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Monday 20 June 2016

Electron-beam processed corn starch: evaluation of physicochemical and structural properties and technical-economic aspects of the processing

Author
M. BraşoveanuI; M. R. NemţanuI, *; D. DuţăII
INational Institute for Laser, Plasma and Radiation Physics, Electron Accelerator Laboratory, 409 Atomistilor St., P.O. Box MG-36, RO-077125, Bucharest-Măgurele, Romania. Phone: + 40-21-457-44-89, Fax: + 40-21-457-42-43, E-mail: monica.nemtanu@inflpr.ro 
IINational Institute for Food Bioresources, 6 Dinu Vintila St., 021102 Bucharest, Romania


ABSTRACT

The properties of starch can be modified by a variety of methods in order to meet desirable technological needs. Electron beam irradiation is able to induce changes in starch properties. The paper deals with investigation of physicochemical and structural modifications of corn starch processed by electron beam up to 50 kGy and evaluation of the technical-economic aspects of starch processing. Paste viscosity, pasting and peak temperatures decreased in a dose-dependent manner, indicating degradation of the macromolecule. Small circular perforations on the granule surface were observed for 50-kGy irradiated sample. Spectral characteristics suffered minor changes, suggesting that the inter- and intramolecular hydrogen bond stability was affected by the electron beam. These modified starches could find applications in foodstuffs requiring low-viscosity starch. A cost estimate of the electron beam processing of corn starch with an average absorbed dose of 30 kGy showed an increase of corn starch price by 16%.

Keywords: Acidity; Pasting properties; Structure; Processing capacity; Processing costs.

INTRODUCTION

Starch is widely used in various industrial food and non-food applications. Its natural properties like swelling, gelatinisation, viscosity, sticking, film formation, biodegradability or hydrophilicity determine the multitude of its industrial applications (Mischnick, 2005). The selection of starches for certain types of processing is very important because it influences the processing, the attributes and the shelf life of the product in which it was incorporated. For instance, native starch may continue to hydrate even after food processing and thus leads to a viscosity increase or can cause a phenomenon of delayed retrogradation and, in both cases, the products become unsuitable for consumption. To solve the inconveniences caused by the limitations of the native starch functionality, modified starch with improved functional properties is generally preferred.
Beside gamma rays, electron beams are often used in material processing by direct and/or indirect action of electrons, generating reactive entities such as free radicals. Electron beam irradiation is also an economically viable, efficient and rapid method to enhance food safety and quality, having several useful effects depending on the irradiation dose. Consequently, the Joint FAO/IAEA/WHO Study Group (WHO, 1999) suggested that any dose above 10 kGy can be considered both safe to consume and nutritionally adequate if it is appropriate to achieve the intended technological objective. At the same time, according to the EU legislation (Directive 1999/2/EC) related to the manufacture, marketing and importation of foods and food ingredients treated with ionizing radiation, the final decision regarding the implementation of a technology is basically made by the producer and it may be authorized in accordance with the legislation in force only if the following conditions are completely fulfilled: (i) there is a reasonable technological need, (ii) it presents no health hazard and is carried out under the conditions proposed, (iii) it is of benefit to the consumer, (iv) it is performed with electrons generated from machine sources operated at or below a nominal energy (maximum quantum energy) level of 10 MeV, and (v) it is not used as a substitute for hygiene and health practices or for good manufacturing or agricultural practice.
In the last five years, the electron beam processing of starches from different botanical sources was reported to be efficient in order to induce different changes in their physicochemical and structural properties, such as an increase of solubility and free acidity, decreases in swelling capacity, pasting properties, intrinsic viscosity, molecular weight and degree of polymerization (Kamal et al., 2007; Nemţanu et al., 2007; Nemtanu and Brasoveanu, 2010; Nemţanu et al., 2010; Pimpa et al., 2007; Shishonok  et al., 2007). Although the electron beam induced modifications have generally similar tendencies, they are different quantitatively, depending on the specificity of each starch and irradiation dose (Nemtanu and Brasoveanu, 2010). Modification of functional properties using electron beams can increase the use of starch in various applications such as in the food, pharmaceutical, paper and textile industries.
Moreover, starch processing using electron accelerator facilities is fast, low cost and environmentally friendly - without any use of polluting agents, catalysts or generation of undesirable residual products (Nemtanu and Brasoveanu, 2010). Therefore, the use of starch modified by electron beam treatment could lead to the opportunity for development of innovative products, reducing product processing costs or extending product shelf life.
The purpose of the present study was: (1) to point out some novel aspects related to the properties of corn starch processed by electron beam in order to be used for varied food applications requiring low-viscosity starch, and (2) to present and discuss an evaluation of technical and economic aspects of electron beam processing of corn starch, which is based on the processing capacity of the electron accelerator technological facility, as well as on the specific cost of processing.

EXPERIMENTAL
Electron Beam Processing
Corn starch from a local commercial market was used for the experiments. The packed starch samples (thickness of 49 mm, area of 50 mm x 80 mm) were treated with a scanned electron beam (EB) using a linear accelerator of mean energy of 6 MeV (NILPRP, Bucharest-Magurele, Romania), at ambient temperature and normal pressure. The absorbed doses (10 - 50 kGy) with mean dose rate of 2 kGy/min were checked using cellulose triacetate film. The configuration of the samples and their positioning in the beam field, at 500 mm distance from the accelerator exit window, allowed us obtain a dose uniformity above 90%.
Physicochemical Characterisation
The moisture content of samples was determined by gravimetric heating (105 ± 2 °C for 4 h) using 4 - 5 g of sample.
The acidity of the samples was determined by titrating starch suspensions against 0.1 M NaOH solution. The starch suspensions were prepared by suspending 5 g of starch in 50 mL of distilled water with continuous shaking to homogenize the suspensions. The results are expressed in terms of acidity degree representing the volume of 0.1 M NaOH solution required to neutralise the acidity of 100 g of starch, according to the SR 90:2007 Romanian standard.
The ash content of starches was determined according to the SR 90:2007 Romanian standard. Briefly, a sample of corn starch (5 g) was accurately weighed into a weighed porcelain crucible. The sample was calcined over a gas burner with low flame initially and then with a strong one. A few drops of hot water or nitric acid were added to the crucible after cooling if coal black dots were still visible. The sample was further evaporated on a water bath and heated until a gray ash without black dots was obtained. The crucible was cooled in a desiccator for 30 min and then weighed. The calcination and weighing were repeated until constant weight. The percentage of ash content was calculated as:

The protein content was determined by the Kjeldahl method according to the SR EN ISO 20483:2007 standard. Starch (2 g) was inserted into a 300 mL Kjeldahl flask. Then 20-25 mL of concentrated sulphuric acid, 2 drops of mercury, some paraffin and 8-10 g potassium sulphate were added. The flask was heated with low flame initially and then gradually higher flame until the liquid became slightly yellowish or colourless and clear. The disaggregation was considered completed after 3-4 hours. The flask was then cooled and the content diluted with 100 ml of distilled water and transferred quantitatively into a 1000 mL Erlenmeyer flask. The volume was completed to 250 mL and a few drops of phenolphthalein were added. Then 80 mL of 30% sodium hydroxide solution, 10 mL of sodium sulphide, several grains of zinc and pumice stone or glass were added. The flask was connected with a distillation apparatus. The conical flask received 30 mL of hydrochloric acid, a few drops of methyl red and distilled water and was then heated until around 200 mL had been distilled. The distillation was interrupted when the amount of 200 mL of distillate was collected. The collector vessel walls were washed with distilled water and the excess hydrochloric acid was titrated with 0.1 M NaOH solution until turning yellow. 1 mL of 0.1 M NaOH solution corresponded to 0.0014 g nitrogen, and 1 g of nitrogen corresponded to 6.25 g of proteins.
The percentage of protein content was calculated as:

where V1 is the volume of 0.1 M HCl introduced into the distillate flask [mL], V2 is the volume of 0.1 M NaOH used for titration [mL] and W is the weight of the starch sample [g].
The fat content of analyzed samples was determined according to the SR 90:2007 Romanian standard. Thus, 10 g of starch were inserted into a paper cartridge filter which was covered with defatted cotton. The cartridge was placed into the tube of a Soxhlet extractor coupled to a weighed flask of the apparatus which was previously filled with 100 ml of ethyl ether and then the refrigerant was fixed. The heating was performed on a water bath for 4 hours. After extraction, the ether was evaporated and the residue was dried in an oven at 95 °C to constant weight. The difference between the final and initial weights of the flask is the amount of fat contained in the starch sample. The percentage of fat content was calculated as:

Pasting properties were monitored with a Brabender Amylograph (Amylograph-E, Brabender, Germany) for starch paste (15%, w/v) in the temperature range of 30-90 °C with a heating rate of 1.5 °C/min, measuring the following parameters: pasting temperature To (°C), peak viscosity temperature Tp (°C), and peak viscosity (BU).
Granule Morphology Evaluation
The analysis of starch granules by scanning electron microscopy (SEM) was performed using a FEI NovaTMNanoSEM 630 microscope.
Fourier Transformed Infrared (FTIR) Spectroscopy
FTIR spectra of starch samples were recorded on a Tensor 27 FTIR spectrometer (Bruker Optik GmbH, Germany) in the frequency range of 4000 - 400 cm-1 using KBr discs prepared from powdered samples mixed with dry KBr and were analyzed with Opus v. 6.5. software.
Statistical Analysis
The results reported are expressed as the means ± standard error of three determinations except for the flow curves. Statistical evaluation of the differences between means was made by using the  t-test with P < 0.05 considered statistically significant.

RESULTS AND DISCUSSION
Physicochemical Characterisation
Moisture content, acidity, ash, protein and fat content are characteristics of great interest, especially when new processing methods are investigated for starch.
From the data displayed in Table 1, it can be noted that the starch samples processed by accelerated EB had no significant changes in the physicochemical properties studied, except the acidity and fat content.
The acidity value of EB treated samples increased as the absorbed dose increased. These results are in accordance with previously reported data on EB irradiated sago starch (Pimpa et al., 2007) or gamma-irradiated dry bean starch (Duarte and Rupnow, 1994). Recently, a few works reported that the carboxyl content increased with increasing absorbed dose for gamma-irradiated corn starch (Chung and Liu, 2009), and potato and bean starches (Chung and Liu, 2010; Gani et al., 2012). Thus, the acidity increase with increasing dose could be assigned to the fragmentation of starch molecules and formation of carboxyl-containing compounds. Moreover, EB processing in presence of oxygen determines the appearance of free radicals, aldehydes, ketones, organic peroxides or other polysaccharide degradation products (Ershov, 1998) that can lead to an increase of starch acidity.
Lipids are common minor constituents found in cereal starches, being present both on the surface and inside granules (Morrison, 1988; Kitahara et al., 1994). Corn starch contains mainly internal lipids (Baszczak et al., 2003) which are composed exclu-sively of the lysophospholipids and free fatty acids (Tester et al., 2004). Changes in lipids due to ionizing radiation occur in two ways: (i) direct (radiolytic), when cation radicals or excited lipid molecules are formed; or (ii) indirect (oxidative), by catalyzing their reaction with molecular oxygen, known as autoxidation (Stewart, 2001). The fat content of EB treated corn starch exhibited no changes up to 30 kGy. Thus, the fat content of starch granules showed low sensitivity to the EB in this range of absorbed dose, most likely due to the protective effect of the granular architecture and the solid physical state of the starch treated, which did not allow the diffusion of the free radicals formed, so that the lipid molecules were not easily subject to attack. However, the fat content values decreased in samples processed with doses higher than 30 kGy, indicating a degradation of lipids. Because the EB processing was performed in the presence of oxygen, this degradation might be due to superimposed oxidative and radiolytic effects, which are important at high irradiation doses. The oxidative effect can be minimized by reducing the presence of oxygen during EB processing.
Pasting Properties
The swelling of starch granules during the pasting process has a major effect on the rheological behaviour of starch paste. During the first stage of granule swelling, water penetrates and soaks the granule amorphous phase without any significant alteration of the crystalline phase. The changes of this limited swelling are reversible in successive drying processes because, at temperatures lower than gelatinization, only a small disturbance of the ordered regions of the granule takes place. With an increase of temperature, the soaking of starch granule occurs simultaneously with the water penetration and lateral or tangential swelling of granule. In this case, the process is irreversible due to the loss of crystalline order in the swelled paste and, consequently, to the strongly distortion of the granule architecture.
Pasting profiles of starch samples treated with accelerated EB analyzed with a Brabender amylograph are shown in Fig. 1 and the results are summarized in Table 2.




The overall profile of amylograms showed similar shape for both control sample and EB treated samples. However, a significant decrease in the paste viscosity could be noted as the absorbed dose increased. Also, a gradual decrease of the initial pasting and peak temperatures was observed for all treated samples as the absorbed dose increased in comparison to the control sample. These changes in a dose-dependent manner show that the starch macromolecule was degraded by EB processing. The reduction of the peak viscosity of starch could be assigned to its weaker water binding capacity, granular rigidity and integrity due to glycosidic bond cleavage (Lee et al., 2006; Liu  et al., 2012). Therefore, according to Pimpa et al. (2006), the decreased viscosity reveals the inability of amylopectin - responsible for promoting swelling - to hold the granule together when water is imbibed as a consequence of the amylopectin branches cleaved by energy transferred to the molecule from the electron beam.
These starches could find applications in foodstuffs like canned or instant soups, dressings and confectionery products that require lower viscosity. Apart from food products, electron beam modified starch could also find use in paper manufacturing, pharmaceuticals and various other industrial applications.
Granule Morphology
SEM investigation showed that native corn starch had granules of 5-15 µm with a polyhedral shape and a slightly roughened surface, rarely concave, having a tendency to agglomerate as a bunch and occasionally presenting small holes on some granule surfaces (Fig. 2 (a)).


The corn starch samples treated with EB showed fewer agglomerated granules than the control sample granules. The granule shape and sizes were apparently unaffected by EB, but even so the appearance of small circular perforations on the granule surface could be observed for sample treated with 50 kGy (Fig. 2 (b)). Shishonok et al.(2007) reported that the electron beam irradiation did not violate the surface structure of potato starch even at relatively high doses (110 - 440 kGy), while Kamal et al. (2007) showed that the shape of corn starch granule was somewhat deformed by both gamma and electron beams for doses between 5 and 100 kGy. However, a recent study reported that gamma irradiated maize starch retained the original shape and size without any granular cracking or roughness occurring on the surface, even for 500 kGy (Liu et al., 2012).
Accelerated electrons are a kind of penetrating radiation which is able to produce effects in the whole volume of the samples, so that the radioinduced changes may occur both in the central regions and in the peripheral regions of the starch granules. However, the fact that microscopic methods reveal no damage to the granule outer layer leads to the conclusion that the radioinduced changes might occur at a more intimate level of matter in the form of structural changes.
FTIR Spectroscopy
FTIR spectra of native corn starch showed complex vibrational modes due to the pyranose ring of the glycosidic unit in the region below 800 cm-1 (Vasco et al., 1972; Kizil and Seetharaman, 2002).
The spectral characteristics of the EB treated samples were similar to native starch (Fig. 3), but with some peaks slightly shifted. These shifts of bands did not indicate any clear and obvious change in the starch structure. However, these minor changes, especially for bands assigned to C-H and O-H bonds, might indicate that the stability of the inter- and intramolecular hydrogen bonds of corn starch structure was affected by EB processing. Liu et al. (2012) also showed that the spectral patterns of highly gamma irradiated maize starches were similar to native starch and no new functional groups were found in the FTIR spectra. Kamal et al. (2007) reported that the IR spectra of the electron beam irradiated corn starch showed that the OH stretching band centred around 3400 cm-1 was affected; an increase in the intensity of the characteristic peak at 1647 cm-1 ascribed to carbonyl groups was also observed.


Most authors have used infrared spectroscopy to estimate the amount of ordered or crystalline regions by analyzing the bands at 1047 and 1022 cm-1. Thus, the crystalline state can be identified by the appearance of a band at 1047 cm-1, while the starch amorphous region is characterized by an absorption band around 1022 cm-1, and their respective ratio indicates the degree of starch order (Cael et al., 1975; Soest et al., 1995; Sevenou et al., 2002; Liu et al., 2004). In our case, the EB processing of corn starch had no influence on this ratio, suggesting that larger crystalline zones might be transformed into small crystallites such that the cristallinity degree was practically unaffected. Abu et al. (2006) reported similar findings on gamma irradiated cowpea starch for which the degree of granule surface order (cristallinity) was not affected up to 50 kGy. On the other hand, the ratio of 1047/1022 decreased with an increase of gamma radiation dose for corn, potato and bean starch, and the granular cristallinity was affected (Chung and Liu, 2009; Chung and Liu, 2010).
Technical and Economic Aspects of Starch Electron Beam Treatment
Due to their construction and functional characteristics, electron accelerators have several advantages, including: full electrical control over the operation and radiation emission; stability and reproducibility of the processing parameters; simplicity of processing control; short duration of processing due to high dose rates; processing in the final stage of the technological flux; clean processing without the use of environmental pollutants, generation of unwanted by-products, and penetration of toxic substances in the treated materials and without any risk of radioactive contamination.
For commercial applications, the most important characteristics of an accelerator are its electron energy and average beam power, parameters that influence the electron path in irradiated material and the processing rate, respectively (Cleland and Parks, 2003). Evaluation of the technical and economic aspects of a technological treatment with electron beam is based on the production capacity of the technological facility and on the specific cost of the processing as well.
Capacity of Starch Processing with Electron Beam
The main technological parameter of an e-beam facility is the processing rate V expressed in kg/s and defined by the following equation:

where η is the yield of beam power use [%], Pbeam is the beam power [kW] and D is the absorbed dose [kGy].
The yield of beam power use is the fraction of the beam energy that turns into used dose:

where Pu is the beam power turned into used dose [kW].
The beam use yield of the technological facilities based on electron accelerators is between 30 and 60% (Korenev, 2004), often around 40% (Morrison, 1989).
At the technological level, other typical parameters for an electron accelerator facility are also used, including:
(i) processing rate per hour and per kGy, Vh,kGy, expressed in :

(ii) processing rate per hour, Vh, expressed in kg/h:

Processing capacity (PC) of an electron accelerator based plant depends both on the beam power and yield of beam power use and on the operating system by the number of hours of use per year (Teodorescu and Fiti, 1979). Thus, the value of annual processing capacity of the facility, expressed in tonnes/year, will be:

in which is the number of hours of accelerator use in order to perform the treatments per year.
To calculate the annual processing capacity, an example is given below by assuming that the treatment is performed with an average absorbed dose of 30 kGy and 6,000 hours per year could be used for treatment, operating three shifts per day. Table 3 shows the values of the most important parameters characterizing electron beam processing in the facility considered.


The processing rate increases with the beam power and the processing capacity of a facility is directly proportional to its processing rate, representing the product of processing rate and the number of hours used for processing. So, when the processing rate is higher, the processing capacity is higher. In our example, the accelerator has a processing rate of 0.80 kg/s and a processing capacity of 17,280 tonnes/year to treat corn starch with an average absorbed dose of 30 kGy. Thus, the beam power determines the amount of product that can be treated in a certain time at the required absorbed dose.
Calculation of Specific Cost of Starch Processing with Electron Beam
Economic profitability of the technological treatment of starch with electron beam is closely related to installation costs, operating costs and specific consumption of electricity. Operating costs depend generally on the required absorbed dose, involving expenses with electricity and the recovery of investment costs (investment amortization), costs of maintenance and regular repair of the facility, staff costs and other specific costs of the envisaged treatment.
Electron accelerators run on electricity, whose cost influences the variable operating costs for a facility based on electron accelerator of great complexity. The amount of electricity used for processing depends on the beam power, yield of beam power use and the conversion factor of the power consumed by the accelerator to obtain an electron beam of a certain power.
For an estimate of specific cost in US$ per ton of corn starch processed with an average absorbed dose of 30 kGy, we considered an annual production of 10,000 tonnes of processed starch and 6,000 operating hours. Table 4presents the percentage values of the operating expenses on which the specific cost of electron beam processing per tonne of product was calculated.


The cost of corn starch processing with electron beam varies from plant to plant depending on the accelerator type and other operating expenses. In our example, it results in a processing cost of 0.08 US$ per kilogram of starch exposed to electron beam with an average absorbed dose of 30 kGy.
Considering that one tonne of native corn starch has a price of approximately 500 US$, after electron beam processing with an average absorbed dose of 30 kGy, the modified starch would cost 580 US$, which means 0.58 US$ per kilogram of corn starch treated with an average absorbed dose of 30 kGy. This estimate showed that EB processing with an average absorbed dose of 30 kGy could increase the price of the native corn starch by about 16%.

CONCLUSIONS
The moisture, protein and ash contents suffered no significant changes upon electron beam processing of corn starch. The acidity increased, while paste viscosity, pasting and peak temperatures decreased as the absorbed dose increased due to the macromolecule degradation. The fat content decreased only for doses higher than 30 kGy. No significant changes appeared in the morphological properties, while the spectral characteristics suffered minor changes, probably because of the sensitivity of inter- and intramolecular hydrogen bonds to the EB processing. Electron beam modified starches could find applications in foodstuffs like canned or instant soups, dressings and confectionery products that require lower viscosity. The irradiation dose required to modify starch to desirable properties can be chosen to meet specific application.
The evaluation of the technical and economic aspects of the corn starch electron beam processing showed that, at a given absorbed dose, the processing capacity of an electron beam facility depended on the processing rate, which was influenced by the beam power and efficiency of its use. The cost of starch electron beam processing depended on the indirect (variable) operating costs and the desired absorbed dose, thus depending on the beam power, the efficiency of its use and the conversion factor of the beam power from the facility consumption power. Our calculation showed that EB processing with an average absorbed dose of 30 kGy increased the price of the native starch by approximately 16%.

ACKNOWLEDGEMENTS
We are grateful to Dr. Adrian Dinescu for the help in recording SEM images of the samples.
This research was partially supported by project PN II 51-007/2007.

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(Submitted: August 2, 2012 ; Revised: October 29, 2012 ; Accepted: November 7, 2012)



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PHYSICOCHEMICAL CHARACTERIZATION OF NATURAL AND ACETYLATED THERMOPLASTIC CASSAVA STARCH

Title
PHYSICOCHEMICAL CHARACTERIZATION OF NATURAL AND ACETYLATED THERMOPLASTIC CASSAVA STARCH 
CARACTERIZACIÓN FISICOQUÍMICA DE ALMIDÓN TERMOPLÁSTICO DE YUCA NATURAL Y ACETILADA 

JOSE MINA  
Escuela de Ingeniería de Materiales, Universidad del Valle, Cali (Colombia), johermin@univalle.edu.co

ALEX VALADEZ-GONZÁLEZ 
Unidad de Materiales, Centro de Investigación Científica de Yucatán A.C. (CICY), Mérida (México), avaladez@cicy.mx

PEDRO HERRERA-FRANCO 
Unidad de Materiales, Centro de Investigación Científica de Yucatán A.C. (CICY), Mérida (México), pherrera@cicy.mx

FABIO ZULUAGA 
Grupo de síntesis y mecanismos en química orgánica,Universidad del Valle, Cali (Colombia), fazulu@univalle.edu.co

SILVIO DELVASTO 
Escuela de Ingeniería de Materiales, Universidad del Valle, Cali (Colombia), delvasto@univalle.edu.co


Received for review November 27th, 2009, accepted April 22th, 2010, final version April, 30th, 2010

ABSTRACT: Thermoplastic starch (TPS) was obtained from natural and acetylated cassava starch using a twin screw extruder and then conditioned at 25 ºC and 54 % of relative humidity. It was found that the crystallinity index, calculated as the ratio of the IR peaks at 1047 (crystalline phase) and 1022 cm-1 (amorphous phase), decreases due to the effect of a plasticization process. Also, as expected, SEM micrographs show that the plasticization process destroyed the starch granular structure almost completely and an amorphous mass was obtained. The TGA results indicated that the activation energy, Ea, was also reduced by the plasticization process. The acetylated TPS shows a decrease in Tg, in tensile strength and in the percentage of moisture absorption compared to natural TPS but a larger strain at the breaking point. This behavior suggests that the chemical modification reduces the secondary interactions between starch chains due to the substitution of the hydroxyl groups by acetates.

KEYWORDS: Thermoplastic starch, cassava starch, acetylated starch, biodegradable materials, starch plasticization, renewable materials.

RESUMEN: Almidón de Yuca natural y acetilado fueron plastificados empleando un extrusor de doble husillo y almacenados a 25 °C y 54 % de humedad relativa. El índice de cristalinidad, estimado como el cociente de las bandas de FTIR a 1047 (fase cristalina) y a 1022 cm-1 (fase amorfa) disminuyó en ambos almidones debido al proceso de plastificación. Mediante las micrografías de SEM se pudo observar que el proceso de plastificación destruyó casi por completo la estructura granular de los almidones. Los resultados de TGA mostraron que la energía de activación, Ea, disminuyó con el proceso de plastificación. Por otra parte se encontró que el almidón termoplástico acetilado presentó una Tg, absorción de agua y resistencia a tensión menores en comparación con el almidón termoplástico natural, mientras que su elongación a la ruptura fue mayor. Este comportamiento sugirió que la esterificación de los grupos hidroxilos del almidón natural redujeron las interacciones entre las cadenas del almidón plastificado.

PALABRAS CLAVE: Almidón termoplástico, almidón de yuca, almidón acetilado, materiales biodegradables, plastificación de almidón, materiales renovables.

1. INTRODUCTION
Recent research efforts in the area of polymeric materials have focused on the development of biodegradable materials obtained from renewable resources such as starch, proteins, hydroxyalkanoates, etc., which are liable to complete biodegradability when subjected to composting conditions [1, 2, 3]. Nowadays, due to the worldwide decrease of oil production, the search for new materials from renewable resources becomes crucial for substituting traditional synthetic polymers. Among these materials stands out thermoplastic starch (TPS) since the starches are the most abundant and cheapest materials that come from renewable resources [1, 4]. 
Starch consists of two major components: amylose, a mostly linear α-D(1,4)- glucan and amylopectin, an α-D-(1-4)-glucan which has α-D(1,6) linkages at the branch point. The linear amylose molecules of starch have a molecular weight of 0.2–2 million, whereas the branched amylopectin molecules have molecular weights as high as 100–400 million [5]. Most often the structure of starch is chemically modified in order to decrease the number of hydroxyl groups in the search for a number of possible applications. Thereby, starch has been treated by hydroxipropylation [6], oxidation [7] and acrylic grafting, mainly. However, in recent years, there has been a considerable research effort with acetylated thermoplastic starch, because of the experience acquired at food applications where acetylation is the second most studied treatment after crosslinking [8]. Acetylation is the esterification of starch with acetic anhydride, used to facilitate the formation of acetates groups, which has been performed mainly to adjust the viscosity of food and to improve its stability and its resistance to degradation as compared to untreated starch. Also, the incorporation of acetate groups reduces the interaction between chains and improves the swelling power and the solubility of starch granules. The change of the physicochemical properties of starch is proportional to the substitution degree of the formed acetate groups [9]. 
In order to change a native starch into a bioplastic material it is necessary to break up its semicrystalline granular structure [10]. A starch without the appropriate additives (plasticizers) does not possess the necessary properties to perform as a thermoplastic. The plasticizers increase the flexibility of starch because of their ability to reduce the formation of hydrogen bonds and increase molecular separation [11]. In the field of food science, water is considered to be the best plasticizer. However, in the case of TPS, water is included as an aid to the main additive, glycerol being the most studied. In order to improve the poor mechanical properties of starch and to reduce its water absorption, several studies found in the literature deal with different aspects concerning the improvement of plasticizers [2, 12], the more stable biodegradable polymer blends [13, 14], the incorporation of natural fibers [15, 16] and as mentioned before, the chemical modifications by acetylation is the most studied. It is worth mentioning that most of these studies were performed on starches obtained from corn, barley, potato, and rice.
The purpose of this paper is to characterize two biodegradable types of thermoplastic starch obtained from natural and acetylated cassava starch (Manihot sculenta crantz) by their physicochemical, thermal and mechanical properties.

2. EXPERIMENTAL METHODOLOGY
2.1 Materials  Cassava starch samples, natural food grade and acetylated industrial grade, were kindly provided by Industrias del Maíz S.A. (Corn Products Andina Colombia), from Cali, Colombia and were used as received. The glycerol used was industrial grade.
2.2 Preparation of thermoplastic starch 
Similar to the process reported by Huang [3] and Ma [17], 2006, both the cassava starch and the acetylated one were first dried for 24 hours at 80°C and then mixed with glycerol in a 70:30 weight ratio using a Black and Decker high speed mixer, until the lumps disappeared (around five minutes). The mixture was then stored in a sealed container for 72 hours. Finally, it was plasticized using a co-rotating twin screw extruder coupled to a Brabender plasticorder Model PLE 330, equipped with a 32 mm conical cylinder and an L/D ratio of 13. The rotating speed was kept at 45 rpm and the temperature profile used was 115, 125, 130 and 135 ºC for all three zones of the screw and the head respectively.

2.3 Fourier Transform Infrared Spectroscopy (FTIR)  Fourier Transform Infrared Spectroscopy analysis was performed on a Nicolet model Protégé 460 magna IR spectrometer. The analysis of the milled starch (natural and acetylated) was carried out with analytical grade KBr pellets using the transmission mode. The spectra were recorded with 4 cm-1 resolution and 100 scans. In the case of the TPS, a microscope with an accessory for attenuated total internal reflectance (ATR) spectroscopy was used.
2.4 Thermogravimetric analysis (TGA) 
Thermal stability was evaluated using a Perkin Elmer TGA 7 in a temperature range between 50 and 650 °C, with a heating rate of 10 ºC/min and an inert atmosphere, nitrogen gas flow of 20 ml/min. The average weight of the evaluated samples was 7 mg. 

2.5 Scanning electron microscopy (SEM) 
The morphology of the milled starch particles and the failure surfaces of both the natural and acetylated thermoplastic starches were observed using a scanning electron microscope JEOL SEM Model LV 5400. The samples were coated with gold prior to the analysis.

2.6 Mechanical properties  Tensile mechanical properties of both, the natural and acetylated thermoplastic starches were determined after the room conditioning of the samples. The testing was carried out only when the hygro-thermal equilibrium had been achieved at 54 % RH and a temperature of 25 ºC. The tensile testing was performed using a Shimadzu Model AG-1 100 KN universal testing machine equipped with a 500 N load cell. Type IV samples were prepared according to the ASTM D-638 standard specifications using a cross-head speed of 5 mm/min.
2.7 Moisture absorption Isotherms  The thermoplastic starch samples were dried in an oven at 80 °C during 12 hours and then placed in a desiccator with an aqueous solution of hexa hydrated magnesium nitrate in order to maintain a relative humidity of approximately 54 ± 2 %. Weight gain data were obtained as a function of time (Pt) at a temperature of 25 ± 2 ºC, moisture absorption (H) was calculated as a percentage, taking the weight obtained right after oven drying (Ps) as the initial value, according to (1).
 (1)
2.8 Dynamical mechanical analysis (DMA)  A Dynamical Mechanical Analyzer (Perkin Elmer DMA 7) was used for the determination of the second order transitions of both the natural thermoplastic starch and the acetylated thermoplastic starch, using the extension mode. The specimen size was 2 mm x 1 mm x 6 mm. The samples were analyzed at a temperature range between -100 and 60 ºC (cooling with liquid nitrogen), using a heating rate of 5 °C/min, a frequency of 1 Hz and a gas nitrogen flow of 20 ml/min.

3. RESULTS AND DISCUSSION
3.1 Fourier transform infrared spectroscopy (FTIR)  The FTIR spectra for both the natural and acetylated starches are shown in Figure 1. The assignment of the corresponding bands for both starches is presented in Table 1. The main difference in the spectra of the milled starches is attributed to the appearance of new bands at 1740, 1375 and 1240 cm-1, characteristic of the acetate group formed in the acetylation process. A typical reaction for such a process is illustrated in Figure 2. Here, the structure of the repeat units where the chemical modification is performed is presented. These bands can also be observed in the spectrum corresponding to the acetylated TPS. In Figure 3, the interval from 800 to 1400 cm-1 of the starches studied is shown. Smits et al. [18] pointed out that the band for 1047 cm-1 is related to the crystalline phase of starch, while the band at 1022 1047 cm-1 is related to the amorphous one. From a reference line drawn between 1180 and 880 cm-1, the index of crystallinity was calculated as the band height ratio (H1047/H1022).
  
Figure 1. FTIR spectra of natural and acetylated starch and natural and acetylated TPS

Table 1. Typical bands in infrared spectroscopy of thermoplastic starch [8, 19] 

  
Figure 2. Scheme of a starch acetylation process

  
Figure 3. FTIR amorphous and crystalline regions for natural and acetylated starch and natural and acetylated TPS

It was found that the crystallinity indexes were 0.911 and 0.821 for the natural and acetylated milled starches respectively, and 0.694 and 0.716 for the natural and acetylated thermoplastic starch. These results indicate, as expected, that the extrusion processes decrease the crystallinity of both types of starch, more noticeable for natural starch compared with the acetylated starch. The effect of the acetylation on the morphology of starches is more complex. On one side, the acetylation, at least at the surface level, partially decreases the arrangement of the starch granules, in agreement with the distortion related to the loss of symmetry upon the replacement of the hydroxyl groups –OH by the acetate groups –CH3OCO, which are larger.
Also, when comparing the crystallinity indexes, between the thermoplastic starches, the acetylation process appears to give place to a highly ordered TPS, when compared to the natural starch. This behavior tends to suggest that the interactions between starch and glycerol play an important role in the plasticization process.
3.2 Thermogravimetric analysis (TGA)  The loss of mass occurring along the temperature range selected for this study, revealed that, with the plasticization process of starch, thermal stability is lost, as shown in Figure 4; such a decrease is mainly attributed to the incorporation of glycerol, a low molecular weight molecule, whose evaporation and/or decomposition occurs at lower temperatures than the starches. Figure 5 shows the derivative of the thermogravimetric analysis curves. 
  
Figure 4. TG thermogram for natural and acetylated starch and natural and acetylated TPS

  
Figure 5. DTG thermograms for natural and acetylated starches and natural and acetylated TPS

The temperature for the higher rate of mass loss of these materials can clearly be observed. It can also be seen that the plasticized starch presents small shifts to the right for the peak and that the acetylation process induces a shift of the peak to the left for both the plasticized starch and the milled-like granular structure. It is observed that for glycerol there is a peak value at 209 °C, where the largest mass loss occurs, as depicted by an almost vertical drop of the thermogram in Figure 4.
The kinetic parameters for the materials can be calculated from the model proposed by Broido [20], as shown in (2).
 (2)
Where:
a = Reaction extent of the component of the sample being degraded 
Ea = Activation energy 
R = Universal Gas Constant 
T = Temperature
Similarly, the reaction extent a is determined from the initial (w0), and final (w¥), and at any given time (wt) weights of the sample, using (3). 
 (3)
The activation energy can be estimated from the calculation of the slope of the curve given by (2); it should be pointed out that, in the graph for Broido’s model there are three different lineal regions in the temperature range which are also associated with three different activation energies.
These regions have also been reported by other researchers [18, 21] in studies of dry and plasticized starch. Figure 6 shows the values of the activation energy for the central temperature region (290 – 340 ºC), which are shown in Table 2 where the peak value of the curve for the derivative occurs.
  
Figure 6. Estimation the activation energy using Broido’s method

Table 2. Mechanical and thermal properties of the natural and the acetylated starches 

The value obtained for the activation energy for natural cassava starch was 129 kJ/mol. A value of 102 kJ/mol was reported by Alvarez and Vazquez [22] for corn starch calculated using the Kissinger method. It should be pointed out that the calculated values for the kinetic parameters vary according to the type 
of atmosphere, the shape and amount of the sample; the flow and heat rate; and the method used for the calculations. In the case of the thermoplastic starch, the secondary interactions between the polymeric chains are smaller, compared with the non-plasticized starch, due to the presence of glycerol. Therefore, it is expected that the activation energy for the thermal degradation process of the TPS should be smaller than the one for the milled starch.
The value of Ea for natural TPS, was 51.3 kJ/mol, a value of 63.3 kJ/mol was reported by Valles et al. [21], for a mixture of thermoplastic starch, also plasticized with glycerol. This value was also calculated using Broido’s model for a temperature interval similar to the one used in this study.
3.3 Scanning electron microscopy  Figures 7 (a) and 7 (c) show micro photographs of the morphology for natural and acetylated cassava starch particles, used in the plasticization process, respectively. As stated by Wurzburg [23], the geometry of this type of starches is sphere-like, as opposed to those from other botanical sources which posses a polygonal shape. The presence of the plasticizer and the resulting shear stress led to the breakage of the granular structure of starch. However, as seen in the microphotographs Figures 7(b) and 7(d), there still are a few granules that are not totally fragmented; this is an indication that the processing conditions can still be improved. The last microphotographs were taken from the failure cross-section of tensile test samples.
  
Figure 7. SEM micrographs of natural milled (a) and acetylated starch (c); tensile fracture surface of natural (b) and acetylated thermoplastic starch (d) 

3.4 Mechanical properties  The tensile strength found for the natural and the thermoplastic starch are low compared to the 5.5 MPa value reported by Huang et al. [2] for corn starch with similar glycerol contents. It should be pointed out that they used a higher strain rate (10 mm/min) and a lower relative humidity (50 %) than those used in this study (5 mm/min y 54 %). Also, as reported by Mathew [24], for a system of corn starch with similar glycerol contents, the tensile strength, elastic modulus and strain at break were 0.23 MPa, 38 MPa, and 14 %, respectively. Ruiz [25] reported a tensile strength of 0.38 MPa, and strain at breaking point of 69 % for a thermoplastic starch with a 65 % modified cassava starch and 35 % glycerol. It is important to point out that the mechanical properties of the TPS vary with time, and with the temperature and relative humidity they are exposed to [26]. This is attributed to the water plasticization and retrogradation phenomena. These are is the main reasons why there are always differences in the reported values for this type of characterization.
The typical stress-strain curves for both natural and acetylated TPS are shown in Figure 8, and the average values of five specimens tested can be seen in Table 2Figure 8 shows that the mechanical behavior of both TPSs is different. The acetylated TPS has lower tensile break strength and a higher tensile break strain compared to natural TPS. This finding suggests that the role of glycerol during the plasticization was more effective for the chemically modified starch than for the natural one. In the case of the tensile modulus, no statistical differences were found.
 
Figure 8. Tensile strength vs strain at breaking point curves for natural and acetylated thermoplastic starch

3.5 Isotherms of moisture absorption  Figure 9 shows the curves for the isotherms of moisture absorption from the thermoplastic starches. It was found that the moisture absorption for the conditions in this study was of the order of 10 %, and a slight decrease in the absorption was evident for the acetylated material. 
  
Figure 9. Moisture absorption isotherm at 25 ºC and 54 % relativity humidity for natural and acetylated thermoplastic starch

As expected, this decrease of the absorption suggests that there is a decrease in the number of hydroxyl groups, attributed to the acetylation of the starch, thus influencing the capacity for the formation of bonds between the material and water molecules from the environment. 
Furthermore, a noticeable decrease in the absorption of moisture could be expected for the starches presenting higher percentages of substitutions. Similar values of moisture absorption have been reported by Curvelo [15] for a starch plasticized with 30 % glycerol; as reported in these studies, an absorption of approximately 9 % is generated for equilibrium conditions when the relative humidity and the temperature are kept at 43 % and 25 ºC respectively.
3.6 Dynamical mechanical analysis  Figure 10 shows the curves for Tan d obtained from a DMA for both, the natural and acetylated TPS. As can be seen, there are two second order transitions β and α, which have been amply reported in the literature [27]. 
  
Figure 10. DMA Tan d curves for natural and acetylated thermoplastic starch

The first peak is associated with local movements attributed to the glycerol molecules, while the second transition is commonly denoted as the glass transition and is related to the movements when the energy is high enough to generate conformational changes at the molecular chain level. In a similar study Da Roz [28] evaluated different plasticizers (ethilenglycol, sorbytol, polyethilenglycol, 1-4 butanodiol, etc.) used as additives for corn starch TPS; he reported transitions in similar intervals to those obtained via DMA and shown in Figure 10. On the other hand, we observe that the chemical modification of starch at low substitution degrees generates a decrease in the associated temperatures related to the transitions, suggesting that, in agreement with the analysis of the moisture absorption isotherms, little effect exist in the decrease of the secondary interactions between the starch molecules, also attributed to the decrease in the number of hydroxyl groups.

4. CONCLUSIONS
The acetylation treatment of cassava starch, contributed to the increase of the amorphous region of the material. This was made evident by the relative decrease of the IR band at 1047 cm-1 associated with the crystalline regions of the material. The water absorption capability of the acetylated starch was smaller than that of the natural one despite the lower acetylation degree. This fact suggests that an increase in the degree of chemical modification could contribute to further diminishing the capability of moisture absorption of the TPS. Although the acetylation process decreases the mechanical strength of the TPS it seems that the better interaction of the glycerol with the modified starch would allow us to decrease it in order to improve the TPS mechanical properties. 

5. ACKNOWLEDGEMENTS 
This work was performed with the support of the Centro de Investigación Científica de Yucatán A.C. (CICY) México, the Universidad del Valle, COLCIENCIAS and the Centro de Excelencia de Nuevos Materiales (CENM), Colombia. The authors also express their gratitude to Industrias del Maíz for having provided the cassava starch and Q.I. Tanit Toledano-Thompson for the SEM micrographs. 

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Retrogradation enthalpy does not always reflect the retrogradation behavior of gelatinized starch

Published Date


Received:
Accepted:
Published online:

Scientific Reports 6, Article number: 20965 (2016)
doi:10.1038/srep20965

Author Information

    • Caili Li
    •  & Xiu Zhang
    These authors contributed equally to this work.

Affiliations

  1. Key Laboratory of Food Nutrition and Safety, Ministry of Education, College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China

    • Shujun Wang
    • Caili Li
    • Xiu Zhang
    •  & Shuo Wang
  2. Faculty of Agriculture and Environment, The University of Sydney, NSW 2006, Australia.

    • Les Copeland

Contributions

Shujun Wang and Shuo Wang conceived and designed the study. C.L. and X. Z. conducted the experiments and data analysis. Shujun Wang and C.L. contributed to the earlier draft of the manuscript. Shujun Wang and L.C. revised and edited the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Shujun Wang or Shuo Wang.

Abstract 

Starch retrogradation is a term used to define the process in which gelatinized starch undergoes a disorder-to-order transition. A thorough understanding of starch retrogradation behavior plays an important role in maintaining the quality of starchy foods during storage. By means of DSC, we have demonstrated for the first time that at low water contents, the enthalpy change of retrograded starch is higher than that of native starch. In terms of FTIR and Raman spectroscopic results, we showed that the molecular order of reheated retrograded starch samples is lower than that of DSC gelatinized starch. These findings have led us to conclude that enthalpy change of retrograded starch at low water contents involves the melting of recrystallized starch during storage and residual starch crystallites after DSC gelatinization, and that the endothermic transition of retrograded starch gels at low water contents does not fully represent the retrogradation behavior of starch. Very low or high water contents do not favor the occurrence of starch retrogradation.

Introduction 
Experimental Section

Materials

The wheat (Zhoumai 18) flour was kindly provided by the Institute of Crop Science, Chinese Academy of Agricultural Science. The rice (Oryza sativa) sample was purchased from a supermarket in Tianjin, China. Amylose (A0512) and amylopectin (A8515) from potato starch were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other chemical reagents were of all analytical grade.

Isolation of Starch

Rice starch (RS) was isolated according to the method of Spigno and De Faveri13, and wheat starch (WS) according to a dough ball method8,14.

Chemical Analysis of Starch

The amount of damaged starch in the isolated starches was determined using the Megazyme Starch Damage Kit (Megazyme International Ireland Ltd. (Bray Co., Wicklow, Ireland). Amylose content was determined by iodine binding according to Chrastil15using a standard curve of 10%, 20%, 25%, 30%, and 35% potato amylose mixed with potato amylopectin. The crude lipid content of starch granules was determined gravimetrically by Soxhlet extraction using petroleum ether. The nitrogen content of wheat grains and starch granules were determined by standard Kjeldahl methodology. Crude protein content was estimated by multiplying the nitrogen content by a conversion factor of 6.25. Moisture content was determined by drying to constant weight at 105 °C and ash content was determined using a muffle furnace at 550 °C.

Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) measurements were performed using a Differential Scanning Calorimeter (200 F3, Netzsch, Germany) equipped with a thermal analysis data station. Starch (approximately 3 mg wet weight) was weighed accurately into an aluminum sample pan. Distilled water was added with a pipette to obtain starch:water ratios of 1:0.5, 1:0.75, 1:1, 1:1.5, 1:2.5 and 1:4 (w/v) in the DSC pans, corresponding to the water content of 33, 43, 50, 60, 71 and 80%, respectively. The pans were sealed and allowed to stand overnight at room temperature before analysis. The pans were heated from 20 to 100 °C at a rate of 10 °C/min. An empty aluminum pan was used as the reference. Starch retrogradation was determined on the same gelatinized samples after storage at 4 °C for 7 days. The retrograded starch samples were re-scanned using the heating profiles described for starch gelatinization. Gelatinization enthalpy change of native starch (ΔHG) and enthalpy change on reheating of retrograded starch gels (ΔHR) were obtained using data recording software. All measurements were performed in triplicate. Degree of retrogradation (%DR) was calculated according to the formula:
After DSC measurements, the sample pans were cooled to room temperature and reweighed. Those pans without any weight loss were collected and starch samples were freeze-dried and used for Fourier-transform infra-red (FTIR) and Raman spectroscopic analysis.
To obtain sufficient material to gain a better understanding of starch retrogradation behavior, retrograded starches were also prepared by simulating the DSC heating conditions without stirring using the RVA to prepare starch gels followed by storage at 4 °C for 7 days. The retrograded starch samples prepared in this way were freeze-dried, ground using a mortar and pestle, and passed through a 100 μm sieve. The resulting retrograded starch powders were mixed with water in ratios of 1:0.5, 1:0.75, 1:1, and 1:1.5 (w/v) in the DSC pans and analyzed using the same procedures for DSC measurements as described above.

Attenuated Total Reflectance (ATR)-FTIR Spectroscopy

The residual molecular order of native starch after DSC heating and of retrograded starch gels after DSC reheating was determined directly using a Thermo Scientific Nicolet IS50 spectrometer (Thermo Fisher Scientific, USA). The ratio of absorbance at 1047/1022 cm−1 was used to estimate the short-range molecular order of starch.

Laser Confocal Micro-Raman (LCM-Raman) Spectroscopy

The molecular order of native starch after DSC heating and of retrograded starch gels after DSC reheating was also determined by using a Renishaw Invia Raman microscope system (Renishaw, Gloucestershire, United Kingdom), which was equipped with a Leica microscope (Leica Biosystems, Wetzlar, Germany) and a 785 nm green diode laser source. Spectra were collected on at least five different spots of gelatinized and retrograded starch samples in the range of 3200–100 cm−1, with a resolution of approximately 7 cm−1. The full width at half height (FWHH) of the band at 480 cm−1, which can be used to characterize the molecular order of starch6, was calculated using the WiRE 2.0 software.

Statistical Analysis

Results are reported as the mean values and standard deviations of at least duplicate measurements. Analyses of variance (ANOVA) by Duncan’s test (p < 0.05) were conducted using the SPSS 17.0 Statistical Software Program (SPSS Inc. Chicago, IL, USA).
Results and Discussion 

Basic Composition of Rice and Wheat starches

Amylose contents of the rice and wheat starches were 11.5% and 25.0%, respectively (Table 1). The amylose content of starch from non-waxy rice varieties varies between 7% and 33%16,17,18, whereas non-waxy wheat starch usually contains 20–30% amylose19. Damaged starch contents of the rice and wheat starches were 0.89% and 1.30%, respectively, whereas ash, protein, lipid and water contents of the two starches were all within the range of values reported in the literature20.

Table 1: Basic composition of wheat and rice starches.

Thermal Properties of Native and Retrograded Starches

The DSC curves of starch gelatinization and from reheated retrograded starch gels are presented in Fig. 1. Rice and wheat starches displayed a typical gelatinization endothermic transition in the temperature ranges of 61.2–73.9 °C and 55.1–67.4 °C, respectively. With increasing water content, the gelatinization endothermic transition became progressively more pronounced and symmetrical (Fig. 1a,c). The area of this gelatinization endotherm increased with increasing water content. On reheating the retrograded starch, much broader but shallower endothermic transitions ranging from 42.9–68.9 °C and from 41.9–65.3 °C were noted for rice and wheat starch, respectively (Fig. 1b,d). The area of this retrogradation endotherm increased initially and then decreased with increasing water content.

Figure 1
Figure 1
DSC curves of RS gelatinization (a), RS retrogradation (b), WS gelatinization (c), WS retrogradation (d).
Thermal transition temperatures of native starch and retrograded starch gels at different water contents are listed in Table 2. There were small differences in the gelatinization transition temperatures of rice starch as water content increased from 31% to 80%. The To, Tp and Tc of rice starch were in the range of 61.2–62.1 °C, 66.9–68.3 °C and 71.0–73.9°, respectively. The transition temperature range (Tc–To) was from 9.8 to 11.7 °C. The To, Tp and Tc of wheat starch ranged from 55.1, 60.8 and 64.6 °C to 56.8, 62.1 and 67.4 °C, respectively. The thermal transition broadened with Tc–To ranging from 9.5 to 11.3 °C. These observations are consistent with previous results, which showed that the conclusion temperature of pea and wheat starches increased gradually with increasing water content7,8.

Table 2: Thermal transition temperatures of rice and wheat starch samples.
After storage for 7 days, the transition temperatures of reheated retrograded starch gels, especially To and Tp, were much lower than those of native starch granules (Table 2), indicating that the melting of crystallites in retrograded starch gels occurred more readily than for native starch crystallites. The To of retrograded starch gels did not vary greatly with different water content, suggesting similarities in the onset of melting behavior of crystallites in retrograded starch gels. However, the Tp and Tc of retrograded rice starch gels decreased from 56.8 to 53.9 °C and from 67.8 to 62.5 °C, respectively, as the water content increased from 33 to 71%. Similarly, the Tp and Tc of retrograded wheat starch gels decreased from 58.3 to 53.6 °C and from 65.3 to 62.7 °C, respectively. With increasing water content, Tc–Todecreased from 25.4 and 22.9 °C to 18.3 and 17.3 °C for rice and wheat starch, respectively.
Gelatinization enthalpy change increased from 2.6 to 14.4 J/g and from 2.9 to 10.7 J/g for rice and wheat starch, respectively, as the water content increased from 33 to 71%, above which the enthalpy change remained essentially the same (Table 3). This observation was consistent with previous results8. Rice starch presented a higher maximum enthalpy change of 14.4 J/g than did wheat starch (10.7 J/g). The maximum enthalpy change of native starch granules has been shown to be variety dependent8.

Table 3: Enthalpy change of native and retrograded starches.
After retrogradation, the enthalpy change of rice and wheat starch gels varied according to water content (Table 3). Enthalpy change of rice starch gels increased from 3.9 J/g at a water content of 33% to a maximum value of 7.0 J/g at a water content of 50%, and then decreased to zero at a water content of 80%. In comparison, the retrogradation enthalpy change of wheat starch gels reached a maximum value of 6.2 J/g at a water content of 33%, and then decreased progressively to 0.5 J/g at a water content of 80%. When the water content was above 80%, no retrogradation of wheat starch gel was observed by DSC (data not shown), consistent with previous reports21,22,23.
Interestingly, the enthalpy change of retrograded starch gels was greater than that of native starch granules when the water content was between 33 and 50% (Table 3), resulting in a calculated value for the degree of retrogradation of the starch gels greater than 100% (Table 3Fig. 2). This suggests that the broad endothermic transition of retrograded starch gels involves not only the melting of recrystallized starch formed during retrogradation, but the further melting of residual crystallites that remained in the retrograded starch gels. As proposed8, the DSC endothermic transition does not represent the full gelatinization of starch granules at DSC water-limited conditions.

Figure 2
Figure 2
Gelatinization enthalpy change of RS (A), retrogradation enthalpy change of RS (B), DR of RS (C), Gelatinization enthalpy change of WS (D), retrogradation enthalpy change of WS (E), DR of WS (F).
To further substantiate the above hypothesis, the thermal transition of freeze-dried retrograded starch powders was determined (Fig. 3). Two separated endothermic transitions were noted in the range of 40~65 °C and 70~100 °C. The lower temperature endotherms are proposed to be the melting of retrograded starch crystallites, whereas the ones at higher temperature are proposed to be due to the melting of residual crystallites remaining after DSC gelatinization. The observation of two separated endotherms indicated that melting of recrystallized starch formed during retrogradation and residual crystallites after DSC gelatinization occurred separately rather than simultaneously. The second endothermic transition is unlikely to be due to the melting of amylose-lipid complexes, which occurs at higher temperatures. Moreover, the lipid content of the starches was very low. The broad endothermic transitions noted for retrograded starch gels are consistent with the consecutive melting of recrystallized starch and residual starch crystallites. However, unlike the freeze-dried material, the endotherms of the reheated starch gels in the DSC pans may overlap because water is likely to migrate more readily from recrystallized starch to residual starch crystallites in gels than in rehydrated powders. Interestingly, the enthalpy change of freeze-dried retrograded starch powers was lower than that of retrograded starch gels (Table 3), consistent with the findings that freeze drying can disrupt the crystalline and molecular order of starch24. At higher water content of 60–80%, the degree of retrogradation was between 0–32.3% and between 4.7–34.1% for rice and wheat starch, respectively.

Figure 3
Figure 3
DSC curves of retrograded rice starch powders (a), retrograded wheat starch powders (b). p1: represents the melting of recrystallized starch during retrogradation; p2: represents the melting of residual starch crystallites after DSC gelatinization.

Short-range Molecular Order of Starch by ATR-FTIR and Raman Spectroscopy

The short-range molecular order of double helices in starch can be characterized by the ratio of absorbances at 1047/1022 cm−1 obtained from FTIR spectroscopy of starch25,26,27,28. To verify the further melting of starch granule crystallites remaining in retrograded starch on DSC reheating, the molecular order of native starch after DSC heating and retrograded starch gels after DSC reheating was determined by ATR-FTIR and Raman spectroscopy. To identify the differences in the molecular order of these starch samples, the deconvoluted FTIR spectra of wheat starch in the range of 1200–800 cm−1 were obtained (Fig. 4). The ratios of 1047/1022 cm−1 of starch after gelatinization decreased with increasing water content (Table 4), indicating that the degree of disruption of molecular order in starch after DSC heating increased with increasing water content. Similar results were also observed with retrograded starch gels after DSC reheating, although in some cases no significant differences were observed. The ratios of 1047/1022 cm−1R were lower than those of 1047/1022 cm−1G over the whole range of water content, although the difference was small in some cases. This result showed that the molecular order of the retrograded starch gels after DSC reheating was lower than that of gelatinized starch, providing evidence to support the conclusion from the DSC data that there was further melting of starch crystallites remaining in retrograded starch gels.

Figure 4
Figure 4
The deconvoluted FTIR spectra of gelatinized WS after DSC heating (a), reheated retrograded WS (b).

Table 4: The ratios of 1047/1022 cm−1 and FWHMs of the band at 480 cm−1 of starch samples.
As the LCM-Raman spectra of starch samples were similar, only those of wheat starch are presented (Fig. 5). Several clear bands can be seen at 480, 865, 943, 1264 and 2900 cm−1, which are related to δ (CH2), νs (C1-O-C4), νs (C1-O-C5), skeletal (C-C-O), and ν (C-H) modes, respectively29,30,31. Of these bands, the ones at 480 and 2900 cm−1 are often used to characterize the molecular order of native starch granules or the changes in molecular order of starch samples during gelatinization or retrogradation6,29,32,33,34. Full width at half maximum height (FWHM) of the strong band at 480 cm−1 is often used to characterize the relative crystallinity of starch samples; this parameter is most responsive to changes in crystallinity29. Values for FWHMR were higher than those for FWHMG over the whole range of water content (although the difference was small at high water content), indicating that the relative crystallinity of retrograded starch gels after DSC reheating was lower than that of gelatinized starch samples, consistent with the ATR-FTIR results.

Figure 5
Figure 5
LCM-Raman spectra of gelatinized WS after DSC heating (a), reheated retrograded WS (b).
The maximum enthalpy change of retrograded starch gels was observed at water content around 50%, which does not correspond to the maximum degree of starch retrogradation. The crystallites formed during retrogradation are less ordered and less stable than crystallites in native starch granules. The observed higher enthalpy change of retrograded starch gels at low water content indicated that the enthalpy change of retrograded starch gels involves not only the melting of new starch crystallites formed on storage, but also further melting of starch crystallites remaining after gelatinization. This conclusion was supported by the ATR-FTIR and Raman spectroscopy analysis of molecular order of starch after gelatinization and after reheating of retrograded starch gels. Several studies investigated the effect of water content on retrogradation of starch21,22,23,35,36,37, but none reported an apparent value greater than 100% for the degree of retrogradation. The effect of water content on starch retrogradation, as determined by measuring DSC enthalpy change of recrystallized amylopectin, displayed a parabolic shape, with maximum retrogradation occurring in starch gels at 40–45% water content21,22,37.
Another interesting finding from the present study is the decreasing Tp and Tc of retrograded starch gels with increasing water content. Two hypotheses, not mutually exclusive, are proposed to interpret this observation. The crystallites formed during retrogradation could become progressively less stable with increasing water content, leading to lower Tpand Tc of retrograded starch gels. Another explanation is that the higher Tp and Tc of retrograded starch gels at low water content is due to further melting of residual starch crystallites that remained in retrograded starch gels. The melting of starch crystallites is not complete at the end of DSC heating, especially at low water content38,39. This interpretation is supported by the observation that the Tc of retrograded rice and wheat starch gels was slightly lower than those of native starch granules at low water content, and that the transition temperature range of retrograded starch gels decreased with increasing water content (Table 2). Taken together, we can conclude that the endothermic transition of retrograded starch gels at low water content does not reflect accurately the retrogradation behavior of gelatinized starch.
It is also interesting to note that the enthalpy change of starch gelatinization remained essentially unchanged at a water content above 71%, whereas the enthalpy change of starch retrogradation decreased progressively up to a water content of 80%. This result indicated that although the enthalpy change reached a maximum value, presumably corresponding to the complete gelatinization of starch, the retrogradation behavior of fully gelatinized starch was different at different water contents. The high water content brought about low degree of retrogradation, indicating that with increasing water content, the coming together and realignment of dispersed starch chains becomes progressively more difficult.
Conclusions

Differential scanning calorimetry studies over a wide range of water content have shown that water strongly influences retrogradation behavior of rice and wheat starches. The DSC thermal transition parameters have demonstrated for the first time that the enthalpy change of retrograded starch gels was greater than that of native starch at water content of 33 to 50%. In combination with ATR-FTIR and Raman results, we conclude that the DSC endothermic transition of retrograded starch gels at low water content does not truly reflect the retrogradation behavior of gelatinized starch. The enthalpy change of retrograded starch gels at low water contents represents the melting of starch crystallites formed by retrogradation and of residual crystallites remaining after gelatinization. At high water content, when complete gelatinization of starch may occur, the degree of retrogradation is influenced by water content, whereas at very low and very high water content, the DSC enthalpy change indicated that little retrogradation of starch occurred.

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