Vol. 88, No. 1, 2011 87 Distribution of Granule Channels, Protein, and Phospholipid in Triticale and Corn Starches as Revealed by Confocal Laser Scanning Microscopy Sabaratnam Naguleswaran, 1 Jihong Li, 1 Thava Vasanthan, 1,2 and David Bressler 1 ABSTRACT Cereal Chem. 88(1):87–94 The morphology and microstructure of starch granules from two culti- vars of triticale and from normal corn were characterized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Compared to numerous pores distributed randomly on the sur- faces of corn starch granules, markedly fewer pores were observed on the surfaces of starch granules isolated from Pronghorn triticale, and even fewer on the surfaces of starch granules isolated from Ultima triticale. CLSM with fluorescence staining revealed that starch-associated protein was predominately distributed on the granule surface and in the internal channels of both triticale and corn starches. However, after triticale starch was treated with SDS or SO 2 , the radially oriented, protein-filled internal channels of the granules were observed more frequently and extended to the central region of granules. Phospholipid was located mainly on the granule surface but also in channels and throughout granules in triticale starches, whereas in corn starch granules, it was mainly in the channels. The amount of protein and phospholipid in chemically and protease- treated starches varied with starch source and treatment conditions. In treated triticale starches, the nitrogen content was positively correlated with the phosphorus content, indicating a close association between protein and phospholipid within starch granules. Starch-associated protein and phos- pholipid may play an important role in maintaining the structural stability of both the granule surface and the internal channels. Triticale (x Triticosecale Wittmack) is a hybrid cereal species developed by crossing wheat (Triticum turgidum or Triticum aes- tivum) with rye (Secale cereale). In little more than a century, triticale has broken through from a biological curiosity to a com- mercially viable crop (Oettler 2005). Its agronomic advantages, which include high grain yield, high test weight, tolerance to cli- matic and soil-related abiotic stresses, resistance to disease and pest-related biotic stresses, and low input requirements compared to other widely grown cereals (Pejin et al 2009) have resulted in its adoption in more than 30 countries and a steady increase in world production. In 2008, triticale was grown on 3,892,789 ha with a total production over 14 million tonnes (FAOSTAT 2010). Triticale is an economically favorable source of carbohydrate for industrial and energy end-uses including the production of bio- chemicals and biofuels because it has demonstrated several indus- trial attributes such as comparable biological value with the most suitable wheat cultivars for bio-ethanol production (Davis-Knight and Weightman 2008); a better reduction in net greenhouse gas emissions than wheat due principally to its lower nitrogen input (Davis-Knight and Weightman 2008); savings in energy and tech- nical enzymes due to its lower temperature requirement for lique- faction and saccharification; and the presence of high levels of autoamylolytic enzymes (Pejin et al 2009). However, the available knowledge on the structural characteristics of triticale starch is very limited, even though some of its physiochemical properties have been characterized (Berry et al 1971; Palasinski et al 1987; Leon et al 1998). It has been reported that surface pores are present in corn, wheat, barley, rye, sorghum, and millet starch granules, while in- ternal channels are found only in corn, wheat, barley, and sorghum starch granules (Fannon et al 1992, 1993; Huber and BeMiller 1997; Li et al 2003; Kim and Huber 2008). Surface pores are ex- terior openings that extend from internal channels that penetrate into the granule interior radially. Channels vary in depth and di- mension, most extending into the central cavity, and appear to facilitate the transfer of chemical reagents into the granule matrix (Huber and BeMiller 2000, 2001; Kim and Huber 2008). Thus, the presence of pores, channels and cavities in starch granules creates a specific surface area for chemical and enzymatic reac- tions (Sujka and Jamroz 2007). Starches with high pore and chan- nel density show higher susceptibility to enzymes than those with low density (Fannon et al 1992; Benmoussa et al 2006). Protein, lipid, and minerals exist in starch granules as minor components. Commercial cereal starches including wheat, corn, barley, and rice typically contain 0.25–0.6% (w/w) of protein and 0.6–1% of lipid, which are either impurities carried over from the isolation process or granule-associated minor components unex- tractable during the regular isolation process (Debet and Gidley 2006). Starch granule-associated proteins are defined as proteins that are distinctly different from storage proteins and are bound to the granule surface or are integral components within the starch granule (Baldwin 2001). Although the quantity and location of granule-associated proteins vary with starch botanical origin and genetic type, they are mainly biosynthetic enzymes with molecular weights of ≈5–149 kDa (Baldwin 2001). Surface proteins mainly are present on the granule surface as aggregates, whereas the in- ternal protein is deposited within granules as separate monomers (Mu-Forster and Wasserman 1998). Phosphorus is present in vari- ous starches in three major forms: starch phosphate monoester, phospholipid, and inorganic phosphate. In most cereal starches, phosphorus is dominantly in the form of phospholipid, that is, essentially all of the phosphorus in wheat starch and 69% of the total phosphorus in normal corn starch (Lim et al 1994; Kasem- suwan and Jane 1996). Thus, the phosphorus content is an index of phospholipid concentration, and its concentration has been used as a measure of lysophospholipid content by multiplying the phos- phorus content by a factor of 16.3 (Morrison 1988, 1995). Surface lipid can be extracted easily by cold solvents without granule swelling, whereas internal lipids can be extracted only with dis- ruption of the granular structure as with hot solvents or treatment with either acid or enzymatic hydrolysis (Morrison 1988). Granule- associated protein and lipid on the granule surface and interior have a significant and disproportionate impact on surface chemis- try and physicochemical properties of starch (swelling, pasting, *The e -Xtra logo stands for “electronic extra” and indicates that Figs. 3 through 6 appear in color online. 1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada. 2 Corresponding author. Phone: 1-780-492-2898. Fax: 1-780-492-8914. E-mail: tv3@ualberta.ca doi:10.1094/CCHEM-04-10-0062 © 2011 AACC International, Inc. e - Xt ra *