Generating high purity and yield of capsanthin and capsorubin carrot germplasm through synthetic metabolic engineering.

Capsanthin and capsorubin are red κ-xanthophylls exclusively found in a handful of other plant species. Currently, capsanthin and capsorubin are only extracted from red pepper. Here, high purity production of capsanthin and capsorubin has been achieved in carrot taproot by synthetic metabolic engineering strategy. Expression of a capsanthin-capsorubin synthase gene (CaCCS) from pepper resulted in dominant production of capsanthin whereas expression of a LiCCS gene from tiger lily resulted in production of both capsanthin and capsorubin in carrot taproot. The highest content of capsanthin and capsorubin was obtained in LiC-1 carrot taproot hosting the LiCCS gene, 150.09 µg/g DW (dry weight). Co-expression of DcBCH1 with CCS could improve the purity of capsanthin and capsorubin by eliminating the non-target carotenoids (eg. α-carotene and ß-carotene). The highest purity of capsanthin and capsorubin was obtained in BLiC-1 carrot taproot hosting DcBCH1+LiCCS genes, 91.10% of total carotenoids. The non-native pigments were esterified partially and stored in the globular chromoplast of carrot taproot. Our results demonstrated the possibility of employing carrot taproot as green factories for high purity production of capsanthin and capsorubin. The capsanthin/capsorubin carrot germplasms were also valuable materials for breeding colorful carrots cultivars.

Effect of Ultrasound-Assisted Extraction of Carotenoids from Papaya (Carica papaya L. cv. Sweet Mary) Using Vegetable Oils.

By-products from fruits and are of great interest for their potential use in the food industry due to their high content of bioactive compounds. Herein, we examined the ultrasound-assisted extraction (UAE) of carotenoid and carotenoid esters from papaya pulp and peel using soybean oil and sunflower oil as alternative green solvents. Response surface methodology (RSM) was established to optimize the UAE process. Three independent variables, ultrasonic amplitude (20-60%), time (10-60 min), and co-solvent percentage (ethanol) (5-20%, v/v), were applied. The highest total carotenoid content in the UAE extracts was obtained from papaya pulp extracts (58.7 ± 1.6 and 56.0 ± 1.5 µg carotenoids/g oil) using soybean oil and sunflower oil, respectively (60% amplitude/ 10 min/ 20% ethanol). On the other hand, the highest carotenoid content (52.0 ± 0.9 µg carotenoids/g oil) was obtained from papaya peel using soybean oil applying the UAE process (20% amplitude/ 77 min/ 20% ethanol); a minor content of 39.3 ± 0.5 µg carotenoids/g oil was obtained from papaya peel using sunflower oil at 60% amplitude/ 60 min/ 5% ethanol. Lycopene was the most abundant carotenoid among all individual carotenoids observed in papaya oil extracts, obtaining the highest yields of this carotenoid when papaya pulp and peel were extracted using soybean oil (94% and 81%, respectively) and sunflower oil (95% and 82%, respectively). Great extraction of xanthophyll esters was detected using 20% of ethanol in the vegetable oil extraction solvent (v/v). High correlations (>0.85) was obtained between total carotenoid content and color determination in the UAE oil extracts. UAE vegetable oil extracts enriched with carotenoids from papaya by-products could be useful to formulate new food ingredients based on emulsions with interesting potential health benefits.

An optimal saponification and extraction method to determine carotenoids in avocado.

Quantification of carotenoids in avocado fruit is a great challenge due to their co-extraction with high-oil concentration and the inherent nature of carotenoids to degrade and undergo cis/trans photoisomerization with prolonged extraction times and high temperatures. The study provides an optimised and validated methodology for quantification of carotenoids in the high-oil avocado matrix, with > 93% recovery of all carotenoids tested being significantly greater than previously published. Saponification with 15% KOH for 60 min was optimal for the avocado matrix. For the first time, this study identified that soap, produced during the saponification reaction, resulted in a significant reduction of carotenoid content from the avocado matrix, due to the production of micelles. A significantly higher carotenoid content (3.58 versus 2.0 mg/100 g DW) was able to be extracted after saponification with acidified phosphate buffer, instead of water as reported previously. Carotenoid profiles of five avocado cultivars were identified and quantified.

The (Bio)chemical Base of Flower Colour in Bidens ferulifolia.

Bidens ferulifolia is a yellow flowering plant, originating from Mexico, which is increasingly popular as an ornamental plant. In the past few years, new colour combinations ranging from pure yellow over yellow-red, white-red, pure white and purple have emerged on the market. We analysed 16 Bidens ferulifolia genotypes to provide insight into the (bio)chemical base underlying the colour formation, which involves flavonoids, anthochlors and carotenoids. In all but purple and white genotypes, anthochlors were the prevalent pigments, primarily derivatives of okanin, a 6'-deoxychalcone carrying an unusual 2'3'4'-hydroxylation pattern in ring A. The presence of a cytochrome-P450-dependent monooxygenase introducing the additional hydroxyl group in position 3' of both isoliquiritigenin and butein was demonstrated for the first time. All genotypes accumulate considerable amounts of the flavone luteolin. Red and purple genotypes additionally accumulate cyanidin-type anthocyanins. Acyanic genotypes lack flavanone 3-hydroxylase and/or dihydroflavonol 4-reductase activity, which creates a bottleneck in the anthocyanin pathway. The carotenoid spectrum was analysed in two Bidens genotypes and showed strong variation between the two cultivars. In comparison to anthochlors, carotenoids were present in much lower concentrations. Carotenoid monoesters, as well as diesters, were determined for the first time in B. ferulifolia flower extracts.

Effect of High Hydrostatic Pressure on the Extractability and Bioaccessibility of Carotenoids and Their Esters from Papaya (Carica papaya L.) and Its Impact on Tissue Microstructure.

High hydrostatic pressure (HHP) is a non-thermal technology widely used in the industry to extend food shelf-life and it has been proven to enhance the extractability of secondary metabolites, such as carotenoids, in plant foods. In this study, fresh-cut papaya pulp of varieties (Sweet Mary, Alicia and Eksotika) from the Canary Islands (Spain) were submitted to the HHP process (pressure: 100, 350 and 600 MPa; time: come-up time (CUT) and 5 min) to evaluate, for the first time, individual carotenoid and carotenoid ester extractability and to assess their bioaccessibility using an in vitro simulated gastrointestinal digestion assay, following the standardized INFOGEST® methodology. In addition, changes in papaya pulp microstructure after HHP treatments and during the different phases of the in vitro digestion were evaluated with optical light microscopy. HPLC-DAD (LC-MS/MS (APCI+)) analyses revealed that HHP treatments increased the carotenoid content, obtaining the highest extractability in pulp of the Sweet Mary papaya variety treated at 350 MPa during 5 min (4469 ± 124 µg/100 g fresh weight) which was an increase of 269% in respect to the HHP-untreated control sample. The highest carotenoid extraction value within each papaya variety among all HHP treatments was observed for (all-E)-lycopene, in a range of 98-1302 µg/100 g fresh weight (23-344%). Light micrographs of HHP-treated pulps showed many microstructural changes associated to carotenoid release related to the observed increase in their content. Carotenoids and carotenoid esters of papaya pulp submitted to in vitro digestion showed great stability; however, their bioaccessibility was very low due to the low content of fatty acids in papaya pulp necessary for the micellarization process. Further studies will be required to improve papaya carotenoid and carotenoid ester bioaccessibility.

Carotenoid and Carotenoid Ester Profile and Their Deposition in Plastids in Fruits of New Papaya (Carica papaya L.) Varieties from the Canary Islands.

The carotenoid profile of non-saponified and saponified extracts of different tissues (pulp and peel) of fruits of three new papaya varieties, Sweet Mary, Alicia, and Eksotika, was characterized for the first time, and almost all carotenoid compounds were quantified. Carotenoids and carotenoid esters were analyzed and characterized using HPLC-photo diode array (PDA-MS with atmospheric pressure chemical ionization with positive ion mode (APCI+) with a C30 reversed-phase column. The carotenoid deposition in collenchyma and chlorenchyma cells of papaya pulp and peel tissues was assessed by optical microscopy, confocal laser scanning microscopy, and transmission electron microscopy. The most abundant carotenoids in the fruit of the three papaya varieties (pulp and peel) were (all-E)-lycopene (230.0-421.2 µg/100 g fresh weight), (all-E)-ß-carotene (120.3-233.2 µg/100 g fresh weight), and (all-E)-ß-cryptoxanthin laurate (74.4-223.2 µg/100 g fresh weight. Moreover, high concentrations of (all-E)-lutein (922.5-1381.1 µg/100 g fresh weight) and its esters, such as (all-E)-lutein-3-O-myristate and (all-E)-lutein dimyristate, were found in peel extracts. The optical microscopy study of papaya pulps showed that carotenoid deposition in all papaya varieties, including Maradol, was mainly localized close to the cell walls, showing the presence of some crystalloids and round-shaped structures, with different sizes and distribution due to the different carotenoid content among varieties. No crystalloids or globular depositions were found in any of the peel sections, and no remarkable differences were found in the papaya peel microstructure of the different papaya varieties.

Carotenoid content of extruded and puffed products made of colored-grain wheats.

Wheat is a relevant source not only of essential macronutrients but also of many other health-promoting phytochemicals (carotenoids, anthocyanins, tocols, phenolic acids, etc.). Colored-grain wheats were used for extrusion and kernel puffing. The total content of carotenoids (sum of lutein, zeaxanthin, antheraxanthin, α- and ß-carotene, and xanthophyll esters) decreased significantly due to extrusion (to 25.7%) and puffing (to 31.6%), compared to the content in the raw material. Zeaxanthin was shown to be the most stable among all detected carotenoids (30.8 and 48.7% was preserved). The results of the performed analyses have not confirmed greater stability of xanthophyll esters against higher temperatures (decrease to 29.5 and 22.1%). Both technologies induced E-to Z-isomerization of all-E-lutein and puffing also of all-E-zeaxanthin. Higher concentrations of 13-Z- and 9-Z-zeaxanthin were identified in puffed grains (2× and 37× on average). To preserve more carotenoids, it is appropriate to look for a more suitable food processing technology.

Prospects for Carotenoid Biofortification Targeting Retention and Catabolism.

Due to the ongoing prevalence of vitamin A deficiency (VAD) in developing countries there has been a large effort towards increasing the carotenoid content of staple foods via biofortification. Common strategies used for carotenoid biofortification include altering flux through the biosynthesis pathway to direct synthesis to a specific product, generally ß-carotene, or via increasing the expression of genes early in the carotenoid biosynthesis pathway. Recently, carotenoid biofortification strategies are turning towards increasing the retention of carotenoids in plant tissues either via altering sequestration within the cell or via downregulating enzymes known to cause degradation of carotenoids. To date, little attention has focused on increasing the stability of carotenoids, which may be a promising method of increasing carotenoid content in staple foods.

Evaluation of the bioaccessibility of carotenoid esters from Lycium barbarum L. in nano-emulsions: A kinetic approach.

Lycium barbarum L., known as goji berry, is a rich source of carotenoid esters, which are mainly composed of zeaxanthin dipalmitate (ZDP), lutein palmitate (LP), ß-cryptoxanthin palmitate (ß-CP), zeaxanthin palmitate (ZP), zeaxanthin myristate palmitate (ZMP), and zeaxanthin palmitate stearate (ZPS). Oil-in-water nano-emulsions containing carotenoid esters from L. barbarum L. with olive oil (ON) and soybean oil (SN) were prepared to investigate the liberation and bioaccessibility (BA) of in vitro digestion. The particle sizes of ON and SN were approximately 160 nm stabilized with sucrose esters and monoacylglyceride as emulsifiers. ON presented an equal liberation of each carotenoid ester as SN, except that LP had a high value. Incorporation of carotenoid esters into the micelle were evaluated using a fractional conversion model, containing two phases, namely, a rapid growth rate for the first phase, and then reaching a plateau for the second phase. The kinetic rate was related to the particle size, oil type and carotenoid ester nature. BA at plateau values for ZDP and ZPS were higher than that of the four other carotenoid esters in SN. Considering the great improvement of the liberation and BA, the excipient nano-emulsion prepared in this study is a good delivery system for carotenoid esters from goji berry.

Characterization and the impact of in vitro simulated digestion on the stability and bioaccessibility of carotenoids and their esters in two Pouteria lucuma varieties.

Lucuma is a starchy orange-yellow fruit native to the Andean region. It is widely consumed in Latin America and has been recently adapted to the agronomical characteristics of the south region of Spain. However, its carotenoid profile has never been reported. The aim of this study was to characterize the carotenoid and carotenoid ester composition of lucuma pulps (var. Molina and Beltran) and assess their bioaccessibility with an in vitro simulated gastrointestinal digestion according to the INFOGEST® methodology. The carotenoid profile in lucuma pulps revealed a high qualitative diversity composed of 33 compounds, corresponding to 9 free xanthophylls, 9 hydrocarbon carotenes and 15 xanthophyll esters. (13Z)-violaxanthin, (all-E)-violaxanthin and (all-E)-antheraxanthin were the most abundant carotenoids in lucuma fruits and were naturally present as xanthophyll esters: (all-E)-antheraxanthin 3-O-palmitate, (all-E)-violaxanthin laurate and (all-E)-violaxanthin palmitate. Carotenoids were stable during in vitro digestion; however, their release from the food matrix was limited which contributed to their low bioaccessibility.

Marigold carotenoids: Much more than lutein esters.

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 ß-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.

Impact of high hydrostatic pressure and thermal treatment on the stability and bioaccessibility of carotenoid and carotenoid esters in astringent persimmon (Diospyros kaki Thunb, var. Rojo Brillante).

The carotenoid and carotenoid ester profile in astringent persimmon (Diospyros kaki Thunb., var. Rojo Brillante) was composed by 13 free xanthophylls, 8 hydrocarbon carotenes and 17 carotenoid esters. The stability and biaoccessibility of these carotenoids was determined by an adaptation of the INFOGEST protocol. Results showed that the stability of persimmon carotenoids ranged from 61 to 74%, depending on the digestion phase, being (all-E)-ß-cryptoxanthin and (all-E)-antheraxanthin 3-O-palmitate the most stable carotenoids. At the final step of the digestion (oral + gastric + duodenal phase), only traces of (all-E)-antheraxanthin, (all-E)-lutein and (all-E)-ß-cryptoxanthin were found in control samples due to the low efficiency of carotenoid micellization, which was affected by the high pectin content naturally present in persimmon tissues. Processing increased the overall carotenoid bioaccessibility to 54% in pressurized samples and to 25% in thermal treated ones. This effect depended on the processing technology as well as on the chemical structure of the carotenoid, being (all-E)-ß-cryptoxanthin and (all-E)-ß-cryptoxanthin laurate the most bioaccessible carotenoids in pressurized samples and (all-E)-ß-cryptoxanthin laurate and (all-E)-antheraxanthin the most bioaccessible ones in pasteurized ones.

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.

Screening of critical factors influencing the efficient hydrolysis of zeaxanthin dipalmitate in an adapted in vitro- digestion model.

As hydrolysis of carotenoid esters is believed to be highly efficient in vivo, their insufficient hydrolysis in in vitro-digestion models, particularly, regarding zeaxanthin diesters, is a current issue. Therefore, in this study, several factors related to the enzymatic hydrolysis were investigated in an adapted version of the standardized INFOGEST in vitro-digestion model, using zeaxanthin dipalmitate (ZDP) as a substrate. The results showed that pancreatic lipase was able to hydrolyze ZDP, whereas carboxyl ester lipase (CEL) substantially contributed to ZDP cleavage. Replacement of commonly used porcine with bovine bile extracts and the substitution of coffee creamer for soybean oil at identical fat contents both significantly improved hydrolysis efficiency and bioaccessibility of total zeaxanthin to better mimic in vivo conditions. Thus, bile and lipids selection for in vitro digestion of carotenoid esters was crucial. The combined use of coffee creamer, pancreatin, CEL, and bovine bile led to the highest hydrolysis efficiency of 29.5%.

Hydrolysis of carotenoid esters from Tagetes erecta by the action of lipases from Yarrowia lipolytica.

The present study was conducted to evaluate the feasibility of enzymatic hydrolysis of carotenoid esters from Tagetes erecta using lipases from the yeast of Yarrowia lipolytica, with the aim of obtaining free lutein. The optimal concentrations of seven nutrients, considering the production of lipases relative to biomass (Yp/x) as the response variable, were determined in flask fermentations. In addition, we studied the effect on hydrolysis of growing Y. lipolytica in the presence of the oleoresin of the marigold flower in flask and stirred tank. Furthermore, hydrolysis of the oleoresin using the lipases from this microorganism was compared with the hydrolysis using lipases from Rhizopus oryzae. Cultured in the presence of marigold oleoresin, Y. lipolytica showed an increase in free carotenoids of 12.41% in flask and 8.8% in stirred tank, representing a fourfold and a threefold increase compared to the initial value in the fermentation, respectively. When lipases from the supernatant from both microorganisms were used for only 14 h hydrolysis experiments, a slight increase was achieved compared to a blank. We concluded that carotenoid esters of the oleoresin could not be completely hydrolyzed in 14 h by these lipases, but that growing Y. lipolytica in the presence of marigold oleoresin gives until fourfold production of free carotenoids in 72 h fermentations.

Composition by LC-MS/MS of New Carotenoid Esters in Mango and Citrus.

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, ß-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 ß-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.

Carotenoid composition of berries and leaves from six Romanian sea buckthorn (Hippophae rhamnoides L.) varieties.

Berries and leaves from six varieties of Carpathians' sea buckthorn (Hippophae rhamnoides L., ssp. Carpatica) were analysed for their carotenoid composition (free and esterified) using a combination of HPLC-PAD, GC-MS and UHPLC-PAD-ESI-MS techniques. GC-MS techniques revealed the fatty acid profile specific for each berry variety, while targeted UHPLC-MS analysis identified the fatty acids involved in carotenoids esterification: palmitic (C16:0), myristic (C14:0) and stearic (C18:0). Total carotenoid content varied between 53 and 97 mg/100g dry weight in berries, and between 3.5 and 4.2mg/100g DW in leaves. The carotenoid di-esters represented the main fraction among berry varieties having zeaxanthin di-palmitate as major compound, while leaves contained only free carotenoids like lutein, ß-carotene, violaxanthin and neoxanthin. Principal component analysis identified the suitable carotenoid biomarkers characteristic for the Carpathians' sea buckthorn from Romania with contribution to their taxonomic classification and authenticity recognition.