- Email: [email protected]
Contribution of zein content and starch characteristics to vitreousness of commercial maize hybrids
Journal of Cereal Science 80 (2018) 57e62
Contents lists available at ScienceDirect
Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Contribution of zein content and starch characteristics to vitreousness of commercial maize hybrids Kristina Kljak*, Marija Duvnjak, Darko Grbesa Department of Animal Nutrition, Faculty of Agriculture, University of Zagreb, Svetosimunska cesta 25, 10 000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 October 2017 Received in revised form 16 January 2018 Accepted 17 January 2018
Fast, simple laboratory methods were used to analyze 22 maize samples varying in kernel vitreousness from 50.23% to 76.41%. Samples were analyzed in terms of zein content (53.86e86.37 g/kg endosperm DM), amylose content (190.76e259.77 g/kg endosperm DM), amylose to amylopectin ratio in starch (0.28 e0.43), as well as starch granule size (10.95e14.89 mm in equivalent diameter) and starch granule shape (circularity, 0.85e0.94). More vitreous samples had higher zein and amylose content, as well as smaller and less circular starch granules. Nearly all grain traits on their own significantly affected vitreousness, and a multiple regression model to account for their combined effects was able to explain 61.8% of variability in kernel vitreousness. Zein content contributed most to the model, followed by starch granule projected area and circularity. In contrast, the amylose content contributed only 5.1% to the model. These results suggest that starch-protein interactions influence maize kernel vitreousness more strongly than starch molecular properties do. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Maize grain Vitreousness Amylose Zein Starch granule
1. Introduction Vitreousness and hardness-associated properties are significantly correlated with end usage of maize and are strongly determined by genotype. Grain vitreousness, which refers to the ratio of vitreous (hard) to floury (soft) endosperm, is a key agronomic trait that influences hardness, post-harvest resistance to insects and fungi, rate of starch digestibility, and semolina yield for food production (Gayral et al., 2015).1 Maize grain is a important source of energy for humans in Africa and South America, and the primary source of energy for domestic animal nutrition. Starch is its key energetic component. Vitreousness strongly affects the energy released when starch is digested by ruminants (Philippeau et al., 2000) and monogastric animals (Giuberti et al., 2013); these studies indicate that lower vitreousness is associated with greater starch digestibility. However, improved starch digestibility does not necessarily imply better energy utilization; in fact, more vitreous maize hybrids have been linked to better feed conversion ratios (Zhao et al., 2016). Higher
* Corresponding author. E-mail address: [email protected] (K. Kljak). 1 A/AP, amylose to amylopectin ratio; DM, dry matter; EqDi, equivalent diameter of starch granule; MinFeret and MaxFeret, minimal and maximal Feret's diameters; TS, total starch. https://doi.org/10.1016/j.jcs.2018.01.010 0733-5210/© 2018 Elsevier Ltd. All rights reserved.
vitreousness may also be associated with superior food quality, leading to slower cooking rates and higher content of resistant starch, which may offer health benefits (Osorio-Díaz et al., 2011). Maize is usually subdefined according to the kernel characteristic of grain vitreousness. Flint maize features a large, continuous volume of vitreous endosperm, while floury maize contains floury endosperm nearly exclusively (Watson, 2003). Dent maize hybrids, which are derivatives of flint-flour classes, differ in their ratio of vitreous to floury endosperm. Vitreousness also varies with the position of kernels on the ear as well as with environmental conditions (Watson, 2003). The texture of vitreous and floury endosperm differs due to the interaction between starch and proteins. In both types of endosperm, protein matrix surrounds starch granules. In vitreous endosperm, the granules are tightly packed, while in floury endosperm, the protein matrix is thinner and features numerous air-filled spaces. The protein matrix itself comprises abundant zein proteins embedded in a matrix of glutelin proteins (Philippeau et al., 2000). The texture differences between vitreous and floury endosperm, therefore, lead to differences in physical properties in maize hybrids varying in vitreousness (Kljak et al., 2011). The potentially substantial effect of vitreousness on maize grain properties means it should be taken into account when selecting hybrids for targeted production. The most important factors affecting maize grain vitreousness are related to starch, the
58
K. Kljak et al. / Journal of Cereal Science 80 (2018) 57e62
dominant constituent in maize endosperm. Starch molecular architecture can be categorized into six levels (Dona et al., 2010) and each of these levels e from individual branches of amylose and amylopectin fractions to endosperm e could influence grain vitreousness. Thus, amylose content and starch granule size and shape should be taken into account, as well as the content of zein, which is the major storage protein in endosperm. Vitreous endosperm has higher zein and amylose content than floury endosperm, as well as smaller starch granules (Cagampang and Kirleis, 1985; DombrinkKurtzman and Bietz, 1993; Gayral et al., 2015; Landry et al., 2004). One would therefore predict similar characteristics in more vitreous hybrids. However, amylose content, zein content and starch granule size do not influence vitreousness independently of one another (Gayral et al., 2016), so they should be analyzed jointly. Previous studies have focused on only amylose or zein content separately (Dombrink-Kurtzman and Bietz, 1993; DombrinkKurtzman and Knutson, 1997; Landry et al., 2004; Mestres and Matencio, 1996). The aim of the present study, therefore, was to explore the combined effect of starch properties and zein content, determined using fast and simple laboratory methods, on kernel vitreousness of commercial maize hybrids. The joint contribution of individual properties to kernel vitreousness was assessed using multiple linear regression. 2. Material and methods 2.1. Plant material
deviations in the results. Kernels were soaked in boiling distilled water for 5 min then dried with a paper towel. The pericarp, germ and endosperm were separated with a scalpel. Floury endosperm was then manually removed from the rest of the endosperm using a scalpel. Endosperm fractions were dried for 24 h at 103 C and weighed. Vitreousness was expressed as a weight proportion of vitreous endosperm in the total endosperm. 2.3. Amylose and zein determination Ground maize samples were defatted by soaking in hexane, then apparent amylose content was determined as described (Knutson, 1986) based on a standard curve obtained using pure amylose (Sigma Aldrich, St. Louis, MO, USA). The apparent amylose was starch fraction dissolved in water-dimethylsulfoxide (1:9) containing 6 103 M iodine after incubation at 50 C for 16 h. True amylose content was calculated by correcting the apparent content for amylopectin as described (Knutson, 1986) and expressed on a dry endosperm basis. The ratio of amylose to amylopectin (A/AP) was calculated based on amylopectin content, which was calculated based on amylose content in total starch. Total starch (TS) content was determined enzymatically (Total Starch Assay Procedure, Megazyme International Ireland, Wicklow, Ireland). Total zeins in maize samples were extracted using 0.0125 M sodium borate (pH 10.0) containing 10 g/kg of sodium dodecyl sulphate and 20 g/kg of 2-mercaptoethanol as described by Wallace et al. (1990). After precipitation of non-zein proteins with ethanol, supernatant was dried and analyzed for nitrogen using Kjeldahl method with modification as described by Kljak et al. (2011). Total nitrogen content in maize grain samples was determined using the Kjeldahl method as described in ISO 5983-2:2009 (ISO, 2009). Protein content in grain samples and zein extracts was calculated using the conversion factor for maize 5.7. Zein was expressed as content in a dry endosperm and total starch.
The selected maize samples are hybrids from different vegetation groups (FAO 200e700) and are known to range widely in kernel vitreousness. The following 11 commercial high-yield maize hybrids (Zea Mays L.) were provided by the Bc Institute (Zagreb, Croatia): Bc 244, Bc 282, Bc 354, Bc 394, Bc 408b, Bc 462, Bc 572, Bc 574, Bc 678, Bc 778 and Pajdas. From them, Bc 354, Bc 394, Bc 408b, Bc 678 and Bc 778 were dent, Bc 244, Bc 282, Bc 572, Bc 574 and Pajdas were dent flint while Bc 462 was a flint hybrid. The samples were grown in-season in the years 2007 (Bc 244, Bc 408b, Bc 462, Bc 678, Bc 778, Pajdas), 2008 (Bc 282, Bc 354, Bc 394, Bc 462, Bc 572, Bc 678, Bc 778, Pajdas) and 2009 (Bc 244, Bc 354, Bc 394, Bc 462, Bc 572, Bc 574, Bc 678, Pajdas). Hybrids grown in the same season were used in experiments conducted yearly in our laboratory. Maize hybrids were grown in a completely randomized design in test fields in central Croatia (2007, 2009) or eastern Croatia (2008) under the same agro-climate and production conditions. Each hybrid was planted on one 560-m2 test lot; when the grain had reached physiological maturity, maize samples of each hybrid were collected from three places (2007) or five places (2008, 2009) across the lot (three or five replicates of each hybrid, respectively). Kernels were removed manually from cobs and stored at 4 C. Maize samples were ground up in a laboratory mill (Cyclotec 1093, Foss Tocator, Hoganas, Sweden) equipped with a 0.3-mm screen for amylose analysis or 1-mm screen for all other analyses, then subjected to chemical analysis. All samples were also analyzed for dry matter (DM) content by drying at 103 C until constant weight.
Maize hybrid samples were subjected to non-starch extraction as described (Mistry and Eckhoff, 1992). The white starchy layer was separated with a spatula after incubation of suspension of ground maize sample and 1 g/kg NaOH solution with mild shaking at 55 C for 90 min; after centrifugation, residue was washed three times with water to remove alkali and dried at room temperature. Starch granules were stained with KI-I2 solution, immobilized in glycerol on a microscope slide, and images were acquired using a high-resolution color camera (ProgRes CT3 3.15 MPix 1/200 CMOS, 2048 1536, 10-bit; Jenoptic, Germany) placed on an adjustable stand. Images were analyzed using NISeELEMENTS 2.3 software a rka (Laboratory Imaging, Prague, Czech Republic) as described (S and Bubník, 2009) in order to determine starch particle size. Seven geometric parameters were measured: projected area, equivalent diameter (EqDi), perimeter, minimal and maximal Feret's diameters (MinFeret, MaxFeret), circularity and elongation. Parameter values were the arithmetic means of at least 1000 granules.
2.2. Maize kernel dissection and determination of vitreousness
2.5. Statistical analysis
Contents of mature maize kernel parts were determined by manual dissection as described (Dombrink-Kurtzman and Bietz, 1993). To achieve a representative sample, 100 kernels from each hybrid were randomly selected and divided into 10 visually homogeneous groups based on kernel size and form, and one kernel from each group was randomly selected for dissection. This process was done in duplicate for each sample, and repeated in case of
Measured traits of analyzed hybrids were subjected to one-way analysis of variance in SAS 9.3 software (SAS, 2011). Effect of maize hybrid and growing season on each measured trait was tested using PROC MIXED procedure; after determination of the effects on nearly every trait, each hybrid from each growing season was considered one sample (n ¼ 22 number of replications) for further assessments of trait relationships. The individual effects of
2.4. Determination of maize starch granule size
K. Kljak et al. / Journal of Cereal Science 80 (2018) 57e62
starch properties and zein content on kernel vitreousness were tested using the PROC MIXED procedure, in which maize hybrid and growing season were treated as fixed effects, as well. Correlations of kernel vitreousness with starch properties and zein content were assessed using the PROC CORR procedure, and these correlations were further explored using the PROC REG multiple regression procedure, during which the optimal model was selected based on the Cp criterion. Statistical significance was achieved if P < 0.05. 3. Results and discussion Maize vitreousness is determined by both genetic background and the environment during the growing season, which contributes to hybrid variability (Lu et al., 1996). Grain samples in the present study were collected over a 3-year period, and several hybrids were sampled from multiple fields and/or multiple growing seasons. This repetitive sampling mimicked to some extent the hybrid variability due to environmental conditions, which was confirmed by the significant effect of growing season on vitreousness. This effect was not examined further since our attention was focused on the relationship between vitreousness and endosperm properties. The relatively large number of hybrids and replicate sampling from different fields and growing seasons ensured that we obtained samples of commercial hybrids ranging widely in kernel vitreousness. 3.1. Starch properties and zein content of maize samples Starch is a major component of maize endosperm, so its properties play an important role in grain texture. Table 1 summarizes determinations of zein and amylose content, A/AP ratio, as well as size and shape of starch granules in maize samples. The 22 maize samples displayed a wide range of amylose content, both in terms of endosperm DM (190.73e259.77 g/kg DM) and in terms of TS (219.10e298.40 g/kg TS). These ranges are typical for dent and flint maize hybrids (Watson, 2003). The A/AP range in our samples is very similar to the range of 0.24e0.35 reported by Blandino et al. (2010) for 33 commercial maize hybrids. Zein content is expressed as content in endosperm DM and TS. Content in TS emphasizes the role of zein in surrounding starch granules in the starch-protein matrix of maize endosperm (Watson, 2003). When expressed in relation to the kernel DM, zein content ranged from 45.92 to 72.13 g/kg, similar to the range of 59.3e73.1 g/ kg reported by Hamaker et al. (1995), who used the same extraction method. The average zein content in TS in our maize samples was 88.50 g/kg TS, very similar to the value of 88.40 reported by
Table 1 Means and ranges for kernel vitreousness, starch properties and zein content for 22 maize samples. Maize trait
Mean
Range
SD
Vitreousness, % Zein, g/kg endosperm DM Zein in TS, g/kg Amylose, g/kg endosperm DM Amylose in TS, g/kg A/AP Starch granule Projected area, mm2 EqDi, mm Perimeter, mm MaxFeret, mm MinFeret, mm Circularity Elongation
62.16 67.48 88.50 226.61 262.76 0.36
50.23e76.41 53.86e86.37 69.04e111.54 190.73e259.77 219.10e298.40 0.28e0.43
6.30 6.71 10.03 16.50 19.58 0.03
125.98 12.16 39.98 13.83 11.12 0.913 1.289
102.89e185.95 10.95e14.89 36.04e49.97 12.56e17.14 9.96e13.56 0.849e0.940 1.216e1.567
16.96 0.81 2.84 0.95 0.75 0.014 0.070
59
Philippeau et al. (2000) for flint maize samples. In contrast, the dent hybrids analyzed by Larson and Hoffman (2008) showed a wider range of zein content (37e95 g/kg DM), resulting in zein in starch content ranging from 58 to 133 g/kg TS. In this study, amylose content in endosperm DM and in TS increased with increasing zein in TS content (Table 3). DombrinkKurtzman and Knutson (1997) reported higher amylose content in vitreous than in floury endosperm, while Dombrink-Kurtzman and Bietz (1993) reported the same for zein content. This similar behavior may help explain why we observed a positive correlation between amylose and zein content in our samples. Maize samples varied in starch granule size and shape, for which the average projected area was 125.98 mm2 and average circularity was 0.913 (Table 1). The average EqDi of our samples (12.16 mm) is slightly lower than the 13.6 mm reported by Dhital et al. (2011), which implies that the genetic backgrounds of our samples biased them toward slightly smaller starch granules. As expected, increases in EqDi of granules in our samples led to increases in perimeter, MaxFeret and MinFeret (data not shown). EqDi correlated positively with circularity and negatively with elongation (Table 3), suggesting that smaller starch granules tended to have more irregular shape. These findings are consistent with previous work showing that maize has irregularly shaped granules with a number of faces (polyhedral) and relatively sharp edges, and that smaller starch granules generally have more irregular, polygonal shape than larger granules (Jane et al., 1994). Both amylose and zein, whether expressed in endosperm DM or TS, correlated negatively with projected area and EqDi (Table 3), implying that smaller starch granules had higher amylose content and were embedded in protein matrix with more protein bodies (Philippeau et al., 2000). The relationship between size and zein content may reflect the effects of greater surface area (Singh et al., 2010), implying that small starch granules are surrounded by more protein matrix. In contrast, the relationship between amylose and starch granule size is more complex. Dhital et al. (2011) reported a nonsignificant tendency for amylose content to increase with starch granule size in commercial maize starch; Cai et al. (2014) found that the same relationship achieved statistical significance in regular maize starch, but then found the relationship to be negative in high-amylose maize starch. Results with our maize samples are consistent with the negative correlation observed in high-amylose maize starches. This may reflect the fact that higher amylose content is associated with greater compressibility of starch granules in vitreous endosperm (Dombrink-Kurtzman and Knutson, 1997). 3.2. Kernel vitreousness and its relationship with zein content and starch properties Vitreousness of the maize samples varied from 50.23 to 76.41% (Table 1), which falls within the range of 66.8e79.1% that Philippeau et al. (2000) reported for flint and which lies at the upper edge of the range of 38.5e57.3% that Philippeau et al. (2000) reported for dent hybrids. Our range for commercial maize hybrid samples is similar to the range of 67.9e74.5% reported for 13 inbred maize lines (Gayral et al., 2015). Light transmission of kernels from maize samples as viewed on a light box is shown in Fig. 1. Variable degree of kernel translucent appearance is in agreement with variable vitreousness obtained by manual dissection. Nearly all maize traits on their own significantly influenced kernel vitreousness (Table 2), supporting our contention that vitreousness cannot be accurately assessed through any one trait alone. Correlations among traits found to have a significant effect on vitreousness are shown in Table 3. Amylose content, whether expressed in endosperm DM or TS, positively correlated with
60
K. Kljak et al. / Journal of Cereal Science 80 (2018) 57e62
Fig. 1. Kernels from maize samples on a light box. Hybrids (from left to right Bc 244, Bc 282, Bc 354, Bc 394, Bc 408b, Bc 462, Bc 572, Bc 574, Bc 678, Pc 778 and Pajdas) are separated in lines by season (2007 e bottom, 2008 e middle, and top e 2009).
Table 2 Effects of individual starch properties and zein content on kernel vitreousness for 22 maize samples, assessed using ANOVA. Maize trait
P
Zein, g/kg DM Zein in TS, g/kg Amylose, g/kg DM Amylose in TS, g/kg A/AP Starch granule Projected area EqDi, mm Perimeter, mm MaxFeret, mm MinFeret, mm Circularity Elongation
*** *** ** * * * * NS NS NS *** *
NS, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.
vitreousness (Table 3). This positive correlation is in agreement with work by Gayral et al. (2015), who found higher amylose content in vitreous than in floury endosperm. In other words, maize samples with a higher proportion of vitreous endosperm are expected to have higher amylose content in the entire kernel. This
relationship between amylose and vitreousness is supported by the positive correlation that we observed between vitreousness and A/ AP. Some have suggested that no simple relationship exists between vitreousness and total zein content (Landry et al., 2004); endosperm containing 610e780 g total zein/kg of total endosperm N contains variable proportions of vitreous region. Nevertheless, the same researchers (Landry et al., 2004) and others (Philippeau et al., 2000) have reported a high positive correlation between vitreousness and zein content. A positive correlation of vitreousness with total zein in endosperm DM and with total zein in TS content was also found in this study (Table 3), consistent with findings from Holding (2014) and Pereira et al. (2008). The larger, more numerous protein bodies in vitreous than in floury endosperm (Holding, 2014) may allow the starch granules to adhere better and to pack more tightly (Pereira et al., 2008). As a result, hybrids with higher vitreousness are expected to have higher zein content. In this study, starch granule size parameters correlated negatively with vitreousness, suggesting that more vitreous maize samples have smaller starch granules. This is in agreement with the work of Cagampang and Kirleis (1985), who found that more floury sorghum cultivars have larger starch granules than more vitreous cultivars. The correlation between vitreousness and starch granule size likely reflects the relationship of vitreousness with amylose
Table 3 Correlation matrix for kernel vitreousness, starch properties and zein content for 22 maize samples. Trait
Zein
Zein in TS
Amylose
Amylose in TS
A/AP
Projected area
EqDi
Circularity
Elongation
Vitreousness Zein Zein in TS Amylose Amylose in TS A/AP Projected area EqDi Circularity
0.50***
0.38*** 0.84***
0.29* 0.09 0.35**
0.24* 0.12 0.42*** 0.97***
0.24* 0.11 0.43*** 0.97*** 0.99***
0.25* 0.35** 0.33** 0.46*** 0.39*** 0.39***
0.24** 0.34** 0.35** 0.47*** 0.42*** 0.42*** 0.98***
0.39*** 0.26* 0.13 0.08 0.07 0.08 0.30** 0.28*
0.10 0.15 0.08 0.22 0.19 0.20 0.34** 0.33** 0.51***
*P < 0.05, **P < 0.01, ***P < 0.001.
K. Kljak et al. / Journal of Cereal Science 80 (2018) 57e62
and zein. Among the shape parameters tested, only circularity correlated significantly with vitreousness, and the correlation was negative. This is consistent with the more polygonal shape of starch granules in vitreous than floury endosperm (Dombrink-Kurtzman and Knutson, 1997). Next we used multiple linear regression to assess relationships among vitreousness, starch properties and zein content in maize samples. Our intent was to determine which properties should be included in a linear model to predict kernel vitreousness. The following properties were identified as significantly affecting kernel vitreousness: amylose content in endosperm DM, total zein content in endosperm DM and TS, and starch granule circularity and projected area (Table 4). A model incorporating these variables explained 61.8% of the variability in kernel vitreousness of our maize samples, with zein content in endosperm DM by itself explaining 24.0% of variability. Similarly, this parameter also showed a higher correlation coefficient with vitreousness than the other parameters analyzed. Starch granule properties explained an additional 28.5% of variability in kernel vitreousness, of which starch granule projected area accounted for 11.2%. This contribution is greater than one might predict from the correlation coefficient between circularity and vitreousness. This may relate to differences in amylose or amylopectin content: in their comparison of starch granule shape between floury and vitreous endosperm, Dombrink-Kurtzman and Knutson (1997) suggested that starch granules may be more spherical in floury endosperm, even when closely packed, due to the higher amylopectin content. Our findings appear to support this hypothesis. This may help explain why projected area accounted for 17.5% of variability in kernel vitreousness; this parameter has already been shown to capture both spherical and polygonal shape of native maize starch granules, making it suitable for multiple lez et al., 2006). regression analysis of vitreousness (Narv aez-Gonza Among the molecular properties we tested, amylose content in endosperm DM accounted for an additional 5.1% of variability in kernel vitreousness. This is in agreement with the observed positive correlation between amylose content and vitreousness. The relatively small contribution from this variable e the only one related to molecular structure in the model e indicates that molecular properties of starch contribute significantly less to kernel vitreousness of our samples than zein content and starch granule size and shape do. These results imply that the extent to which starch granules are embedded in the surrounding protein matrix and engage in starch-protein interactions influences kernel vitreousness more than the molecular structure of starch does. The linear regression model we obtained showed a relatively high degree of fit to the data, but it failed to explain completely the variation in maize kernel vitreousness across our samples. It may be possible to explain more of this variability by analyzing additional parameters, such as zein composition, amylopectin branch chain length, starch crystallinity, or lipid content and composition (Cai et al., 2014; Dhital et al., 2011; Dombrink-Kurtzman and Bietz, 1993; Gayral et al., 2016; Mestres and Matencio, 1996). Our goal in
Table 4 Regression model relating kernel vitreousness, starch properties and zein content in 22 maize samples. Variable
Parameter estimate Partial R2 Cumulative R2
Intercept 220.93 Zein 0.99 Circularity 216.37 Starch granule projected area 0.14 Zein in TS 4.27 Amylose 1.23
e 0.240 0.112 0.175 0.040 0.051
e 0.240 0.352 0.527 0.567 0.618
61
the present work was to apply simple, fast methods to a relatively large number of samples in order to explore the combined effects of endosperm properties on kernel vitreousness of commercial maize hybrids. For the purpose of estimating hybrid vitreousness, we recommend using Eq. (1) (R2 ¼ 0.614), in which contents of amylose and zein are expressed in relation to whole-kernel DM.
Vitreousness ð%Þ ¼ 184:85 þ 1:23 total zein
g DM kg
186:16 circularity 0:13
starch granule projected area mm2 g þ 1:62 4:13 zein in TS kg g amylose DM kg
(1)
4. Conclusions The results of this study confirm that starch properties and zein content influence maize kernel vitreousness. In our samples, vitreousness was higher with greater zein and amylose content as well as with smaller and less circular starch granules. The results further show that the complex behavior of maize vitreousness is due to interactions between the starch granules and proteinaceous matrix in endosperm, and that parameters reflecting these interactions should be considered together in order to accurately account for vitreousness. The combination of simple, fast determination of amylose and zein content together with image analysis for determination of starch granule size and shape led to a multiple regression model involving several variables, which explained 61.8% of observed variability in kernel vitreousness. The most important factors in this model are zein content and starch granule size and shape. This implies that kernel vitreousness depends more on starch-protein interactions than on the molecular properties of starch, such as those measured through amylose content. Acknowledgments This research was financed by a grant from the Ministry of Science, Education and Sports of the Republic of Croatia (“Nutritional, antioxidant and prebiotic attribute of corn for domestic animals”, 178-1780496-0368). References Blandino, M., Mancini, M.C., Peila, A., Rolle, L., Vanara, F., Reyneri, A., 2010. Determination of maize kernel hardness: comparison of different laboratory tests to predict dry-milling performance. J. Sci. Food Agric. 90, 1870e1878. Cagampang, G.B., Kirleis, A.W., 1985. Properties of starches isolated from sorghum €rke 37, 253e257. floury and corneous endosperm. Starch-Sta Cai, C., Lin, L., Man, J., Zhao, L., Wang, Z., Wei, C., 2014. Different structural properties of high-amylose maize starch fractions varying in granule size. J. Agric. Food Chem. 62, 11711e11721. Dhital, S., Shrestha, A.K., Hasjim, J., Gidley, M.J., 2011. Physicochemical and structural properties of maize and potato starches as a function of granule size. J. Agric. Food Chem. 59, 10151e10161. Dombrink-Kurtzman, M.A., Bietz, J.A., 1993. Zein composition in hard and soft endosperm of maize. Cereal Chem. 70, 105e108. Dombrink-Kurtzman, M.A., Knutson, C.A., 1997. A study of maize endosperm hardness in relation to amylose content and susceptibility to damage. Cereal Chem. 74, 776e780. Dona, A.C., Pages, G., Gilbert, R.G., Kuchel, P.W., 2010. Digestion of starch: in vivo and in vitro kinetic models used to characterise oligosaccharide or glucose release. Carbohydr. Polym. 80, 599e617. Gayral, M., Bakan, B., Dalgalarrondo, M., Elmorjani, K., Delluc, C., Brunet, S., Linossier, L., Morel, M.-H., Marion, D., 2015. Lipid partitioning in maize (Zea
62
K. Kljak et al. / Journal of Cereal Science 80 (2018) 57e62
mays L.) endosperm highlights relationships among starch lipids, amylose, and vitreousness. J. Agric. Food Chem. 63, 3551e3558. Gayral, M., Gaillard, C., Bakan, B., Dalgalarrondo, M., Elmorjani, K., Delluc, C., Brunet, S., Linossier, L., Morel, M.-H., Marion, D., 2016. Transition from vitreous to floury endosperm in maize (Zea mays L.) kernels is related to protein and starch gradients. J. Cereal. Sci. 68, 148e154. Giuberti, G., Gallo, A., Moschini, M., Cerioli, C., Masoero, F., 2013. Evaluation of the impact of maize endosperm vitreousness on in vitro starch digestion, dry matter digestibility and fermentation characteristics for pigs. Anim. Feed Sci. Technol. 186, 71e80. Hamaker, B.R., Mohamed, A.A., Habben, J.E., Huang, C.P., Larkins, B.A., 1995. Efficient procedure for extracting maize and sorghum kernel proteins reveals higher prolamin contents than the conventional method. Cereal Chem. 72, 583e588. Holding, D.R., 2014. Recent advances in the study of prolamin storage protein organization and function. Front. Plant Sci. 5, 1e9. ISO, 2009. Animal Feeding Stuffs e Determination of Nitrogen Content and Calculation of Crude Protein Content e Part 2: Block Digestion and Steam Distillation Method (ISO 5983e2:2009). International Organization for Standardization. Jane, J.L., Kasemsuwan, T., Leas, S., Zobel, H., Robyt, J.F., 1994. Anthology of starch granule morphology by scanning electron microscopy. Starch-St€ arke 46, 121e129. Kljak, K., Grbesa, D., Aleus, D., 2011. Relationships between kernel physical properties and zein content in corn hybrids. Bull. Univ. Agric. Sci. Vet. Med. ClujNapoca - Agric. 68, 188e194. Knutson, C.A., 1986. A simplified colorimetric procedure for determination of amylose in maize starches. Cereal Chem. 63, 89e92. Landry, J., Delhaye, S., Damerval, C., 2004. Protein distribution pattern in floury and vitreous endosperm of maize grain. Cereal Chem. 81, 153e158. Larson, J., Hoffman, P.C., 2008. A method to quantify prolamin proteins in corn that are negatively related to starch digestibility in ruminants. J. Dairy Sci. 91, 4834e4839. Lu, T.J., Jane, J.L., Keeling, P.L., Singletary, G.W., 1996. Maize starch fine structures affected by ear developmental temperature. Carbohydr. Res. 282, 157e170.
Mistry, A.H., Eckhoff, S.R., 1992. Characteristics of alkali-extracted starch obtained from corn flour. Cereal Chem. 69, 296e303. Mestres, C., Matencio, F., 1996. Biochemical basis of kernel milling characteristics and endosperm vitreousness of maize. J. Cereal. Sci. 24, 283e290. ez-Gonza lez, E.D., de Dios Figueroa-Ca rdenas, J., Taba, S., Tostado, E.C., Narva Peniche, R.A.M., S anchez, F.R., 2006. Relationships between the microstructure, physical features, and chemical composition of different maize accessions from Latin America. Cereal Chem. 83, 595e604. rez, L.A., Islas-Herna ndez, J.J., GomezOsorio-Díaz, P., Agama-Acevedo, E., Bello-Pe pez, O., 2011. Effect of endosperm type on texture and Montiel, N.O., Paredes-Lo in vitro starch digestibility of maize tortillas. LWT-Food Sci. Technol. 44, 611e615. Pereira, R.C., Davide, L.C., Pedrozo, C.A., Carneiro, N.P., Souza, I.R.P., Paiva, E., 2008. Relationship between structural and biochemical characteristics and texture of corn grains. Genet. Mol. Res. 7, 498e508. Philippeau, C., Landry, J., Michalet-Doreau, B., 2000. Influence of the protein distribution of maize endosperm on ruminal starch degradability. J. Sci. Food Agric. 80, 404e408. Singh, J., Dartois, A., Kaur, L., 2010. Starch digestibility in food matrix: a review. Trends Food Sci. Technol. 21, 168e180. a rka, E., Bubník, Z., 2009. Using image analysis to identify acetylated distarch S €rke 61, 457e462. adipate in a mixture. Starch-Sta Wallace, J.C., Lopes, M.A., Paiva, E., Larkins, B.A., 1990. New method for extraction and quantitation of zeins reveal a high content of g-zein in modified opaque-2 maize. Plant Physiol. 92, 191e196. Watson, S.A., 2003. Description, development, structure and composition of the corn kernel. In: White, P.J., Johnson, L.A. (Eds.), Corn: Chemistry and Technology. American Association of Cereal Chemists, St. Paul, pp. 69e106. Zhao, J.P., Cui, D.P., Zhang, Z.Y., Jiao, H.C., Song, Z.G., Lin, H., 2016. Live performance, carcass characteristic and blood metabolite responses of broilers to two distinct corn types with different extent of grinding. J. Anim. Physiol. Anim. Nutr. 101, 378e388.
Contents lists available at ScienceDirect
Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Contribution of zein content and starch characteristics to vitreousness of commercial maize hybrids Kristina Kljak*, Marija Duvnjak, Darko Grbesa Department of Animal Nutrition, Faculty of Agriculture, University of Zagreb, Svetosimunska cesta 25, 10 000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 October 2017 Received in revised form 16 January 2018 Accepted 17 January 2018
Fast, simple laboratory methods were used to analyze 22 maize samples varying in kernel vitreousness from 50.23% to 76.41%. Samples were analyzed in terms of zein content (53.86e86.37 g/kg endosperm DM), amylose content (190.76e259.77 g/kg endosperm DM), amylose to amylopectin ratio in starch (0.28 e0.43), as well as starch granule size (10.95e14.89 mm in equivalent diameter) and starch granule shape (circularity, 0.85e0.94). More vitreous samples had higher zein and amylose content, as well as smaller and less circular starch granules. Nearly all grain traits on their own significantly affected vitreousness, and a multiple regression model to account for their combined effects was able to explain 61.8% of variability in kernel vitreousness. Zein content contributed most to the model, followed by starch granule projected area and circularity. In contrast, the amylose content contributed only 5.1% to the model. These results suggest that starch-protein interactions influence maize kernel vitreousness more strongly than starch molecular properties do. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Maize grain Vitreousness Amylose Zein Starch granule
1. Introduction Vitreousness and hardness-associated properties are significantly correlated with end usage of maize and are strongly determined by genotype. Grain vitreousness, which refers to the ratio of vitreous (hard) to floury (soft) endosperm, is a key agronomic trait that influences hardness, post-harvest resistance to insects and fungi, rate of starch digestibility, and semolina yield for food production (Gayral et al., 2015).1 Maize grain is a important source of energy for humans in Africa and South America, and the primary source of energy for domestic animal nutrition. Starch is its key energetic component. Vitreousness strongly affects the energy released when starch is digested by ruminants (Philippeau et al., 2000) and monogastric animals (Giuberti et al., 2013); these studies indicate that lower vitreousness is associated with greater starch digestibility. However, improved starch digestibility does not necessarily imply better energy utilization; in fact, more vitreous maize hybrids have been linked to better feed conversion ratios (Zhao et al., 2016). Higher
* Corresponding author. E-mail address: [email protected] (K. Kljak). 1 A/AP, amylose to amylopectin ratio; DM, dry matter; EqDi, equivalent diameter of starch granule; MinFeret and MaxFeret, minimal and maximal Feret's diameters; TS, total starch. https://doi.org/10.1016/j.jcs.2018.01.010 0733-5210/© 2018 Elsevier Ltd. All rights reserved.
vitreousness may also be associated with superior food quality, leading to slower cooking rates and higher content of resistant starch, which may offer health benefits (Osorio-Díaz et al., 2011). Maize is usually subdefined according to the kernel characteristic of grain vitreousness. Flint maize features a large, continuous volume of vitreous endosperm, while floury maize contains floury endosperm nearly exclusively (Watson, 2003). Dent maize hybrids, which are derivatives of flint-flour classes, differ in their ratio of vitreous to floury endosperm. Vitreousness also varies with the position of kernels on the ear as well as with environmental conditions (Watson, 2003). The texture of vitreous and floury endosperm differs due to the interaction between starch and proteins. In both types of endosperm, protein matrix surrounds starch granules. In vitreous endosperm, the granules are tightly packed, while in floury endosperm, the protein matrix is thinner and features numerous air-filled spaces. The protein matrix itself comprises abundant zein proteins embedded in a matrix of glutelin proteins (Philippeau et al., 2000). The texture differences between vitreous and floury endosperm, therefore, lead to differences in physical properties in maize hybrids varying in vitreousness (Kljak et al., 2011). The potentially substantial effect of vitreousness on maize grain properties means it should be taken into account when selecting hybrids for targeted production. The most important factors affecting maize grain vitreousness are related to starch, the
58
K. Kljak et al. / Journal of Cereal Science 80 (2018) 57e62
dominant constituent in maize endosperm. Starch molecular architecture can be categorized into six levels (Dona et al., 2010) and each of these levels e from individual branches of amylose and amylopectin fractions to endosperm e could influence grain vitreousness. Thus, amylose content and starch granule size and shape should be taken into account, as well as the content of zein, which is the major storage protein in endosperm. Vitreous endosperm has higher zein and amylose content than floury endosperm, as well as smaller starch granules (Cagampang and Kirleis, 1985; DombrinkKurtzman and Bietz, 1993; Gayral et al., 2015; Landry et al., 2004). One would therefore predict similar characteristics in more vitreous hybrids. However, amylose content, zein content and starch granule size do not influence vitreousness independently of one another (Gayral et al., 2016), so they should be analyzed jointly. Previous studies have focused on only amylose or zein content separately (Dombrink-Kurtzman and Bietz, 1993; DombrinkKurtzman and Knutson, 1997; Landry et al., 2004; Mestres and Matencio, 1996). The aim of the present study, therefore, was to explore the combined effect of starch properties and zein content, determined using fast and simple laboratory methods, on kernel vitreousness of commercial maize hybrids. The joint contribution of individual properties to kernel vitreousness was assessed using multiple linear regression. 2. Material and methods 2.1. Plant material
deviations in the results. Kernels were soaked in boiling distilled water for 5 min then dried with a paper towel. The pericarp, germ and endosperm were separated with a scalpel. Floury endosperm was then manually removed from the rest of the endosperm using a scalpel. Endosperm fractions were dried for 24 h at 103 C and weighed. Vitreousness was expressed as a weight proportion of vitreous endosperm in the total endosperm. 2.3. Amylose and zein determination Ground maize samples were defatted by soaking in hexane, then apparent amylose content was determined as described (Knutson, 1986) based on a standard curve obtained using pure amylose (Sigma Aldrich, St. Louis, MO, USA). The apparent amylose was starch fraction dissolved in water-dimethylsulfoxide (1:9) containing 6 103 M iodine after incubation at 50 C for 16 h. True amylose content was calculated by correcting the apparent content for amylopectin as described (Knutson, 1986) and expressed on a dry endosperm basis. The ratio of amylose to amylopectin (A/AP) was calculated based on amylopectin content, which was calculated based on amylose content in total starch. Total starch (TS) content was determined enzymatically (Total Starch Assay Procedure, Megazyme International Ireland, Wicklow, Ireland). Total zeins in maize samples were extracted using 0.0125 M sodium borate (pH 10.0) containing 10 g/kg of sodium dodecyl sulphate and 20 g/kg of 2-mercaptoethanol as described by Wallace et al. (1990). After precipitation of non-zein proteins with ethanol, supernatant was dried and analyzed for nitrogen using Kjeldahl method with modification as described by Kljak et al. (2011). Total nitrogen content in maize grain samples was determined using the Kjeldahl method as described in ISO 5983-2:2009 (ISO, 2009). Protein content in grain samples and zein extracts was calculated using the conversion factor for maize 5.7. Zein was expressed as content in a dry endosperm and total starch.
The selected maize samples are hybrids from different vegetation groups (FAO 200e700) and are known to range widely in kernel vitreousness. The following 11 commercial high-yield maize hybrids (Zea Mays L.) were provided by the Bc Institute (Zagreb, Croatia): Bc 244, Bc 282, Bc 354, Bc 394, Bc 408b, Bc 462, Bc 572, Bc 574, Bc 678, Bc 778 and Pajdas. From them, Bc 354, Bc 394, Bc 408b, Bc 678 and Bc 778 were dent, Bc 244, Bc 282, Bc 572, Bc 574 and Pajdas were dent flint while Bc 462 was a flint hybrid. The samples were grown in-season in the years 2007 (Bc 244, Bc 408b, Bc 462, Bc 678, Bc 778, Pajdas), 2008 (Bc 282, Bc 354, Bc 394, Bc 462, Bc 572, Bc 678, Bc 778, Pajdas) and 2009 (Bc 244, Bc 354, Bc 394, Bc 462, Bc 572, Bc 574, Bc 678, Pajdas). Hybrids grown in the same season were used in experiments conducted yearly in our laboratory. Maize hybrids were grown in a completely randomized design in test fields in central Croatia (2007, 2009) or eastern Croatia (2008) under the same agro-climate and production conditions. Each hybrid was planted on one 560-m2 test lot; when the grain had reached physiological maturity, maize samples of each hybrid were collected from three places (2007) or five places (2008, 2009) across the lot (three or five replicates of each hybrid, respectively). Kernels were removed manually from cobs and stored at 4 C. Maize samples were ground up in a laboratory mill (Cyclotec 1093, Foss Tocator, Hoganas, Sweden) equipped with a 0.3-mm screen for amylose analysis or 1-mm screen for all other analyses, then subjected to chemical analysis. All samples were also analyzed for dry matter (DM) content by drying at 103 C until constant weight.
Maize hybrid samples were subjected to non-starch extraction as described (Mistry and Eckhoff, 1992). The white starchy layer was separated with a spatula after incubation of suspension of ground maize sample and 1 g/kg NaOH solution with mild shaking at 55 C for 90 min; after centrifugation, residue was washed three times with water to remove alkali and dried at room temperature. Starch granules were stained with KI-I2 solution, immobilized in glycerol on a microscope slide, and images were acquired using a high-resolution color camera (ProgRes CT3 3.15 MPix 1/200 CMOS, 2048 1536, 10-bit; Jenoptic, Germany) placed on an adjustable stand. Images were analyzed using NISeELEMENTS 2.3 software a rka (Laboratory Imaging, Prague, Czech Republic) as described (S and Bubník, 2009) in order to determine starch particle size. Seven geometric parameters were measured: projected area, equivalent diameter (EqDi), perimeter, minimal and maximal Feret's diameters (MinFeret, MaxFeret), circularity and elongation. Parameter values were the arithmetic means of at least 1000 granules.
2.2. Maize kernel dissection and determination of vitreousness
2.5. Statistical analysis
Contents of mature maize kernel parts were determined by manual dissection as described (Dombrink-Kurtzman and Bietz, 1993). To achieve a representative sample, 100 kernels from each hybrid were randomly selected and divided into 10 visually homogeneous groups based on kernel size and form, and one kernel from each group was randomly selected for dissection. This process was done in duplicate for each sample, and repeated in case of
Measured traits of analyzed hybrids were subjected to one-way analysis of variance in SAS 9.3 software (SAS, 2011). Effect of maize hybrid and growing season on each measured trait was tested using PROC MIXED procedure; after determination of the effects on nearly every trait, each hybrid from each growing season was considered one sample (n ¼ 22 number of replications) for further assessments of trait relationships. The individual effects of
2.4. Determination of maize starch granule size
K. Kljak et al. / Journal of Cereal Science 80 (2018) 57e62
starch properties and zein content on kernel vitreousness were tested using the PROC MIXED procedure, in which maize hybrid and growing season were treated as fixed effects, as well. Correlations of kernel vitreousness with starch properties and zein content were assessed using the PROC CORR procedure, and these correlations were further explored using the PROC REG multiple regression procedure, during which the optimal model was selected based on the Cp criterion. Statistical significance was achieved if P < 0.05. 3. Results and discussion Maize vitreousness is determined by both genetic background and the environment during the growing season, which contributes to hybrid variability (Lu et al., 1996). Grain samples in the present study were collected over a 3-year period, and several hybrids were sampled from multiple fields and/or multiple growing seasons. This repetitive sampling mimicked to some extent the hybrid variability due to environmental conditions, which was confirmed by the significant effect of growing season on vitreousness. This effect was not examined further since our attention was focused on the relationship between vitreousness and endosperm properties. The relatively large number of hybrids and replicate sampling from different fields and growing seasons ensured that we obtained samples of commercial hybrids ranging widely in kernel vitreousness. 3.1. Starch properties and zein content of maize samples Starch is a major component of maize endosperm, so its properties play an important role in grain texture. Table 1 summarizes determinations of zein and amylose content, A/AP ratio, as well as size and shape of starch granules in maize samples. The 22 maize samples displayed a wide range of amylose content, both in terms of endosperm DM (190.73e259.77 g/kg DM) and in terms of TS (219.10e298.40 g/kg TS). These ranges are typical for dent and flint maize hybrids (Watson, 2003). The A/AP range in our samples is very similar to the range of 0.24e0.35 reported by Blandino et al. (2010) for 33 commercial maize hybrids. Zein content is expressed as content in endosperm DM and TS. Content in TS emphasizes the role of zein in surrounding starch granules in the starch-protein matrix of maize endosperm (Watson, 2003). When expressed in relation to the kernel DM, zein content ranged from 45.92 to 72.13 g/kg, similar to the range of 59.3e73.1 g/ kg reported by Hamaker et al. (1995), who used the same extraction method. The average zein content in TS in our maize samples was 88.50 g/kg TS, very similar to the value of 88.40 reported by
Table 1 Means and ranges for kernel vitreousness, starch properties and zein content for 22 maize samples. Maize trait
Mean
Range
SD
Vitreousness, % Zein, g/kg endosperm DM Zein in TS, g/kg Amylose, g/kg endosperm DM Amylose in TS, g/kg A/AP Starch granule Projected area, mm2 EqDi, mm Perimeter, mm MaxFeret, mm MinFeret, mm Circularity Elongation
62.16 67.48 88.50 226.61 262.76 0.36
50.23e76.41 53.86e86.37 69.04e111.54 190.73e259.77 219.10e298.40 0.28e0.43
6.30 6.71 10.03 16.50 19.58 0.03
125.98 12.16 39.98 13.83 11.12 0.913 1.289
102.89e185.95 10.95e14.89 36.04e49.97 12.56e17.14 9.96e13.56 0.849e0.940 1.216e1.567
16.96 0.81 2.84 0.95 0.75 0.014 0.070
59
Philippeau et al. (2000) for flint maize samples. In contrast, the dent hybrids analyzed by Larson and Hoffman (2008) showed a wider range of zein content (37e95 g/kg DM), resulting in zein in starch content ranging from 58 to 133 g/kg TS. In this study, amylose content in endosperm DM and in TS increased with increasing zein in TS content (Table 3). DombrinkKurtzman and Knutson (1997) reported higher amylose content in vitreous than in floury endosperm, while Dombrink-Kurtzman and Bietz (1993) reported the same for zein content. This similar behavior may help explain why we observed a positive correlation between amylose and zein content in our samples. Maize samples varied in starch granule size and shape, for which the average projected area was 125.98 mm2 and average circularity was 0.913 (Table 1). The average EqDi of our samples (12.16 mm) is slightly lower than the 13.6 mm reported by Dhital et al. (2011), which implies that the genetic backgrounds of our samples biased them toward slightly smaller starch granules. As expected, increases in EqDi of granules in our samples led to increases in perimeter, MaxFeret and MinFeret (data not shown). EqDi correlated positively with circularity and negatively with elongation (Table 3), suggesting that smaller starch granules tended to have more irregular shape. These findings are consistent with previous work showing that maize has irregularly shaped granules with a number of faces (polyhedral) and relatively sharp edges, and that smaller starch granules generally have more irregular, polygonal shape than larger granules (Jane et al., 1994). Both amylose and zein, whether expressed in endosperm DM or TS, correlated negatively with projected area and EqDi (Table 3), implying that smaller starch granules had higher amylose content and were embedded in protein matrix with more protein bodies (Philippeau et al., 2000). The relationship between size and zein content may reflect the effects of greater surface area (Singh et al., 2010), implying that small starch granules are surrounded by more protein matrix. In contrast, the relationship between amylose and starch granule size is more complex. Dhital et al. (2011) reported a nonsignificant tendency for amylose content to increase with starch granule size in commercial maize starch; Cai et al. (2014) found that the same relationship achieved statistical significance in regular maize starch, but then found the relationship to be negative in high-amylose maize starch. Results with our maize samples are consistent with the negative correlation observed in high-amylose maize starches. This may reflect the fact that higher amylose content is associated with greater compressibility of starch granules in vitreous endosperm (Dombrink-Kurtzman and Knutson, 1997). 3.2. Kernel vitreousness and its relationship with zein content and starch properties Vitreousness of the maize samples varied from 50.23 to 76.41% (Table 1), which falls within the range of 66.8e79.1% that Philippeau et al. (2000) reported for flint and which lies at the upper edge of the range of 38.5e57.3% that Philippeau et al. (2000) reported for dent hybrids. Our range for commercial maize hybrid samples is similar to the range of 67.9e74.5% reported for 13 inbred maize lines (Gayral et al., 2015). Light transmission of kernels from maize samples as viewed on a light box is shown in Fig. 1. Variable degree of kernel translucent appearance is in agreement with variable vitreousness obtained by manual dissection. Nearly all maize traits on their own significantly influenced kernel vitreousness (Table 2), supporting our contention that vitreousness cannot be accurately assessed through any one trait alone. Correlations among traits found to have a significant effect on vitreousness are shown in Table 3. Amylose content, whether expressed in endosperm DM or TS, positively correlated with
60
K. Kljak et al. / Journal of Cereal Science 80 (2018) 57e62
Fig. 1. Kernels from maize samples on a light box. Hybrids (from left to right Bc 244, Bc 282, Bc 354, Bc 394, Bc 408b, Bc 462, Bc 572, Bc 574, Bc 678, Pc 778 and Pajdas) are separated in lines by season (2007 e bottom, 2008 e middle, and top e 2009).
Table 2 Effects of individual starch properties and zein content on kernel vitreousness for 22 maize samples, assessed using ANOVA. Maize trait
P
Zein, g/kg DM Zein in TS, g/kg Amylose, g/kg DM Amylose in TS, g/kg A/AP Starch granule Projected area EqDi, mm Perimeter, mm MaxFeret, mm MinFeret, mm Circularity Elongation
*** *** ** * * * * NS NS NS *** *
NS, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.
vitreousness (Table 3). This positive correlation is in agreement with work by Gayral et al. (2015), who found higher amylose content in vitreous than in floury endosperm. In other words, maize samples with a higher proportion of vitreous endosperm are expected to have higher amylose content in the entire kernel. This
relationship between amylose and vitreousness is supported by the positive correlation that we observed between vitreousness and A/ AP. Some have suggested that no simple relationship exists between vitreousness and total zein content (Landry et al., 2004); endosperm containing 610e780 g total zein/kg of total endosperm N contains variable proportions of vitreous region. Nevertheless, the same researchers (Landry et al., 2004) and others (Philippeau et al., 2000) have reported a high positive correlation between vitreousness and zein content. A positive correlation of vitreousness with total zein in endosperm DM and with total zein in TS content was also found in this study (Table 3), consistent with findings from Holding (2014) and Pereira et al. (2008). The larger, more numerous protein bodies in vitreous than in floury endosperm (Holding, 2014) may allow the starch granules to adhere better and to pack more tightly (Pereira et al., 2008). As a result, hybrids with higher vitreousness are expected to have higher zein content. In this study, starch granule size parameters correlated negatively with vitreousness, suggesting that more vitreous maize samples have smaller starch granules. This is in agreement with the work of Cagampang and Kirleis (1985), who found that more floury sorghum cultivars have larger starch granules than more vitreous cultivars. The correlation between vitreousness and starch granule size likely reflects the relationship of vitreousness with amylose
Table 3 Correlation matrix for kernel vitreousness, starch properties and zein content for 22 maize samples. Trait
Zein
Zein in TS
Amylose
Amylose in TS
A/AP
Projected area
EqDi
Circularity
Elongation
Vitreousness Zein Zein in TS Amylose Amylose in TS A/AP Projected area EqDi Circularity
0.50***
0.38*** 0.84***
0.29* 0.09 0.35**
0.24* 0.12 0.42*** 0.97***
0.24* 0.11 0.43*** 0.97*** 0.99***
0.25* 0.35** 0.33** 0.46*** 0.39*** 0.39***
0.24** 0.34** 0.35** 0.47*** 0.42*** 0.42*** 0.98***
0.39*** 0.26* 0.13 0.08 0.07 0.08 0.30** 0.28*
0.10 0.15 0.08 0.22 0.19 0.20 0.34** 0.33** 0.51***
*P < 0.05, **P < 0.01, ***P < 0.001.
K. Kljak et al. / Journal of Cereal Science 80 (2018) 57e62
and zein. Among the shape parameters tested, only circularity correlated significantly with vitreousness, and the correlation was negative. This is consistent with the more polygonal shape of starch granules in vitreous than floury endosperm (Dombrink-Kurtzman and Knutson, 1997). Next we used multiple linear regression to assess relationships among vitreousness, starch properties and zein content in maize samples. Our intent was to determine which properties should be included in a linear model to predict kernel vitreousness. The following properties were identified as significantly affecting kernel vitreousness: amylose content in endosperm DM, total zein content in endosperm DM and TS, and starch granule circularity and projected area (Table 4). A model incorporating these variables explained 61.8% of the variability in kernel vitreousness of our maize samples, with zein content in endosperm DM by itself explaining 24.0% of variability. Similarly, this parameter also showed a higher correlation coefficient with vitreousness than the other parameters analyzed. Starch granule properties explained an additional 28.5% of variability in kernel vitreousness, of which starch granule projected area accounted for 11.2%. This contribution is greater than one might predict from the correlation coefficient between circularity and vitreousness. This may relate to differences in amylose or amylopectin content: in their comparison of starch granule shape between floury and vitreous endosperm, Dombrink-Kurtzman and Knutson (1997) suggested that starch granules may be more spherical in floury endosperm, even when closely packed, due to the higher amylopectin content. Our findings appear to support this hypothesis. This may help explain why projected area accounted for 17.5% of variability in kernel vitreousness; this parameter has already been shown to capture both spherical and polygonal shape of native maize starch granules, making it suitable for multiple lez et al., 2006). regression analysis of vitreousness (Narv aez-Gonza Among the molecular properties we tested, amylose content in endosperm DM accounted for an additional 5.1% of variability in kernel vitreousness. This is in agreement with the observed positive correlation between amylose content and vitreousness. The relatively small contribution from this variable e the only one related to molecular structure in the model e indicates that molecular properties of starch contribute significantly less to kernel vitreousness of our samples than zein content and starch granule size and shape do. These results imply that the extent to which starch granules are embedded in the surrounding protein matrix and engage in starch-protein interactions influences kernel vitreousness more than the molecular structure of starch does. The linear regression model we obtained showed a relatively high degree of fit to the data, but it failed to explain completely the variation in maize kernel vitreousness across our samples. It may be possible to explain more of this variability by analyzing additional parameters, such as zein composition, amylopectin branch chain length, starch crystallinity, or lipid content and composition (Cai et al., 2014; Dhital et al., 2011; Dombrink-Kurtzman and Bietz, 1993; Gayral et al., 2016; Mestres and Matencio, 1996). Our goal in
Table 4 Regression model relating kernel vitreousness, starch properties and zein content in 22 maize samples. Variable
Parameter estimate Partial R2 Cumulative R2
Intercept 220.93 Zein 0.99 Circularity 216.37 Starch granule projected area 0.14 Zein in TS 4.27 Amylose 1.23
e 0.240 0.112 0.175 0.040 0.051
e 0.240 0.352 0.527 0.567 0.618
61
the present work was to apply simple, fast methods to a relatively large number of samples in order to explore the combined effects of endosperm properties on kernel vitreousness of commercial maize hybrids. For the purpose of estimating hybrid vitreousness, we recommend using Eq. (1) (R2 ¼ 0.614), in which contents of amylose and zein are expressed in relation to whole-kernel DM.
Vitreousness ð%Þ ¼ 184:85 þ 1:23 total zein
g DM kg
186:16 circularity 0:13
starch granule projected area mm2 g þ 1:62 4:13 zein in TS kg g amylose DM kg
(1)
4. Conclusions The results of this study confirm that starch properties and zein content influence maize kernel vitreousness. In our samples, vitreousness was higher with greater zein and amylose content as well as with smaller and less circular starch granules. The results further show that the complex behavior of maize vitreousness is due to interactions between the starch granules and proteinaceous matrix in endosperm, and that parameters reflecting these interactions should be considered together in order to accurately account for vitreousness. The combination of simple, fast determination of amylose and zein content together with image analysis for determination of starch granule size and shape led to a multiple regression model involving several variables, which explained 61.8% of observed variability in kernel vitreousness. The most important factors in this model are zein content and starch granule size and shape. This implies that kernel vitreousness depends more on starch-protein interactions than on the molecular properties of starch, such as those measured through amylose content. Acknowledgments This research was financed by a grant from the Ministry of Science, Education and Sports of the Republic of Croatia (“Nutritional, antioxidant and prebiotic attribute of corn for domestic animals”, 178-1780496-0368). References Blandino, M., Mancini, M.C., Peila, A., Rolle, L., Vanara, F., Reyneri, A., 2010. Determination of maize kernel hardness: comparison of different laboratory tests to predict dry-milling performance. J. Sci. Food Agric. 90, 1870e1878. Cagampang, G.B., Kirleis, A.W., 1985. Properties of starches isolated from sorghum €rke 37, 253e257. floury and corneous endosperm. Starch-Sta Cai, C., Lin, L., Man, J., Zhao, L., Wang, Z., Wei, C., 2014. Different structural properties of high-amylose maize starch fractions varying in granule size. J. Agric. Food Chem. 62, 11711e11721. Dhital, S., Shrestha, A.K., Hasjim, J., Gidley, M.J., 2011. Physicochemical and structural properties of maize and potato starches as a function of granule size. J. Agric. Food Chem. 59, 10151e10161. Dombrink-Kurtzman, M.A., Bietz, J.A., 1993. Zein composition in hard and soft endosperm of maize. Cereal Chem. 70, 105e108. Dombrink-Kurtzman, M.A., Knutson, C.A., 1997. A study of maize endosperm hardness in relation to amylose content and susceptibility to damage. Cereal Chem. 74, 776e780. Dona, A.C., Pages, G., Gilbert, R.G., Kuchel, P.W., 2010. Digestion of starch: in vivo and in vitro kinetic models used to characterise oligosaccharide or glucose release. Carbohydr. Polym. 80, 599e617. Gayral, M., Bakan, B., Dalgalarrondo, M., Elmorjani, K., Delluc, C., Brunet, S., Linossier, L., Morel, M.-H., Marion, D., 2015. Lipid partitioning in maize (Zea
62
K. Kljak et al. / Journal of Cereal Science 80 (2018) 57e62
mays L.) endosperm highlights relationships among starch lipids, amylose, and vitreousness. J. Agric. Food Chem. 63, 3551e3558. Gayral, M., Gaillard, C., Bakan, B., Dalgalarrondo, M., Elmorjani, K., Delluc, C., Brunet, S., Linossier, L., Morel, M.-H., Marion, D., 2016. Transition from vitreous to floury endosperm in maize (Zea mays L.) kernels is related to protein and starch gradients. J. Cereal. Sci. 68, 148e154. Giuberti, G., Gallo, A., Moschini, M., Cerioli, C., Masoero, F., 2013. Evaluation of the impact of maize endosperm vitreousness on in vitro starch digestion, dry matter digestibility and fermentation characteristics for pigs. Anim. Feed Sci. Technol. 186, 71e80. Hamaker, B.R., Mohamed, A.A., Habben, J.E., Huang, C.P., Larkins, B.A., 1995. Efficient procedure for extracting maize and sorghum kernel proteins reveals higher prolamin contents than the conventional method. Cereal Chem. 72, 583e588. Holding, D.R., 2014. Recent advances in the study of prolamin storage protein organization and function. Front. Plant Sci. 5, 1e9. ISO, 2009. Animal Feeding Stuffs e Determination of Nitrogen Content and Calculation of Crude Protein Content e Part 2: Block Digestion and Steam Distillation Method (ISO 5983e2:2009). International Organization for Standardization. Jane, J.L., Kasemsuwan, T., Leas, S., Zobel, H., Robyt, J.F., 1994. Anthology of starch granule morphology by scanning electron microscopy. Starch-St€ arke 46, 121e129. Kljak, K., Grbesa, D., Aleus, D., 2011. Relationships between kernel physical properties and zein content in corn hybrids. Bull. Univ. Agric. Sci. Vet. Med. ClujNapoca - Agric. 68, 188e194. Knutson, C.A., 1986. A simplified colorimetric procedure for determination of amylose in maize starches. Cereal Chem. 63, 89e92. Landry, J., Delhaye, S., Damerval, C., 2004. Protein distribution pattern in floury and vitreous endosperm of maize grain. Cereal Chem. 81, 153e158. Larson, J., Hoffman, P.C., 2008. A method to quantify prolamin proteins in corn that are negatively related to starch digestibility in ruminants. J. Dairy Sci. 91, 4834e4839. Lu, T.J., Jane, J.L., Keeling, P.L., Singletary, G.W., 1996. Maize starch fine structures affected by ear developmental temperature. Carbohydr. Res. 282, 157e170.
Mistry, A.H., Eckhoff, S.R., 1992. Characteristics of alkali-extracted starch obtained from corn flour. Cereal Chem. 69, 296e303. Mestres, C., Matencio, F., 1996. Biochemical basis of kernel milling characteristics and endosperm vitreousness of maize. J. Cereal. Sci. 24, 283e290. ez-Gonza lez, E.D., de Dios Figueroa-Ca rdenas, J., Taba, S., Tostado, E.C., Narva Peniche, R.A.M., S anchez, F.R., 2006. Relationships between the microstructure, physical features, and chemical composition of different maize accessions from Latin America. Cereal Chem. 83, 595e604. rez, L.A., Islas-Herna ndez, J.J., GomezOsorio-Díaz, P., Agama-Acevedo, E., Bello-Pe pez, O., 2011. Effect of endosperm type on texture and Montiel, N.O., Paredes-Lo in vitro starch digestibility of maize tortillas. LWT-Food Sci. Technol. 44, 611e615. Pereira, R.C., Davide, L.C., Pedrozo, C.A., Carneiro, N.P., Souza, I.R.P., Paiva, E., 2008. Relationship between structural and biochemical characteristics and texture of corn grains. Genet. Mol. Res. 7, 498e508. Philippeau, C., Landry, J., Michalet-Doreau, B., 2000. Influence of the protein distribution of maize endosperm on ruminal starch degradability. J. Sci. Food Agric. 80, 404e408. Singh, J., Dartois, A., Kaur, L., 2010. Starch digestibility in food matrix: a review. Trends Food Sci. Technol. 21, 168e180. a rka, E., Bubník, Z., 2009. Using image analysis to identify acetylated distarch S €rke 61, 457e462. adipate in a mixture. Starch-Sta Wallace, J.C., Lopes, M.A., Paiva, E., Larkins, B.A., 1990. New method for extraction and quantitation of zeins reveal a high content of g-zein in modified opaque-2 maize. Plant Physiol. 92, 191e196. Watson, S.A., 2003. Description, development, structure and composition of the corn kernel. In: White, P.J., Johnson, L.A. (Eds.), Corn: Chemistry and Technology. American Association of Cereal Chemists, St. Paul, pp. 69e106. Zhao, J.P., Cui, D.P., Zhang, Z.Y., Jiao, H.C., Song, Z.G., Lin, H., 2016. Live performance, carcass characteristic and blood metabolite responses of broilers to two distinct corn types with different extent of grinding. J. Anim. Physiol. Anim. Nutr. 101, 378e388.