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Effect of traditional nixtamalization on anthocyanin content and profile in Mexican blue maize (Zea mays L.) landraces
Accepted Manuscript Effect of Traditional Nixtamalization on Anthocyanin Content and Profile in Mexican Blue Maize (Zea mays L.) Landraces Saraid Mora-Rochín, Nalleli Gaxiola-Cuevas, Janet Alejandra Gutiérrez-Uribe, Jorge Milán-Carrillo, Evelia María Milán-Noris, Cuauhtémoc Reyes-Moreno, Sergio Othon Serna-Saldivar, Edith Oliva Cuevas-Rodríguez PII:
S0023-6438(16)30009-3
DOI:
10.1016/j.lwt.2016.01.009
Reference:
YFSTL 5212
To appear in:
LWT - Food Science and Technology
Received Date: 1 July 2015 Revised Date:
1 January 2016
Accepted Date: 5 January 2016
Please cite this article as: Mora-Rochín, S., Gaxiola-Cuevas, N., Gutiérrez-Uribe, J.A., Milán-Carrillo, J., Milán-Noris, E.M., Reyes-Moreno, C., Serna-Saldivar, S.O., Cuevas-Rodríguez, E.O., Effect of Traditional Nixtamalization on Anthocyanin Content and Profile in Mexican Blue Maize (Zea mays L.) Landraces, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2016.01.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of Traditional Nixtamalization on Anthocyanin Content and Profile in Mexican
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Blue Maize (Zea mays L.) Landraces
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Saraid Mora-Rochína, b, Nalleli Gaxiola-Cuevasb, Janet Alejandra Gutiérrez-Uribec, Jorge
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Milán-Carrilloa,b, Evelia María Milán-Norisb, Cuauhtémoc Reyes-Morenoa, b, Sergio Othon
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Serna-Saldivarc, Edith Oliva Cuevas-Rodrígueza, b*
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a
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(FCQB), Universidad Autónoma de Sinaloa (UAS); bDoctorado en Ciencias, Especialidad
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Biotecnología (Programa Regional de
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Biotecnología-FEMSA. Escuela de Ingeniería y Ciencias. Tecnológico de Monterrey-Campus
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Monterrey, Monterrey, Nuevo León, CP 64849 México.
Maestría en Ciencia y Tecnología de Alimentos, Facultad de Ciencias Químico Biológicas
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Centro de
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Biotecnología), FCQB-UAS;
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*Corresponding author: Edith Oliva Cuevas-Rodríguez, Blvd de la Americas y Josefa Ortiz
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de Dominguez s/n Ciudad Universitaria, C.P. 80010, Culiacan, Sinaloa, México. Tel/Fax:
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+1526677137860
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E-mail address: [email protected]
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ABSTRACT
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Mexican blue maize (Zea mays L.) grains have been poorly evaluated regarding their
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potential as functional food ingredients. The aims of this research were to identify and
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quantify anthocyanins from fifteen Mexican blue maize accessions of Elotero Sinaloa
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landrace recollected in the northwestern region of Mexico. Additionally, the effect of
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traditional nixtamalization processing on these compounds was evaluated. The acyl type
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anthocyanins, such as cyanidin-3-(6”-succinylglucoside) (Cy-Suc-Glu) and cyanidin-3-(6”-
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disuccinylglucoside) (Cy-diSuc-Glu) were the most abundant compounds in blue maize,
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accounting for 52.1% and 15.6% the total anthocyanins, respectively. Other predominant
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anthocyanins included cyanidin-3-glucoside (Cy-3-Glu), pelargonidin-3-glucoside (Pg-3-
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Glu),
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malonyglucoside) (Cy-Mal-Glu). The raw blue maize presented a similar anthocyanins
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profile dominated by cyanidin derivatives on (86.9% on average). Nixtamalization
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processing increased the relative percentage of glycosylated anthocyanins (Cy-3-Glu, and
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Pg-3-Glu) and decreased the acylated anthocyanins (Cy-Suc-Glu, and Cy-diSuc-Glu) when
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compared to raw kernels. Results obtained indicate that the studied Mexican native blue
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maize contained anthocyanin patterns predominated by acylated cyanide derivatives.
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This information could be useful to select the best pigmented maize for the derivation of
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food products with nutraceutical potential.
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(Pg-Mal-Glu)
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cyanidin-3-(6”-
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pelargonidin-3-(6”-malonylglucoside)
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Keywords: Anthocyanin, Nixtamalization, Blue Maize and Tortilla
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1.
Introduction Mexico, considered center of origin and domestication of maize (Zea mays L.), has
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the largest diversity of genetic resources in the world with approximately 59 different
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native landraces, which have been classified based on morphological characters, and
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isozyme frequencies (Sánchez, Goodman, & Stuber, 2000). Native pigmented maize are
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cultivated in different regions of Mexico; Chalqueño, Bolita, and Elotes Conico landraces
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prevail in High Valleys of the Central Mesa. However, recent recollections of more than
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300 maize accessions from Northwest México (state of Sinaloa) have been identified and
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classified into 13 maize landraces, being Tabloncillo, and Elotero Sinaloa the distinctive
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landraces of this region (Pineda-Hidalgo et al., 2013).
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Pigmented maize tortilla is the main basic daily staple in small communities in
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Mexico and Central America. The term nixtamalization refers to the alkaline cooking
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process of converting maize into foodstuffs such as tortillas and snacks (maize chips,
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tortillas chips and tacos) (Serna-Saldívar, Gomez, & Rooney, 1990). Currently, demand of
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nixtamalized products of blue maize has received an increased attention from a
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nutraceutical perspective owing to their potential health benefits and unique flavor and
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color properties (Urias-Peraldí et al., 2013). Blue maize is an important source of
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anthocyanins; these bioactive compounds from maize kernels have shown multiple
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functional roles such as protection against oxidative stress, increased antimutagenic
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activity, and inhibition of colorectal carcinogenesis (Tsuda, 2012; Long et al., 2013; López-
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Martínez, Parkin, & Garcia, 2014). However, maize kernels need to be processed prior to
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human consumption, which may modify or degrade their natural phytochemicals. More
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information regarding the impact of nixtamalization processing conditions on the
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anthocyanin profiles of blue maize grains is needed especially in terms of the fate of their
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antioxidant compounds in nixtamalized products (Nayar, Liu, & Tang, 2015). Anthocyanins belong to the widespread class of phenolic compounds collectively
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named flavonoids. The chemical structure of the anthocyanin determines its stability,
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color intensity, and potential biological activity. While monomeric anthocyanins possess
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limited stability against hydration and pH changes whereas acylated anthocyanins show
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noteworthy stability to pH changes, temperature and light exposure (Dangles, Saito, &
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Brouillard, 1993). Such stability has been attributed to intramolecular and/or
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intermolecular copigmentation and self-association reactions. Accordingly, sources of
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acylated anthocyanins may provide the desirable stability for food applications (Giusti &
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Wrolstad, 2003).
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Recent studies have shown that the main anthocyanins present in Andean
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pigmented maize have been previously identified as cyanidin-3-glucoside (Cy-3-Glu),
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pelargonidin-3-glucoside (Pg-3-Glu), and peonidin-3-glucoside (Pn-3-Glu), along with their
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corresponding malonyl derivatives. In these previous studies it has been found that
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proportion of acylated anthocyanins varied from 35.6% to 53.9% of total monomeric
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moieties (Jing, Noriega, Schwartz, & Giusti, 2007; Montilla, Hillebrand, Antezana, &
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Winterhalter, 2011; Pedreschi & Cisneros-Zevallos, 2007). Nevertheless, only few studies
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on anthocyanins have focused on Mexican blue maize grains, and even fewer showed
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their detailed anthocyanin profile. In Mexican blue/purple maize landraces, Cy-Mal-Glu
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and Cy-diMal-Glu were the major anthocyanins identified. The cyanidin is considered the
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main aglycone, accounting for 73-75.7% of all the anthocyanins (Salinas-Moreno, Pérez-
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Alonso, Vazquez-Carrillo, Aragón-Cuevas, & Velázquez-Cardenas, 2012). During processing of pigmented maize kernels into tortillas, the nixtamalization
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condition (alkaline pH) and high temperature are two of the main factors that affect the
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amount of anthocyanins originally present in kernels. Additionally the thermal lime
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treatment makes these compounds more unstable and susceptible to degradation
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(Sanchez-Madrigal et al., 2015). Several studies have been reported on the anthocyanin
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content and their degradation products during alkaline cooking process (Del Pozo-Insfran,
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Brenes, Serna-Saldivar, & Talcott, 2006; De la Parra, Serna-Saldivar, & Liu, 2007; Mora-
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Rochín et al., 2010; Aguayo-Rojas et al., 2012), but these studies were conducted with a
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limited number of contrasting maize grains. Unfortunately, there is little information
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available regarding the individual anthocyanins profile within groups of the same landrace
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and pigmented maize. On the other hand, the present study was undertaken to evaluated
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anthocyanin content and profile of fifteen different blue maize accessions correspond to
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Elotero Sinaloa landrace of northwest region of México. The effect of traditional
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nixtamalization process on the fate of these compounds was also investigated.
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2.
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2.1. Chemicals
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Materials and methods
Sodium hydroxide, hexane, methanol, ethyl acetate, HPLC grade acetonitrile and
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HPLC grade trifluoroacetic acid were obtained from Sigma Aldrich Chemical Co. (St Louis,
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MO, USA). Analytical (HPLC) standards of cyanidin-3-glucoside (purity > 95%) and
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pelargonidin-3-glucoside (purity > 97%) were obtained from Sigma Aldrich Chemical Co.
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(St Louis, MO, USA). A syringe filter unit was supplied by Pall Gelman Laboratory (MI,
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USA).
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2.2. Materials The study was performed on 15 contrasting blue kernels correspond to typical
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landrace of northwestern region of Mexico: Elotero Sinaloa. These pigmented maize
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accessions were collected from open-pollinated maintained by traditional farmers at their
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villages in the municipality of Concordia located at (23° 17′ 18″ N, 106° 4′ 3″ W), state of
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Sinaloa México. All materials were grown and harvested during 2013. Crops were
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managed following standard recommendations. Maize samples were stored at -4 °C until
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further processing.
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2.3.
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Determination of biophysical properties
Grain physical characteristics were determined using standard procedures: Kernel
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test weight (TW) according to Official US Grain Standard Procedures (AACC, 2000; Method
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55-10) and thousand-kernel weight (TKT) by weighing 100 randomly selected kernels at
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13% of grain moisture. Flotation index (FI), as an indirect measure of hardness, was
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assessed using the procedure proposed by Vazquez-Carrillo, Santiago-Ramos, Gaytán-
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Martínez, Morales-Sánchez, & Guerrero-Herrera. (2015).
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2.4. Production of nixtamalized maize flours
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Nixtamalized maize flour was obtained according to Milán-Carrillo, Gutierrez-
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Dorado, Cuevas-Rodríguez, Garzón-Tiznado, & Reyes-Moreno. (2004). Briefly, maize
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kernels (1000 g lots) was added in a medium kettle containing 3 L of distilled water (1:3,
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maize grains/water) and either to 5.4 g of Ca(OH)2/L water. The samples were cooked for
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31 min at approximately 85 °C, followed by a steep time of 8.1 h. After steeping, the
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cooking liquor (nejayote) was drained and discarded and the nixtamal (alkaline-cooked
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maize) washed with running tap water for 40 s and blotted between paper towels. Wet
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nixtamal was dried at 55 °C/12 h in a forced air oven and then cooled at room
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temperature. Finally, the dried nixtamal was milled (UD Cyclone Sample Mill, UD Corp.
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Boulder, CO, USA) to pass through an 80-US mesh (0.180 mm) screen, and packed in
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plastic bags. Nixtamalized maize flours were stored at -20 °C until use.
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2.5. Tortilla preparation from nixtamalized maize flours
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Tortillas were prepared by mixing 400 g of nixtamalized maize flours with 400 mL of
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water to achieve an adequate masa consistency for the production of table tortillas.
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Pieces of fresh dough (30 g) were pressed and shaped into flat disks (15 cm) using a
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manual machine (Casa Herrera, México DF, México). The dough disks were baked on a hot
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griddle at 270 ± 10 °C for 15 s on one side, followed by 30 s on the other side, and then
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again on the first side until puffing of the tortilla occurred. The fresh tortillas were dried,
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milled (UD Cyclone Sample Mill, UD Corp. Boulder, CO, USA) to pass through an 80-US
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mesh (0.180 mm) sieve and packed in plastic bags. Tortillas from nixtamalized flours were
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stored at -20 °C until use further.
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2.6. Total anthocyanin content
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Extractions were performed using ground sample (250 g) and mixing with 10 mL of
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acidified methanol solution with 36.5 g/L HCl (95:5, mL:mL). The samples were then
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shaken for 30 min and centrifuged at 3000g for 5 min (Sorvall RC5C, Sorvall Instruments,
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Dupont, Wilmington, DE, USA), and the supernatants were collected. Absorbance readings
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at 535 nm were taken and corrected for background absorbance at 700 nm using a
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Microplate Reader (Synergy HT, Bio-Tek Instruments, Inc., Winooski VT, USA). Using the
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molar extinction coefficients of 25,965 Abs/M x cm and a molecular weight of 449.2 g/mol
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the total anthocyanin content was calculated and expressed as mg of cyanidin 3-glucoside
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equivalent (CGE) per 100 g of dry weight DW (Abdel-Aal & Hucl, 1999)
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2.7. Identification and quantification of anthocyanins
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Anthocyanins extracts were obtained according to procedure described by Abdel-
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Aal, Young, & Rabalski. (2006). Briefly, 250 mg of sample was mixing with 10 mL of
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acidified methanol with 36.5 g/L HCl (95:5, mL:mL) and continuous shaking for 30 min a
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room temperature. After the pH was adjusted to 1.0 by 6 mol/L HCl. The samples were
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then centrifuged (1,538g, 10 min, 4 °C) in a Sorvall RC 5C superspeed centrifuge (Sorvall
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Instruments, Dupont, Wilmington, DE, USA); the supernatants were collected and the
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residue submitted twice to the same extraction conditions described above. The
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supernatants were combined and concentrated under vacuum in a Büchi K124 rotary
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evaporator (Büchi Labortechnik AG, Switzerland) a 30 °C until the methanol was removed.
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The concentrated extracts were lyophilized and stored at -20°C prior to analysis. For HPLC
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analyses of anthocyanins, lyophilized extracts were re-dissolved in methanol concentrated
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(5 mg/mL), filtered through a syringe filter with 0.45 µm nylon membrane (Pall Gelman
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Laboratory, Ann Arbor, MI, USA).
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The HPLC analyzed were carrier out with an HPLC-PDA system (1200 Series, Agilent
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Technologies, Santa Clara, CA, USA). Chromatograms were obtained at 525 nm after
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injection of 2.0 µL of sample. Separation was performed in a Zorbax-SB Eclipse XDB-C18
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column (4.6 x 150 mm, 5 µm; Agilent Technologies, Santa Clara, CA, USA) at 45 °C. The
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mobile phase used was (A) trifluoroacetic acid (TFA) with HPLC-grade water (TFA/H2O,
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1:99 mL:mL) and (B) HPLC-grade acetonitrile, establishing the following gradient: isocratic
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A/B (85:15, mL:mL) for 5 min, A/B (85:15-81:19, mL:mL) over 7 min, isocratic A/B (81:19,
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mL:mL) for 10 min, A/B (81:19-60:40, mL:mL) over 15 min and A/B (60:40-100:0, mL:mL)
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over 25 min, using a flow rate of 0.8 mL/min. Pure anthocyanin compounds (Cy-3-Glu and
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Pg-3-Glu) were used as standards. Ultraviolent-visible (UV-vis) absorption spectra were
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recorded for the predominant peaks.
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The identification of each peak was confirmed by HPLC-MS-TOF (Agilent, Santa
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Clara, CA, USA). Chromatographic conditions used were the same as those described for
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HPLC-PDA analysis. Mass spectra were collected using an electrospray source in positive
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mode (ESI+) under the following conditions: m/z range, 200-1000; nitrogen gas; gas
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temperature, 350 °C; drying gas flow rate, 10 L/min; nebulizer pressure, 127.9 kPa;
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capillary voltage, 4.0 KV; and fragment voltage, 100 V. Compounds were characterized and
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identified by their MS, MS/MS spectra and LC retention times and by comparison with
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available references samples.
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Anthocyanins were quantified using a linear calibration curves, generated with
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commercial authentic standards of Cy-3-Glu and Pg-3-Glu at seven different
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concentrations (0.1 to 5.0 mg/mL) at 520 nm. For each compounds, the regression
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equations were Cy-3-Glu (y = 5.83-117.99x; R2 = 0.997) and Pg-3-Glu (y = 0.69-3.89x; R2 =
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0.998). The results were expressed as micrograms of commercial standards per g of dry
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weight (DW).
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2.8
Statistical analysis ANOVA procedures were used for the analysis of the experimental data. Differences
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among treatments were determined using Duncan’s comparison test. Data were reported
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as mean ± standard deviation (SD) in triplicate.
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3.
Results and discussion
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3.1
Biophysical kernel properties
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Biophysical traits of Mexican blue maize showed a significant variation (p < 0.05)
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among genotypes (Table 1). Serna-Saldivar et al. (2008) indicated that the preferred
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properties for the commercial nixtamalization process are test weight (TW) and thousand
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kernels weight (TKW) greater than 72 kg/hL and 320 g, respectively. Interestingly, the
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majority of the Mexican blue maize accessions analyzed possessed these attributes (Table
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1). Among raw blue grains, FAUAS-290 accession followed by FAUAS-419 and FAUAS-429
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had higher (p < 0.05) TW and TKW, compared to the other blue maize accessions; but
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FAUAS-230 blue grain followed by FAUAS-249, FAUAS-252 and FAUAS-485 had lower TKW
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than 320 g.
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Grain hardness (measured indirectly from Flotation index, FI) showed that FAUAS-
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230 (FI = 14.7%) blue maize was the hardest, while FAUAS-485 (FI = 94.7%) was the softest
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one (Table 1). Moreover, some Mexican blue maize such as FAUAS-230 followed by
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FAUAS-290, FAUAS-419, FAUAS-437, FAUAS-491 and FAUAS-512 had FI ≤ 40%,
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characteristic which make them adequate for the nixtamalized flour industry. The rest of
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the maize accessions FAUAS-220, FAUAS-252, FAUAS-387, FAUAS-429, FAUAS-447,
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FAUAS-457 and FAUAS-488 had FI > 40%, indicating that their kernels had soft or
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intermediate endosperm hardness and readily uptake water during nixtmalization which
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positively affects masa and tortillas yields (Vázquez-Carrillo et al., 2015). Therefore, this
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blue maize Elotero Sinaloa landrace endemic of Northwest Mexico should yield masa
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according to the Mexican normative, NMX-FF-034/1-SCFI-2002 (SAGARPA, 2002).
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Total anthocyanins content
Total anthocyanin content (TAC) in raw blue maize genotypes and their tortillas
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produced following the traditional process is shown in Table 2. Among raw blue kernels
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FAUAS-485 (34.3 mg CGE/100 g DW) and FAUAS-512 (33.7 mg CGE/100 g DW) contained
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the highest (p < 0.05) TAC concentration compared to the rest of the blue maize. On the
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other hand, the FAUAS-429 (14.1 mg CGE/100 g DW) kernels contained the lowest (p <
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0.05) TAC. The assayed concentration were close to TAC previously obtained for American
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and Mexican blue maize (Del Pozo-Insfran et al., 2006), American blue maize (De la Parra
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et al., 2007) and different native populations of Mexican pigmented genotypes (Espinosa-
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Trujillo, Mendoza-Castillo, Castillo-González, Ortiz-Cereceres, & Delgado-Alvarado, 2010),
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but were lower compared to 25 Mexican blue maize hybrids (Urias-Peraldí et al., 2013)
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and native Mexican pigmented maize populations belonging to the Chalqueño, Elotes
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Cónicos and Bolita landraces (Salinas-Moreno et al., 2012). The observed differences in
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TAC could be attributed to the variability in the blue maize genotypes including genetic
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background, grain physical properties and particularly the relative ratio of the anatomical
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parts of the kernel since the pericarp and aleurone the layer are the structures richer in
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anthocyanins (Jing et al., 2007; Salinas-Moreno, Martínez-Bustos, Soto-Hernández,
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Ortega-Paczka, & Arellano-Vázquez, 2003).
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As expected, the traditional nixtamalization process caused significant (p < 0.05)
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losses of total anthocyanins. Less than 46.0% of TAC was retained in tortillas produced by
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traditional nixtamalization (Table 2). The remaining amounts of anthocyanins in tortillas
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produced from Mexican blue maize varied from 2.1 to 15.5 mg CGE/100 g DW. The
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FAUAS-512 maize tortillas prepared from blue maize nixtamalized flour retained 46.0%
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TAC and therefore contained the highest amount of anthocyanins among the array of the
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blue maize tortillas. These significant losses occurred during nixtamalization process as a
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result of the combined effect of alkaline pH (approximately 10) and thermal processing
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which enhanced physical losses of the pericarp and leaching of anthocyanins into the
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cooking liquor or nejayote (Mora-Rochín et al., 2010). De la Parra et al. (2007) reported
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anthocyanin losses of 83% during alkaline cooking with different types of maize
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genotypes. Likewise, Salinas-Moreno et al. (2003) reported relevant anthocyanin losses in
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a pigmented maize landrace during traditional nixtamalization process.
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3.3. Identification and quantification of anthocyanins
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Anthocyanin profiles observed in raw blue grains of Elotero Sinaloa landrace and
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their respective tortillas were very similar, with the presence of 10 peaks (Table 3). For
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reasons of space, representative chromatograms of only two of the samples are presented
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(Fig 1A, Fig 1B, Table 3). Anthocyanin compounds were determined by comparison of the
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spectroscopic and chromatographic properties with those of authentic anthocyanin
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standards (Cy-3-Glu and Pg-3-Glu). The remaining compounds were tentatively identified
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on the basis of mass identification using a combination of the retention time, peak
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spectra, mass-to-charge ratio and MS fragmentation (Abdel-Aal et al., 2006). Six major
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anthocyanins were identified, including cyanidin-3-glucoside (Cy-3-Glu; peak 1, m/z 449),
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pelargonidin-3-glucoside (Pg-3-Glu; peak 3, m/z 433), cyanidin-3-(6”-malonylglucoside)
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(Cy-Mal-Glu; peak 2, peak 4 and peak 5, m/z 535), cyanidin-3-(6”-succinylglucoside) (Cy-
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Suc-Glu; peak 6 and peak 8, m/z 549), pelargonidin-3-(6”-malonylglucoside) (Pg-Mal-Glu;
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peak 7, m/z 519) and cyanidin-3-(6”-disuccinylglucoside) (Cy-diSuc-Glu; peak 9 and peak
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10, m/z 649).
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Some derived compounds had similar mass spectra and spectroscopic properties,
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but different retention times (Table 3). This could be explained by the presence of three
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isomers of Cy-Mal-Glu, two isomers of Cy-Suc-Glu and two isomers of Cy-diSuc-Glu. These
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results are consistent with the previous research that reported the presence of two
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isomers of Cy-diSuc-Glu and three isomers of Cy-mal-Glu associated with Mexican native
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and hybrid blue maize genotypes (Urias-Lugo et al., 2015). In investigations of American
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pigmented maize it has been shown the presence of malonylglucoside isomers of cyanidin,
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which was distinguished mainly on the basis of LC elution profiles (Collison, Yang, Dykes,
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Murray, & Awika, 2015). Therefore, the analysis of nuclear magnetic resonance (NMR) is
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necessary to identify unambiguously the nature of the compounds that have different
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retention time and the same mass and fragmentation in the samples in order to establish
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different isomers of anthocyanins in blue maize (Abdel-Aal et al., 2006).
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The cyanidin was the main aglycone in the raw Mexican blue maize and constituted
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from 71.7% to 93.1% of the total anthocyanin. The highest concentration was assayed in
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the FAUAS-437 maize. The most abundant anthocyanins were Cy-Suc-Glu, Cy-diSuc-Glu
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and Cy-3-Glu accounting for 52.1%, 15.6%, and 9.4% respectively of the total anthocyanins
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(Table 4). The composition of individual anthocyanins, respectively, of the blue maize
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accessions of Elotero Sinaloa landrace showed remarkable differences with those
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reported by Salinas-Moreno et al. (2012), who found Cy-Mal-Glu and Cy-diMal-Glu as the
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most abundant anthocyanins for blue/purple grain of Chalqueño, Elotes Cónicos, and
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Bolita Mexican maize landraces, while other researchers found for Andean purple maize
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three major anthocyanins Cy-diMal-Glu, Cy-3-Glu, Pg-3-Glu (Pedreschi & Cisneros-Zavala,
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2007; Jing et al., 2007). Recently, Collison et al. (2015) identified Cy-3-Glu and Cy-Mal-Glu
294
as the major anthocyanins in American red/blue maize.
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Interestingly, seven of nine anthocyanins found in the raw Mexican blue maize
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studied herein were acylated. The acylated anthocyanin in maize Elotero Sinaloa landrace
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varied from 66.4 to 94.7% (average 82.9%) of the total anthocyanins. The highest
298
predominance of acylated anthocyanins in the blue maize analyzed was in FAUAS-437,
299
followed by FAUAS-457, FAUAS-419 and FAUAS-447. These maize accessions contained
300
from 91.7% to 94.7% of total anthocyanins, respectively. These values were significantly
301
higher compared to those previously reported in Andean purple/magenta maize kernels
302
(Pedreschi & Cisneros-Zavala, 2007; Jing et al., 2007; Montilla et al., 2011). Salinas-
303
Moreno et al. (2012) reported an average of 63.4% of acylated anthocyanins in
304
blue/purple Mexican maize landraces of the highlands of the central Mexico, whereas, the
305
American red/blue maize contained average of 60.0% acylated anthocyanins (Collison et
306
al., 2015). The acylated anthocyanins are considered more stable to pH and temperature
307
changes compared to the non-acylated ones. Therefore, sources of acylated anthocyanins
308
may provide the desirable stability for food applications (De Pascual-Teresa, Santos-
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Buelga, & Rivas-Gonzalo, 2002; Giusti & Wrolstad, 2003). The traditional nixtamalization process lowered the amount of anthocyanins in blue
311
maize tortillas. Given that all of the blue genotypes were nixtamalized under the same
312
conditions, the differences in the percentage of loss are mainly due to the anthocyanins
313
profile present in each blue maize. The group of six blue maize (FAUAS-220, FAUAS-419,
314
FAUAS-437, FAUAS-447, FAUAS-485 and FAUAS-512) tortillas retained the highest (45.0%
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to 60.1%) amounts of total anthocyanins when compared to raw grains.
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The changes in the anthocyanin profile between the respective blue raw kernels and
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their tortillas were mainly in the higher relative percentage of Cy-3-Glu (9.4% to 15.6%)
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and Pg-3-Glu (7.7% to 16.6%), and lower relative percentage of the acyl types such as Cy-
319
Suc-Glu (52.1% to 38.6%) and Cy-diSuc-Glu (15.7% to 13.1%). These changes could be
320
attributed to the thermal treatment under alkaline or high pH conditions that occur during
321
lime cooking. The effect was especially noticeable in the acylated anthocyanins. According
322
to the reports of Fossen et al. (1998) and De Pascual-Teresa et al. (2002) probably the
323
ester link that binds the acyl (malonyl and succinyl) radical with the sugar is very unstable
324
under alkaline pH, and thus may break, liberating the acyl radical and remaining only the
325
simple anthocyanin.
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4.
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Conclusions
The current study showed substantial differences in anthocyanin content and
327 328
composition profiles among Mexican native blue maize. It also contained high
329
predominance of acylated anthocyanins such with cyanidin-3-(6”-succinylglucoside) and
330
cyanidin-3-(6”-disuccinylglucoside). Interestingly, the highest relative percentage of
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acylated anthocyanins in relation to total anthocyanins it was found 82.9%. It is
332
noteworthy that tortillas obtained from nixtamalized blue maize (FAUAS-220, FAUAS-419,
333
FAUAS-437, FAUAS-447, FAUAS-485 and FAUAS-512) flours retained between 45.0% to
334
60.1% of total anthocyanins when compared to their respective raw kernels. On the basis
335
of results the presented herein the different Mexican native blue maize kernels may hold
336
promise for the development of functional foods or as a source of natural colorants.
337
Acknowledgements
338
This research was partially supported by the Universidad Autónoma de Sinaloa (Project
339
PROFAPI-2012), PROMEP/SEP (Project 2012, Thematic Networks for Cooperation, food
340
Biotechnology) and Consejo Nacional de Ciencia y Tecnología (CONACYT, Project 168279).
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Figure captions.
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Fig. 1. Representative HPLC chromatograms for anthocyanins (A) of raw grains and theirs
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corresponding (B) tortilla produced of nixtamalized flour of the FAUAS-512 blue maize.
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Peaks numbers refer to those indicated in Table 3.
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Table 1. a
Maize Accessions
Test weight (kg/hL)
1,000 Kernel weight (g)
Flotation index
FAUAS-220
76.4 ± 0.3b
340.7 ± 7.4f
60.0 ± 4.0e
Intermediate
FAUAS-230
78.7 ± 0.2a
280.8 ± 1.2i
14.7 ± 3.1h
Hard
FAUAS-249
76.8 ± 0.3b
247.5 ± 2.5k
78.0 ± 4.0b
Very soft
FAUAS-252
73.7 ± 0.2c
204.0 ± 3.9l
82.7 ± 3.1c
Soft
FAUAS-290
78.9 ± 0.5a
418.8 ± 4.9a
37.3 ± 2.3f
Intermediate
FAUAS-387
77.6 ± 0.6a,b
331.5 ± 1.8g
SC
Biophysical characteristics of fifteen Blue Mexican maize .
68.0 ± 4.0d
Soft
FAUAS-419
77.4 ± 0.3a,b
407.1 ± 7.7b
26.0 ± 3.9f
Intermediate
FAUAS-429
79.1 ± 0.6a
417.7 ± 2.7a
68.0 ± 2.0d
Soft
FAUAS-437
79.9 ± 0.3a
368.0 ± 6.8j
36.7 ± 2.1g
Intermediate
FAUAS-447
78.6 ± 0.2a
365.1 ± 4.6e
80.0 ± 3.0c
Soft
FAUAS-485
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75.1 ± 0.1b
362.5 ± 3.2e
82.7 ± 3.3c
Soft
68.1 ± 0.2d
264.7 ± 3.2j
94.7 ± 2.3a
Very soft
78.9 ± 0.4a
301.9 ± 2.3h
66.7 ± 3.3d
Soft
EP
FAUAS-488
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FAUAS-457
Hardness
78.2 ± 0.3a
387.9 ± 5.1c
24.7 ± 4.6f
Intermediate
FAUAS-512
78.9 ± 0.4a
324.4 ± 4.4g
36.0 ± 4.0f
Intermediate
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FAUAS-491
a
Means values ± standard error. Means by column and treatment with different letters show significant difference, (p < 0.05).
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Table 2. Anthocyanin content and percent of retention in different raw blue Mexican maize and their tortillas produced throughout nixtamalization process
a
Process Anthocyanin content % Retention Raw 25.9 ± 3.8c TNF 10.4 ± 0.5j 40.9 ± 2.3c FAUAS-230 Raw 17.7 ± 0.5g TNF 5.1 ± 0.1m 28.8 ± 0.9f f FAUAS-249 Raw 19.7 ± 0.3 TNF 2.1 ± 0.1p 10.7 ± 0.4k FAUAS-252 Raw 21.3 ± 0.7e TNF 2.5 ± 0.1o 11.7 ± 0.4j FAUAS-290 Raw 23.7 ± 0.6d TNF 5.0 ± 0.1m 21.1 ± 0.4h e FAUAS-387 Raw 22.1 ± 1.4 TNF 3.9 ± 0.1n 17.6 ± 0.3i b FAUAS-419 Raw 28.2 ± 0.2 TNF 8.7 ± 0.1k 30.9 ± 0.2f FAUAS-429 Raw 14.1 ± 0.9i TNF 3.3 ± 0.2n 23.4 ± 1.3 FAUAS-437 Raw 27.2 ± 0.9n TNF 8.7 ± 0.1k 32.7 ± 0.6e FAUAS-447 Raw 15.2 ± 0.2h TNF 5.4 ± 0.4m 35.5 ± 2.4de c FAUAS-457 Raw 24.8 ± 1.9 TNF 6.5 ± 0.5l 26.2 ± 1.9g FAUAS-485 Raw 34.3 ± 1.1a 36.2 ± 0.5d TNF 12.4 ± 0.2j FAUAS-488 Raw 25.9 ± 0.8c TNF 7.5 ± 0.1k 29.0 ± 0.5f cd FAUAS-491 Raw 24.1 ± 2.7 TNF 10.6 ± 0.4j 44.0 ± 0.9b a FAUAS-512 Raw 33.7 ± 3.1 TNF 15.5 ± 1.1h 46.0 ± 1.1a a Means values ± standard error. Means by column and treatment with different letters
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Maize Accessions FAUAS-220
show significant difference, (p < 0.05). Anthocyanin content is given in mg cyanindin 3glucoside equivalent (CGE)/100g dry weight (DW); TNF = Tortillas produced from nixtamalized flour
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Table 3. Mass spectrometric data for identification of anthocyanins of raw blue Mexican maize and their tortillas produced throughout nixtamalization process RT (Min)
λmax (nm)
1
Cy-3-Glu
3.1
517
3.5
512
4.2
502
a
2
1
Pg-3-Glu
4
Cy-Mal-Glu
5
Cy-Mal-Glu
6
Cy-Suc-Glu
1
MS/MS fragments (m/z)
449
287
535
287
433
287
4.9
515
535
287
5.3
513
535
287
2
6.1
514
549
287
7
Pg-Mal-Glu
6.9
504
519
271
8
Cy-Suc-Glu
7.8
516
549
287
9
Cy-diSuc-Glu
8.5
520
649
287
10
Cy-diSuc-Glu
9.0
519
649
287
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1
2
3
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Cy-Mal-Glu
Major ions (m/z)
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Anthocyanin
SC
Peak
3
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Cy, cyanidin; Glu, glucoside; Mal, malonyl; Pg, pelargonidin; Suc, succinyl.
1-3
Compounds with identical molecular mass within each superscript
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Table 4. Individual anthocyanin concentrations (µ µg/g DW) from fifteen Mexican blue maize and their tortillas produced throughout nixtamalization a process Cy-3-Glu
Cy-Mal-Glu c
l
Cy-Suc-Glu
l
f
Raw Tortilla
16.1 ± 0.4 i 6.8 ± 0.1
Raw Tortilla
10.5 ± 0.1 m 3.6 ± 0.0
4.5 ± 0.1 q 2.2 ± 0.0
10.4± 0.1 m 3.61± 0.0
29.6 ± 0.1 o 6.7 ± 0.0
FAUAS-249
Raw Tortilla
11.8 ± 0.1 l 4.1 ± 0.0
d
5.8 ± 0.10 q 2.5 ± 0.0
k
12.9± 0.1 mn 3.5± 0.0
c
39.3 ± 0.1 n 8.2 ± 0.0
FAUAS-252
Raw Tortilla
19.8 ± 0.1 n 3.1 ± 0.0
a
6.5± 0.1 s 1.1 ± 0.0
k
66.1 ± 0.1 p 4.1 ± 0.0
Raw Tortilla
15.4 ± 0.1 i 6.8 ± 0.1
c
19.2 ± 0.1 p 3.7 ± 0.0
g
Raw Tortilla
9.3 ± 0.1 m 3.4 ± 0.0
FAUAS-419
Raw Tortilla
4.4 ± 0.1 k 4.3 ± 0.0
FAUAS-429
Raw Tortilla Raw Tortilla
3.9 ± 0.1 l 6.2 ± 0.1
Raw Tortilla
2.6 ± 0.0 o 3.1 ± 0.0
FAUAS-457
Raw Tortilla
8.3 ± 0.1 i 6.7 ± 0.2
FAUAS-485
Raw Tortilla
16.5 ± 0.0 h 7.8 ± 0.0
FAUAS-437 FAUAS-447
FAUAS-488 FAUAS-491 FAUAS-512 Total
f
i
b
e
13.3 ± 0.1 s 1.0 ± 0.00
d
5.6± 0.1 n 2.3± 0.0 8.1± 0.1 hi 7.4± 0.1 a
22.4± 0.1 m 3.7± 0.04
k
8.5 ± 0.0 no 4.3 ± 0.0
g
2.6± 0.11 i 7.2± 0.3
8.5 ± 0.1 n 3.0 ± 0.0
g
7.9 ± 0.1 r 0.6 ± 0.0
h
j
7.6± 0.1 hi 8.5± 0.1
g
p
n
3.4 ± 0.0 ND a 26.0 ± 0.1 h 7.8 ± 0.0
g
e
b
g
8.4 ± 0.0 j 6.1 ± 0.1
d
Raw Tortilla
8.2 ± 0.1 g 10.9 ± 0.1
Raw Tortilla
7.1 ± 0.1 j 6.3 ± 0.1
9.4 ± 0.0 r 1.9 ± 0.0
Raw Tortilla Raw Tortilla
7.9 ± 0.1 i 6.9 ± 0.0 150.2 83.1
h
15.4 ± 0.2 i 7.4 ± 0.0 156.2 55.7
i
62.0 ± 0.4 op 17.9 ± 0.1
i
82.5 ± 0.1 n 20.9 ± 0.2
i
12.5 ± 0.1 x 0.8± 0.0
g
115.1 ± 0.6 q 12.1 ± 0.1
e
138.9 ± 1.2 m 34.4 ± 0.1
m
op
10.6± 0.7 u 2.2± 0.0
94.9 ± 0.4 k 42.7 ± 0.2
63.7 ± 0.3 mn 9.8 ± 0.1
cd
15.9 ± 0.2 jk 3.3 ± 0.0
16.6 ± 0.0 q 3.5 ± 0.0
e
1.8 ± 0.0 o 0.7 ± 0.0
m
11.9 ± 0.1 su 2.4 ± 0.0
115.5 ± 0.4 no 19.9 ± 0.1
a
56.8 ± 0.0 n 8.7 ± 0.1
b
44.1 ± 0.2 o 6.3 ± 0.0
0.9± 0.0 fg 8.2± 0.0
o
55.0 ± 0.1 j 22.5 ± 0.2
0.7± 0.0 m 3.8± 0.0
o
23.0 ± 0.1 m 10.6 ± 0.1
8.1± 0.2 h 7.5± 0.1
g
105.1 ± 0.6 k 17.1 ± 0.1
n
56.9 ± 0.2 j 20.2 ± 0.1
e
9.2 ± 0.0 i 4.5 ± 0.1
d
k
gh
22.2± 0.1 m 7.0 ± 0.1
c
88.8 ± 0.7 k 45.3 ± 0.2
g
1.2 ± 0.1
n
6.3 ± 0.0 s 2.8 ± 0.0
n
83.3 ± 0.4 pq 14.9 ± 0.1
d
91.0 ± 0.4 i 54.7 ± 0.3
j
a
15.7 ± 0.0 h 7.7 ± 0.1
62.5 ± 0.2 m 11.7 ± 0.1
f
4.1 ± 0.1 j 6.4 ± 0.1
l
57.3 ± 0.5 k 18.5 ± 0.0
e
75.7 ± 0.6 i 29.8 ± 0.2 830.9 206.2
e
b
c
mn
6.7 ± 0.0 q 3.7± 0.0
18.5 ± 0.1 u 2.3± 0.1
j
11.5 ± 0.1 n 6.6 ± 0.1
3.5 ± 0.0
ND e 7.7 ± 0.1 h 4.9 ± 0.1 85.8 30.3
h
b
25.4 ± 0.2 k 8.7 ± 0.0 248.4 69.7
17.3
72.6 64.6
51.6
92.1 74.7
17.9
71.5 65.1
60.1
94.7 73.6
53.7
91.7 67.4
23.9
92.5 72.8
49.2
82.7 68.5
31.0
80.4 51.2
42.8
88.0 68.0
l
39.5 ± 0.1 n 21.2 ± 0.1
108.6 ± 0.4 j 53.5 ± 0.4
2.6 ± 0.1 n 1.0 ± 0.0
83.1 58.8
fg
f
d
24.8
g
217.9 ± 1.5 j 52.1 ± 0.3
15.3± 0.1 o 5.8 ± 0.0
10.5
77.9 55.8
f
a
58.9± 0.3 l 7.8± 0.0
25.4
70.0 64.2
c
f
18.1± 0.2 j 9.2± 0.1
66.4 59.7
b
6.9 ± 0.1 g 5.6 ± 0.1
ND c 5.4 ± 0.g ND k 3.0 ± 0.0 ND b 11.3 ± 0.2 h 5.2 ± 0.0
28.9
c
g
e
78.4 65.3
g
4.6 ± 0.0 o 0.8 ± 0.0
a
d
c
122.1 ± 0.6 l 37.9 ± 0.1
e
92.9 ± 0.2 l 39.8 ± 0.3
b
141.9 ± 1.0 h 66.4 ± 0.2 1,594.7 533.7
PAC (%)
45.0
l
c
15.3± 0.0 n 2.2± 0.0
9.8 ± 0.4 f 8.6 ± 0.0 123.3 88.9
AR (%) e
5.2± 0.0 v 1.8± 0.0
h
8.8± 0.3 p 5.1± 0.0
Total
1.9 ± 0.0 ND l 2.2 ± 0.0 o 0.6 ± 0.0
44.2 ± 0.3 ki 16.9 ± 0.1
b
jk
i
n
2.2± 0.0 f 8.9 ± 0.1
Cy-diSuc-Glu
8.5 ± 0.4 j 3.6 ± 0.0
14. 6± 0.1 n 4.3 ± 0.0
c
d
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FAUAS-387
m
Pg-Mal-Glu
51.6 ± 0.8 l 15.0 ± 0.1
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FAUAS-290
e
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FAUAS-230
4.5 ± 0.5 g 8.0± 0.0
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FAUAS-220
5.5 ± 0.3 o 4.2 ± 0.0
Pg-3-Glu
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Process
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Maize Accessions
46.8 33.5
87.5 76.6 82.9 67.8
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Means values ± standard error. Means by column and treatment with different letters show significant difference, (p < 0.05). Cy-3-Glu: Cyanidin-3-glucoside; Pg-3-Glu: Pelargonidin-3-Glucoside; Cy-Mal-Glu: Cyanidin-Malonil-Glucoside; Pg-Mal-Glu: Pelargonidin-Malonil-Glucoside; Cy-Suc-Glu: Cyanidin-Succinil-Glucoside; Cy-diSuc-Glu: Cyanidin-Disuccinil-Glucoside; ND: Not detected; AR = Anthocyanin retention (%), PAA = Proportion of Acyl Anthocyanins (%).
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1
HIGHLIGHTS
2 Anthocyanin profiles of 15 Mexican blue maize landraces from Sinaloa were estimated.
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Acylated anthocyanins were predominant in blue landrace from northwest of Mexico.
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Nixtamalization process of blue maize diminished anthocyanins.
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Anthocyanins retention of tortillas from six blue maize ranged from 45 to 70%.
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S0023-6438(16)30009-3
DOI:
10.1016/j.lwt.2016.01.009
Reference:
YFSTL 5212
To appear in:
LWT - Food Science and Technology
Received Date: 1 July 2015 Revised Date:
1 January 2016
Accepted Date: 5 January 2016
Please cite this article as: Mora-Rochín, S., Gaxiola-Cuevas, N., Gutiérrez-Uribe, J.A., Milán-Carrillo, J., Milán-Noris, E.M., Reyes-Moreno, C., Serna-Saldivar, S.O., Cuevas-Rodríguez, E.O., Effect of Traditional Nixtamalization on Anthocyanin Content and Profile in Mexican Blue Maize (Zea mays L.) Landraces, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2016.01.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of Traditional Nixtamalization on Anthocyanin Content and Profile in Mexican
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Blue Maize (Zea mays L.) Landraces
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Saraid Mora-Rochína, b, Nalleli Gaxiola-Cuevasb, Janet Alejandra Gutiérrez-Uribec, Jorge
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Milán-Carrilloa,b, Evelia María Milán-Norisb, Cuauhtémoc Reyes-Morenoa, b, Sergio Othon
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Serna-Saldivarc, Edith Oliva Cuevas-Rodrígueza, b*
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a
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(FCQB), Universidad Autónoma de Sinaloa (UAS); bDoctorado en Ciencias, Especialidad
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Biotecnología (Programa Regional de
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Biotecnología-FEMSA. Escuela de Ingeniería y Ciencias. Tecnológico de Monterrey-Campus
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Monterrey, Monterrey, Nuevo León, CP 64849 México.
Maestría en Ciencia y Tecnología de Alimentos, Facultad de Ciencias Químico Biológicas
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Centro de
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Biotecnología), FCQB-UAS;
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*Corresponding author: Edith Oliva Cuevas-Rodríguez, Blvd de la Americas y Josefa Ortiz
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de Dominguez s/n Ciudad Universitaria, C.P. 80010, Culiacan, Sinaloa, México. Tel/Fax:
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+1526677137860
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E-mail address: [email protected]
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ABSTRACT
24
Mexican blue maize (Zea mays L.) grains have been poorly evaluated regarding their
25
potential as functional food ingredients. The aims of this research were to identify and
26
quantify anthocyanins from fifteen Mexican blue maize accessions of Elotero Sinaloa
27
landrace recollected in the northwestern region of Mexico. Additionally, the effect of
28
traditional nixtamalization processing on these compounds was evaluated. The acyl type
29
anthocyanins, such as cyanidin-3-(6”-succinylglucoside) (Cy-Suc-Glu) and cyanidin-3-(6”-
30
disuccinylglucoside) (Cy-diSuc-Glu) were the most abundant compounds in blue maize,
31
accounting for 52.1% and 15.6% the total anthocyanins, respectively. Other predominant
32
anthocyanins included cyanidin-3-glucoside (Cy-3-Glu), pelargonidin-3-glucoside (Pg-3-
33
Glu),
34
malonyglucoside) (Cy-Mal-Glu). The raw blue maize presented a similar anthocyanins
35
profile dominated by cyanidin derivatives on (86.9% on average). Nixtamalization
36
processing increased the relative percentage of glycosylated anthocyanins (Cy-3-Glu, and
37
Pg-3-Glu) and decreased the acylated anthocyanins (Cy-Suc-Glu, and Cy-diSuc-Glu) when
38
compared to raw kernels. Results obtained indicate that the studied Mexican native blue
39
maize contained anthocyanin patterns predominated by acylated cyanide derivatives.
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This information could be useful to select the best pigmented maize for the derivation of
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food products with nutraceutical potential.
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(Pg-Mal-Glu)
and
cyanidin-3-(6”-
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pelargonidin-3-(6”-malonylglucoside)
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Keywords: Anthocyanin, Nixtamalization, Blue Maize and Tortilla
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1.
Introduction Mexico, considered center of origin and domestication of maize (Zea mays L.), has
47
the largest diversity of genetic resources in the world with approximately 59 different
48
native landraces, which have been classified based on morphological characters, and
49
isozyme frequencies (Sánchez, Goodman, & Stuber, 2000). Native pigmented maize are
50
cultivated in different regions of Mexico; Chalqueño, Bolita, and Elotes Conico landraces
51
prevail in High Valleys of the Central Mesa. However, recent recollections of more than
52
300 maize accessions from Northwest México (state of Sinaloa) have been identified and
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classified into 13 maize landraces, being Tabloncillo, and Elotero Sinaloa the distinctive
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landraces of this region (Pineda-Hidalgo et al., 2013).
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Pigmented maize tortilla is the main basic daily staple in small communities in
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Mexico and Central America. The term nixtamalization refers to the alkaline cooking
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process of converting maize into foodstuffs such as tortillas and snacks (maize chips,
58
tortillas chips and tacos) (Serna-Saldívar, Gomez, & Rooney, 1990). Currently, demand of
59
nixtamalized products of blue maize has received an increased attention from a
60
nutraceutical perspective owing to their potential health benefits and unique flavor and
61
color properties (Urias-Peraldí et al., 2013). Blue maize is an important source of
62
anthocyanins; these bioactive compounds from maize kernels have shown multiple
63
functional roles such as protection against oxidative stress, increased antimutagenic
64
activity, and inhibition of colorectal carcinogenesis (Tsuda, 2012; Long et al., 2013; López-
65
Martínez, Parkin, & Garcia, 2014). However, maize kernels need to be processed prior to
66
human consumption, which may modify or degrade their natural phytochemicals. More
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information regarding the impact of nixtamalization processing conditions on the
68
anthocyanin profiles of blue maize grains is needed especially in terms of the fate of their
69
antioxidant compounds in nixtamalized products (Nayar, Liu, & Tang, 2015). Anthocyanins belong to the widespread class of phenolic compounds collectively
71
named flavonoids. The chemical structure of the anthocyanin determines its stability,
72
color intensity, and potential biological activity. While monomeric anthocyanins possess
73
limited stability against hydration and pH changes whereas acylated anthocyanins show
74
noteworthy stability to pH changes, temperature and light exposure (Dangles, Saito, &
75
Brouillard, 1993). Such stability has been attributed to intramolecular and/or
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intermolecular copigmentation and self-association reactions. Accordingly, sources of
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acylated anthocyanins may provide the desirable stability for food applications (Giusti &
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Wrolstad, 2003).
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Recent studies have shown that the main anthocyanins present in Andean
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pigmented maize have been previously identified as cyanidin-3-glucoside (Cy-3-Glu),
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pelargonidin-3-glucoside (Pg-3-Glu), and peonidin-3-glucoside (Pn-3-Glu), along with their
82
corresponding malonyl derivatives. In these previous studies it has been found that
83
proportion of acylated anthocyanins varied from 35.6% to 53.9% of total monomeric
84
moieties (Jing, Noriega, Schwartz, & Giusti, 2007; Montilla, Hillebrand, Antezana, &
85
Winterhalter, 2011; Pedreschi & Cisneros-Zevallos, 2007). Nevertheless, only few studies
86
on anthocyanins have focused on Mexican blue maize grains, and even fewer showed
87
their detailed anthocyanin profile. In Mexican blue/purple maize landraces, Cy-Mal-Glu
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and Cy-diMal-Glu were the major anthocyanins identified. The cyanidin is considered the
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main aglycone, accounting for 73-75.7% of all the anthocyanins (Salinas-Moreno, Pérez-
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Alonso, Vazquez-Carrillo, Aragón-Cuevas, & Velázquez-Cardenas, 2012). During processing of pigmented maize kernels into tortillas, the nixtamalization
92
condition (alkaline pH) and high temperature are two of the main factors that affect the
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amount of anthocyanins originally present in kernels. Additionally the thermal lime
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treatment makes these compounds more unstable and susceptible to degradation
95
(Sanchez-Madrigal et al., 2015). Several studies have been reported on the anthocyanin
96
content and their degradation products during alkaline cooking process (Del Pozo-Insfran,
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Brenes, Serna-Saldivar, & Talcott, 2006; De la Parra, Serna-Saldivar, & Liu, 2007; Mora-
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Rochín et al., 2010; Aguayo-Rojas et al., 2012), but these studies were conducted with a
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limited number of contrasting maize grains. Unfortunately, there is little information
100
available regarding the individual anthocyanins profile within groups of the same landrace
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and pigmented maize. On the other hand, the present study was undertaken to evaluated
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anthocyanin content and profile of fifteen different blue maize accessions correspond to
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Elotero Sinaloa landrace of northwest region of México. The effect of traditional
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nixtamalization process on the fate of these compounds was also investigated.
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2.
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2.1. Chemicals
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Materials and methods
Sodium hydroxide, hexane, methanol, ethyl acetate, HPLC grade acetonitrile and
107 108
HPLC grade trifluoroacetic acid were obtained from Sigma Aldrich Chemical Co. (St Louis,
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MO, USA). Analytical (HPLC) standards of cyanidin-3-glucoside (purity > 95%) and
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pelargonidin-3-glucoside (purity > 97%) were obtained from Sigma Aldrich Chemical Co.
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(St Louis, MO, USA). A syringe filter unit was supplied by Pall Gelman Laboratory (MI,
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USA).
113
2.2. Materials The study was performed on 15 contrasting blue kernels correspond to typical
115
landrace of northwestern region of Mexico: Elotero Sinaloa. These pigmented maize
116
accessions were collected from open-pollinated maintained by traditional farmers at their
117
villages in the municipality of Concordia located at (23° 17′ 18″ N, 106° 4′ 3″ W), state of
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Sinaloa México. All materials were grown and harvested during 2013. Crops were
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managed following standard recommendations. Maize samples were stored at -4 °C until
120
further processing.
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2.3.
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Determination of biophysical properties
Grain physical characteristics were determined using standard procedures: Kernel
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test weight (TW) according to Official US Grain Standard Procedures (AACC, 2000; Method
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55-10) and thousand-kernel weight (TKT) by weighing 100 randomly selected kernels at
125
13% of grain moisture. Flotation index (FI), as an indirect measure of hardness, was
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assessed using the procedure proposed by Vazquez-Carrillo, Santiago-Ramos, Gaytán-
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Martínez, Morales-Sánchez, & Guerrero-Herrera. (2015).
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2.4. Production of nixtamalized maize flours
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Nixtamalized maize flour was obtained according to Milán-Carrillo, Gutierrez-
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Dorado, Cuevas-Rodríguez, Garzón-Tiznado, & Reyes-Moreno. (2004). Briefly, maize
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kernels (1000 g lots) was added in a medium kettle containing 3 L of distilled water (1:3,
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maize grains/water) and either to 5.4 g of Ca(OH)2/L water. The samples were cooked for
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31 min at approximately 85 °C, followed by a steep time of 8.1 h. After steeping, the
134
cooking liquor (nejayote) was drained and discarded and the nixtamal (alkaline-cooked
135
maize) washed with running tap water for 40 s and blotted between paper towels. Wet
136
nixtamal was dried at 55 °C/12 h in a forced air oven and then cooled at room
137
temperature. Finally, the dried nixtamal was milled (UD Cyclone Sample Mill, UD Corp.
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Boulder, CO, USA) to pass through an 80-US mesh (0.180 mm) screen, and packed in
139
plastic bags. Nixtamalized maize flours were stored at -20 °C until use.
140
2.5. Tortilla preparation from nixtamalized maize flours
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Tortillas were prepared by mixing 400 g of nixtamalized maize flours with 400 mL of
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water to achieve an adequate masa consistency for the production of table tortillas.
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Pieces of fresh dough (30 g) were pressed and shaped into flat disks (15 cm) using a
144
manual machine (Casa Herrera, México DF, México). The dough disks were baked on a hot
145
griddle at 270 ± 10 °C for 15 s on one side, followed by 30 s on the other side, and then
146
again on the first side until puffing of the tortilla occurred. The fresh tortillas were dried,
147
milled (UD Cyclone Sample Mill, UD Corp. Boulder, CO, USA) to pass through an 80-US
148
mesh (0.180 mm) sieve and packed in plastic bags. Tortillas from nixtamalized flours were
149
stored at -20 °C until use further.
150
2.6. Total anthocyanin content
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Extractions were performed using ground sample (250 g) and mixing with 10 mL of
151 152
acidified methanol solution with 36.5 g/L HCl (95:5, mL:mL). The samples were then
153
shaken for 30 min and centrifuged at 3000g for 5 min (Sorvall RC5C, Sorvall Instruments,
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Dupont, Wilmington, DE, USA), and the supernatants were collected. Absorbance readings
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at 535 nm were taken and corrected for background absorbance at 700 nm using a
156
Microplate Reader (Synergy HT, Bio-Tek Instruments, Inc., Winooski VT, USA). Using the
157
molar extinction coefficients of 25,965 Abs/M x cm and a molecular weight of 449.2 g/mol
158
the total anthocyanin content was calculated and expressed as mg of cyanidin 3-glucoside
159
equivalent (CGE) per 100 g of dry weight DW (Abdel-Aal & Hucl, 1999)
160
2.7. Identification and quantification of anthocyanins
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Anthocyanins extracts were obtained according to procedure described by Abdel-
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Aal, Young, & Rabalski. (2006). Briefly, 250 mg of sample was mixing with 10 mL of
163
acidified methanol with 36.5 g/L HCl (95:5, mL:mL) and continuous shaking for 30 min a
164
room temperature. After the pH was adjusted to 1.0 by 6 mol/L HCl. The samples were
165
then centrifuged (1,538g, 10 min, 4 °C) in a Sorvall RC 5C superspeed centrifuge (Sorvall
166
Instruments, Dupont, Wilmington, DE, USA); the supernatants were collected and the
167
residue submitted twice to the same extraction conditions described above. The
168
supernatants were combined and concentrated under vacuum in a Büchi K124 rotary
169
evaporator (Büchi Labortechnik AG, Switzerland) a 30 °C until the methanol was removed.
170
The concentrated extracts were lyophilized and stored at -20°C prior to analysis. For HPLC
171
analyses of anthocyanins, lyophilized extracts were re-dissolved in methanol concentrated
172
(5 mg/mL), filtered through a syringe filter with 0.45 µm nylon membrane (Pall Gelman
173
Laboratory, Ann Arbor, MI, USA).
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The HPLC analyzed were carrier out with an HPLC-PDA system (1200 Series, Agilent
175
Technologies, Santa Clara, CA, USA). Chromatograms were obtained at 525 nm after
176
injection of 2.0 µL of sample. Separation was performed in a Zorbax-SB Eclipse XDB-C18
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column (4.6 x 150 mm, 5 µm; Agilent Technologies, Santa Clara, CA, USA) at 45 °C. The
178
mobile phase used was (A) trifluoroacetic acid (TFA) with HPLC-grade water (TFA/H2O,
179
1:99 mL:mL) and (B) HPLC-grade acetonitrile, establishing the following gradient: isocratic
180
A/B (85:15, mL:mL) for 5 min, A/B (85:15-81:19, mL:mL) over 7 min, isocratic A/B (81:19,
181
mL:mL) for 10 min, A/B (81:19-60:40, mL:mL) over 15 min and A/B (60:40-100:0, mL:mL)
182
over 25 min, using a flow rate of 0.8 mL/min. Pure anthocyanin compounds (Cy-3-Glu and
183
Pg-3-Glu) were used as standards. Ultraviolent-visible (UV-vis) absorption spectra were
184
recorded for the predominant peaks.
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The identification of each peak was confirmed by HPLC-MS-TOF (Agilent, Santa
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Clara, CA, USA). Chromatographic conditions used were the same as those described for
187
HPLC-PDA analysis. Mass spectra were collected using an electrospray source in positive
188
mode (ESI+) under the following conditions: m/z range, 200-1000; nitrogen gas; gas
189
temperature, 350 °C; drying gas flow rate, 10 L/min; nebulizer pressure, 127.9 kPa;
190
capillary voltage, 4.0 KV; and fragment voltage, 100 V. Compounds were characterized and
191
identified by their MS, MS/MS spectra and LC retention times and by comparison with
192
available references samples.
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Anthocyanins were quantified using a linear calibration curves, generated with
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commercial authentic standards of Cy-3-Glu and Pg-3-Glu at seven different
195
concentrations (0.1 to 5.0 mg/mL) at 520 nm. For each compounds, the regression
196
equations were Cy-3-Glu (y = 5.83-117.99x; R2 = 0.997) and Pg-3-Glu (y = 0.69-3.89x; R2 =
197
0.998). The results were expressed as micrograms of commercial standards per g of dry
198
weight (DW).
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2.8
Statistical analysis ANOVA procedures were used for the analysis of the experimental data. Differences
201
among treatments were determined using Duncan’s comparison test. Data were reported
202
as mean ± standard deviation (SD) in triplicate.
203
3.
Results and discussion
204
3.1
Biophysical kernel properties
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Biophysical traits of Mexican blue maize showed a significant variation (p < 0.05)
206
among genotypes (Table 1). Serna-Saldivar et al. (2008) indicated that the preferred
207
properties for the commercial nixtamalization process are test weight (TW) and thousand
208
kernels weight (TKW) greater than 72 kg/hL and 320 g, respectively. Interestingly, the
209
majority of the Mexican blue maize accessions analyzed possessed these attributes (Table
210
1). Among raw blue grains, FAUAS-290 accession followed by FAUAS-419 and FAUAS-429
211
had higher (p < 0.05) TW and TKW, compared to the other blue maize accessions; but
212
FAUAS-230 blue grain followed by FAUAS-249, FAUAS-252 and FAUAS-485 had lower TKW
213
than 320 g.
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Grain hardness (measured indirectly from Flotation index, FI) showed that FAUAS-
215
230 (FI = 14.7%) blue maize was the hardest, while FAUAS-485 (FI = 94.7%) was the softest
216
one (Table 1). Moreover, some Mexican blue maize such as FAUAS-230 followed by
217
FAUAS-290, FAUAS-419, FAUAS-437, FAUAS-491 and FAUAS-512 had FI ≤ 40%,
218
characteristic which make them adequate for the nixtamalized flour industry. The rest of
219
the maize accessions FAUAS-220, FAUAS-252, FAUAS-387, FAUAS-429, FAUAS-447,
220
FAUAS-457 and FAUAS-488 had FI > 40%, indicating that their kernels had soft or
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intermediate endosperm hardness and readily uptake water during nixtmalization which
222
positively affects masa and tortillas yields (Vázquez-Carrillo et al., 2015). Therefore, this
223
blue maize Elotero Sinaloa landrace endemic of Northwest Mexico should yield masa
224
according to the Mexican normative, NMX-FF-034/1-SCFI-2002 (SAGARPA, 2002).
225
3.2
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Total anthocyanins content
Total anthocyanin content (TAC) in raw blue maize genotypes and their tortillas
227
produced following the traditional process is shown in Table 2. Among raw blue kernels
228
FAUAS-485 (34.3 mg CGE/100 g DW) and FAUAS-512 (33.7 mg CGE/100 g DW) contained
229
the highest (p < 0.05) TAC concentration compared to the rest of the blue maize. On the
230
other hand, the FAUAS-429 (14.1 mg CGE/100 g DW) kernels contained the lowest (p <
231
0.05) TAC. The assayed concentration were close to TAC previously obtained for American
232
and Mexican blue maize (Del Pozo-Insfran et al., 2006), American blue maize (De la Parra
233
et al., 2007) and different native populations of Mexican pigmented genotypes (Espinosa-
234
Trujillo, Mendoza-Castillo, Castillo-González, Ortiz-Cereceres, & Delgado-Alvarado, 2010),
235
but were lower compared to 25 Mexican blue maize hybrids (Urias-Peraldí et al., 2013)
236
and native Mexican pigmented maize populations belonging to the Chalqueño, Elotes
237
Cónicos and Bolita landraces (Salinas-Moreno et al., 2012). The observed differences in
238
TAC could be attributed to the variability in the blue maize genotypes including genetic
239
background, grain physical properties and particularly the relative ratio of the anatomical
240
parts of the kernel since the pericarp and aleurone the layer are the structures richer in
241
anthocyanins (Jing et al., 2007; Salinas-Moreno, Martínez-Bustos, Soto-Hernández,
242
Ortega-Paczka, & Arellano-Vázquez, 2003).
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As expected, the traditional nixtamalization process caused significant (p < 0.05)
244
losses of total anthocyanins. Less than 46.0% of TAC was retained in tortillas produced by
245
traditional nixtamalization (Table 2). The remaining amounts of anthocyanins in tortillas
246
produced from Mexican blue maize varied from 2.1 to 15.5 mg CGE/100 g DW. The
247
FAUAS-512 maize tortillas prepared from blue maize nixtamalized flour retained 46.0%
248
TAC and therefore contained the highest amount of anthocyanins among the array of the
249
blue maize tortillas. These significant losses occurred during nixtamalization process as a
250
result of the combined effect of alkaline pH (approximately 10) and thermal processing
251
which enhanced physical losses of the pericarp and leaching of anthocyanins into the
252
cooking liquor or nejayote (Mora-Rochín et al., 2010). De la Parra et al. (2007) reported
253
anthocyanin losses of 83% during alkaline cooking with different types of maize
254
genotypes. Likewise, Salinas-Moreno et al. (2003) reported relevant anthocyanin losses in
255
a pigmented maize landrace during traditional nixtamalization process.
256
3.3. Identification and quantification of anthocyanins
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Anthocyanin profiles observed in raw blue grains of Elotero Sinaloa landrace and
258
their respective tortillas were very similar, with the presence of 10 peaks (Table 3). For
259
reasons of space, representative chromatograms of only two of the samples are presented
260
(Fig 1A, Fig 1B, Table 3). Anthocyanin compounds were determined by comparison of the
261
spectroscopic and chromatographic properties with those of authentic anthocyanin
262
standards (Cy-3-Glu and Pg-3-Glu). The remaining compounds were tentatively identified
263
on the basis of mass identification using a combination of the retention time, peak
264
spectra, mass-to-charge ratio and MS fragmentation (Abdel-Aal et al., 2006). Six major
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anthocyanins were identified, including cyanidin-3-glucoside (Cy-3-Glu; peak 1, m/z 449),
266
pelargonidin-3-glucoside (Pg-3-Glu; peak 3, m/z 433), cyanidin-3-(6”-malonylglucoside)
267
(Cy-Mal-Glu; peak 2, peak 4 and peak 5, m/z 535), cyanidin-3-(6”-succinylglucoside) (Cy-
268
Suc-Glu; peak 6 and peak 8, m/z 549), pelargonidin-3-(6”-malonylglucoside) (Pg-Mal-Glu;
269
peak 7, m/z 519) and cyanidin-3-(6”-disuccinylglucoside) (Cy-diSuc-Glu; peak 9 and peak
270
10, m/z 649).
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Some derived compounds had similar mass spectra and spectroscopic properties,
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but different retention times (Table 3). This could be explained by the presence of three
273
isomers of Cy-Mal-Glu, two isomers of Cy-Suc-Glu and two isomers of Cy-diSuc-Glu. These
274
results are consistent with the previous research that reported the presence of two
275
isomers of Cy-diSuc-Glu and three isomers of Cy-mal-Glu associated with Mexican native
276
and hybrid blue maize genotypes (Urias-Lugo et al., 2015). In investigations of American
277
pigmented maize it has been shown the presence of malonylglucoside isomers of cyanidin,
278
which was distinguished mainly on the basis of LC elution profiles (Collison, Yang, Dykes,
279
Murray, & Awika, 2015). Therefore, the analysis of nuclear magnetic resonance (NMR) is
280
necessary to identify unambiguously the nature of the compounds that have different
281
retention time and the same mass and fragmentation in the samples in order to establish
282
different isomers of anthocyanins in blue maize (Abdel-Aal et al., 2006).
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The cyanidin was the main aglycone in the raw Mexican blue maize and constituted
283 284
from 71.7% to 93.1% of the total anthocyanin. The highest concentration was assayed in
285
the FAUAS-437 maize. The most abundant anthocyanins were Cy-Suc-Glu, Cy-diSuc-Glu
286
and Cy-3-Glu accounting for 52.1%, 15.6%, and 9.4% respectively of the total anthocyanins
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(Table 4). The composition of individual anthocyanins, respectively, of the blue maize
288
accessions of Elotero Sinaloa landrace showed remarkable differences with those
289
reported by Salinas-Moreno et al. (2012), who found Cy-Mal-Glu and Cy-diMal-Glu as the
290
most abundant anthocyanins for blue/purple grain of Chalqueño, Elotes Cónicos, and
291
Bolita Mexican maize landraces, while other researchers found for Andean purple maize
292
three major anthocyanins Cy-diMal-Glu, Cy-3-Glu, Pg-3-Glu (Pedreschi & Cisneros-Zavala,
293
2007; Jing et al., 2007). Recently, Collison et al. (2015) identified Cy-3-Glu and Cy-Mal-Glu
294
as the major anthocyanins in American red/blue maize.
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Interestingly, seven of nine anthocyanins found in the raw Mexican blue maize
296
studied herein were acylated. The acylated anthocyanin in maize Elotero Sinaloa landrace
297
varied from 66.4 to 94.7% (average 82.9%) of the total anthocyanins. The highest
298
predominance of acylated anthocyanins in the blue maize analyzed was in FAUAS-437,
299
followed by FAUAS-457, FAUAS-419 and FAUAS-447. These maize accessions contained
300
from 91.7% to 94.7% of total anthocyanins, respectively. These values were significantly
301
higher compared to those previously reported in Andean purple/magenta maize kernels
302
(Pedreschi & Cisneros-Zavala, 2007; Jing et al., 2007; Montilla et al., 2011). Salinas-
303
Moreno et al. (2012) reported an average of 63.4% of acylated anthocyanins in
304
blue/purple Mexican maize landraces of the highlands of the central Mexico, whereas, the
305
American red/blue maize contained average of 60.0% acylated anthocyanins (Collison et
306
al., 2015). The acylated anthocyanins are considered more stable to pH and temperature
307
changes compared to the non-acylated ones. Therefore, sources of acylated anthocyanins
308
may provide the desirable stability for food applications (De Pascual-Teresa, Santos-
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Buelga, & Rivas-Gonzalo, 2002; Giusti & Wrolstad, 2003). The traditional nixtamalization process lowered the amount of anthocyanins in blue
311
maize tortillas. Given that all of the blue genotypes were nixtamalized under the same
312
conditions, the differences in the percentage of loss are mainly due to the anthocyanins
313
profile present in each blue maize. The group of six blue maize (FAUAS-220, FAUAS-419,
314
FAUAS-437, FAUAS-447, FAUAS-485 and FAUAS-512) tortillas retained the highest (45.0%
315
to 60.1%) amounts of total anthocyanins when compared to raw grains.
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The changes in the anthocyanin profile between the respective blue raw kernels and
317
their tortillas were mainly in the higher relative percentage of Cy-3-Glu (9.4% to 15.6%)
318
and Pg-3-Glu (7.7% to 16.6%), and lower relative percentage of the acyl types such as Cy-
319
Suc-Glu (52.1% to 38.6%) and Cy-diSuc-Glu (15.7% to 13.1%). These changes could be
320
attributed to the thermal treatment under alkaline or high pH conditions that occur during
321
lime cooking. The effect was especially noticeable in the acylated anthocyanins. According
322
to the reports of Fossen et al. (1998) and De Pascual-Teresa et al. (2002) probably the
323
ester link that binds the acyl (malonyl and succinyl) radical with the sugar is very unstable
324
under alkaline pH, and thus may break, liberating the acyl radical and remaining only the
325
simple anthocyanin.
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4.
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Conclusions
The current study showed substantial differences in anthocyanin content and
327 328
composition profiles among Mexican native blue maize. It also contained high
329
predominance of acylated anthocyanins such with cyanidin-3-(6”-succinylglucoside) and
330
cyanidin-3-(6”-disuccinylglucoside). Interestingly, the highest relative percentage of
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acylated anthocyanins in relation to total anthocyanins it was found 82.9%. It is
332
noteworthy that tortillas obtained from nixtamalized blue maize (FAUAS-220, FAUAS-419,
333
FAUAS-437, FAUAS-447, FAUAS-485 and FAUAS-512) flours retained between 45.0% to
334
60.1% of total anthocyanins when compared to their respective raw kernels. On the basis
335
of results the presented herein the different Mexican native blue maize kernels may hold
336
promise for the development of functional foods or as a source of natural colorants.
337
Acknowledgements
338
This research was partially supported by the Universidad Autónoma de Sinaloa (Project
339
PROFAPI-2012), PROMEP/SEP (Project 2012, Thematic Networks for Cooperation, food
340
Biotechnology) and Consejo Nacional de Ciencia y Tecnología (CONACYT, Project 168279).
341
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Figure captions.
463
Fig. 1. Representative HPLC chromatograms for anthocyanins (A) of raw grains and theirs
464
corresponding (B) tortilla produced of nixtamalized flour of the FAUAS-512 blue maize.
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Peaks numbers refer to those indicated in Table 3.
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Table 1. a
Maize Accessions
Test weight (kg/hL)
1,000 Kernel weight (g)
Flotation index
FAUAS-220
76.4 ± 0.3b
340.7 ± 7.4f
60.0 ± 4.0e
Intermediate
FAUAS-230
78.7 ± 0.2a
280.8 ± 1.2i
14.7 ± 3.1h
Hard
FAUAS-249
76.8 ± 0.3b
247.5 ± 2.5k
78.0 ± 4.0b
Very soft
FAUAS-252
73.7 ± 0.2c
204.0 ± 3.9l
82.7 ± 3.1c
Soft
FAUAS-290
78.9 ± 0.5a
418.8 ± 4.9a
37.3 ± 2.3f
Intermediate
FAUAS-387
77.6 ± 0.6a,b
331.5 ± 1.8g
SC
Biophysical characteristics of fifteen Blue Mexican maize .
68.0 ± 4.0d
Soft
FAUAS-419
77.4 ± 0.3a,b
407.1 ± 7.7b
26.0 ± 3.9f
Intermediate
FAUAS-429
79.1 ± 0.6a
417.7 ± 2.7a
68.0 ± 2.0d
Soft
FAUAS-437
79.9 ± 0.3a
368.0 ± 6.8j
36.7 ± 2.1g
Intermediate
FAUAS-447
78.6 ± 0.2a
365.1 ± 4.6e
80.0 ± 3.0c
Soft
FAUAS-485
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75.1 ± 0.1b
362.5 ± 3.2e
82.7 ± 3.3c
Soft
68.1 ± 0.2d
264.7 ± 3.2j
94.7 ± 2.3a
Very soft
78.9 ± 0.4a
301.9 ± 2.3h
66.7 ± 3.3d
Soft
EP
FAUAS-488
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FAUAS-457
Hardness
78.2 ± 0.3a
387.9 ± 5.1c
24.7 ± 4.6f
Intermediate
FAUAS-512
78.9 ± 0.4a
324.4 ± 4.4g
36.0 ± 4.0f
Intermediate
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FAUAS-491
a
Means values ± standard error. Means by column and treatment with different letters show significant difference, (p < 0.05).
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Table 2. Anthocyanin content and percent of retention in different raw blue Mexican maize and their tortillas produced throughout nixtamalization process
a
Process Anthocyanin content % Retention Raw 25.9 ± 3.8c TNF 10.4 ± 0.5j 40.9 ± 2.3c FAUAS-230 Raw 17.7 ± 0.5g TNF 5.1 ± 0.1m 28.8 ± 0.9f f FAUAS-249 Raw 19.7 ± 0.3 TNF 2.1 ± 0.1p 10.7 ± 0.4k FAUAS-252 Raw 21.3 ± 0.7e TNF 2.5 ± 0.1o 11.7 ± 0.4j FAUAS-290 Raw 23.7 ± 0.6d TNF 5.0 ± 0.1m 21.1 ± 0.4h e FAUAS-387 Raw 22.1 ± 1.4 TNF 3.9 ± 0.1n 17.6 ± 0.3i b FAUAS-419 Raw 28.2 ± 0.2 TNF 8.7 ± 0.1k 30.9 ± 0.2f FAUAS-429 Raw 14.1 ± 0.9i TNF 3.3 ± 0.2n 23.4 ± 1.3 FAUAS-437 Raw 27.2 ± 0.9n TNF 8.7 ± 0.1k 32.7 ± 0.6e FAUAS-447 Raw 15.2 ± 0.2h TNF 5.4 ± 0.4m 35.5 ± 2.4de c FAUAS-457 Raw 24.8 ± 1.9 TNF 6.5 ± 0.5l 26.2 ± 1.9g FAUAS-485 Raw 34.3 ± 1.1a 36.2 ± 0.5d TNF 12.4 ± 0.2j FAUAS-488 Raw 25.9 ± 0.8c TNF 7.5 ± 0.1k 29.0 ± 0.5f cd FAUAS-491 Raw 24.1 ± 2.7 TNF 10.6 ± 0.4j 44.0 ± 0.9b a FAUAS-512 Raw 33.7 ± 3.1 TNF 15.5 ± 1.1h 46.0 ± 1.1a a Means values ± standard error. Means by column and treatment with different letters
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Maize Accessions FAUAS-220
show significant difference, (p < 0.05). Anthocyanin content is given in mg cyanindin 3glucoside equivalent (CGE)/100g dry weight (DW); TNF = Tortillas produced from nixtamalized flour
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Table 3. Mass spectrometric data for identification of anthocyanins of raw blue Mexican maize and their tortillas produced throughout nixtamalization process RT (Min)
λmax (nm)
1
Cy-3-Glu
3.1
517
3.5
512
4.2
502
a
2
1
Pg-3-Glu
4
Cy-Mal-Glu
5
Cy-Mal-Glu
6
Cy-Suc-Glu
1
MS/MS fragments (m/z)
449
287
535
287
433
287
4.9
515
535
287
5.3
513
535
287
2
6.1
514
549
287
7
Pg-Mal-Glu
6.9
504
519
271
8
Cy-Suc-Glu
7.8
516
549
287
9
Cy-diSuc-Glu
8.5
520
649
287
10
Cy-diSuc-Glu
9.0
519
649
287
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1
2
3
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3
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Cy-Mal-Glu
Major ions (m/z)
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Anthocyanin
SC
Peak
3
AC C
Cy, cyanidin; Glu, glucoside; Mal, malonyl; Pg, pelargonidin; Suc, succinyl.
1-3
Compounds with identical molecular mass within each superscript
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Table 4. Individual anthocyanin concentrations (µ µg/g DW) from fifteen Mexican blue maize and their tortillas produced throughout nixtamalization a process Cy-3-Glu
Cy-Mal-Glu c
l
Cy-Suc-Glu
l
f
Raw Tortilla
16.1 ± 0.4 i 6.8 ± 0.1
Raw Tortilla
10.5 ± 0.1 m 3.6 ± 0.0
4.5 ± 0.1 q 2.2 ± 0.0
10.4± 0.1 m 3.61± 0.0
29.6 ± 0.1 o 6.7 ± 0.0
FAUAS-249
Raw Tortilla
11.8 ± 0.1 l 4.1 ± 0.0
d
5.8 ± 0.10 q 2.5 ± 0.0
k
12.9± 0.1 mn 3.5± 0.0
c
39.3 ± 0.1 n 8.2 ± 0.0
FAUAS-252
Raw Tortilla
19.8 ± 0.1 n 3.1 ± 0.0
a
6.5± 0.1 s 1.1 ± 0.0
k
66.1 ± 0.1 p 4.1 ± 0.0
Raw Tortilla
15.4 ± 0.1 i 6.8 ± 0.1
c
19.2 ± 0.1 p 3.7 ± 0.0
g
Raw Tortilla
9.3 ± 0.1 m 3.4 ± 0.0
FAUAS-419
Raw Tortilla
4.4 ± 0.1 k 4.3 ± 0.0
FAUAS-429
Raw Tortilla Raw Tortilla
3.9 ± 0.1 l 6.2 ± 0.1
Raw Tortilla
2.6 ± 0.0 o 3.1 ± 0.0
FAUAS-457
Raw Tortilla
8.3 ± 0.1 i 6.7 ± 0.2
FAUAS-485
Raw Tortilla
16.5 ± 0.0 h 7.8 ± 0.0
FAUAS-437 FAUAS-447
FAUAS-488 FAUAS-491 FAUAS-512 Total
f
i
b
e
13.3 ± 0.1 s 1.0 ± 0.00
d
5.6± 0.1 n 2.3± 0.0 8.1± 0.1 hi 7.4± 0.1 a
22.4± 0.1 m 3.7± 0.04
k
8.5 ± 0.0 no 4.3 ± 0.0
g
2.6± 0.11 i 7.2± 0.3
8.5 ± 0.1 n 3.0 ± 0.0
g
7.9 ± 0.1 r 0.6 ± 0.0
h
j
7.6± 0.1 hi 8.5± 0.1
g
p
n
3.4 ± 0.0 ND a 26.0 ± 0.1 h 7.8 ± 0.0
g
e
b
g
8.4 ± 0.0 j 6.1 ± 0.1
d
Raw Tortilla
8.2 ± 0.1 g 10.9 ± 0.1
Raw Tortilla
7.1 ± 0.1 j 6.3 ± 0.1
9.4 ± 0.0 r 1.9 ± 0.0
Raw Tortilla Raw Tortilla
7.9 ± 0.1 i 6.9 ± 0.0 150.2 83.1
h
15.4 ± 0.2 i 7.4 ± 0.0 156.2 55.7
i
62.0 ± 0.4 op 17.9 ± 0.1
i
82.5 ± 0.1 n 20.9 ± 0.2
i
12.5 ± 0.1 x 0.8± 0.0
g
115.1 ± 0.6 q 12.1 ± 0.1
e
138.9 ± 1.2 m 34.4 ± 0.1
m
op
10.6± 0.7 u 2.2± 0.0
94.9 ± 0.4 k 42.7 ± 0.2
63.7 ± 0.3 mn 9.8 ± 0.1
cd
15.9 ± 0.2 jk 3.3 ± 0.0
16.6 ± 0.0 q 3.5 ± 0.0
e
1.8 ± 0.0 o 0.7 ± 0.0
m
11.9 ± 0.1 su 2.4 ± 0.0
115.5 ± 0.4 no 19.9 ± 0.1
a
56.8 ± 0.0 n 8.7 ± 0.1
b
44.1 ± 0.2 o 6.3 ± 0.0
0.9± 0.0 fg 8.2± 0.0
o
55.0 ± 0.1 j 22.5 ± 0.2
0.7± 0.0 m 3.8± 0.0
o
23.0 ± 0.1 m 10.6 ± 0.1
8.1± 0.2 h 7.5± 0.1
g
105.1 ± 0.6 k 17.1 ± 0.1
n
56.9 ± 0.2 j 20.2 ± 0.1
e
9.2 ± 0.0 i 4.5 ± 0.1
d
k
gh
22.2± 0.1 m 7.0 ± 0.1
c
88.8 ± 0.7 k 45.3 ± 0.2
g
1.2 ± 0.1
n
6.3 ± 0.0 s 2.8 ± 0.0
n
83.3 ± 0.4 pq 14.9 ± 0.1
d
91.0 ± 0.4 i 54.7 ± 0.3
j
a
15.7 ± 0.0 h 7.7 ± 0.1
62.5 ± 0.2 m 11.7 ± 0.1
f
4.1 ± 0.1 j 6.4 ± 0.1
l
57.3 ± 0.5 k 18.5 ± 0.0
e
75.7 ± 0.6 i 29.8 ± 0.2 830.9 206.2
e
b
c
mn
6.7 ± 0.0 q 3.7± 0.0
18.5 ± 0.1 u 2.3± 0.1
j
11.5 ± 0.1 n 6.6 ± 0.1
3.5 ± 0.0
ND e 7.7 ± 0.1 h 4.9 ± 0.1 85.8 30.3
h
b
25.4 ± 0.2 k 8.7 ± 0.0 248.4 69.7
17.3
72.6 64.6
51.6
92.1 74.7
17.9
71.5 65.1
60.1
94.7 73.6
53.7
91.7 67.4
23.9
92.5 72.8
49.2
82.7 68.5
31.0
80.4 51.2
42.8
88.0 68.0
l
39.5 ± 0.1 n 21.2 ± 0.1
108.6 ± 0.4 j 53.5 ± 0.4
2.6 ± 0.1 n 1.0 ± 0.0
83.1 58.8
fg
f
d
24.8
g
217.9 ± 1.5 j 52.1 ± 0.3
15.3± 0.1 o 5.8 ± 0.0
10.5
77.9 55.8
f
a
58.9± 0.3 l 7.8± 0.0
25.4
70.0 64.2
c
f
18.1± 0.2 j 9.2± 0.1
66.4 59.7
b
6.9 ± 0.1 g 5.6 ± 0.1
ND c 5.4 ± 0.g ND k 3.0 ± 0.0 ND b 11.3 ± 0.2 h 5.2 ± 0.0
28.9
c
g
e
78.4 65.3
g
4.6 ± 0.0 o 0.8 ± 0.0
a
d
c
122.1 ± 0.6 l 37.9 ± 0.1
e
92.9 ± 0.2 l 39.8 ± 0.3
b
141.9 ± 1.0 h 66.4 ± 0.2 1,594.7 533.7
PAC (%)
45.0
l
c
15.3± 0.0 n 2.2± 0.0
9.8 ± 0.4 f 8.6 ± 0.0 123.3 88.9
AR (%) e
5.2± 0.0 v 1.8± 0.0
h
8.8± 0.3 p 5.1± 0.0
Total
1.9 ± 0.0 ND l 2.2 ± 0.0 o 0.6 ± 0.0
44.2 ± 0.3 ki 16.9 ± 0.1
b
jk
i
n
2.2± 0.0 f 8.9 ± 0.1
Cy-diSuc-Glu
8.5 ± 0.4 j 3.6 ± 0.0
14. 6± 0.1 n 4.3 ± 0.0
c
d
M AN U
FAUAS-387
m
Pg-Mal-Glu
51.6 ± 0.8 l 15.0 ± 0.1
TE D
FAUAS-290
e
EP
FAUAS-230
4.5 ± 0.5 g 8.0± 0.0
AC C
FAUAS-220
5.5 ± 0.3 o 4.2 ± 0.0
Pg-3-Glu
RI PT
Process
SC
Maize Accessions
46.8 33.5
87.5 76.6 82.9 67.8
ACCEPTED MANUSCRIPT a
AC C
EP
TE D
M AN U
SC
RI PT
Means values ± standard error. Means by column and treatment with different letters show significant difference, (p < 0.05). Cy-3-Glu: Cyanidin-3-glucoside; Pg-3-Glu: Pelargonidin-3-Glucoside; Cy-Mal-Glu: Cyanidin-Malonil-Glucoside; Pg-Mal-Glu: Pelargonidin-Malonil-Glucoside; Cy-Suc-Glu: Cyanidin-Succinil-Glucoside; Cy-diSuc-Glu: Cyanidin-Disuccinil-Glucoside; ND: Not detected; AR = Anthocyanin retention (%), PAA = Proportion of Acyl Anthocyanins (%).
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
1
HIGHLIGHTS
2 Anthocyanin profiles of 15 Mexican blue maize landraces from Sinaloa were estimated.
4
Acylated anthocyanins were predominant in blue landrace from northwest of Mexico.
5
Nixtamalization process of blue maize diminished anthocyanins.
6
Anthocyanins retention of tortillas from six blue maize ranged from 45 to 70%.
SC
RI PT
3
7
M AN U
8
AC C
EP
TE D
9