Food Chemistry 191 (2016) 2–6

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Food Chemistry

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Determination of some minerals and b-carotene contents in aromatic indica ( L.) germplasm ⇑ N. Renuka, Sarika V. Mathure, Rahul L. Zanan, Ratnakar J. Thengane, Altafhusain B. Nadaf

Department of Botany, Savitribai Phule Pune University, Pune 411007, India article info abstract

Article history: 39 aromatic indica rice (Oryza sativa L.) cultivars were characterized for Iron, Zinc, Calcium, Magnesium, Received 4 December 2014 Copper and b-carotene contents. The b-carotene contents were ranging from 1.23 to 9.9 lg/g in brown Received in revised form 2 May 2015 and 0.08 to 1.99 lg/g in milled rice. Among the mineral contents, Magnesium was found ranging from Accepted 11 May 2015 855 lg/g (Gham) to maximum of 1636 lg/g (Badshahbhog) followed by Iron in 32 lg/g (Jirga) to Available online 19 May 2015 218 lg/g (Kalsal), Copper content from 2 lg/g (Girga) to 1004 lg/g (Gham), Zinc content from 25 (Gham) to 165 lg/g (Ambemohar-157) and Calcium ranged from 14 lg/g (Ambemohar pandhara) to Keywords: 67 lg/g (Kate chinoor). The study showed that the germplasm assessed is a good source of micronutrients b-Carotene and can be further exploited in breeding programme. Minerals landraces Ó 2015 Elsevier Ltd. All rights reserved. Oryza sativa L. spp. indica

1. Introduction conventional or genetic engineering to accumulate micronutrients in edible portion (Stein, 2010). Choice of potential food fortification Rice (Oryza sativa L.) is the staple food of over half the world’s vehicles depends on food commonly consumed by the target population. It is the predominant dietary energy source for 17 group, its affordability and availability (Latfi, Venkatesh Mannar, countries in Asia and the Pacific, 9 countries in North and South Merx, & Heuvel, 1996). Identification of genetic resources with America and 8 countries in Africa. Rice provides 20% of the world’s high levels of targeted micronutrients is a necessary step to dietary energy supply (Donald, 2002). Rice is low in fat and high in enhance micronutrient levels through conventional plant starchy carbohydrate, packed full of vitamins and minerals and breeding (Graham, Senadhira, Beebe, Iglesias, & Monasterio, provides an excellent source of vitamin E, B vitamins (thiamin, 1999; Ortiz-Monasterio et al., 2007). niacin) and Potassium. Dietary minerals and trace elements play Micronutrient concentrations in rice are not sufficient to meet a significant role in maintenance of optimal health. These minerals the recommended daily dietary allowances to sustain good health. and vitamins are limiting in diets (McGloughlin, 2010) and its Milled rice is deficient in many essential micronutrients like Fe, Zn, ingestion in inadequate amount or due to poor bioavailability has vitamin E and vitamin A (Tan et al., 2005; Vasconcelos et al., 2003). negative impacts on the health (Stein, 2010). Main source of all To develop micronutrient enriched staple foods, traditional plant nutrients for people comes from agricultural products (Welch & breeding methods or biotechnological techniques were adopted Graham, 2004). According to Grusak and Cakmak (2005), average by researchers (Bouis, 2000) to combat micronutrient malnutrition mineral requirement from plant food source to human is as fol- (Graham, Welch, & Bouis, 2001). The most effective approach for lows: Calcium (Ca) (12 mg/g food), Copper (Cu) (0.015–0.03 mg/g solving the problem of mineral nutrient deficiencies in humans is food), Iron (Fe) (0.15 mg/g food), Magnesium (Mg) (3.5 mg/g food) to develop rice cultivars with abundant mineral nutrients. Many and Zinc (Zn) (0.15 mg/g food). Deficiencies of these have negative attempts have been made to enhance mineral and b-carotene impact on public health at regional as well as at global level (BC) content through transgenic approaches. However the regula- (Stein, 2010). tory measures have still not allowed these transgenics for commer- Malnutrition and hidden hunger due to deficiency of micronu- cial cultivation (Ramjoue, 2008). The elite landrace in terms of trients is becoming a severe problem in the world, especially in better minerals and BC contents can serve as a promising source developing countries (Datta et al., 2006). One of the interventions as an alternative for GMOs. India is considered as one of the centers against micronutrient malnutrition is breeding of crops through of origin of rice (O. sativa L. ssp. indica) and has also remained as a center of its diversity (Khush, 2000). Our earlier studies on aro- matic rice cultivars from Maharashtra reveled that these cultivars ⇑ Corresponding author. possess superior agronomic traits (Mathure et al., 2011) and have E-mail address: [email protected] (A.B. Nadaf). http://dx.doi.org/10.1016/j.foodchem.2015.05.045 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved. N. Renuka et al. / Food Chemistry 191 (2016) 2–6 3

2. Material and methods

2.1. Rice samples

39 rice cultivars representing 33 non aromatic, 3 bas- mati (long grain 6.61–7.5 mm, 2 mm breadth, length-to-width ratio over 3.0) and 3 non aromatic were selected for the study. These rice cultivars were previously collected by us during 2007 to 2012 and stored at the Department of Botany, Savitribai Phule Pune University, Pune, Maharashtra, India. The seedlings of these cultivars were raised in the experimental field at Karjat rice research station, Karjat, District Raigadh, Maharashtra state, India following routine practice. At maturity the panicles were har- vested, dried in shade, threshed and seeds were used for analyses.

2.2. Quantification of BC and mineral contents

BC content in brown and milled rice cultivars were estimated using approved AACC protocol described by Santra, Rao, and Tamhankar (2003). The brown and milled rice grain samples were ground to fine powder and sieved through 100 mesh size sieve. A homogenous suspension was prepared by dispersing 1 g powdered sample in 5 ml water saturated n-butanol, mixed gently and allowed to stand in dark for overnight at room temperature. The samples were centrifuged at 10,000 rpm for 10 min (Eppendorf-5418R, Germany) to obtain the supernatant. The optical density of the supernatant was measured at 440 nm using the UV–visible spec- trophotometer (Shimadzu-1601, Japan) against water saturated n-butanol as blank. A calibration curve was developed using stan- dard BC (Sigma, Bangalore, India) and BC content was estimated. Elements Ca, Cu, Fe, Mg and Zn were estimated following the method of Bhargava and Raghupathi (1993) using diacid mixture method. In 1 g powdered unpolished rice sample, 10 ml

HNO3:HClO4 acid mixture (9:4) was added and mixed by swirling.

Fig. 1. Heat map representation of nutrient contents in rice grains of aromatic rice The mixture was placed in a digestion chamber and heated at cultivars. higher temperature until fuming of red nitrogen dioxide ceased. The contents were evaporated to reduce the volume to 3–5 ml. The completion of digestion was confirmed by the formation of colorless liquid. After cooling, 20 ml of de-ionized water was added market potential owing to aroma contents (Mathure, Wakte, and the solution was filtered through Whatman filter paper 1. The Nadaf, & Jawali, 2011). These cultivars also exhibited high genetic aliquots of this solution were used for determination of mineral diversity (Mathure, Nadaf, & Jawali, 2010). As these cultivars elements. Individual standard stock solutions (1 mg/L) for each have potential to effectively contribute towards the gene pool, a mineral element were prepared for quantification. Different set of 39 aromatic rice cultivars was characterized for minerals dilutions ranging from low to high concentrations were made and BC contents. and standard graph was plotted. The mineral contents were

Fig. 2. Distribution of b-carotene content (BC) in brown and milled rice grains of aromatic rice cultivars. 4 N. Renuka et al. / Food Chemistry 191 (2016) 2–6 estimated in triplicate using atomic absorption spectrophotometer 2.3. Data analyses (Perkin-Elmer 3110; Perkin Elmer, Waltham, MA). The operating parameters for working these elements were set as per the The descriptive analysis including mean, standard deviation, manufacturer’s protocol. Four external standard curves were frequencies and minimum and maximum values of variables was constructed using reference standard to quantify the metals performed. Paired t test was conducted to determine significance content in all samples. Calibration curves were performed with between BC and minerals contents using SPSS software (version 5–6 different concentrations. For elemental analyses, a 10 cm 11, Chicago, IL, USA). 1-slot burner head with standard air-acetylene flame and single element hollow cathode lamps were employed. A spoiler nebulizer 3. Results and discussion was applied for all the elements and for the Mg determination impact bead nebulizer was used. IUPAC criteria were followed to The BC content in (BC brown) and milled rice (BC calculate the detection limit (Long & Winefordner, 1983). milled), Ca, Cu, Fe, Mg and Zn are depicted in Fig. 1 and

Fig. 3. Distribution of mineral content in rice grains of aromatic rice cultivars. N. Renuka et al. / Food Chemistry 191 (2016) 2–6 5

Supplementary 1. Maximum BC was recorded in brown rice of The values of Fe, Zn and Mg are in agreement to that of the Belgaum basmati (9.9 lg/g) followed by Girga (9.56 lg/g), earlier reports except Ca (Graham et al. 1999; Itani, Tamaki, Ambemohar (7.87 lg/g) and the least was recorded in Kaligajvili Arai, & Horino, 2002; Heinemann, Fagundes, Pinto, Penteado, & (1.23 lg/g). In majority of the cultivars, BC content in brown rice Lanfer-Marquez, 2005; Shabbir, Anjum, Zahoor, & Nawaz, 2008; was less than 6 lg/g whereas, in milled rice, it varied in a narrow Jiang et al., 2008; Krishnan et al., 2009; Zeng et al., 2004). The range (0.08 lg/g (Jirga) – 1.99 lg/g (Kamod) (Fig. 2). BC content cultivars possessed higher Cu content than the values reported reduced to a greater extent in milled samples. Krishnan, Datta, for the fragrant rice (Heinemann et al., 2005; Jiang et al., 2008; Parkhi, and Datta (2009) reported that during milling process, aleu- Zeng et al., 2004). Among the transgenics developed by Krishnan rone layer and part of the embryo is removed which are the main et al. (2009), 21 mg/kg Fe content was recorded in the unpolished storage site for major micronutrients and about 70% of micronutri- IR68144 and 15 mg/kg in BR29. Fe content in polished rice grains of ents are lost. Lamberts and Delcour (2008) recorded significant IR68144 and BR29 were 15 and 8.9 mg/kg, respectively. Zn content reduction (2–3-fold) in BC after milling in 5 rice cultivars (0.020 of unpolished transgenic rice seeds of IR68144 was 40 mg/kg, to 0.085 lg/g). This indicated that brown rice is a better source while in BR29 36 mg/kg. Polished rice grains of IR68144 and of BC. BR29 recorded 37 and 32 mg/kg of Zn respectively. The differences Vitamin A deficiency is an important nutritional problem in the in Fe content may be affected by their growing environments and developing world (World Bank, 1993). Vitamin A is an essential genetic differences (Meng, Wei, & Yang, 2005). Oko, Ubi, Efisue, and micronutrient, deficiencies of which in the diet can lead in xeroph- Dambaba (2012) reported no significant correlation among the Ca, thalmia, blindness and premature death (West, 2002). World Mg and Potassium hence these mineral elements can be indepen- widely, 250 million preschool children are vitamin A deficient, dently selected in a breeding program for improvement of rice. and 250,000–500,000 face blindness every year associated with Among 39 cultivars, with respect to BC and minerals contents, 8 50% mortality within 12 months of losing their sight (West & top ranking cultivars were identified (Table 1). Velchi, Makarand Darnton-Hill, 2001). BC is the major source of vitamin A for and Ambemohar ajara were superior for brown BC where as majority of the people in the world (Sommer & West, 1996). A Indrayani, Manila, Kondhekar chinnor and Velchi were signifi- genetically engineered rice variety ‘’ having higher BC cantly superior for milled BC. Ambemohar ajara, Indrayani, in the endosperm of grain (1.6 lg/g) was developed to combat Lalbhath and Makarand were significantly superior for Ca content. vitamin A deficiency (Ye et al., 2000). Further, Hoa, Al-Babili, Tarori basmati and Ambemohar ajara were significantly superior Schaub, Potrykus, and Beyer (2003) developed three novel indica for Cu content. Kondhekar chinnor, Ambemohar ajara and and japonica golden rice lines (0.4–1.2 lg/g carotenoid in T2 lines). Lalbhath were superior for Fe content. Lalbhath, Tarori basmati, An improved version of this ‘Golden rice 2’ with BC up to 35 lg/g Manila, Indrayani and Kondhekar chinnor were significantly supe- was also developed (Paine et al., 2005). Datta et al. (2006) rior for Mg content. Velchi, Makarand and Tarori basmati were sig- developed improved golden rice (variety BR29) through nificantly superior for Zn content. Based on overall contents of BC post-transgeneration enhancement for higher BC. They recorded and minerals, Velchi followed by Kondhekar chinnor, Indrayani, 9.34 lg/g total carotenoids and 3.92 lg/g BC in milled grains. Lalbhath and Tarori basmati can be taken as superior cultivars. Krishnan et al. (2009) developed transgenic indica rice lines of These rice cultivars could be used for further improvement in IR68144 and BR29, and were analyzed for their Fe, Zn and BC hybridization programme to achieve desired varieties. content. The analysis revealed the presence of higher accumulation The study demonstrated that the cultivars assessed are not only of Fe, Zn and BC (0.4–0.96 lg/g) in transgenic rice grains in diverse in terms of agronomic traits but also a good source of comparison with control. Datta, Sahoo, Krishnan, Ganguly, and micronutrients and hence can be further exploited in breeding pro- Datta (2014) reported 0.257–3.188 lg/g total carotenoids in the gramme. In spite of increasing response to genetically modified endosperm of transgenic double haploid rice cultivar BR29. In (GM) crops obtained through genetic engineering worldwide, acti- view of these, the cultivars under study can be taken as a better vist groups in many countries in general and in Europe in particu- source for BC. lar have continued to fight the introduction of GM foods (Paarlberg, Among the elements studied, average Mg content was highest 2002). The new European Union regulation set strict labeling and (1273.92 lg/g) followed by Fe (105.82 lg/g), Cu (102.59 lg/g), Zn traceability on all GM-derived foods and feeds (requiring a costly (69.67 lg/g) and Ca (23.49 lg/g) (Fig. 3, Supplementary 1). This physical segregation of GM from non-GM all the way up the mar- variation in mineral element contents can be directly or indirectly keting chain) that will further discourage the planting of GM crops related to varietal characteristics. Majority of the cultivars recorded in poor countries (Paarlberg, 2002). In this context, the indigenous Ca content below 34 lg/g with the maximum quantity of 67 lg/g in cultivars possessing superior nutritional traits will open up new Kate-chinoor. The Cu content varied from 2 lg/g (Girga) to avenues in the cultivar developmental programme. 1004 lg/g (Gham). 80% cultivars recorded less than 100 lg/g Cu content. Gham, Jiri, Chimasal, Raibhog and Kadkya can be taken Acknowledgement as the good sources of Cu. The Fe content varied from 32 to The authors are thankful to the authorities of Karjat rice 218 lg/g, Mg from 855 to 1636 lg/g and Zn from 25 to 165 lg/g. research station, Karjat for providing field facilities.

Table 1 Superior aromatic rice (Oryza sativa L.) cultivars with respect to BC and mineral elements from the predominant group.

Name of accessions BC-brown (lg/g) BC-milled (lg/g) Ca (lg/g) Cu (lg/g) Fe (lg/g) Mg (lg/g) Zn (lg/g) Ambemohar ajara 2.97 ± 0.16c 0.86 ± 0.06e 28 ± 0.05a 35 ± 0.02b 128 ± 1.03b 1006 ± 2.09h 33 ± 0.05h Kondhekar chinnor 2.21 ± 0.24d 1.17 ± 0.20b 15 ± 0.02e 20 ± 0.46c 138 ± 2.03a 1224 ± 1.58e 71 ± 0.35f Lalbhath 2.15 ± 0.18e 0.47 ± 0.00f 22 ± 0.46c 11 ± 0.09e 120 ± 0.98c 1414 ± 2.25a 99 ± 2.68d Velchi 3.92 ± 0.04a 1.13 ± 0.01c 19 ± 0.01d 2 ± 0.01f 87 ± 0.07d 1213 ± 1.52f 140 ± 0.01a Indrayani 2.08 ± 0.15f 1.20 ± 0.10a 25 ± 0.03b 13 ± 0.13d 74 ± 0.23e 1229 ± 1.90d 91 ± 0.05e Makarand 3.59 ± 0.11b 0.33 ± 0.04g 22 ± 1.00c 190.16 ± c 65 ± 0.13f 1130 ± 1.11g 132 ± 1.01b Taraori basmati 1.28 ± 0.29h 1.02 ± 0.05d 18 ± 0.02d 75 ± 1.05a 59 ± 1.05g 1323 ± 3.79b 109 ± 0.08c Manila 1.74 ± 0.03g 1.16 ± 0.03b 16 ± 0.09e 19 ± 0.09c 45 ± 1.74h 1235 ± 2.27c 48 ± 0.08g

± Standard Error, Values with the same letter in each rice type for each BC/mineral content are not significantly different at p = 0.05. 6 N. Renuka et al. / Food Chemistry 191 (2016) 2–6

Appendix A. Supplementary data Mathure, S. V., Wakte, K. V., Nadaf, A. B., & Jawali, N. (2011). Quantification of 2- acetyl-1-pyrroline and other rice aroma volatiles among Indian scented rice cultivars by HS-SPME/GC-FID. Food Analytical Methods, 4(3), 326–333. Supplementary data associated with this article can be found, in Mathure, S., Nadaf, A., & Jawali, N. (2010). Diversity analysis in selected non- the online version, at http://dx.doi.org/10.1016/j.foodchem.2015. basmati scented rice collection. Rice Science, 17(1), 35–42. 05.045. Mathure, S., Shaikh, A., Renuka, N., Wakte, K., Nadaf, A., & Jawali, N. (2011). Characterisation of aromatic rice (Oryza sativa L.) germplasm and correlation between their agronomic and quality traits. Euphytica, 179, 237–246. References McGloughlin, M. N. (2010). Modifying agricultural crops for improved nutrition. New Biotechnology, 27(5), 494–504. Bhargava, B. S., & Raghupathi, H. B. (1993). 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