**3. Mechanism of TPS retrogradation**

Zhang & Han (2010) showed that gelatinized and plasticized TPS contains about 10% crystallinity, meaning 90% of TPS is amorphous region. Starch polymers in the amorphous region are metastable and consequently they automatically retrogradate over time during storage. Mechanism of starch retrogradation has been intensively investigated. Liu & Han (2005) observed starch crystal formation of amylose and amylopectin film respectively under an inverted phase-contrast microscope. They found amylose film featured a layer of dendrites with 90-degree branching (Fig. 2a), while amylopectin film showed a network of interlinked clusters with an amorphous background (Fig. 2b). Lian et al. (2011) observed the starch retrogradation under a microscope. They found starch nuclei in 2 h and crystals in 48 h (Fig. 3a & b). Russell (1987) postulated amylose formed nuclei by congregating, and amylopectin formed crystalline lamellae by prolongating the rod-like growth of crystals. Delville et al. (2003) proposed a mechanism for formation of starch crystalline lamellae shown in Fig.4, presenting the crystalline cluster formation of amylopectin. The cluster formation begins with the formation of crystalline lamellae composed of double helices of amylopectin short chains (symbolized with rectangular boxes). Then, the packing of double helices forms crystalline clusters (Delville et al., 2003).

Tako & Hizukuri (2002) proposed starch retrogradation mechanisms at a molecular level, in which starch retrogradation occurs as a result of intermolecular hydrogen bonding between O-6 of D-glucosyl residues of amylose molecules and OH-2 of D-glucosyl residues of short side-chains of amylopectin molecules (Fig. 5). The starch retrogradation was also attributed to intermolecular hydrogen bonding between OH-2 of D-glucosyl residues of amylose molecules and O-6 of D-glucosyl residues of short side-chains of amylopectin molecules (Fig. 6). In addition to intermolecular hydrogen bonding between amylose and amylopectin, hydrogen bonding between O-3 and OH-3 of D-glucosyl residues on different amylopectin molecules also occurs (Fig. 7). The intramolecular hydrogen bonding might occur between OH-6 and adjacent hemiacetal oxygen atom of the D-glucosyl residues within an amylose molecule (Fig. 5 and 6), while the intramolecular association within an amylopectin molecule was not suggested to exist (Tako & Hizukuri, 2002).

Retrogradation and Antiplasticization of Thermoplastic Starch 121

Fig. 4. Schematics of amylopectin retrogradation at the rubbery state (amylopectin double

Fig. 5. Hydrogen bonding between amylose and amylopectin molecules (Dotted lines represent hydrogen bond. AY, amylose; AP, short side-chain of amylopectin molecules)

helices are represented as rectangles). (From Delville et al., 2003)

(From Tako & Hizukuri, 2002)

Fig. 2. Photomicrographs of amylose film (a) and amylopectin film (b). Amylose film shows a dendrite feature, while amylopectin film shows a cluster network with an amorphous background. (From Liu & Han, 2005)

Fig. 3. Optical micrographs of retrograded starch of sweet potato aging 2 h (a) and aging 48 h (b). (From Lian et al., 2011)

Fig. 2. Photomicrographs of amylose film (a) and amylopectin film (b). Amylose film shows a dendrite feature, while amylopectin film shows a cluster network with an amorphous

Fig. 3. Optical micrographs of retrograded starch of sweet potato aging 2 h (a) and aging 48

background. (From Liu & Han, 2005)

h (b). (From Lian et al., 2011)

Fig. 4. Schematics of amylopectin retrogradation at the rubbery state (amylopectin double helices are represented as rectangles). (From Delville et al., 2003)

Fig. 5. Hydrogen bonding between amylose and amylopectin molecules (Dotted lines represent hydrogen bond. AY, amylose; AP, short side-chain of amylopectin molecules) (From Tako & Hizukuri, 2002)

Retrogradation and Antiplasticization of Thermoplastic Starch 123

Properties of TPS products depend upon its microstructure. Zhang & Han (2010) studied the effect of crystallinity on the properties of the starch film, including moisture content, gas permeability, and tensile properties, etc. When crystallinity of glycerol-starch film increased from 6.0% to 8.0%, moisture content decreased from 11.0% to 8.0%. Similar phenomena were observed in sorbitol-, fructose-, and mannose-films. During the retrogradation process, starch chains aligned to form crystalline lamellas. Water molecules, as well as plasticizer molecules, are pushed out and evaporate resulting in reduced moisture content. Retrogradation of starch was also observed to have negative effect on gas permeability, including oxygen permeability (OP) and water vapor permeability (WVP) (Zhang and Han, 2010). Sorbitol-film had a reduction in OP from 20×10-7 to 0.3×10-7 cc mm h-1 kPa-1 m-2 when its crystallinity increased from 7.0% to 12.0%, while OP in glycerol-film was reduced from 6×10-7 to 4×10-7 cc mm h-1 kPa-1 m-2 when its crystallinity increased from 5.0% to 9.0%. A similar trend was also found in WVP. When crystallinity of glycerol-film increased from 6.0% to 9.0%, its WVP decreased from 1.2 to 0.9 g mm m-2 h-1 kPa-1. Pushpadass & Hanna (2009) explained that the increase in crystallinity with time decreased the free volume in the TPS network and resulted in decrease in WVP. Zhang & Han (2010) proposed that crystallite in the films results in difficulty for gas molecules to diffuse within the starch films, leading to low permeability. Crystallinity also resulted in decreased elongation of the film. Elongation of mannose-film decreased from 4.0% to 0.2% when its crystallinity increased from 6.0% to 19.0%, elongation of fructose-film from 4.0% to 1.5% when its crystallinity increased fom 6.0% to 18.0%. Pushpadass & Hanna (2009) reported similar findings. They reported the relative crystalline content of TPS samples increased from 3 % (after 4 h extrusion) to 7%, 14%, and 17% after 3, 30, and 120 d of storage. As a result, tensile strength of TPS samples increased by 39.3 – 134.1%, elongation decreased by 48.0 – 81.1%, and WVP decreased by 6.1 % – 19.3%. A lower E value means that TPS becomes stiffer, less flexible and more difficult to handle. Crystallites may act as physical crosslinking points which generate internal stresses of TPS, leading to the increase in tensile strength and decrease in

**4. Effect of retrogradation on the property of thermoplastic starch** 

elongation (Delville et al. 2003)

**5.1 Differential scanning calorimetry** 

**5. Technologies to study the starch retrogradation** 

therefore valuable tools to quantify retrograded starches.

Many analytical techniques have been developed to monitor the starch retrogradation based on the TPS property changes. These methods include differential scanning calorimetry (DSC), differential thermal analysis (DTA), X-ray diffraction (XRD), and enzymatic susceptibility, and others. Karin et al. (2000) summarized some of the methods. For TPS crystallinity study, DSC and X-ray diffraction have proven to be extremely sensitive and

Differential scanning calorimetry (DSC) is a thermo-analytical technique. It measures temperatures and heat flows associated with thermal transitions in a material sample. A reference with a well-defined heat capacity over the range of temperature is identically

Fig. 6. Retrogradation mechanism of starch (Dotted lines represent hydrogen bond. AY, amylose; AP, short side-chain of maylopectin molecules) (From Tako & Hizukuri, 2002)

Fig. 7. Association between amylose and amylopectin molecules (Dotted lines, represent the hydrogen bonding sites). Two or more short-chains of amylopectin molecules may interact with one amylose molecule. Self-association within amylopectin molecules may also take place (From Tako & Hizukuri, 2002)

Fig. 6. Retrogradation mechanism of starch (Dotted lines represent hydrogen bond. AY, amylose; AP, short side-chain of maylopectin molecules) (From Tako & Hizukuri, 2002)

Fig. 7. Association between amylose and amylopectin molecules (Dotted lines, represent the hydrogen bonding sites). Two or more short-chains of amylopectin molecules may interact with one amylose molecule. Self-association within amylopectin molecules may also take

place (From Tako & Hizukuri, 2002)
