70 80 90 100

Retention time (min)

70 80 90 100

Retention time (min)

DR. differential refractometer.

Figure 3.1 (A) Mode of Interconnection of clusters. (B) Cluster models of amylopectin.

tion in some starch double helices. However, although amylose readily forms double helices in solution, it readily leaches out of hydrated starch at granule gelatinization temperatures (68). This observation shows that amylose in starch granules does not associate in the same manner inside granules as it does outside of the granule. This observation additionally raises some intriguing questions about the nature of amylose synthesis and the kinetic trapping of amylose within starch granules.

While normal starch can be readily separated into two components, amylose and amy-lopectin, studies of high amylose starches have revealed what some authors describe as a third starch component, sometimes termed intermediate starch. For normal starches containing ~25% amylose, the results from quantification by iodine inclusion complex formation

(11,139,181,127), or precipitation in the presence of complexing agents (191,192,126,240,241) are in general agreement. However, for high amylose starches, these methods give different results. Thus, from detailed observations, high amylose starches were initially considered to contain either normal amylopectin and short chain linear amylose (1,2,75) or longer chain amylopectin compared with amylopectin from normal starch (148,149). Liquid chromatographic studies (140) have confirmed the latter. Additional studies of high amylose starches by differential precipitation with 1-butanol combined with Sepharose 2B chromatography (245) revealed an inability to clearly separate amylose from amylopectin. Further fraction-ation revealed high molecular weight material in what traditionally was the amylose fraction (8,9,209). Later studies showed that this material can be removed by repeated precipitation with 1-butanol and was most likely contaminating amylopectin (209,213), while removal of the low molecular weight material in the amylopectin fraction using differential precipitation techniques has not been successful. In summary, starch from high amylose mutants appears to contain a significant amount of an amylopectin-like component having an altered architecture. This intermediate starch component is characterized by:

1. An inability to precipitate with 1-butanol

2. An ability to eluete within the same molecular size range as amylose

3. An ability to bind iodine and having a lambda maximum between amylose and amylopectin

Estimates of intermediate material defined in this way have exceeded 55% (w/w) of the total starch of high amylose mutants (10).

Other workers (116,209,239) have defined intermediate starch to be the material that has the ability to precipitate in the presence of some complexing agents (e.g., 2-nitropro-pane) or mixtures of complexing agents (1-butanol with isoamyl alcohol) but not others (e.g., 1-butanol, 1-nitropropane). This type of intermediate material obtained by differential precipitation is a relatively small proportion of normal starches (<10%) (116,209,220,239) irrespective of the amylose content of the high amylose starch. The molecules have been considered by some as amylopectin molecules with long external chains and a limited capacity to form clathrate complexes and precipitate (116,209,220,239). It has also been suggested (209) that this type of intermediate material could be a mixture of amylopectin and a small amount of contaminating amylose, which might be the case if the amylopectin from high amylose starch has an overall molecular size similar to that of amylose from normal starch (116).

Amylopectin molecules within granules are believed to be organized radially, with the long C-chain innermost (84). As a result of high magnification microscopy studies it was proposed that the radially oriented amylopectin clusters are organized into super helices, which may relate to the blocklets seen in microscopy studies (166). In turn the super helices may be responsible for the formation of concentric spherical rings. Although these growth rings are a characteristic of all starch granules, the mechanisms determining their formation are still not well understood (60,170). Recently, Bertoft proposed a bidirectional backbone model, where the super helix could be organized so that the longer amy-lopectin chains are oriented in line with the super helix, while the amylopectin clusters may be oriented radially (18) (Figure 3.2).

X-ray crystallography has shown that there are three distinct types of crystalline order in starch: A-type, B-type and V-type. V-type crystallinity is often associated with crystalline packing of amylose lipid complexes. The A-type and B-type starches differ in the organization of helices: A-type crystals are densely packed hexagonal arrays of double helices, B-type crystals, though also double helices packed in a hexagonal array, are unlike A-type

9 nm

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