Bioactive compounds in rice on Italian market: pigmented varieties as a source of carotenoids, total phenolic compounds and anthocyanins, before and after cooking

Accepted Manuscript Bioactive compounds in rice on italian market: pigmented varieties as a source of carotenoids, total phenolic compounds and anthocyanins, before and after cooking Valentina Melini, Gianfranco Panfili, Alessandra Fratianni, Rita Acquistucci PII: DOI: Reference:

S0308-8146(18)31818-1 https://doi.org/10.1016/j.foodchem.2018.10.053 FOCH 23714

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

11 September 2017 31 January 2018 10 October 2018

Please cite this article as: Melini, V., Panfili, G., Fratianni, A., Acquistucci, R., Bioactive compounds in rice on italian market: pigmented varieties as a source of carotenoids, total phenolic compounds and anthocyanins, before and after cooking, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.10.053

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BIOACTIVE COMPOUNDS IN RICE ON ITALIAN MARKET: PIGMENTED VARIETIES AS A SOURCE OF CAROTENOIDS, TOTAL PHENOLIC COMPOUNDS AND ANTHOCYANINS, BEFORE AND AFTER COOKING

(SUGGESTION FOR ABBREVIATED TITLE: BIOACTIVE COMPOUNDS IN PIGMENTED RICE AND EFFECT OF COOKING)

Valentina Melini*1, Gianfranco Panfili2, Alessandra Fratianni2, Rita Acquistucci1 1

CREA Research Centre for Food and Nutrition Via Ardeatina 546 I-00178 Rome Italy 2

University of Molise, Department of Agricultural, Environmental and Food Sciences Via Francesco De Sanctis I-86100 Campobasso Italy *corresponding author e-mail: [email protected] Tel. +39 06 51494656 Fax. +39 06 51494550

Abstract The aim of this study was to fully characterize the main pigmented rice varieties, available to consumers on the Italian market, in terms of carotenoids, total phenolic compounds and anthocyanins, and to investigate the effect of cooking on these components. Lutein was the main carotenoid in all samples under investigation (0.33-4.11 µg/g d.m.), while anthocyanins were observed only in black genotypes. Phenolic compounds were found mainly in free form, and values ranged between 544.1 and 1508.3 mg/100 g (d.m.) in raw samples. Cooking decreased significantly (p<0.05) total lutein, free phenolic compound and anthocyanin content. In contrast, the increase of insoluble-bound phenolic compounds was observed in some samples, after cooking. The study provides data contributing to gain a better knowledge in novel food composition and enabling the estimation of dietary intake of health-promoting components. Keywords: pigmented rice; carotenoids; phenolic compounds; anthocyanins; cooking 1

1

Introduction

Pigmented rice refers to rice varieties whose grain qualities fall outside the range of common types of rice in terms of physical appearance, chemical composition and aroma. Its distinctive quality is a grain pigmentation varying from deep-purple to brown-reddish, due to the accumulation of natural pigments in the pericarp, seed coat and aleurone (Chaudhary, 2003). It differs from “Golden rice”, which is characterized by a pleasant yellow colour, as it encompasses natural germoplasms, while the latter is a “high-tech” rice obtained in 2000 by genetic engineering (Ye et al., 2000). Pigmented rice varieties are indigenous to Asian countries, where rice is a major supply of energy, thanks to its carbohydrate content, and is also an important source of dietary proteins, vitamins and minerals. In Asia, pigmented rice is also used for purposes other than nutrition: it is applied in therapeutic treatments, such as in Ayurveda medicine, and in religious rites and ceremonies (Ahuja, Ahuja, Thakrar, & Singh, 2008). In Italy, there is rather a more rooted tradition of growing and consuming rice varieties with white kernels which are very appreciated by consumers worldwide thanks to their quality traits. Some of them have been also awarded Protected Designation of Origin (PDO) or Protected Geographical Indication (PGI) recognition by the European Union (European Commission. Agriculture and Rural Development – DOOR, 2017). However, pigmented rice has recently picked up popularity among Italian consumers, and it is popping up even at larger chain supermarkets. Some of the pigmented varieties currently available on the Italian market have been obtained from Asian varieties and made suitable to the growing conditions of Italian rice fields. Venere and Ermes varieties are examples of pigmented rice made in Italy: the former was the first black rice constituted in Italy in 1997 and it is currently grown and processed in Piedmont and Sardinia (Italy), the latter is an aromatic red rice, exclusively born, cultivated and processed in Italy. Other pigmented rice varieties now marketed in Italy are from France where they are grown under conventional and/or organic systems.

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An in-depth insight into current literature shows that so far a body of studies has focused on Asian pigmented rice varieties, deeply engraved in their culture and tradition, that were target of a comprehensive investigation (Kim et al., 2010; Kong & Lee, 2010; Sompong, Siebenhandl-Ehn, LinsbergerMartin, & Berghofer, 2011; Chen, Nagao, Itani, & Irifune, 2012; Gunaratne, Wu, Li, Bentota, Corke, & Cai, 2013; Pereira-Caro, Watanabe, Crozier, Fujimura, Yokota, & Ashihara, 2013), while the nutritional and nutraceutical characteristics of Italian pigmented rice varieties have been poorly investigated. In 2007, Finocchiaro and colleagues determined the content of some bioactive compounds and the total antioxidant capacity in one Italian red rice variety grown in the Novara area (Italy) (Finocchiaro et al., 2007). Few years later, the content of total phenolic compounds, proanthocyanidins and anthocyanins was determined in some red and black Italian varieties (Finocchiaro, Ferrari, & Gianinetti, 2010). Bordiga and colleagues determined the phenolic compound content and the antioxidant activity of three black and two red Italian rice varieties (Bordiga et al., 2014) and more recently, Zaupa and colleagues characterized samples of two rice varieties in terms of anthocyanins and phenolic compounds, and also studied the effect of domestic cooking on content thereof (Zaupa, Calani, Del Rio, Brighenti, & Pellegrini, 2015). These studies mainly focused on the determination of anthocyanins and total phenolic compounds, while the content of carotenoids, acting both as antioxidants and pigments alongside anthocyanins, has been so far disregarded. Based on that, the detection of this lipophilic pigment class in pigmented rice urged. In addition, most papers report only on the free fraction of phenolic compounds, whereas the insolublebound one has been neglected. Assessing the content of the latter fraction of phenolic compounds is, actually, very important from a nutritional point of view. Insoluble-bound phenolic compounds are polyphenols bound to the cell wall polysaccharides or proteins forming stable complexes. Most of them arrive nearly intact in the colon where they become accessible thanks to fermentation of polysaccharides or proteins they are bound to, by colonic microflora, or thanks to the activity of intestinal enzymes such as esterases. They also show a significantly higher antioxidant capacity than free phenolics (Pérez-Jiménez, Díaz-Rubio, & Saura-Calixto, 2013).The lack of an extensive investigation of the cooking effect on both hydrophilic and lipophilic bioactive molecules, in rice available on Italian market, is an additional gap in the 3

nutritional characterization of rice, since this cereal is actually consumed after cooking. In Italy, rice is usually prepared as “risotto”. It consists in addition of an aqueous medium (broth/water) at its boiling point (about 100°C) to rice and in boiling till complete absorption of added water. Despite it is well known that bioactive molecules are sensitive to thermal treatments, the food matrix where they are integrated into, can affect their transformation. It must be also underlined that currently available composition data on some Italian pigmented rice varieties are difficult to compare, since scientists used different procedures for the extraction of bioactive molecules, each one tailored for one or two rice varieties. Based on the above-mentioned considerations, this study investigated the content of bioactive compounds, such as carotenoids, anthocyanins, free and insoluble-bound phenolic compounds, in seven samples of pigmented rice varieties, available to consumers on the Italian market, in the raw and cooked form. The rationale of the study was to investigate the rice varieties which are more easily accessible to the Italian population and influence its diet and possibly health. The analysis of cooked samples also enabled to assess the retention of bioactive compounds, and provided an estimation of the real intake of these health-promoting components and of the real nutritional value of pigmented rice.

2 2.1

Materials and Methods Chemicals and reagents

Pyrogallol, sodium hydroxide, sodium carbonate, sodium chloride, chloride acid, Folin-Ciocalteu Reagent, formic acid, methanol, acetone, ethanol, hexane, ethyl acetate, tetrahydrofuran and gallic acid were purchased from Carlo Erba Reagenti (Milan, Italy). Pure Lutein, zeaxanthin, β-carotene, cyanidin-3-O-glucoside, peonidin-3-O-glucoside and malvidin were purchased from Extrasynthèse (Geney, France). HPLC grade solvents and water purified by a Milli-Q system (Millipore Corp., Billerica, MA, USA) were used in HPLC analysis. 4

2.2

Sampling

Samples of pigmented rice (Oryza sativa L.) varieties from Italy (Otello, Venere, Nerone, Artemide, Ermes) and France (Riz Rouge de Camargue) were analyzed and labeled as shown in Table 1. Venere and Ermes samples were provided by Ente Nazionale Risi, an Italian public institution with the mandate to implement and coordinate research on rice. The left-over ones were collected in Italian shops and/or supermarkets. A bulk sample of each rice variety was prepared by combining at least three units of commercially available products. Subsamples were obtained by quartering and were stored at 4°C in the dark. All the analyses were carried out within two months from the collection.

2.3

Preparation of raw and cooked samples for analysis

Immediately prior to analyses, raw rice grains were reduced to coarse particles and then pulverized to fine particles (< 0.5 mm) by using a laboratory mill (Janke & Kunkel IKA LabortechniK, Stanfen, Germany), provided with a water-cooling system. Test samples for analyses were thus obtained. Cooked rice samples were prepared by simulating the “risotto” preparation, as described in Melini & Acquistucci (2017a). Cooking was replicated twice for each rice variety and analysis were performed on pooled samples. Preliminary tests were performed in order to assess the rice-to-water ratio enabling both an optimal and uniform cooking (the disappearance of the starch central core was tested by pressing the cooked kernel between two transparent glass slides), and a complete absorption of cooking water.

2.4

Proximate composition analysis

Moisture content was determined according to the ICC Standard method No. 110/1 (ICC Standard Methods, 2003) and reported as g/100 g fresh weight (f.w.). Protein content was determined on the basis of total nitrogen content (N x 5.95) according to the ICC standard method No. 105/2 (ICC Standard Methods, 2003), crude fat was assessed according to the AACC official method 30-25.01 (AACC Approved Method of Analysis, 1999) and ash content was evaluated 5

according to the AOAC official method 923.03 (AOAC Official Methods of Analysis, 2005). Results were expressed as g/100 g dry matter basis (d.m.).

2.5

Extraction and determination of carotenoids

Carotenoids were extracted according to the procedure reported in Panfili, Fratianni, & Irano (2004), with some modifications. Briefly, samples (2 g) were saponified by potassium hydroxide 600 g/L (2 mL) under nitrogen for 60 min at 70 °C in presence of 5 mL of ethanolic pyrogallol (120 g/L), 2 mL of ethanol (95%) and 2 mL of sodium chloride (10 g/L). They were extracted by 30 mL of hexane:ethyl acetate (9:1 v/v) and the extraction was repeated till an uncoloured upper organic phase was obtained. Pooled supernatants were vacuum dried and stored overnight at -40 °C. Then, they were analyzed for carotenoid content by RP-HPLC, as described in Melini & Acquistucci (2017a). Results were expressed as µg of carotenoid per g of sample on a dry matter basis (d.m.).

2.6

Extraction and determination of free lutein

Free lutein (FL) was extracted as reported in Fratianni, Mignogna, Niro, & Panfili (2015), with few modifications. Briefly, sample (2 g) was placed in a screw-capped tube, then ethanolic pyrogallol (120 g/L), ethanol (95%), sodium chloride (10 g/L) and water (2 mL) were added. Tubes (under nitrogen) were placed in a water bath at 70 °C for 60 min and mixing was performed every 5-10 min. Then, they were cooled down and sodium chloride (10 g/L) was added. FL was extracted by using hexane:ethyl acetate (9:1 v/v) till an uncoloured upper organic phase was obtained. Pooled supernatants were vacuum dried and the residues were dissolved in methanol:tetrahydrofuran (95:5 v/v) immediately before HPLC analysis. Lutein separation was performed by RP-HPLC as reported in Melini & Acquistucci (2017a).

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2.7

Extraction and determination of free phenolic compounds

Free phenolic compounds (FPCs) were isolated as reported by Arranz & Saura Calixto (2010) by a two-step extraction with acidified aqueous methanol (50:50 v/v) and acetone:water (70:30 v/v) under magnetic stirring. They were quantified on pooled extracts by the Folin-Ciocalteu colorimetric assay, as detailed in Melini & Acquistucci (2017a). Results were expressed as mg of Gallic Acid Equivalents (GAE) per 100 g of sample on a dry matter basis.

2.8

Extraction and determination of insoluble-bound phenolic compounds

Insoluble-bound phenolic compounds (BPCs) were determined as reported in Melini & Acquistucci (2017a). Briefly, they were extracted by ethyl acetate from the pellet left over after the isolation of FPCs and that had undergone saponification with sodium hydroxide 2M under ultrasonic irradiation at 40 °C for 90 min. They were quantified by the Folin-Ciocalteu colorimetric assay, after the dried extracts were reconstituted with methanol:water 50:50 (v/v). Results were expressed as mg of Gallic Acid Equivalents (GAE) per 100 g of sample on a dry matter basis.

2.9

Extraction and determination of anthocyanins

Anthocyanins were extracted and determined as described in Melini & Acquistucci (2017a). Briefly, samples were extracted by acidified aqueous methanol (85:15 v/v), and then an aliquot of the obtained fresh extract (5 mL) was vacuum evaporated and stored at -40 °C till HPLC analysis. Anthocyanins separation was obtained by a Phenomenex Luna® (250x4.6 mm i.d., 5 µm) column and detected by a photodiode array detector. Results were expressed as mg/100 g dry matter basis (d.m.).

2.10 Statistical analysis

Results are reported as mean ± standard deviation (SD) in tables. Extractions were performed at least twice on each samples and analytical analyses of each extract were carried out at least in triplicate. Tukey’s HSD

7

test was performed by MathWorks® Matlab 8.2 software and used in conjunction with ANOVA to compare mean values among samples. A value of p < 0.05 was considered statistically significant.

3 3.1

Results and discussion Proximate composition of raw samples

The proximate composition of the raw samples under investigation is shown in Table 1. The moisture content ranged between 11.8% and 13.5%. The determination of this parameter is important in managing and marketing rice, and also in correcting grain composition data, thus enabling the comparison among rice samples with different moisture content. Protein content varied between 7.4 and 11.3 g/100 g (d.m.). The lowest content, detected in VNR sample, was comparable to the average protein content of white rice and also in keeping with Champagne (2004) that quoted protein content in rice ranging between 5.8 and 7.7g/100 g. In contrast, some samples, namely NRN and ATM, showed a protein content appreciably above those reported for white rice. Such a high content was also found by Fabian & Ju (2011) in rice bran, and it is common to whole grains. It is worth highlighting that a serving of 80 g of pigmented rice showing 10 g/ 100 g (d.m.) of protein content provides about 7 g of proteins that contribute to the 12.5% of the Population Reference Intake (PRI) in adults male and to the 17% of PRI in adults female, established for the healthy Italian Population. As a matter of fact, the daily protein PRI in adults male is 63 g and in adults female is 54 g (LARN, 2014). The significant differences detected among samples are possibly due to genetic traits, environmental factors and production systems, especially nitrogen fertilizers ( Chandel, Banerjee, See, Meena, Sharma, & Verulkar, 2010). Crude fat ranged between 2.8 and 3.7 g/100 g (d.m.). Comparable values are commonly found in brown rice (Juliano, 1985). Lipid extraction was carried out by a Soxhlet apparatus that enables the extraction of “non-starch lipids”. They are free or bound to proteins in the starch-bearing tissues and are distributed throughout the grain (Choudhury & Juliano, 1980).

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Ash content ranged between 1.55 and 1.88 g/100 g (d.m.). These values are common to brown rice (Lamberts et al., 2007) and suggest that samples under investigation might serve as a good source of minerals, such as potassium and phosphorus.

3.2

Carotenoid content in raw and cooked samples

Mankind, like other animals, is not able to synthetize ex novo carotenoids and dietary intake is the unique source of these molecules. The need for an adequate dietary intake of carotenoids in human nutrition is due to their health effects, namely protection against chronic pathologies, such as cardiovascular diseases, cancer, cataract and macular degeneration (Rodriguez-Amaya, 2016). Compared to fruits and vegetables, cereals contain a relatively small amount of carotenoids almost exclusively concentrated in the grain bran layer, and the common practice of milling and stripping results in a loss of these pigments (Tan et al., 2005). As far as rice is concerned, in white rice little or no bran remains on the endosperm where the biosynthesis of carotenoids is blocked at the first enzymatic step (Bai et al., 2016). As a consequence, white rice is not a source of carotenoids which to pay attention to. In contrast, pigmented rice are consumed with their bran layer intact, hence they might contribute to the daily intake of these bioactive molecules. The main carotenoids detected in the rice samples under investigation were all-trans-(E)-lutein, all-trans(E)-zeaxanthin and β-carotene (Table 2). The xanthophylls all-trans-(E)-lutein and all-trans-(E)-zeaxanthin will be referred to hereafter as lutein and zeaxanthin. In raw samples, lutein was found the main carotenoid and it accounted for more than 82% of the total carotenoids (sum of lutein, zeaxanthin and β-carotene). Values ranged from 0.33 µg/g (d.m.) to 4.11 µg/g (d.m.): the lowest content was detected in EMS sample and the highest one in ATM. Lutein content detected in ATM sample is in keeping with Pereira-Caro, Cros, Yokota, & Crozier (2013) that detected a concentration of 4.3 µg/g (d.m.) in a rice sample of the same Italian cultivar. Moreover, Kim et al. (2010) observed, in some Korean black grain rice samples, a lutein content comparable to ATM sample, and Pereira-Caroet al. (2013a) detected, in a Japanese black-purple rice sample belonging to the cultivar 9

Asamurasaki, a lutein content matching the OTL and NRN one. Generally speaking, lutein content in blackpurple rice varieties was higher than in red ones, except for VNR whose value was not statistically (p<0.05) different from FRR. Differences among samples might be due to variety genetic traits; however, other factors such as production climate and geographic site, harvesting and post-harvesting handling and/or storage might affect carotenoid content (Rodriguez-Amaya, 2016). As concerns zeaxanthin, values varied between 0.01 µg/g (d.m.) and 0.07 µg/g (d.m.), and it accounted for less than 4% of total carotenoids, except for RRC sample where zeaxanthin content was 12% of total carotenoids. The highest concentration was found in OTL and RRC samples, while lower values were detected in the other samples. In some Korean pigmented rice cultivars, Kim et al. (2010) detected a zeaxanthin content ranging from 0.02 µg/g (d.m.) to 0.15 µg/g (d.m.), while higher values were detected by Pereira-Caro et al. (2013a) in a Japanese black rice sample, and a comparable content was found by Melini & Acquistucci (2017a) in pigmented Thai varieties. As concerns β-carotene content, values ranged from 0.02 µg/g (d.m.) to 0.45 µg/g (d.m.), and no statistically significant (p<0.05) differences were detected among samples, except for NRN and ATM. The latter showed the highest concentration and this matched the values found by Kim et al. (2010) in Korean black rice varieties. Rice is actually consumed after cooking, a process that determines favourable changes in texture, aroma and flavour, and affects the patterns of macro- and micro-nutrients, as well. Therefore, the effect of cooking on carotenoid profile was also studied in order to obtain reliable composition values that might be used by epidemiologists dealing with the role of food components and their interactions in human health and diseases. In detail, samples were processed by simulating the preparation of risotto, the most common way to consume rice in Italy. It consists in the addition of boiling broth/cooking water, that is completely reabsorbed by rice kernels over the time necessary for an optimal cooking. In the cooked samples under investigation, lutein content ranged between 0.17 µg/g (d.m.) and 2.89 µg/g (d.m.), zeaxanthin varied between 0.01 µg/g (d.m.) and 0.03 µg/g (d.m.), while β-carotene was detected only in ATM (Table 2). In 10

most samples, cooking caused a significant (p < 0.05) loss of both xanthophylls. As concerns lutein, the loss due to cooking ranged from 30% to 69%. ATM sample was the most resistant to cooking stress, while FRR was the most affected. Comparable lutein losses due to cooking are also reported by Melini & Acquistucci (2017a) in Thai black (44%) and in Thai red rice (57%). Nevertheless, it must be underlined that lutein content detected in the cooked pigmented rice varieties under investigation was comparable to or even higher than other commonly consumed cereal-based products. The Food Composition Databases of the United States Department of Agriculture (USDA) report a lutein+zeaxanthin content of 7.00 µg/100 g in cooked pasta and 97 µg/100 g in multigrain toasted bread.

3.3

After cooking, a significant decrease of zeaxanthin content was also observed in all samples except for the black-purple ATM sample. As far as β-carotene is concerned, the loss in ATM was 47%. ATM exhibited a behaviour different from other samples under investigation in retaining carotenoids after cooking. Generally speaking, carotenoid losses are common during food processing and they are mainly caused by enzymatic and non-enzymatic oxidation (Rodriguez-Amaya, 2016) that, at its turn, depends on the availability of oxygen and on the carotenoid structure. However, other factors should be considered: light, heat, metals, enzymes and peroxide stimulate oxidative reactions, while antioxidants inhibit them. During thermal processing, oxidative enzymes are inactivated; hence, it might be supposed that differences in the thermal stability of oxidative enzymes, as well as the presence of metals and the co-oxidation of lipids (Sajilata, Singhal, & Kamat, 2008), might be responsible for the different behaviour of the ATM sample.Free lutein content in raw and cooked samples

In foods, xanthophylls occur in free form (unesterified) and/or esterified to fatty acids (Saini, Nile, & Park, 2015). Esterification occurs progressively during maturation of vegetables and fruits (Rodriguez-Amaya, 2016), and although it does not alter the chromophore properties of the carotenoid (Britton et al., 2008), it modifies the chemical and biological properties thereof, and hence their bioaccessibility and bioavailability (Saini, Nile, & Park, 2015). In detail, esterified xanthophylls are more fat-soluble than their free forms, and 11

this structural difference may alter the efficiency of micellization and thus the absorption of these antioxidants in human gut ( Fernández-García, Carvajal-Lérida, Jarén-Galán, Garrido-Fernández, PérezGálvez, & Hornero-Méndez, 2012). Therefore, from a nutritional point of view, it is recommended to determine also free lutein (in addition to total lutein) to fully characterize a food matrix. In the raw samples under investigation, FL content ranged between 0.28 µg/g (d.m.) and 4.35 µg/g (d.m.) (Table 2). Significant (p<0.05) differences were found among black rice varieties, and all red genotypes showed a FL content lower than black ones. FL accounted for at least 50% of total lutein. Moreover, in ATM and EMS varieties lutein was found only in free form. Bunea, Socaciu, & Pintea (2014) also observed that in some foods, such as corn, spinach and broccoli, xanthophylls occur exclusively in unesterified form. FL was also determined in cooked samples and values ranged between 0.04 µg/g (d.m.) and 1.11 µg/g (d.m.): OTL and VNR samples showed a FL content lower than NRN and ATM, while in red varieties FL amount was significantly (p<0.05) lower than black genotypes. Moreover, it was observed that cooking process caused a significant decrease (p<0.05) of FL in all samples under investigation. The variability among samples for free lutein degradation could be due to the occurrance of enzymes, peroxides and metals that can stimulate carotenoid oxidation. However, the presence of antioxidants other than carotenoids inhibiting carotenoid oxidation might be responsible for differences among samples in carotenoid loss (Rodriguez-Amaya, 2016). As the esterification could affect the absorption of these antioxidants, in-vitro and in-vivo studies on the bioavailability of free lutein from rice should also be performed in order to reliably assess the nutritional value of samples under investigation.

3.4

FPCs and BPCs in raw and cooked samples

In this study, both FPCs and BPCs were determined (in raw and cooked samples) in view of their nutritional importance as antioxidants contributing to the prevention of a number of chronic diseases, such as cardiovascular diseases, cancer and neurodegenerative diseases ( Del Rio, Rodriguez-Mateos, Spencer, Tognolini, Borges, & Crozier, 2013). Data are reported in Table 3.

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In raw samples, FPC content ranged from 544.1 mg/100g (d.m.) to 1508.3 mg/100g (d.m.), and the statistical analysis showed that the concentration of FPCs was significantly (p<0.05) higher in black-purple varieties than in red ones. In detail, the ATM sample showed the highest FPC content, while no significant difference was found between VNR and NRN. Red varieties did not differ significantly in FPCs, as well. Data are in keeping with Bordiga et al. (2014). Paiva and colleagues (Paiva et al., 2014) found in a black genotype grown in Brazil a FPC content comparable to that detected in ATM sample (1368 mg GAE/100 g d.m.). They found similar FPC content also in a red genotype from Brazil (1285 mg/100 g d.m.). The great variability of phenolic compound content among rice samples may be due to the genotype, but also to factors such as grain type, part of the grain sampled, grain handling, and processing (Ragaee, Seetharaman, & Abdel-Aal, 2014). In addition, the sample preparation for the analytical determination considerably affects the content of phenolic compounds, and the lack of a unique procedure might contribute to the differences in phenolic compound content observed in several studies (Melini & Acquistucci, 2017b). The BPC content was also assessed in raw samples: values ranged between 66.0 µg/g (d.m.) and 116.2 µg/g (d.m.). The highest value was found in NRN sample, followed by OTL and ATM. In contrast, BPC content in the black VNR variety was not statistically different from the red varieties. BPCs detected in samples under investigation were in keeping with values observed by Paiva et al. (2014) in two pigmented rice samples from Brazil.The percentage of FPCs and BPCs in the raw samples under investigations was also expressed with respect to TPCs (determined as sum of FPCs and BPCs) (Table 3). FPCs were found the major fraction and ranged between 88% and 95% of TPCs, while BPC percentage varied between 5% and 12%. Data are in keeping with Min, Gu, McClung, Bergman, & Chen (2012), that found 11%–27% of BPCs in bran coloured rice cultivars grown in USA; with Massaretto, Madureira Alves, Mussi de Mira, Carmona, & Lanfer Marquez (2011) that reported 8%–26% of BPCs in pigmented rice genotypes grown in Brazil; and with Kong and Lee (2010) that observed 10%–11% of BPCs in Korean pigmented rice. Recently, Melini & Acquistucci (2017a) also found in Thai pigmented rice a BPC content ranging between 11% and 15%. Therefore, experimental 13

data confirm that, in pigmented rice, phenolic compounds are mostly in free form, while in non-pigmented rice and other cereals they occur mainly in bound form (Acosta-Estrada, Gutiérrez-Uribe, & Serna-Saldívar, 2014). This might be explained by the occurrence of phenolic compounds, such as anthocyanins and proanthocyanidins, that are peculiar to pigmented rice. They might be extracted by organic mixtures and detected as FPCs, as they are not esterified to cellular components. However, the use of chloride acid to acidify the organic mixture for the extraction of FPCs might also promote the hydrolysis of BPCs (Goufo & Trindade, 2014), hence a higher content of the free form might be observed. In cooked samples, FPCs ranged between 325.0 mg/100 g (d.m.) and 1384.8 mg/100 g (d.m.) (Table 3). Red varieties showed a FPC content lower than black-purple ones (p<0.05). Among these latter, the highest amount was found in ATM sample, while no significant (p<0.05) difference was found between OTL and VNR. BPC content varied from 65.1 mg/100 g (d.m.) to 114.4 mg/100 g (d.m.) (Table 3). Interestingly, no significant differences were found among black varieties, except ATM that showed, once again, the highest amount of phenolic compounds. In addition, red samples showed a BPC content comparable to ATM and higher than the other black-purple genotypes. FPCs were found the major fraction even after cooking and they ranged between 77% and 94% of TPCs, while BPC percentage varied between 6% and 23% (Table 3). The comparison of phenolic content between raw and cooked samples enabled to assess the effect of cooking on these bioactive molecules, and to evaluate the resistance of genotypes under investigation. As concerns FPC content, it was significantly (p<0.05) affected by cooking; nevertheless, black rice varieties were found more resistant to this treatment than red ones (p<0.05). About 95% of FPCs was preserved in NRN, 92% in ATM and about 78% in OTL and ATM samples. On the contrary, in the red samples under investigation 60%-67% of FPCs was retained after cooking. EMS variety showed the lowest resistance to cooking treatment. In general, the loss of FPCs might be caused by i) their migration from the food matrix into other medium, namely the leaching into cooking water, ii) by their chemical degradation or transformation into other molecules and finally iii) by their interactions with other food components that make them less extractable 14

(Duodu, 2011). As far as this study is concerned, a decrease of FPC level due to leaching is unlikely, since the cooking procedure that samples underwent, ensured the re-absorption of cooking water (and thus of leached components). A reduction of FPCs by 83% after hydrothermal treatment was also observed by Massaretto and collegues (2011) in some rice samples from Brazil. As far as BPCs are concerned, significant differences (p<0.05) were observed in all samples, except for VNR and ATM. In detail, it was found that 79% of phenolic compounds was retained in OTL variety and 59% in NRN. In VNR and ATM, the occurrence of components with antioxidant activity in the food matrix may protect BPCs from oxidative degradation. In contrast, in all red samples under investigation a significant increase of BPCs was found: in EMS the amount increased to 151%, in RRC to 159% and in FRR to 135%. This trend might be explained by taking into account that FPCs may migrate into the endosperm with absorbed water, and form BPCs by binding proteins and/or other macromolecules (Duodu, 2011). This hypothesis might be confirmed by the decrease in FPCs that has been observed. Nevertheless, the increase of BPC content might also be explained by the softening or disintegrating effect of boiling water on rice kernel tissue that promotes the release of BPCs from the food matrix (Duodu, 2011). The hypothesis of a possible effect of cell wall-degrading enzymes, such as esterases, in facilitating the extraction of BPCs (Kadiri, 2017) can be discarded, since rice bran esterases are inactivated by temperature. The inactivation kinetic depends on the temperature itself, on the holding time and on rice moisture content (Brunschwiler, Heine, Kappeler, Conde-Petit, & Nyström, 2013). Min, McClung, & Chen (2014) observed an increase of insoluble-bound phenolic compounds in brown, purple and red bran whole grain rice, as well.

3.5

Anthocyanin content in raw and cooked samples

Anthocyanins are phenolic compounds that occur in many pigmented plant foods, such as fruits, vegetables and cereals, and contribute to the colour thereof. They are also responsible for a number of beneficial effects on human health, such as vision improvements, protection against metabolic, degenerative and cardiovascular diseases, and anti-inflammatory and anti-cancer activities (Haq, Riaz, & Saad, 2016). All rice varieties underwent anthocyanins analysis, and results are shown in Table 4. 15

In black rice genotypes, cyanidin-3-O-glucoside (C3G) and peonidin-3-O-glucoside (P3G) were identified. C3G values ranged between 71.5 mg/100 g (d.m.) and 198.6 mg/100 g (d.m.): the lowest content was found in OTL and the highest in ATM. As far as red varieties are concerned, traces of malvidin were found in FRR samples, while the other genotypes showed no anthocyanins. The occurrence of anthocyanins in red rice is still under debate: Kim et al. (2010) reported no anthocyanins in red rice varieties from Korea, and Gunaratne et al. (2013) did not observe any anthocyanins in red rice varieties from Sri Lanka. In contrast, Chen et al. (2012) found malvidin in four Japanese red rice varieties, and Pereira-Caro et al. (2013b) observed C3G in a red rice cultivar from France. As far as P3G is concerned, differences among samples were significant (p<0.05). Values ranged between 5.0 and 16.2 mg/100 g (d.m.): the highest content was found in NRN sample, while VNR showed the lowest one. Kim et al. (2010) observed in Korean black varieties similar P3G values, ranging between 1.28 mg/100 g (d.m.) and 11.87 mg/100 g (d.m.); however, in Artemide, Nerone e Venere samples, Bordiga et al. (2014) detected P3G values higher than those obtained in this study. This might be due to differences in the procedures used for the extraction of anthocyanins from the food matrix and/or to pre- and post-harvest conditions of rice samples. C3G accounted for more than 87% of total anthocyanins (calculated as sum of C3G and P3G), therefore it was confirmed the main anthocyanin in pigmented rice, in keeping with Goufo & Trindade (2014). In cooked samples, C3G ranged between 63.1 mg/100 g (d.m.) and 100.6 mg/100 g (d.m.): ATM and VNR showed the highest value, while NRN and OTL did not differ significantly (p<0.05). As far as the evaluation of processing on individual anthocyanins is concerned, a significant (p<0.05) loss (48-50%) of C3G due to cooking was observed only in NRN and ATM samples. In contrast, OTL and VNR varieties showed no significant (p<0.05) differences in C3G content prior to and after cooking. Min et al. (2014) also observed a decrease of C3G from approximately 1261 µg/g of sample (d.m.) to 435 µg/g of sample (d.m.) in a whole grain purple rice variety from USA due to wet-cooking, while Zaupa et al. (2015) reported a loss of C3G (28%) in an Italian black rice variety undergone to risotto-like cooking. The decrease of C3G content might be due to the transformation of this component into other molecules.

16

As an example, Hiemori, Koh, & Mitchell (2009) observed the degradation of C3G into protocatechuic acid (during cooking) in a black rice from California. Moreover, in all cooking treatments involving the addition of water to the food matrix, anthocyanins might be leached into water, as they are soluble in this solvent. However, in this study, the cooking process enabled absorption of water, avoiding anthocyanin loss due to leaching. Other factors, such as the presence of several antioxidants, might be responsible of anthocyanin retention (Cortez, Luna-Vital, Margulis, & Gonzalez de Mejia, 2017). As far as P3G is concerned, in cooked samples values ranged between 3.3 mg/100 g (d.m.) and 6.7 mg/100 g (d.m.). Except for OTL, no significant (p<0.05) differences were found among samples. In contrast, cooking treatment caused a significant (p<0.05) decrease of P3G in all samples except for VNR. The loss ranged between 39% and 64%.

3.6

Comprehensive bioactive compound content: comparing samples

In this study, the main bioactive compounds, both lipophilic and hydrophilic, were determined, and results were discussed in specific sections. Nevertheless, single data do not enable a comprehensive estimation of the whole bioactive compound potential of each sample and do not allow an overall comparison of the pigmented varieties under investigation. Therefore, radar plots were used in order to fully characterize samples, and also as a smart indicator of their nutraceutical potentialities. In Figure 1, the radar charts of bioactive compounds in the raw and cooked pigmented rice varieties under investigation are shown. The content of each bioactive compound or group of antioxidants was normalized and displayed in axis starting from the centre. The normalization enabled to compare values that are rather different. The scores for the variables were connected to form a “spider web” whose area can be assumed as a measure of the total bioactive compound content of each pigmented rice variety, and thus of the nutraceutical potentialities thereof. As far as raw samples are concerned (Fig. 1a), ATM showed the greatest area and it provides the highest amount of FPCs, total lutein, FL, β-carotene and C3G. All red varieties showed an area lower than black ones. 17

The radar chart of cooked samples (Fig. 1b) shows that even after cooking, ATM variety had the greatest content of bioactive compounds amongst samples under investigation, and it had the highest content of all antioxidants, except for zeaxanthin, FL and BPCs. Radar charts also underline that, after cooking, red rice varieties under investigation provide a higher amount of BPCs than black rice genotypes, because of the above-mentioned reasons.

4

Conclusions

This study provides a comprehensive portrait of the main pigmented rice varieties at Italian consumers’ disposal, in terms of bioactive compound content. Both hydrophilic and lipophilic antioxidants were determined: lutein was found to be the main carotenoid, anthocyanins were detected only in black samples and in contrast to other cereals, phenolic compounds were found mainly in free form. Also lutein was found mainly in free form, in raw samples. It emerged that black rice varieties have a greater nutraceutical potential than red ones. The special value of black rice varieties relies on the fact that the antioxidants they provide are both hydrophilic and lipophilic. As a matter of fact, fruits like blueberries are rich in water-soluble antioxidants, and vegetables, such as spinach, are rich in carotenoids. In contrast, black rice contains a mixture of both classes. The effect of cooking on bioactive compounds was also evaluated to more accurately assess the true dietary intake of health-promoting compounds through the consumption of pigmented rice available in Italian shops. Therefore, results from this study also provide reliable data for inclusion in comprehensive food composition databases and for the assessment of the intake of bioactive compounds.

Acknowledgements This study was performed within the project QUALIFU-FINALE funded by the Italian Ministry of Agricultural, Food and Forestry Policies (MiPAAF) and it was a part of a PhD thesis. The authors would like to thank Dr Francesca Melini (CREA Research Centre for Food and Nutrition) for the linguistic revision and editing of this paper. 18

Conflict of interest Authors report no conflict of interest.

19

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Table 1: Sample identification and proximate composition Table 2: Carotenoid content (µg/g d.m.) in raw and cooked rice samples Table 3: FPCs, BPCs and TPCs (mg/100 g d.m.) in raw and cooked samples and ratio thereof (%) Table 4: Anthocyanin content (mg/100g d.m.) in raw and cooked rice samples, and loss (%) due to cooking Fig. 1: Radar chart of raw (a) and cooked (b) black rice samples. Fig. 2: Radar chart of raw (a) and cooked (b) red rice samples.

26

(a)

(b)

Fig. 2: Radar chart of raw (a) and cooked (b) black rice samples. OTL, Otello; VNR, Venere; NRN, Nerone; ATM, Artemide; FPCs, Free Phenolic compounds; BPCs, Insoluble-bound phenolic compounds; C3G, Cyanidin-3-O-glucoside; P3G, Peonidin-3-O-glucoside.

27

(a)

(b)

Fig. 3: Radar chart of raw (a) and cooked (b) red rice samples. EMS, Ermes; RRC, Riz de Camargue rouge/PGI; FRR, Riz de Camargue rouge/organic; FPCs, Free Phenolic compounds; BPCs, Insoluble-bound phenolic compounds; C3G, Cyanidin-3-O-glucoside; P3G, Peonidin-3-O-glucoside.

28

Table 5: Sample identification and proximate composition

Country Sample Variety/certification origin

Kernel colour

Moisture content g/100 g

Protein content g/100 g (d.m.)

Crude fat content g/100 d (d.m.)

Ash content g/100 g (d.m.)

OTL

Otello

Italy

Black

11.8±0.0a

9.9±0.1a

3.1±0.2a

1.88±0.00a

VNR

Venere

Italy

Black

13.5±0.1b

7.4±0.0b

3.0±0.1a

1.63±0.00b

NRN

Nerone

Italy

Black

13.4±0.0b

11.3±0.0c

3.7±0.3b

1.83±0.01c

ATM

Artemide

Italy

Black

13.3±0.1b

11.2±0.1c

3.2±0.1a

1.84±0.00c

EMS

Ermes

Italy

Red

13.0±0.0c

8.2±0.1d

2.8±0.0c

1.55±0.01d

RRC

Riz de Camargue rouge/PGI

France

Red

12.9±0.0c

9.4±0.0e

3.3±0.1a

1.84±0.02c

FRR

Riz de Camargue rouge/organic

France

Red

13.3±0.0b

10.2±0.2a

3.0±0.2a

1.86±0.01c

Mean values within a column superscripted by the same small letter are not significantly different at p < 0.05. OTL, Otello; VNR, Venere; NRN, Nerone; ATM, Artemide; EMS, Ermes; RRC, Riz de Camargue rouge/PGI; FRR, Riz de Camargue rouge/organic; d.m., dry matter.

29

Table 6: Carotenoid content (µg/g d.m.) in raw and cooked rice samples and loss (%)

Sam ple

Lutein raw

OTL VNR NRN ATM EMS RRC FRR

2.81±0.0 0aA 1.03±0.1 6bA 2.74±0.3 1aA 4.11±0.7 2cA 0.33±0.0 0dA 0.42±0.0 3dA 0.88±0.0 5bA

Zeaxanthin

lo raw ss 1.19±0.1 58 0.07±0.0 1aB 2aA 0.55±0.0 46 0.01±0.0 6bB 0bA 0.91±0.0 67 0.03±0.0 8cB 0bA 2.89±0.0 30 0.04±0.0 9dB 1bA 0.17±0.0 48 0.01±0.0 3eB 0bA 0.2±0.01 53 0.07±0.0 eB 2aA 0.27±0.0 69 0.02±0.0 1fB 0bA cooked

β-carotene

FL

lo lo lo raw cooked raw cooked ss ss ss 0.02±0.0 72 0.03±0.0 - 1.42±0.0 0.68±0.0 n.d. 0aB 0a 8aA 6aB 52 0.01±0.0 19 0.04±0.0 - 0.78±0.0 0.18±0.0 n.d. 0bB 1a 1bA 2bB 77 0.01±0.0 53 0.11±0.0 - 2.06±0.1 1.11±0.0 n.d. 0bB 3b 0cA 8cB 46 0.03±0.0 33 0.45±0.1 0.24±0. 47 4.35±0.1 0.99±0.0 0aA 5cA 05B 8dA 6cB 77 0.01±0.0 24 0.02±0.0 - 0.35±0.0 0.04±0.0 n.d. 0bA 0a 0eA 0eB 89 0.01±0.0 80 0.03±0.0 - 0.28±0.0 0.07±0.0 n.d. 0abB 1a 4eA 2eB 75 0.01±0.0 60 0.03±0.0 - 0.50±0.0 0.09±0.0 n.d. 0bB 1a 1fA 0eB 82 cooked

Mean values of raw or cooked samples within a column superscripted by the same small letter are not significantly different at p < 0.05. Mean values of raw samples and the equivalent one superscripted by the same capital letter are not significantly different at p < 0.05. OTL, Otello; VNR, Venere; NRN, Nerone; ATM, Artemide; EMS, Ermes; RRC, Riz de Camargue rouge/PGI; FRR, Riz de Camargue rouge/organic; FL, free lutein; d.m., dry matter; nd, not detectable.

30

Table 7: FPCs, BPCs and TPCs (mg/100 g d.m.) in raw and cooked samples and ratio thereof (%) Sample Raw samples OTL VNR NRN ATM EMS RRC FRR Cooked samples

FPCs mg/100 g d.m.

BPCs mg/100 g d.m.

TPCs mg/100 g d.m.

FPCs/TPCs %

BPCs/TPCs %

679.3±9.4aA 796.7±11.2bA 849.7±21.7bA 1508.3±29.2cA 583.3±9.8dA 544.1±14.8dA 580.5±10.8dA

92.8±1.5 aA 67.0±2.0bA 116.2±2.2cA 84.0±1.6.aA 66.0±2.4bA 72.2±6.9bA 71.2±3.1bA

772.1±10.2 863.7±11.4 965.9±21.8 1592.3±29.2 649.3±10.1 616.3±15.1 651.8±11.2

88 92 88 95 90 88 89

12 8 12 5 10 12 11

528.4±51.9aB (22%) 73.0±5.7aB (21%) 601.4±52.2 88 12 624.4±60.2aB VNR (22%) 65.1±4.5aA (ns) 689.5±60.4 91 9 803.7±1.6bB NRN (5%) 68.7±4.7aB (41%) 872.3±5.0 92 8 1384.8±19.9cB ATM (8%) 83.8±11.5bA (ns) 1468.6±21.7 94 6 325.0±4.3dB 99.5±3.8bB (EMS (40%) 51%) 424.6±5.7 77 23 376.5±7.6eB 114.4±6.2bB (RRC (35%) 61%) 490.9±9.8 77 23 388.5±3.0eB 96.0±2.3bB (80 FRR (33%) 33%) 484.5±3.8 20 Mean values of raw or cooked samples within a column superscripted by the same small letter are not significantly different at p < 0.05. Mean values of raw samples and the equivalent one superscripted by the same capital letter are not significantly different at p < 0.05. OTL, Otello; VNR, Venere; NRN, Nerone; ATM, Artemide; EMS, Ermes; RRC, Riz de Camargue rouge/PGI; FRR, Riz de Camargue rouge/organic; FPCs, free phenolic compounds; BPCs, insoluble-bound phenolic compounds; TPCs, total phenolic compounds; d.m., dry matter. In brackets the loss (%) of FPCs and BPCs due to cooking. ns: not significant OTL

31

Table 8: Anthocyanin content (mg/100g d.m.) in raw and cooked rice samples, and loss (%) due to cooking Sample

C3G

P3G Loss raw cooked raw cooked loss (%) OTL 71.5±8.0aA 65.5±5.4aA ns 9.2±0.7aA 3.3±0.3aB 64 VNR 81.7±16.5aA 93.5±8.2bA ns 5.0±0.3.0bA 4.2±0.6abA ns NRN 130.5±7.9bA 63.1±7.0aB 48 16.2±2.3cA 5.8±0.5bB 64 ATM 198.6±13.2cA 100.6±3.8bB 50 10.9±1.1dA 6.7±0.8bB 39 EMS nd nd nd nd RRC nd nd nd nd FRR traces nd nd nd Mean values within a column superscripted by the same small letter are not significantly different at p < 0.05. Mean values within a row superscripted by the same capital letter are not significantly different at p < 0.05. OTL, Otello; VNR, Venere; NRN, Nerone; ATM, Artemide; EMS, Ermes; RRC, Riz de Camargue rouge/PGI; FRR, Riz de Camargue rouge/organic; nd, not detactable; ns, not significant; C3G, Cyanidin-3-Oglucoside; P3G, Peonidin-3-O-glucoside; d.m., dry matter.

32

Highlights

1. 2. 3. 4. 5.

Total lutein (TL) was the main carotenoid and accounted for more than 82%. Phenolic compounds were 88-95% in free form (FPCs) and 5-12% in bound form (BPCs). Cyanidin-3-O-glucoside (C3G) accounted for more than 87% in black rice. Cooking decreased TL by 30-69% and FPCs by 5-40%. BPCs increased (33-61%) in red rice. After cooking, more than 48% of C3G was retained by two black varieties.

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Chemical compounds

Lutein (PubChem CID: 6433159) Zeaxanthin (PubChem CID: 5280899) 5

β-Carotene (PubChem CID: 5280489)

6

Cyanidin-3-O-glucoside chloride (PubChem CID: 68247)

7

Peonidin-3-O-glucoside chloride (PubChem CID: 14311151)

34