% to 48.8% in peach pulp (p 0.05). The addition of CEL produced lower hydrolysis extents, i.e., 58.5% in mandarin (not statistically significant) and 28.4% in peach (p0.05), compared to those obtained with RGL. The hydrolysis extent positively correlated with the carotenoid ester concentration in both matrices. Bioaccessibility values were higher in mandarin pulp (range 32-34%) compared to those in peach pulp (range 16-21%), and were associated with the hydrolysis extent of the carotenoid esters during digestion. Addition of RGL and CEL produced no significant (p 0.05) effect on the overall carotenoid bioaccessibility values of mandarin, while positively affected those in peach. Altogether these results corroborate that the hydrolysis extent of xanthophyll esters limits bioaccessibility. Additionally, hydrophobicity of the carotenoid inversely correlates with micellarization, as free (all-E)-xanthophylls micellarized in a higher extent compared to (all-E)-β-carotene and xanthophyll esters. The new information of our results is that the addition of rabbit gastric lipase substantially contributes to the hydrolysis of β-cryptoxanthin esters from fruit pulps, and consequently, to increase carotenoid bioaccessibility, being even more effective than CEL.

Carotenoid esters are present in considerable amounts in most fruits, such as in citrus. Although the bioavailability of carotenoid esters is similar or even higher compared to that of free carotenoids, these molecules are generally detected only in the free form in human plasma, suggesting that hydrolysis of carotenoid esters occurs in vivo. However, the available in vitro digestion methods were not able to achieve satisfactory carotenoid ester hydrolysis so far. As bile salts play an essential role in the hydrolytic action of lipolytic enzymes from pancreatin, we evaluated the effect of increasing the bile extract/food ratio from 0.045 to 0.12 (g g-1) on the hydrolysis of β-cryptoxanthin esters from mandarin pulp during in vitro digestion. Additionally, considering the positive effect of lipids on carotenoid bioavailability, the impact of soybean oil addition on carotenoid ester hydrolysis was studied. Finally, bioaccessibility and recovery of 33 carotenoids were assessed by LC-DAD-MS. The hydrolysis extent of β-cryptoxanthin esters enhanced from 29% to 55% by increasing the bile extract/food ratio, but reduced respectively to 28% and 11% by the addition of 1% and 10% oil (p0.05). The bioaccessibility of overall carotenoids improved from 19% to 35% by increasing the bile extract/food ratio, along with that of (all-E)-β-carotene (from 19 to 31%) and total (all-E)-β-cryptoxanthin (17% to 49%). Soybean oil addition reduced carotenoid micellarization, regardless of the concentration (p0.05). Irrespective of the bile extract amount and oil addition, the bioaccessibility of carotenoids was inversely related to its hydrophobicity, with respect to the following ranking: free xanthophyllscarotenes ≥ xanthophyll esters. Altogether, these results indicate that increasing the bile extract amount is a simple and inexpensive option to improve carotenoid ester hydrolysis in in vitro digestion protocols. Additionally, the constant amounts of bile (and possibly enzymes) of static methods, such as INFOGEST, should be further optimized for experiments involving lipid addition in which carotenoid bioaccessibility is evaluated.

Caja ( Spondias mombin L.) fruit, native to Latin American tropical forests, is consumed either in natura or its pulp is used to prepare beverages. Aside from important physiological actions, carotenoids are responsible for the yellow skin and pulp color of caja. As xanthophylls are often acylated to fatty acids in fruits, the native carotenoid composition of caja frozen pulp and of its beverages was evaluated by high performance liquid chromatography coupled to diode array detector and tandem mass spectrometry. The main carotenoids in caja frozen pulp were (all- E )-β-cryptoxanthin (6.5 μg g −1 fresh weight (fw)) and (all- E )-zeinoxanthin (3.5 μg g −1 fw); esters of lutein, β-cryptoxanthin and zeinoxanthin were also identified. As compounds need to be released from the matrix to be available for absorption, in vitro bioaccessibility of carotenoids in caja water and milk based beverages was investigated. Addition of 7% sugar enhanced bioaccessibility to a similar extent of all carotenoids, in the order water > skimmed milk > whole milk based beverages. The increase in carotenoid bioaccessibility accompanied the increase in the lipid amount in milk used in beverages. Milk calcium did not affect carotenoid bioaccessibility while milk proteins were responsible for the four times increased bioaccessibility of carotenoids with free hydroxy group compared to those of carotenes and totally esterified xanthophylls.

Peach palm fruits are native from the Amazonia biome and available as different varieties with distinct colour of the fruit peel. Although peach palm fruits have been known to exhibit high bioactive compound content, the carotenoid profile of some varieties, but not all, was reported, while the phenolic compounds of all varieties remain unknown until now. In our study, the carotenoid and phenolic compound profiles were scrutinized for the first time in cooked pulps of orange and yellow peach palm fruits by HPLC-DAD-MS. The major carotenoid in pulps of both orange and yellow peach palm fruits was β-carotene (20% and 24%, respectively), and lutein accounted for 14% in the yellow variety, which may explain the colour difference between the fruits. Both pulps of peach palm fruits presented the same phenolic compounds profile, mostly composed by di-C-glycosyl flavones, with schaftoside the major compound in yellow (45%) and orange (32%) varieties, while vicenin-2 was detected at high relative concentration (21%) in the pulp of orange peach palm fruits, in contrast to 6% found in the yellow fruits. Therefore, orange and yellow peach palm fruits exhibited a promising composition of bioactive compounds for the research and development of high-quality derived food products with claimed health benefits.

Carotenoids from cyanobacteria Aphanothece microscopica Nageli and green microalgae Chlorella vulgaris and Scenedesmus obliquus were identified. The total carotenoid content, based on dry weight of biomass, of A. microscopica Nägeli, C. vulgaris and S. obliquus were 1398.88μg/g, 1977.02μg/g and 2650.70μg/g, respectively. A total of 23 different carotenoids were separated in all the extracts, the major ones being all-trans-β-carotene (29.3%) and all-trans-lutein (28.1%) in Scenedesmus; all-trans-echinenone (22.8%) and all-trans-β-carotene (17.7%) in Chlorella; all-trans-echinenone (28.3%) and all-trans-β-carotene (26.2%) in Aphanothece. The carotenoid extracts were shown to be a potent scavenger of peroxyl radical, with values of 31.1 (Chlorella), 14.0 (Scenedesmus) and 7.3 (Aphanothece) times more potent than α-tocopherol.

Carotenoids are naturally found in both free form and esterified with fatty acids in most fruits and some vegetables; however, up to now the great majority of studies presents data on carotenoid composition only after saponification. The reasons for this approach are that a single xanthophyll can be esterified with several different fatty acids, generating a great number of different compounds with similar chemical and structural characteristics, thus, increasing the complexity of analysis compared to the respective saponified extract. This means that since UV/Vis spectrum does not change due to esterification, differentiation between free and acylated xanthophylls is dependent at least on elution order and mass spectrometry (MS) features. The presence of interfering compounds, especially triacylglycerides (TAGs), in the non-saponified extract of carotenoids can also impair carotenoid ester analyses by MS due to high background noise and ionization suppression since TAGs can be present in much higher concentrations than the carotenoid esters. This leads to the need of development of new and effective clean-up procedures to remove the potential interferents. In addition, only few standards of xanthophyll esters are commercially available, making identification and quantification of such compounds even more difficult. Xanthophyll esterification may also alter some properties of these compounds, including solubility, thermostability and bioavailability. Considering that commonly consumed foods are dietary sources of xanthophyll esters and that it is the actual form of ingestion of such compounds, an increasing interest on the native carotenoid composition of foods is observed nowadays. This review presents a compilation of the current available information about xanthophyll ester analyses and occurrence and a practical guide for extraction, pre-chromatographic procedures, separation and identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Orange juice is considered an excellent dietary source of several bioactive compounds with beneficial properties for human health. Citrus sinensis Osbeck cv. 'Cara Cara' is a bud mutation originated from 'Washington' navel orange, also known as 'Bahia' navel orange. The ascorbic acid, flavonoid, and carotenoid contents in pasteurized and nonpasteurized Bahia and Cara Cara juices using two LC-MS/MS platforms were investigated. Higher ascorbic acid content was observed in Bahia compared to Cara Cara in both pasteurized and nonpasteurized juices. Total flavanones content as well as hesperidin levels were higher in Cara Cara with respect to Bahia pasteurized juice. Cara Cara was also characterized by a significantly higher and diversified carotenoid content compared to Bahia juice with a mixture of (Z)-isomers of lycopene, all-E-β-carotene, phytoene, and phytofluene isomers accounting for the highest carotenoid proportion. The exceptionally high carotenoid content of Cara Cara may be particularly interesting for nutritional or functional studies of uncommon carotenes in a citrus food matrix.

The composition of carotenoids (carotenes and free and acylated xanthophylls) and their bioaccessibilities were determined for the first time in pulps of mandarins cultivated in Brazil. Two cultivars of mandarin, Citrus reticulata Blanco cv. 'Ponkan' and Citrus reticulata × C. sinensis cv. 'Murcott', showed higher contents of most carotenoids compared to those found in C. deliciosa Tenore cv. 'Rio'. The major carotenoids in mandarin cv. 'Ponkan' and 'Murcott' were (all-E)-β-cryptoxanthin laurate (19-21%), (all-E)-β-cryptoxanthin myristate (15-17%) and (Z)-ζ-carotene (7-12%), followed by (all-E)-β-cryptoxanthin palmitate (4-7%), free (all-E)-β-cryptoxanthin (5-6%) and (all-E)-β-carotene (4-5%), while in mandarin cv. 'Rio' (all-E)-β-cryptoxanthin myristate (22%) was the major compound, followed by (all-E)-β-cryptoxanthin laurate (16%), (all-E)-β-cryptoxanthin palmitate (11%), (all-E)-β-cryptoxanthin (9%) and (all-E)-β-carotene (6%). After in vitro digestion, the qualitative carotenoid profile of the supernatant containing the micellarized carotenoids was similar to that of fresh fruits, but the contents were significantly lower. Carotenoid and mandarin physico-chemical properties influenced the bioaccessibility of carotenoids. Free (all-E)-β-cryptoxanthin showed the highest bioaccessibility in all mandarin cultivars (33-42%), while the bioaccessibilities of β-carotene (16-36%) and the major carotenoid esters (18-33%) were lower. The overall recovery of carotenoids during in vitro digestion was around 98% after the oral phase, 79% after oral + gastric phases and 77% after oral + gastric + duodenal phases, with free (all-E)-β-cryptoxanthin and (all-E)-β-carotene being the most stable ones. Besides possible E-Z isomerization and ester hydrolysis, evident losses occurred in total carotenoid contents and also in the most individual carotenoids and they were not compensated for by the former reactions.

The objective of this study was to evaluate the effect of carotenoids intake of Scenedesmus obliquus, on lipid peroxidation, the endogenous antioxidant defense system as well as the serum lipid profile in vivo. Male mice were divided into control groups and supplemented with different doses of microalgae carotenoids: 0.25 (MC1) and 2.5 (MC2) mg·kg-1 bodyweight. The lipid profile (total cholesterol, triglycerides, low and high-density lipoprotein) and markers of hepatic toxicity were determined in serum samples. Antioxidant enzymes and thiobarbituric acid reactive substances were determined in the heart, liver, kidneys, and spleen. Both doses used to treat the animals did not show adverse effects by markers of hepatic toxicity. MC1 did not cause significant changes in the serum lipid profile. In contrast, it created a significant reduction in lipid peroxidation of the spleen (46%) as well as an increase in the GR in the heart (40%) and GPx in the kidneys (79%) activity. The MC2 treatment also increased GR (49%) in the heart and GPx (243%) in the heart and kidneys (58%) activity, however, significantly increased levels of lipid peroxidation in the liver (160%) as well as serum triglycerides (60%). According to results, it is suggested that the consumption of S. obliquus carotenoids at the MC1 dose was safe to the animals and could be explored as an alternative to improve the activity of antioxidant enzymes and reduce lipid peroxidation.

Arrabidaea chica (Brazilian name = pariri) is a plant species that belongs to the Bignoniaceae family, occurring in tropical America and widespread in the Amazonian region of Brazil. In this study, the phenolic compound and carotenoid profiles of A. chica leaves were determined by HPLC-DAD-MS. Scutellarin was identified as the main phenolic compound (15,147.22 μg/g, dry basis, d.b.) and lutein (204.28 μg/g, d.b.), β-carotene (129.5 μg/g, d.b.) and α-carotene (79.86 μg/g, d.b.) as the major carotenoids. Moreover, A. chica leaves presented 152.7 μg/g of ascorbic acid (fresh weight). For the first time, the carotenoid profile and ascorbic acid contents were reported for A. chica leaves. Regarding the antioxidant capacity, A. chica extract was able to scavenge ABTS radical (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)), and for the first time, the protective effect against singlet oxygen (1O2) was reported. The A. chica extract was efficient as ABTS radical scavenger (86.81 μM Trolox/g of fresh leaves) and inhibited the degradation of tryptophan by 1O2 in a concentration-dependent manner with an IC50 at 177 μg/mL. Thus, A. chica leaves can be exploited as a promising source of bioactive compounds that may be useful to human health or food systems against oxidative damage.

The reliable identification of carotenoid esters requires expertise on diverse analytical tools, such as liquid chromatography, UV–visible and mass spectrometry. In this chapter, the main UV–visible and mass spectrometry characteristics of free carotenoids and carotenoid esters are presented with the aim of providing a simple guideline containing the key aspects of the carotenoid structure that can be revealed or deduced from this combined information. General aspects of the main mass spectrometer instruments used so far for carotenoid ester analyses are also presented.

Interest in the composition of carotenoid esters of fruits is growing because esterification may affect their bioavailability. Thus, the aim was to provide a detailed identification of carotenoid esters in citrus and mango. Orange cv. 'Valencia' and cv. 'Pera' presented 9 free carotenoids, 38 monoesters, and 60 diesters. Violaxanthin and luteoxanthin derivatives were the major ones, followed by antheraxanthin, lutein, zeaxanthin, β-cryptoxanthin, and zeinoxanthin esters, many of them reported for the first time in orange pulp. The carotenoid ester composition of tangor cv. 'Murcott', reported for the first time, showed 8 free carotenoids, 34 monoesters, and 33 diesters, with β-cryptoxanthin esters as major compounds, followed by violaxanthin and zeaxanthin esters. In citrus, carotenoids were acylated mainly with capric, lauric, myristic, myristoleic, palmitic, palmitoleic, and oleic acids. In mango, 5 free carotenoids, 2 monoesters, and 19 diesters were identified, from which many violaxanthin and neoxanthin esters were reported for the first time.

The aim of this study is to investigate the effects of different home cooking techniques (boiling, steaming, and stir-frying) in kale and red cabbage, on the levels of bioactive compounds (carotenoids, anthocyanins and phenolic compounds) determined by high-performance liquid chromatography coupled with photodiode array and mass spectrometry detectors (HPLC-DAD-MS(n)), and on the antioxidant activity evaluated by ABTS, ORAC and cellular antioxidant activity (CAA) assays. The steaming technique resulted in a significant increase in phenolic content in kale (86.1%; p

In vitro digestion methods are a useful approach to predict the bioaccessibility of food components and overcome some limitations or disadvantages associated with in vivo methodologies. Recently, the INFOGEST network published a static method of in vitro digestion with a proposal for assay standardization. The INFOGEST method is not specific for any food component; therefore, we aimed to adapt this method to assess the in vitro bioaccessibility of carotenoids and carotenoid esters in a model fruit (Byrsonima crassifolia). Two additional steps were coupled to the in vitro digestion procedure, centrifugation at 20 000g for the separation of the aqueous phase containing mixed micelles and exhaustive carotenoid extraction with an organic solvent. The effect of electrolytes, enzymes and bile acids on carotenoid micellarization and stability was also tested. The results were compared with those found with a simpler method that has already been used for carotenoid bioaccessibility analysis. These values were in the expected range for free carotenoids (5–29%), monoesters (9–26%) and diesters (4–28%). In general, the in vitro bioaccessibility of carotenoids assessed by the adapted INFOGEST method was significantly higher (p < 0.05) than those assessed by the simplest protocol, with or without the addition of simulated fluids. Although no trend was observed, differences in bioaccessibility values depended on the carotenoid form (free, monoester or diester), isomerization (Z/E) and the in vitro digestion protocol. To the best of our knowledge, it was the first time that a systematic identification of carotenoid esters by HPLC-DAD-MS/MS after in vitro digestion using the INFOGEST protocol was carried out.

The production of superoxide anion radical (O2•(-)) is essential for the life of aerobic organisms. This free radical acts as a signaling molecule, regulating numerous biological processes including apoptosis, aging, and senescence. Nevertheless, when overproduction of O2•(-) occurs and/or antioxidant defences are deficient, oxidative stress may develop, damaging important biomolecules and altering their physiological function. These effects have been associated to the development of several diseases. Scavenging of O2•(-) has been used as a hallmark to test the antioxidant capacity of several types of compounds in cellular and non-cellular systems. However, despite the pathophysiological importance of O2•(-), the information about its endogenous and/or chemical generation and detection is dispersed and there are no reports that concisely cover the information in an integrated form. This gap can explain the limitations attributed to the currently used systems, namely in what concerns the selectivity, specificity and validation. This review attempts to provide a critical assessment of the available O2•(-) generating and detection, both in endogenous and chemical systems, scrutinizing its advantages and limitations in order to facilitate the choice and implementation of the O2•(-) generator and/or detection method that better fits the researchers' objectives.

The profile of the three classes of pigments (carotenoids, chlorophylls and phycobiliproteins) and the peroxyl radical scavenger capacity of extracts from microalgae Phormidium autumnale were determined. Twenty-four carotenoids, three phycobiliproteins and two chlorophylls were identified. The major carotenoids of biomass were all-trans-β-carotene (225.44 μg·g− 1), all-trans-lutein (117.56 μg·g− 1) and all-trans-zeaxanthin (88.46 μg·g− 1). Furthermore, characteristic carotenoids in cyanobacteria such as echinenone (79.07 μg.g− 1), myxoxanthophyll (18.83 μg.g− 1) and canthaxanthin (1.89 μg·g− 1) were found. The chlorophyll a (2.700 μg·g− 1) and C-phycocyanin (2.05 × 105 μg·g− 1) were predominant pigments in the other fractions of color compounds from biomass. The carotenoid and chlorophyll extracts were shown to be potent scavengers of peroxyl radical, being almost 28 and 85 times more potent α-tocopherol respectively, and for phycobiliproteins, the antioxidant capacity was 274 μmol trolox·g− 1 (dry wt).

The aim of this study was to develop a new method for carotenoid extraction from orange peel, using ionic liquid (IL) to replace conventional organic solvents, assisted by ultrasound. Four different IL were tested: 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]), 1-n-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]), 1-n-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), and 1-hexyl-3-methylimidazolium chloride ([HMIM][Cl]). Response surface methodology was applied in order to optimize the carotenoid extraction conditions, and Amberlite XAD-7HP resin was used to separate the carotenoids from the IL, allowing their recovery. Determination of carotenoids was carried out by high-performance liquid chromatography coupled to photodiode array and mass spectrometry detectors (HPLC–DAD–MSn). Thermal stability at different temperatures (60 °C and 90 °C) and peroxyl radical scavenging activity of the carotenoid extracts obtained with acetone and IL were evaluated. [BMIM][Cl] was the most effective IL, leading to a total carotenoid content of 32.08 ± 2.05 μg/g, while 7.88 ± 0.59 μg/g of dry matter was obtained by acetone extraction. IL and carotenoid recoveries using XAD-7HP resin were in the range of 59.5–63.8% and 52.2–58.7%, respectively. A carotenoid extract was successfully obtained with IL, finally isolated just by using ethanol, besides being more stable and presenting higher antioxidant activity than that obtained with acetone.

Carotenoids constitute a large group of lipophilic pigments whose health-promoting benefits have been widely recognized. Hydroxy-containing carotenoids can be found in both free form or esterified with fatty acids in several plant matrices, but the native carotenoid profile is overall poorly explored due to the difficulty of analyzing carotenoid esters. One of the main natural sources of carotenoids is the marigold flower, which has been extensively used by the industry for the production of food colorants or supplements, both often manufactured with no saponification process. Although lutein esters are well established as the major compounds naturally found in marigold petals and their products, carotenoid esters other than the lutein ones have not been extensively examined. We carried out a comprehensive identification of carotenoids and carotenoid esters from marigold petals by LC-DAD-(APCI+)MS/MS. Whereas 18 carotenoids were identified in the saponified extract, 56 were identified when no saponification procedure was carried out: 6 free carotenoids, 20 monoesters and 30 diesters. This is the first time that esters of zeaxanthin, violaxanthin, auroxanthin, zeinoxanthin and β-cryptoxanthin are identified in marigold. The structural information obtained through characteristic fragmentation patterns and diagnostic fragments in MS and MS/MS spectra (APCI+) sustained the differentiation between carotenoid esters with similar characteristics. Therefore, the separation of carotenoids by reversed-phase liquid chromatography using C30 columns in combination with DAD and APCI-MS/MS detection allowed high sensitivity and selectivity for carotenoid ester analysis.

Tucuma (Astrocaryum vulgare) and peach palm (Bactris gasipaes) are Amazonian fruits with high potential to be used as source of carotenoids due to the remarkable yellow-orange colour of their pulp and peel. In this study, for the first time, both the extraction of carotenoids from the peel of tucuma and peach palm fruits and their carotenoid profile were investigated. The carotenoid contents of the peel of both fruits were higher than those found in the pulp. The highest yield of carotenoids from both fruit peels were obtained at the solid-liquid ratio of 1:7 (w/v), 80 min of shaking and by adding 30% of ethyl acetate in acetone, being β-carotene the major carotenoid (7.8 and 7.3 mg/100 g for tucuma and peach palm, respectively), followed by γ-carotene and δ-carotene. Thus, peels of tucuma and peach palm fruits were classified as very high carotenoid sources to be used by any potential interested industry.

Pitanga, a fruit of the pitangueira tree (Eugenia uniflora L.), is native to Brazil and has a high antioxidant capacity due to the elevated amount of anthocyanins. The present study aimed to investigate the chemical composition of the purple pitanga fruit and to evaluate its antioxidant effect in the nematode Caenorhabditis elegans. We observed that the ethanolic extract of purple pitanga did not cause any toxic effects but notably increased worm lifespan. The extract improved the survival, reproduction and lifespan of the worms in pre- and post-exposure to stressors H2O2 and juglone, as well as improved the lifespan of the oxidative stress hypersensitive strain mev-1. Notably, PPE extract decreased reactive oxygen species by DCF-DA probe and protein carbonyl content from worms stressed with H2O2. The extract also affected the expression of superoxide dismutase SOD-3 and heat shock protein HSP-16.2 levels, daf 16 target genes that modulate lifespan and antioxidant metabolism. In addition, we demonstrate that these effects are dependent on DAF-16, as PPE extract did not provide protection in daf-16 mutants. Therefore, these results suggest that PPE significantly protected against oxidative stress modulating daf-16 target genes.

Carotenoids possessing hydroxyl groups can be found in nature both as free xanthophylls and as carotenoid esters, i.e. acylated with fatty acids. A single carotenoid can be acylated with different fatty acids, resulting in a high number of possible structures of carotenoid esters. The analysis of carotenoid esters can be challenging; although extraction techniques are essentially the same as those used for free carotenoids, depending on the sample composition, interfering compounds such as triacylglycerides are also present in the extract in significant amounts to cause high background noise and ionization suppression in mass spectrometric analysis. Besides analysis of UV-visible spectrum features, identification of carotenoid esters must include tandem mass spectrometry (MSn) because no changes in the carotenoid molecule chromophore occur due to acylation with fatty acids. Although carotenoid esters are widespread found in foods, especially in fruits, and their bioavailability is equivalent or even higher than that of the free carotenoid, occurrence of xanthophyll esters is very limited in human plasma and tissues. Challenges and further investigations needed on the topics above are given in this review.

In vitro digestion methods are routinely used to assess the bioaccessibility of carotenoids and other dietary lipophilic compounds. Here, we compared the recovery of carotenoids and their efficiency of micellarization in digested fruits, vegetables, egg yolk, and salmon and also in mixed-vegetable salads with and without either egg yolk or salmon using the static INFOGEST method22 and the procedure of Failla et al.16 Carotenoid stability during the simulated digestion was ≥70%. The efficiencies of the partitioning of carotenoids into mixed micelles were similar when individual plant foods and salad meals were digested using the two static methods. Furthermore, the addition of cooked egg or salmon to vegetable salads increased the bioaccessibility of some carotenoids. Our findings showed that the two methods of in vitro digestion generated similar estimates of carotenoid retention and bioaccessibility for diverse foods.

The aim of this work was to evaluate the production of carotenoids from microalgae Phormidium autumnale cultivated using agroindustrial wastes. The carotenoid profile from microalgae biomass was assessed by high performance liquid chromatography coupled with a photodiode array and mass spectrometry detectors. A total of twenty carotenoids were separated from the biomass. The all-trans-β-carotene (70.22 μg/g), all-trans-zeaxanthin (26.25 μg/g), all-trans-lutein (21.92 μg/g), all-trans-echinenone (19.87 μg/g) and cis-echinenone (15.70 μg/g) were the major ones. For the production of single-cell carotenoids it is possible to estimate a total carotenoid production of up to 107,902.5 kg/year in an industrial scale. Based on these results, we observed the potential of P. autumnale to the production of microalgal carotenoid in agroindustrial wastewater.

© 2014 Elsevier Ltd. Addition of lutein to dairy products is an alternative that widens the range of foods which could be lutein sources. However, bioaccessibility is an essential aspect to be considered during the development of products with added bioactive substances. We evaluated the in vitro bioaccessibility of lutein esters added to milk and yogurt with different fat contents, and determined the efficiency of enzymatic hydrolysis of the esters during digestion. Bioaccessibility of lutein and efficiency of hydrolysis were significantly lower in skimmed products than semi-skimmed and whole products, indicating that a minimal amount of fat is required to allow micellization and hydrolysis. The efficiency of ester hydrolysis ranged between 12 and 35%, which was attributed to pancreatic lipase. Whole and semi-skimmed samples were shown to be good vehicles for the addition of lutein, since presented bioaccessibility indices (38.3-47.5%) are similar to those found in natural food sources of xanthophylls., The authors are grateful to CAPES, FAPESP and CNPq for their financial support

Carotenoid analysis in citrus is still a challenge due to their complex carotenoid profile and matrix acidity, which can promote isomerization and epoxide-furanoid rearrangements of these compounds. A new method for carotenoid extraction from freeze-dried citrus was developed. Extraction was carried out by magnetic stirring using ethyl acetate and methanol as solvents and Na2CO3 to neutralize organic acids. Liquid-liquid partition was achieved by centrifugation. Carotenoid quantification was accomplished by both UV-Vis and high-performance liquid chromatography-diode array detection (HPLC-DAD). Method repeatability (2.9 to 8.5%) and recovery (82 to 88%) were similar to reported values for procedures including all the carotenoid analysis steps. In 'Valencia' orange pulp, xanthophylls accounted for 98% of the total carotenoid content and the predominant carotenoids were (9Z)-violaxanthin, (all-E)-lutein and (9Z)-antheraxanthin. This method was further applied for carotenoid analysis in freeze-dried 'Tommy Atkins' mango pulp. The developed method is precise, accurate, fast and able to maintain epoxy-carotenoids stability during analysis.

A hydrophilic extract of murici (Byrsonima crassifolia), a fruit native to the North and Northeast regions of Brazil, was evaluated in relation to its phenolic composition and in vitro antioxidant potential against some physiologically relevant reactive oxygen and nitrogen species. Additionally, the protective effect of murici extract against peroxyl radical (ROO )-induced toxicity to human erythrocytes was also determined. The major phenolic compound, determined by HPLC–DAD–MSn, was quercetin (2.72 ± 0.35 μg/mL). The extract was able to scavenge ROO (0.30 ± 0.04 μmol trolox equivalent/mg), hypochlorous acid (IC50 = 10.0 ± 0.1 μg/mL), hydroxyl radical (IC50 = 7 ± 1 μg/mL) and peroxynitrite anion (IC50 = 21.0 ± 0.6 μg/mL and 17.0 ± 1.6 μg/mL, respectively, in absence and presence of NaHCO3). Human erythrocytes were subjected to oxidative damage, but murici extract was not able to inhibit hemolysis, even at the highest tested concentration. On the other hand, the extract inhibited hemoglobin oxidation (IC50 = 271 ± 44 μg/mL), lipid peroxidation (1000 μg/mL) by 48 ± 5%, depletion of glutathione (100 μg/mL) by 49 ± 2% and formation of its oxidized form (100 μg/mL) by 96 ± 4%.

β-Carotene, zeaxanthin, lutein, β-cryptoxanthin, and lycopene are liposoluble pigments widely distributed in vegetables and fruits and, after ingestion, these compounds are usually detected in human blood plasma. In this study, we evaluated their potential to inhibit hemolysis of human erythrocytes, as mediated by the toxicity of peroxyl radicals (ROO•). Thus, 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH) was used as ROO• generator and the hemolysis assay was carried out in experimental conditions optimized by response surface methodology, and successfully adapted to microplate assay. The optimized conditions were verified at 30 × 106 cells/mL, 17 mM of AAPH for 3 h, at which 48 ± 5% of hemolysis was achieved in freshly isolated erythrocytes. Among the tested carotenoids, lycopene (IC50 = 0.24 ± 0.05 μM) was the most efficient to prevent the hemolysis, followed by β-carotene (0.32 ± 0.02 μM), lutein (0.38 ± 0.02 μM), and zeaxanthin (0.43 ± 0.02 μM). These carotenoids were at least 5 times more effective than quercetin, trolox, and ascorbic acid (positive controls). β-Cryptoxanthin did not present any erythroprotective effect, but rather induced a hemolytic effect at the highest tested concentration (3 μM). These results suggest that selected carotenoids may have potential to act as important erythroprotective agents by preventing ROO•-induced toxicity in human erythrocytes. Practical Application Selected carotenoids may have potential to be used in the development of phytopharmaceutical products as important erythroprotective agents by preventing ROO•-induced toxicity in human erythrocytes.

Chlorophyll a and carotenoids are important pigments in photosynthesis. Several studies have been published describing extraction and analysis protocols of these pigments, mainly in vascular plant species. This study standardizes an extraction and analysis protocol of these substances in Gracilaria tenuistipitata var. liui, a red seaweed. Apical portions grown in vitro were triturated in liquid nitrogen. Extracts were prepared in 1.5 mL solvent and centrifuged. Quantitative and qualitative analyses of pigments were performed by UV/visible light spectrophotometry and high performance liquid chromatography (HPLC) and HPLC coupled to mass spectrometry (HPLC-MS). The parameters assessed were: minimum biomass, best extraction solvent, and number of extraction steps. Methanol was the most efficient solvent, and 50 mg fresh biomass was the amount of sample indicated, submitted to one single extraction step. No significant differences were observed in levels of these pigments by UV-visible light spectrophotometry and HPLC. However, HPLC or HPLC-MS are required to identify the different carotenoids present in this seaweed species. Clorofila a e carotenoides são importantes pigmentos da fotossíntese. Na literatura são encontrados vários protocolos de extração e análise desses pigmentos utilizando, principalmente, plantas vasculares. O objetivo deste estudo foi padronizar uma metodologia de extração e análise dessas substâncias em uma macroalga vermelha, Gracilaria tenuistipitata var. liui. Amostras de talos gametofíticos cultivados in vitro foram trituradas em nitrogênio líquido, extraídas em 1,5 mL de solvente, centrifugadas e os pigmentos analisados quantitativamente e qualitativamente através de espectrofotometria de UV/visível, cromatografia liquida de alta eficiência (CLAE) e CLAE-acoplada a espectrometria de massas (CLAE-EM). Foram testados os parâmetros massa mínima, solvente para extração e número de extrações. Dentre os solventes testados, o metanol foi o mais eficiente, sendo 50mg de material fresco a massa mínima indicada para ser submetida a somente uma extração. Não foram encontradas diferenças significativas na quantificação desses pigmentos comparando-se os dados obtidos em espectrofotometria de UV/visível com os de CLAE. No entanto, para a identificação dos diferentes carotenoides e suas quantificações são necessárias CLAE ou CLAE-EM.

The thermal degradation of carotenoids in cashew apple (Anacardium occidentale L.) juice leads to changes in the beverage colour, and possibly in the aroma and flavour, although the latter hypothesis has not yet been properly investigated. Thus the objective of this study was to investigate the formation of odour active volatiles derived from the thermal degradation of carotenoids in a cashew apple juice model. A carotenoid extract in an acidic aqueous medium was submitted to 60 and 90 °C for 1 and 2 h. The non-volatile compounds were identified by high performance liquid chromatography coupled with a photodiode array and mass spectrometry detectors (HPLC-PDA). The volatiles were isolated by headspace-solid phase micro-extraction, separated by gas chromatography, identified by mass spectrometry (SPME-GC–MS) and their odour significance assessed by GC-Olfactometry. Thirty-three odour active volatiles were identified in the heated system, amongst which 1,2,3,5-tetramethylbenzene, naphthalene and p-xylene. The results indicated that the volatiles formed from the thermal degradation of the carotenoids influence the aroma and flavour of thermally processed cashew apple products.

Introduction Caryocar villosum has been reported as a source of bioactive compounds that can be used as a potential product against oxidative damage in foods or biological systems. Objective To obtain extracts from fruit pulps of C. villosum with high levels of bioactive compounds that have both antioxidant and colour properties. Method The contents of bioactive compounds (total phenolic compounds, flavonoids, tannins, carotenoids and tocopherols), the colour parameters, the scavenging capacity against peroxyl radicals (ROO•) and the quenching activity against singlet oxygen (1O2) were determined. All data were used for extract classification by applying principal components analysis and hierarchical cluster analysis. Results The water and ethanol:water (1:1, v/v) extracts presented the highest levels of total phenolic compounds (9.2 and 6.3 mg gallic acid equivalent/g extract, respectively), total flavonoids (3.8 and 2.5 mg catechin equivalent/g extract, respectively) and total tannins (7.6 and 2.4 mg tannic acid equivalent/g extract, respectively). The ethanol:water (1:1, v/v) extract also showed the highest scavenging capacity against ROO• (0.3 mmol trolox equivalent/g extract) and the highest protection against 1O2 (12.5%). On the other hand, the ethanol extracts, which were the most vivid and yellow colour (C*ab = 13.7 and b* = 13.3), presented the highest level of total carotenoids (0.1 mg/g), but low scavenging capacity against ROO• (0.01 mmol trolox equivalent/g extract). Conclusion Based on these results and depending on the applicability, the ethanol:water, water and ethanol are the most promising solvents to obtain C. villosum extracts with high contents of bioactive compounds, ROO• scavenging capacity and protection against 1O2. Copyright © 2013 John Wiley & Sons, Ltd.

This work investigated the effects of irradiation (0, 1 and 2 kGy) on the content of bioactive compounds such as vitamin C and carotenoids with provitamin A activity in arugula during the storage at 5±1 °C for up to 13 and 16 days, respectively. The vitamin C content decreased in non-irradiated as well as irradiated (1 and 2 kGy) samples during the storage period. On the other hand, no significant change in the content of carotenoids with provitamin A activity was observed after irradiation or storage period. Thus, the irradiation had minimal detrimental effects on the contents of carotenoids in arugula.

The composition of carotenoids from murici fruit ( Byrsonima crassifolia ), a fruit native to North and Northeast regions of Brazil, was determined by high-performance liquid chromatography coupled to diode array and mass spectrometry detectors (HPLC-DAD-MS/MS). Sixteen compounds were found in murici and 13 carotenoids were identified or tentatively identified. The major carotenoids were (all- E )-lutein (17.3 ± 1.2 μg/g of dry weight) and (all- E )-zeaxanthin (3.5 ± 0.2 μg/g of dry weight). Xanthophylls were the predominant carotenoids in murici, representing 94% (w/w) of the total carotenoids. The carotenoid extract was shown to be a potent scavenger of peroxyl radical, being almost 13 times more potent than α-tocopherol. To the best of our knowledge, it is the first time that the detailed description of the carotenoid composition of murici is reported.

Various species of the genus Passiflora have been extensively used in traditional medicine as sedatives, anxiolytics, diuretics and analgesics. In the present study, after the identification and quantification of phytochemical compounds from yellow passion fruit pulp by liquid chromatography-photodiode array-mass spectrometry (HPLC-PDA-MS/MS), its antihypertensive effect was investigated on spontaneously hypertensive rats. Additionally, the renal function, evaluated by kidney/body weight, serum creatinine, proteinuria, urinary flow, reduced glutathione (GSH) levels and thiobarbituric acid-reactive substances (TBARS) and mutagenicity in bone marrow cells were assessed to evaluate the safety of passion fruit consumption. Yellow passion fruit pulp (5, 6 or 8 g/kg b.w.) was administered by gavage once a day for 5 consecutive days. HLPC-PDA-MS/MS analysis revealed that yellow passion fruit pulp contains phenolic compounds, ascorbic acid, carotenoids and flavonoids. The highest dose of passion fruit pulp significantly reduced the systolic blood pressure, increased the GSH levels and decreased TBARS. There were no changes in renal function parameters or the frequency of micronuclei in bone marrow cells. In conclusion, the antihypertensive effect of yellow passion fruit pulp, at least in part, might be due to the enhancement of the antioxidant status. The exact mechanisms responsible by this effect need further investigation.

The consumption of lutein is associated with the prevention and reduction of age-related macular degeneration. Its incorporation into Prato cheese as a yellowish food coloring is a valid alternative to increase the daily intake of this compound. However, part of the lutein added may be lost in the whey during the cheese making, or it can be degraded by light during storage, resulting in color changes reducing the sensory acceptance of the cheese. The objectives of this study were to determine the transference of the lutein (dye), added to the milk, in the whey, and cheese, to evaluate the effect of the lutein addition, light exposure, and storage time on the cheese color, and to verify the sensory acceptance of Prato cheese with addition of lutein. The lutein recovery of cheese was 95.25%. Color saturation (chrome) increased during storage time resulting in a cheese with more intense color, but there were no changes in the hue of the cheese. Adjusting the amount of lutein added to Prato cheese may lead to greater acceptance. The high recovery of lutein in the cheese and the fact that the hue remained unchanged during storage under light showed that the incorporation of lutein into Prato cheese is feasible from a technical point of view. A ingestão de luteína está associada à prevenção e redução da degeneração macular relacionada à idade e a sua adição em queijo Prato, como corante de tom amarelado, é uma alternativa válida para aumentar a ingestão diária desse composto. Entretanto, parte da luteína adicionada pode ser perdida no soro durante a fabricação do queijo e também, ser degradada pela ação da luz durante armazenamento, resultando em mudanças na cor dos queijos, diminuindo a aceitação sensorial. O objetivo do trabalho foi determinar a transferência do corante luteína, adicionado ao leite, no soro e queijo, avaliar o efeito da adição de luteína, exposição à luz e tempo de armazenamento na cor do queijo e verificar a aceitação sensorial do queijo com luteína. A recuperação de luteína no queijo foi de 95,25%. A saturação da cor aumentou com o tempo de armazenamento, resultando em queijos com coloração mais intensa, porém a tonalidade dos queijos não sofreu alteração. Ajustes no teor de luteína adicionado ao queijo Prato pode levar a uma maior aceitação. A alta recuperação de luteína no queijo e a manutenção da cor durante o armazenamento sob luz mostrou que a adição de luteína ao queijo Prato é viável do ponto de vista técnico.

A micro-assay was developed and validated, using a microplate reader in 96-well format, C(11)-BODIPY(581/591) as fluorescent probe and AIBN as ROO() generator. The structure-activity relationship was established for 15 carotenoid standards, indicating that the opening of the β-ionone ring and the increase of chromophore extension in the carotenoid structure were the major factors leading to the increase of ROO() scavenging capacity. The values for ROO() scavenging capacity were calculated using α-tocopherol as reference compound. Among the studied carotenoids, all-trans-lycopene was the most efficient ROO() scavenger (8.67±0.74) followed by all-trans-astaxanthin (6.50±0.62). All the carotenoids showed to be more effective ROO() scavengers than α-tocopherol and some hydrophilic compounds. Finally, the method was successfully applied to assay the ROO() scavenging capacity of carotenoid extracts from two Amazonian fruits, peach palm (7.83±0.21) and mamey (6.90±0.44).

Caryocar villosum (piquia) is a native fruit from the Amazonian region, considered to be an interesting source of bioactive compounds. In this paper, five extracts of C. villosum pulp were obtained, using solvents with different polarities and their in vitro scavenging capacity against reactive oxygen species (ROS) and reactive nitrogen species (RNS) was determined. Additionally, the phenolic compounds and carotenoids in each extract were identified and quantified by a high performance liquid chromatography coupled to diode array and mass spectrometer detectors (HPLC–DAD–MS/MS). The ethanol/water and water extracts, which presented the highest phenolic contents (5163 and 1745 μg/g extract, respectively), with ellagic acid as the major phenolic compound, proved to have the highest ROS and RNS scavenging potential. Nevertheless, in general, ellagic acid was less effective in scavenging ROS (IC 50 from 1.7 to 108 μg/ml) and RNS (IC 50 from 0.05 to 0.59 μg/ml), when compared to gallic acid (IC 50 from 0.4 to 226 μg/ml for ROS and IC 50 from 0.04 to 0.12 μg/ml for RNS). The results obtained in the present study clearly demonstrated that the in vitro antioxidant efficiency of C. villosum extracts was closely related to their contents of phenolic compounds.

Carotenoids are naturally found in both free form and esterified with fatty acids in most fruits; however, up to now the great majority of studies only evaluated their composition after saponification. This fact is easily explained by the difficult to analyze carotenoid esters. Preliminary studies showed that cleanup procedures in the extract are necessary for further analysis by LC-MS/MS since triacylglycerols (TAGs) impair the MS detection. Considering these facts, we developed a new cleanup procedure to remove TAGs and other lipids from carotenoid fruit extracts. This procedure is based on physical removal of solid lipids at low temperature followed by open column chromatography on MgO and diatomaceous earth. Before cleanup, four carotenoid diesters and two free xanthophylls were identified in murici (Byrsonyma crassifolia), corresponding to about 65% of the total chromatogram area. After carrying out the two-step cleanup procedure, 35 carotenoids were identified, being 14 monoesters, six free carotenoids and 15 carotenoid diesters. We can conclude that this two-step procedure was successfully applied to murici, an Amazonian fruit, which contains high amounts of lipids.

Chrysobalanus icaco L. is an underexplored plant found in tropical areas around the globe. Currently, there is no apparent information regarding the effects C. icaco fruits may exert in vivo or potential role in health promotion. This study aimed at providing evidence regarding the in vivo influence of this fruit on antigenotoxicity, antimutagenicity, and oxidative stress in rats. Male Wistar rats were treated with 100, 200, or 400 mg/kg body weight (bw)/d C. icaco fruit for 14 d. Doxorubicin (DXR, 15 mg/kg bw, ip) was used for DNA damaging and as an oxidant to generate reactive oxygen species (ROS). Genomic instability was assessed by the comet assay and micronucleus (MN) test, while antioxidant activity was determined by oxidative burst of neutrophils. Chrysobalanus icaco fruit polyphenols were quantified and characterized by high-performance liquid chromatography coupled to a diode array detector and tandem mass spectrometer (HPLC-DAD-MS/MS). The concentrations of 19 chemical elements were determined by inductively coupled plasma–mass spectroscopy (ICP-MS). Significant amounts of polyphenols, magnesium, and selenium were found in C. icaco fruit. This fruit displayed in vivo antioxidant activity against DXR-induced damage in rat peripheral blood neutrophils, antigenotoxicity in peripheral blood cells, and antimutagenicity in bone-marrow cells and peripheral blood cells. Correlation analyses between endpoints examined indicated that the mechanism underlying chemopreventive actions of C. icaco fruit was attributed to inhibition of NADPH oxidase complex manifested as low levels of DNA damage in animals exposed to DXR. Data indicate that phytochemicals and minerals in C. icaco fruit protect DNA against damage in vivo associated with their antioxidant properties.

The β-carotene, α-carotene, β-cryptoxanthin, lycopene, lutein, zeaxanthin, neoxanthin, violaxanthin concentrations in raw fruits and vegetables were compiled (19 fruits, 24 vegetables, and mixed fruit and salad dishes), using data from foods collected and analyzed in Brazil, by means of high-performance liquid chromatography with diode array detection. The food intake was obtained from the Food Consumption Module of the Household Budget Survey (2008–2009), recorded in 13569 households, representative of all Brazilian regions. Vegetables contributed more to vitamin A intake than fruits (70.3% vs 21.5% retinol equivalents), raw salads, pumpkin, kale and carrot being the main contributors. The mean dietary intakes of fruits (86 g/d) and vegetables (64 g/d) did not meet the recommendation of the WHO/FAO. All food items supplied β-carotene (0.9 mg/p/d), the major contributors being kale, pumpkin and mango. β-Cryptoxanthin (0.1 mg/p/d) was mostly supplied by orange, tangerine and papaya (87.4% of its intake). Lycopene (0.7 mg/p/d) was found only in guava, watermelon and tomato. Lutein (0.8 mg/p/d), violaxanthin and neoxanthin (0.6 and 0.2 mg/p/d, respectively), were mainly supplied by green leafy vegetables and α-carotene (0.16 mg/p/d) and zeaxanthin (0.06 mg/p/d) by a small number of foods., AZM and BOA acknowledge the financial support from São Paulo Research Foundation (FAPESP, proc. 13/50789-0) from Brasil and the program iLINK+ for the promotion of scientific international collaboration of CSIC with foreign institutions (I-LINK0770) from the Spanish National Research Council (CSIC). A. Z. Mercadante, B. Olmedilla-Alonso and L. Vargas-Murga are members of the IBERCAROT network, financed by the Ciencia y Tecnología para el Desarrollo (CYTED) program (grant no. P111RT0247).

The antioxidant capacities of gum arabic and maltodextrin microcapsules containing antioxidant molecules (trolox, α-tocopherol, β-carotene, apo-8'-carotenal and apo-12'-carotenal) against reactive oxygen and nitrogen species were evaluated. The scavenging capacities were influenced by the wall material, the reactive species, namely ROO(), H(2)O(2), HO(), HOCl and ONOO(-), and the antioxidant molecule. In general, a more pronounced enhancement of the antioxidant capacity due to incorporation of antioxidant molecules was observed in gum arabic microcapsules. The empty microcapsules showed capacity to scavenge all the studied ROS and RNS, being gum arabic a more potent antioxidant than maltodextrin. Apo-8'-carotenal incorporation promoted the highest increase in the scavenging capacities among the evaluated antioxidants, varying from 50% to 132% and from 39% to 85% for gum arabic and maltodextrin microcapsules, respectively, suggesting that this carotenoid presented the best balance between the molecule localization inside the microcapsules and the reactivity against the specific reactive species.