A comprehensive review on carotenoids in foods and feeds: status quo, applications, patents, and research needs.

Carotenoids are isoprenoids widely distributed in foods that have been always part of the diet of humans. Unlike the other so-called food bioactives, some carotenoids can be converted into retinoids exhibiting vitamin A activity, which is essential for humans. Furthermore, they are much more versatile as they are relevant in foods not only as sources of vitamin A, but also as natural pigments, antioxidants, and health-promoting compounds. Lately, they are also attracting interest in the context of nutricosmetics, as they have been shown to provide cosmetic benefits when ingested in appropriate amounts. In this work, resulting from the collaborative work of participants of the COST Action European network to advance carotenoid research and applications in agro-food and health (EUROCAROTEN, www.eurocaroten.eu, https://www.cost.eu/actions/CA15136/#tabs|Name:overview) research on carotenoids in foods and feeds is thoroughly reviewed covering aspects such as analysis, carotenoid food sources, carotenoid databases, effect of processing and storage conditions, new trends in carotenoid extraction, daily intakes, use as human, and feed additives are addressed. Furthermore, classical and recent patents regarding the obtaining and formulation of carotenoids for several purposes are pinpointed and briefly discussed. Lastly, emerging research lines as well as research needs are highlighted.

The first harmonised total diet study in Portugal: Nitrate occurrence and exposure assessment.

A harmonised TDS methodology was used to assess the Portuguese population's nitrate baseline dietary exposure and the risk of exceeding the Acceptable Daily Intake (ADI). Nitrate occurrence in 164 TDS samples pooled based on 20 FoodEx2 groups, representative of 18 to 75 years old population diet, was determined using UV-HPLC. The 'vegetables' group had the highest mean nitrate concentration (353 mg/kg), followed by 'starchy roots' (62 mg/kg), 'composite dishes' (53 mg/kg) and 'fruits' (46 mg/kg). Lettuce contained the most nitrates (1729 mg/kg). The estimated mean nitrate exposure of 1.17 mg/kg bw/day, with 3.18% of individuals exceeding the ADI, was assessed on a semi-probabilistic approach using the MCRA software. Lettuce (28%) contributed most to nitrate exposure followed by vegetable soup (13%). Processed meat contribution to exposure was 0.86% and 2.44% (overall population and consumers only). At population level the nitrate estimated exposure (mean and 95th percentile) revealed no reason for concern.

The first harmonised total diet study in Portugal: Planning, sample collection and sample preparation.

The first Total Diet Study (TDS) was carried out in Portugal using harmonised procedures developed under the TDS-Exposure project. This publication describes in detail the TDS planning, sample collection and preparation, filling a gap found in literature. TDS food list (1070 items) by food group for 18-74 years olds was derived using 24 h recall food consumption survey data. Of the 164 TDS samples defined based on FoodEx2, the most represented belonged to 'Composite dishes' (21%) and 'Fish and Fish products' (15%). A total of 227 TDS samples (12 subsamples each), were collected (2014-2016) in Greater Lisbon region as 126 'national', 17 'regional non-seasonal' and 21 'regional seasonal'. Seasonal samples were mostly fruits, vegetables and some fish. Subsamples were prepared as consumed, pooled, homogenised and stored at -18 °C. The harmonised procedures used allow comparison of exposure assessment with other countries and evaluation of exposure trends if TDS are used.

European Database of Carotenoid Levels in Foods. Factors Affecting Carotenoid Content.

Many studies indicate that diets including carotenoid-rich foods have positive effects on human health. Some of these compounds are precursors of the essential nutrient vitamin A. The present work is aimed at implementing a database of carotenoid contents of foods available in the European market. Factors affecting carotenoid content were also discussed. Analytical data available in peer-reviewed scientific literature from 1990 to 2018 and obtained by HPLC/UHPLC were considered. The database includes foods classified according to the FoodEx2 system and will benefit compilers, nutritionists and other professionals in areas related to food and human health. The results show the importance of food characterization to ensure its intercomparability, as large variations in carotenoid levels are observed between species and among varieties/cultivars/landraces. This highlights the significance of integrating nutritional criteria into agricultural choices and of promoting biodiversity. The uncertainty quantification associated with the measurements of the carotenoid content was very rarely evaluated in the literature consulted. According to the EuroFIR data quality evaluation system for food composition tables, the total data quality index mean was 24 in 35, reflecting efforts by researchers in the analytical methods, and less resources in the sampling plan documentation.

Uncertainty estimation and in-house method validation of HPLC analysis of carotenoids for food composition data production.

The method for separation and quantitative determination of the main carotenoids in food by high-performance liquid chromatography (HPLC) was in-house validated. Tomato (Lycopersicon esculentum M.) as food matrix was used to demonstrate its linearity, repeatability, intermediate precision, detection and quantification limits, sensitivity and bias. In addition, stability of carotenoids was studied in function of temperature and time. Method accuracy was quantified through measurement uncertainties estimate based on this validation study. Furthermore, a study was conducted to evaluate variability coming from location in an experimental field composed by 12 subfields. The use of two metal free reverse phase columns and an organic mobile phase based on acetonitrile, methanol and dichloromethane enabled the separation of the six target compounds (trans-α-carotene, trans-ß-carotene, ß-cryptoxanthin, all-lycopene, lutein, zeaxanthin) within a 30min run; detection at 450nm and external calibration allowed the quantification of the analytes. Carotenoids concentration and measurement uncertainty, in mg/100g, in tomato varieties "lido" and "for salad" were, respectively, 1.0±0.14 and 0.39±0.056 for trans-ß-carotene, 8±2.0 and 2.3±0.57 for all-lycopene and 0.10±0.017 and 0.08±0.015 for lutein; trans-α-carotene, ß-cryptoxanthin and zeaxanthin were not detected in both varieties (detection limits, in µg/100g, 0.81, 0.57 and 0.77, respectively). For ß-carotene and lutein, uncertainty associated with the entire process including small-scale within-region variation was statistically different, at a significance level of 5%, from measurement uncertainty (which includes sampling in the laboratory).

Comprehensive Database of Carotenoid Contents in Ibero-American Foods. A Valuable Tool in the Context of Functional Foods and the Establishment of Recommended Intakes of Bioactives.

Foods that are commonly consumed in the diet are considered to provide more than 40 different carotenoids. However, the content in carotenoids varies considerably in both qualitative and quantitative terms as a consequence of different genotypes, climatic conditions of the production area, and agronomic factors, among others. In this paper, analytical data, obtained by HPLC or UHPLC, of carotenoids in fruits and vegetables produced in Ibero-America have been compiled from peer-reviewed journals, organized in food categories, and documented in relation to the sampling and analytical quality system used. In addition to common products of the diet of the Ibero-American countries, other wild or little used fruit and vegetables have been included with the aim of contributing to promote and to value species and local varieties. The importance of the commodities containing carotenoids in food, health, agriculture, and biodiversity, and the need of their preservation, was evidenced in this work namely by the large differences in carotenoid content related to the locals of production and varieties, and the high levels of carotenoids in native fruits and vegetables. The contribution of these compounds to meet the needs of vitamin A as well as the necessity of establishing recommendation for the daily intakes of theses bioactive compounds were also discussed.

Carotenoid stability in fruits, vegetables and working standards - effect of storage temperature and time.

The effects of freezing and storage temperature on the mass fraction of α- and ß-carotene, ß-cryptoxanthin, lutein, lycopene and zeaxanthin in minimally processed fresh food products, were evaluated after sample preparation, extraction and saponification (only when strictly necessary). Effects of freezing and long-term stability were studied at two temperatures, -20 and -70°C, using high performance liquid chromatography (reversed phase columns, UV-Vis diode array detector) at time points during storage; measurement uncertainty was included in the evaluation. Stability of working standard solutions was also examined. Freezing did not affect the carotenoid mass fraction under the conditions studied. Carotenoids in orange, cherry, peach, apple, and kale were stable (except α-carotene and zeaxanthin in peach) for 13, 9.7, 5.7, 2.5 and 7.5months, respectively. For these food sample matrices, no significant difference between the freezing/storage at -20 and -70°C was observed. Standard solutions (0.05-5µg/mL) were stable for at least 6months at -70°C, except lycopene which at 0.05µg/mL was apparently stable only for six weeks.

Critical assessment of three high performance liquid chromatography analytical methods for food carotenoid quantification.

Three sets of extraction/saponification/HPLC conditions for food carotenoid quantification were technically and economically compared. Samples were analysed for carotenoids alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein, lycopene, and zeaxanthin. All methods demonstrated good performance in the analysis of a composite food standard reference material for the analytes they are applicable to. Methods using two serial connected C(18) columns and a mobile phase based on acetonitrile, achieved a better carotenoid separation than the method using a mobile phase based on methanol and one C(18)-column. Carotenoids from leafy green vegetable matrices appeared to be better extracted with a mixture of methanol and tetrahydrofuran than with tetrahydrofuran alone. Costs of carotenoid determination in foods were lower for the method with mobile phase based on methanol. However for some food matrices and in the case of E-Z isomer separations, this was not technically satisfactory. Food extraction with methanol and tetrahydrofuran with direct evaporation of these solvents, and saponification (when needed) using pyrogallol as antioxidant, combined with a HPLC system using a slight gradient mobile phase based on acetonitrile and a stationary phase composed by two serial connected C(18) columns was the most technically and economically favourable method.