Enhanced water dispersibility and permeability through a Caco-2 cell monolayer of ß-cryptoxanthin extracted from kumquats by complexation with casein.

ß-Cryptoxanthin (BCX) possesses potential therapeutic and health benefits. However, BCX absorption is low because of its poor aqueous solubility. In this study, a complex between BCX and casein (Cas) was prepared to improve the water dispersibility and bioavailability of BCX. BCX was recovered quantitatively from freeze-dried kumquat powder through solid-liquid extraction and saponification. The complexation significantly improved the apparent solubility of BCX under acidic and neutral conditions. A cell membrane permeation test using a Caco-2 cell monolayer was performed to evaluate the bioavailability of the BCX-Cas complex. This complex and a blank sample were digested in vitro and added to the apical side of the Caco-2 cell membrane. The quantity of BCX that permeated using the BCX-Cas complex after 24 h was 22.7 times greater than that of the blank. Thus, complexation of BCX with Cas improved dramatically the bioavailability of BCX from a kumquat extract.

Low expression of carotenoids cleavage dioxygenase 1 (ccd1) gene improves the retention of provitamin-A in maize grains during storage.

Provitamin-A (proA) is essentially required for vision in humans but its deficiency affects children and pregnant women especially in the developing world. Biofortified maize rich in proA provides new opportunity for sustainable and cost-effective solution to alleviate malnutrition, however, significant loss of carotenoids during storage reduces its efficacy. Here, we studied the role of carotenoid cleavage dioxygenase 1 (ccd1) gene on degradation of carotenoids in maize. A set of 24 maize inbreds was analyzed for retention of proA during storage. At harvest, crtRB1-based maize inbreds possessed significantly high proA (ß-carotene: 12.30 µg/g, ß-cryptoxanthin: 4.36 µg/g) than the traditional inbreds (ß-carotene: 1.74 µg/g, ß-cryptoxanthin: 1.28 µg/g). However, crtRB1-based inbreds experienced significant degradation of proA carotenoids (ß-carotene: 20%, ß-cryptoxanthin: 32% retention) following 5 months. Among the crtRB1-based genotypes, V335PV had the lowest retention of proA (ß-carotene: 1.63 µg/g, ß-cryptoxanthin: 0.82 µg/g), while HKI161PV had the highest retention of proA (ß-carotene: 4.17 µg/g, ß-cryptoxanthin: 2.32 µg/g). Periodical analysis revealed that ~ 60-70% of proA degraded during the first three months. Expression analysis revealed that high expression of ccd1 led to low retention of proA carotenoids in V335PV, whereas proA retention in HKI161PV was higher due to lower expression. Highest expression of ccd1 was observed during first 3 months of storage. Copy number of ccd1 gene varied among yellow maize (1-6 copies) and white maize (7-35 copies) while wild relatives contained 1-4 copies of ccd1 gene per genome. However, copy number of ccd1 gene did not exhibit any correlation with proA carotenoids. We concluded that lower expression of ccd1 gene increased the retention of proA during storage in maize. Favourable allele of ccd1 can be introgressed into elite maize inbreds for higher retention of proA during storage.

Characterization of ß-Cryptoxanthin and Other Carotenoid Derivatives from Rhodotorula taiwanensis, A Novel Yeast Isolated from Traditional Starter Culture of Assam.

The present investigation was intended to characterize pigments for the first time from Rhodotorula taiwanensis (LC011412) yeast isolated from the ethic fermentation starter culture source meant to evaluate its carotenoid contents for beneficial applications. The pigments were extracted by an optimized solvent system, purified by flash chromatography and were identified by TLC and UV/VIS spectroscopy. The absorbance spectra confirmed the presence of ß-carotene, ß-cryptoxanthin, torulene and torularhodin that showed maximum absorbance (λmax ) within the ranges. The fractions were further characterized by LC/MS and analyzed through FT-IR and NMR for structure elucidation. Spectral analyses also confirmed the presence of the compounds mentioned above. These compounds promise great commercial value and could be useful for large scale production anticipated for potential applications in food, nutraceutical, pharmaceutical and cosmetic sectors. It is pertinent that the characterized carotenoid pigments from the isolate have incredible prospects in industrial applications which require profound attention.

Free carotenoids and carotenoids esters composition in Spanish orange and mandarin juices from diverse varieties.

The carotenoid profiles of citrus fruit have been well studied, but knowledge about the xanthophylls esters pattern is more limited. In this study, the native carotenoid profiles of mandarin and orange juices were studied. Xanthophylls in oranges were esterified with lauric, myristic, palmitic and stearic acids. The fatty acids esterifying ß-cryptoxanthin in mandarins were lauric, myristic, palmitic and capric acid. In oranges, the average relationships of total free carotenoids to the sum of mono-esters and di-esters were 0.82 and 1.67, respectively, and the average relationships total mono-esters to total of di-esters was 2.51. In general, the mono-ester fraction was the most significant quantitatively. In mandarins, the average relationship of total free carotenoids to the sum of mono-esters was 0.17, which is in line with the fact that ß-cryptoxanthin was mostly esterified and not free (unesterified).

Esterified carotenoids as new food components in cyanobacteria.

Among the nutritional properties of microalgae, this study is focused in the presence of carotenoid esters in prokaryote microalgae, an event that has not been shown so far. Three carotenoid esters that accumulate in non-stressful culture conditions are identified in Aphanotece microscopica Nägeli and Phormidum autumnale Gomont, what may provide an extra value to the quality attributes of the carotenoid profile in cyanobacteria as functional foods. In addition, new data on the carotenoid characterization added quality criteria for the identification of the esterified metabolites, enabling the monitoring of these food components. Specifically, the metabolomic approach applied to the food composition analysis, has allowed to differentiate between the esters of zeinoxanthin and ß-cryptoxanthin, which were undifferentiated to date during the MS characterization of carotenoids in other food sources. We propose a new qualifier product ion specific for zeinoxanthin ester, which it is not present in the MS2 spectrum of ß-cryptoxanthin esters.

Immunostimulatory effect of kumquat (Fortunella crassifolia) and its constituents, ß-cryptoxanthin and R-limonene.

Natural killer (NK) cells play an important role in the innate immune system by eliminating cancer cells and virally infected cells. Aging and stress attenuate the activity of NK cells, thereby increasing the risk of various diseases. In this study, we demonstrated that the consumption of a small number of kumquats in an in vivo model could suppress elevated plasma corticosterone levels and reverse the decline in splenocyte cytotoxicity caused by restraint stress. Our results identified ß-cryptoxanthin (BCX) as an active kumquat component with a NK cell-activating effect, and R-limonene as an active component that mediates not only the anti-stress effect but also NK cell activation by oral administration. In addition, BCX, R-limonene, and R-limonene metabolites were found to enhance IFN-γ production in KHYG-1 cells, a human NK cell line. Collectively, our findings suggest that the ingestion of a few kumquats on a daily basis can help to combat stress and enhance NK cell activity.

Transformation of naturally occurring (3R,3'R,6'R)-lutein and its fatty acid esters to (3 R)-ß-cryptoxanthin and (3R,6'R)-α-cryptoxanthin.

The objective of this study was to develop straightforward processes that could be applied to the large-scale production of ß-cryptoxanthin in an attempt to facilitate investigation of its biological activity. An oleoresin obtained from crude extracts of marigold flowers (Tagetes erecta) with approximately 24% total lutein fatty acid ester content was directly used as starting material for partial synthesis of (3 R)-ß-cryptoxanthin under mild reaction conditions at ambient temperature. Therefore, acid-catalyzed deoxygenation of lutein esters from marigold oleoresin followed by hydrogenation in the presence of catalytic amount of platinum (Pt) supported on alumina (5%) at ambient temperature gave a mixture of (3 R)-ß-cryptoxanthin fatty acid esters (major) and (3 R,6'R)-α-cryptoxanthin fatty acid esters (minor). Saponification and Z-to-E isomerization of the product followed by crystallization gave a mixture of (3 R)-ß-cryptoxanthin as the major product. Similarly, acid-catalyzed hydrogenation of unesterified (3 R,3'R,6'R)-lutein with Pt/alumina in ethyl acetate gave a mixture of (3 R,6'R)-α-cryptoxanthin acetate (minor) in a one-pot reaction. Alkaline hydrolysis and Z-to-E isomerization of the mixture followed by crystallization provided (3 R)-ß-cryptoxanthin.

The Biochemical Basis of Vitamin A Production from the Asymmetric Carotenoid ß-Cryptoxanthin.

Vitamin A serves essential functions in mammalian biology as a signaling molecule and chromophore. This lipid can be synthesized from more than 50 putative dietary provitamin A precursor molecules which contain at least one unsubstituted ß-ionone ring. We here scrutinized the enzymatic properties and substrate specificities of the two structurally related carotenoid cleavage dioxygenases (CCDs) which catalyze this synthesis. Recombinant BCO1 split substrates across the C15,C15' double bond adjacent to a canonical ß-ionone ring site to vitamin A aldehyde. Substitution of the ring with a hydroxyl group prevented this conversion. The removal of methyl groups from the polyene carbon backbone of the substrate did not impede enzyme activity. Homology modeling and site-directed mutagenesis identified amino acid residues at the entrance of the substrate tunnel, which determined BCO1's specificity for the canonical ß-ionone ring site. In contrast, BCO2 split substrates across the C9,C10 double bond adjacent to assorted ionone ring sites. Kinetic analysis revealed a higher catalytic efficiency of BCO2 with substrates bearing 3-hydroxy-ß-ionone rings. In the mouse intestine, the asymmetric carotenoid ß-cryptoxanthin with one canonical and one 3-hydroxy-ß-ionone ring site was meticulously converted to vitamin A. The tailoring of this asymmetric substrate occurred by a stepwise processing of the carotenoid substrate by both CCDs and involved a ß-apo-10'-carotenal intermediate. Thus, opposite selectivity for ionone ring sites of the two mammalian CCDs complement each other in the metabolic challenge of vitamin A production from a chemically diverse set of precursor molecules.

Bioaccessibility of provitamin A carotenoids from fruits: application of a standardised static in vitro digestion method.

Provitamin A carotenoids (ß-carotene, α-carotene, and ß-cryptoxanthin) contribute to the dietary intake of vitamin A and are associated with decreased risk of many chronic diseases. Besides their contents in foods, their bioaccessibility is of great interest since it represents the amount that will be absorbed in the gut. The aim of this study was to adopt, for the first time, the in vitro digestion model suitable for food, proposed in a consensus paper by Minekus et al. (2014), to assess the bioaccessibility of carotenoids from the fruits that are the major contributors to the intake of ß-cryptoxanthin in Spain (orange, tangerine, red pepper, peach, watermelon, and persimmon) and loquat. The highest ß-cryptoxanthin content and the lowest bioaccessibility was found in mandarin and loquat (13331.6 and 929.2 µg per 100 g respectively), whereas the highest contents of ß-carotene and α-carotene were recorded in red pepper (1135.3 and 90.4 µg per 100 g respectively). The bioaccessibility of ß-cryptoxanthin was similar to that of ß-carotene (0.02-9.8% and 1-9.1%, respectively) and was higher than that of ß-carotene in red pepper, watermelon and peach. α-Carotene bioaccessibility ranged between 0% and 4.6%. We discuss the critical factors for comparing our data: the form of the food being analyzed (raw/cooked/previously frozen, in the presence or absence of oil/fat) and the protocol for bioaccessibility assessment. Different food processing techniques may increase carotenoid bioaccessibility compared to raw food. However, given the difficulties encountered when comparing the results of studies on bioaccessibility, it seems logical to propose the application of the previously mentioned standardized in vitro protocol.