Ultrasonic investigation of wheat starch retrogradation

ARTICLE IN PRESS

www.elsevier.com/locate/jfoodeng

Journal of Food Engineering xxx (2005) xxx–xxx

Ultrasonic investigation of wheat starch retrogradation

Francesca Lionetto a,*, Alfonso Maffezzoli a, Marie-Astrid Ottenhof b,Imad A. Farhat b, John R. Mitchell b

a Department of Innovation Engineering, University of Lecce, via Monteroni, 73100 Lecce, Italyb Division of Food Sciences, School of Biosciences, University of Nottingham, Loughborough LE12 5RD, UK

Received 1 November 2004; accepted 18 April 2005

Abstract

A recently developed technique, based on ultrasonic wave propagation, was applied to study the effect of storage time on the

retrogradation process of wheat starch extrudates during storage at room temperature. A specifically designed experimental set-

up enabled to propagate in situ ultrasonic waves continuously for several days at constant water content and storage temperature

(�34% water and 25 �C). The velocity and the attenuation of the ultrasonic waves changed as a result of the recrystallisation process

of amylopectin molecules of wheat starch samples during storage.

The propagation of ultrasonic waves, acting as a dynamic mechanical deformation at high frequency (10 MHz), gave access to

the complex longitudinal modulus that, compared to the complex Young modulus obtained from low frequency DMA, enabled a

better insight of the changes in viscoelastic behaviour of starch extrudates during retrogradation. The results of the ultrasonic mon-

itoring of starch retrogradation were correlated with those obtained from X-ray diffraction and differential scanning calorimetry.

The present study demonstrated the potential of the ultrasonic technique in detecting the changes in physical properties of con-

centrated starch systems.

� 2005 Elsevier Ltd. All rights reserved.

Keywords: Ultrasonic wave propagation; Wheat starch; Retrogradation; DSC; XRD; DMA

1. Introduction

Ultrasonic techniques are finding an increasing num-

ber of applications in the food industry for both the

analysis and modification of foods (Knorr, Zenker,

Heinz, & Lee, 2004; McClements, 1995; Mulet, Bened-

ito, Bon, & Sanjuan, 1999; Povey & Mason, 1998).

Low intensity ultrasound, which involves low power le-

vel of ultrasonic waves, is a non-destructive technique

applied to monitor processes, e.g. thickness and liquidlevel measurements or detection of extraneous matter

in foods (Knorr et al., 2004; McClements, 1995; McCle-

0260-8774/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jfoodeng.2005.04.015

* Corresponding author. Tel.: +39 0832 297387; fax: +39 0832

297525.

E-mail address: francesca.lionetto@unile.it (F. Lionetto).

ments, 1997; Mulet, Benedito, Golas, & Carcel, 2002) or

to evaluate the textural properties of fruits, cheese andfatty tissues (Benedito, Carcel, Clemente, & Mulet,

2000; Benedito, Carcel, Sanjuan, & Mulet, 1999; Miz-

rach, Flitsanov, Akerman, & Zauberman, 2000) and

the rheological properties of honey, oils and dairy prod-

ucts (Buckin & Kudryashov, 2001; Greenwood &

Bamberger, 2002; Kulmyrzaev & McClements, 2000;

Saggin & Coupland, 2001). Another field, where ultra-

sound has been shown to be a good alternative to tradi-tional analytical techniques, is the quantitative

assessment of food composition, such as the sugar con-

tent of some fruit juices and drinks (Contreras, Fairley,

McClements, & Povey, 1992), the solid fat content of

oils and adipose tissues (McClements & Povey, 1987),

the composition of fish and meat based products (Ghae-

2 F. Lionetto et al. / Journal of Food Engineering xxx (2005) xxx–xxx

ARTICLE IN PRESS

dian, Coupland, Decker, & McClements, 1998; Simal,

Benedito, Clemente, Femenia, & Rossello, 2003). Never-

theless, the application of ultrasonic analysis has been

successful only in the case of food products with two

phases, showing distinct physical properties (Benedito,

Mulet, Clemente, & Garcia-Perez, 2004).One of the most important advantages of the ultra-

sonic technique consists in its capacity to monitor

non-destructively the phase transitions occurring in a

material as the changes in material density and elastic

properties, associated with the transition, affect signifi-

cantly the material acoustic response. In polymer sci-

ence, for example, ultrasonic wave propagation has

been recently applied to study time dependent kineticprocesses, such as the polymerisation kinetic of thermo-

setting resins (Lionetto, Rizzo, Luprano, & Maffezzoli,

2004; Maffezzoli, Quarta, Luprano, Montagna, &

Nicolais, 1999a, Maffezzoli, Tarzia, Cannoletta, Monta-

gna, & Luprano, 1999b; Matsukawa & Nagai, 1996), the

water sorption in dry hydrogels (Maffezzoli, Luprano, &

Montagna, 1998; Maffezzoli et al., 1999a, 1999b) and

the crystallisation process, for example, in poly(ethyleneoxide) systems (Alig, Tadjbakhsch, Floudas, & Tsitsili-

anis, 1998).

The possibility to monitor non-destructively the

phase transitions of many food components during

manufacturing or storage is of crucial importance for

process control and optimisation. From this necessity,

widely present in the food engineering field, arose the

idea of applying low intensity ultrasound to follow theretrogradation process of starch during storage at room

temperature.

Starch, the storage polysaccharide of many plants, is

found in the form of partially crystalline water-insoluble

granules, the size and composition of which depends on

the botanical source. Native starch is a mixture of two

polysaccharides, amylose and amylopectin, and contains

also small amounts of non-carbohydrate constituentssuch as lipids, phosphates and proteins. Amylose is

essentially a linear polymer, while amylopectin is a

highly branched macromolecule. Amylopectin, whose

content varies from 70 to 85% depending on starch

botanical source, is assumed to be the main responsible

of the crystallinity of the native starch granules (Buleon,

Colonna, Planchot, & Ball, 1998a, 1998b; Jenkins,

Cameron, & Donald, 1993; Miles, Morris, Orford, &Ring, 1985).

During processing, starch undergoes a conversion

from its native partially crystalline granular structure

to a polymeric solution or melt, depending on the

amount of water and the type of process used. In the

most general case, when heated in presence of excess

water, starch granules undergo an order-disorder transi-

tion, called gelatinisation. During cooling and storage ofgelatinised starch at temperature lower than the gelatin-

isation temperature, amylose retrogradation rapidly

occurs (Goodfellow & Wilson, 1990) while, on longer

time scale storage (hours or weeks or even months,

depending on composition and storage conditions),

amylopectin retrogradation occurs.

Starch retrogradation, which essentially involves

molecular packing and recrystallisation phenomena,leads generally to significant changes in the mechanical

properties of starch-based products and thus greatly

affects their sensory (e.g. texture and flavour perception

(Buleon et al., 1998a, Buleon, Le Bail, Colonna, &

Bizot, 1998b)), nutritional (susceptibility to enzymic

hydrolysis (Farhat et al., 2001)) and processing (shred-

ding, cutting, etc.) characteristics. However, not always

the recrystallisation process of starch leads to texturefirming. Other factors are known to be involved such

as gluten emulsifiers, fat, cellular structures, etc.

(Hallberg & Chinachoti, 2002).

Retrogradation happens because gelatinised starch is

supercooled and stored below its melting temperature.

Therefore, the drive towards thermodynamic equilib-

rium during storage leads re-ordering and crystallisa-

tion. Since starch retrogradation is accompanied bychanges in its viscoelastic properties, it may be expected

that it is also paralleled by corresponding changes in its

acoustic behaviour.

Due to the great importance of starch retrogradation

from a scientific and technological point of view, a large

number of studies have been carried out on this area

using different analytical methods (Farhat et al., 2001;

Fredriksson, Silverio, Andersson, Eliasson, & Aman,1998; Miles et al., 1985; Ortega-Ojeda & Eliasson,

2001; Zeleznak & Hoseney, 1986). The few applications,

found in the literature, of the ultrasonic wave propaga-

tion on starch based products are devoted to the mea-

surement of the rheological properties of dough

(Letang, Piau, & Verdier, 1999; Letang, Piau, Verdier,

& Lefebvre, 2001; Ross, Pyrak-Nolte, & Campanella,

2004). To our knowledge, there are no reports of studieson the monitoring of starch retrogradation using ultra-

sonics. The lack on the market of ultrasonic instruments

suitable for this task has probably hindered the applica-

tion of ultrasound to starch based products. The re-

sponse of the material to small deformations applied

very rapidly could offer a better characterization of the

mechanical behaviour in starch based products, which

is of great interest for food scientists. Moreover, theuse of ultrasound to characterize starch retrogradation

could be very promising since ultrasonic technique is ra-

pid and non-destructive, can be used in optically opaque

systems and can be easily adapted as an on-line tool for

process monitoring.

The aim of this work was to apply ultrasonic wave

propagation to the study of the changes occurring in a

model concentrated wheat starch system during storageat room temperature, with the material passing from a

completely rubbery state to a biphasic one, in which

F. Lionetto et al. / Journal of Food Engineering xxx (2005) xxx–xxx 3

ARTICLE IN PRESS

the crystalline phase acts like a rigid inclusion in the

amorphous rubbery matrix. In order to carry out the

ultrasonic measurements, a purpose designed experi-

mental set-up was developed.

Fig. 1. Experimental set-up for ultrasonic measurements.

Fig. 2. Waveforms of the reflected signals from the interface between

transducer delay line and starch sample (echo A0) and from the

interface between the sample and the aluminium wall (echo A1).

2. Experimental techniques

2.1. Sample preparation

Non-expanded wheat starch/water gel ribbons were

prepared by extrusion through a 1 · 30 mm slit die using

a Clextral BC-21 co-rotating intermeshing twin extru-

der. The extrusion temperature profile in the four zonesextruder barrel was 40, 90, 120, 75 �C, the feed rate of

solids was 5 kg/h and the screw speed was 300 rpm. Dis-

tilled water was introduced into the second zone of the

extruder barrel. The extruded samples contained �34%

water (wet weight basis, w.w.b.). The water content

was determined directly after extrusion by drying the

samples at 105 �C for 24 h. The samples were collected

in the form of strips, sealed in airtight aluminium foilbags to prevent loss of water and stored in an incubator

at 25 ± 1 �C.

2.2. Ultrasonic analysis

The experimental set-up for ultrasonic measurements

consisted of a narrow-band longitudinal wave contact

transducer (V611-RB, Panametrics, USA) operating a10 MHz (13 mm crystal diameter), a pulser-receiver card

(model SFT 4001H PCI, Sofratest, France) with an A/D

flash converter at 60 MHz, a custom-made measurement

cell and a PC with a data analysis software (LABVIEW

6, National Instruments, USA). The starch sample (in a

form of a disc of 12 mm diameter and 1.8 mm thickness)

was wrapped by a thin PVC film to avoid moisture loss

during the test. The sample was then loaded in the mea-surement cell, which consisted of an aluminium box,

specifically developed to enable the continuous monitor-

ing of the changes in the starch acoustic properties with-

out sample dehydration over a storage time of �9 days.

Four bolts were fastened sufficiently to provide a good

contact pressure between the starch specimen and the

walls of the aluminium box. A schematic diagram of

the ultrasonic set-up is reported in Fig. 1.A thin film of silicon oil was used as a coupling agent

between the sample (wrapped by PVC) and the trans-

ducer, in order to ensure a good transmission of ultra-

sonic waves, which propagate very poorly in air.

Finally, to prevent water loss, which could significantly

modify (by affecting the retrogradation kinetics) the vis-

co-elastic properties of the sample, the measurement cell

was entirely sealed with silicon. The samples wereweighed before and after the ultrasonic measurement,

but no significant moisture loss was observed. All the

experiments were replicated three times. Ultrasonic data

were collected continuously each 20 s for the first 58 h of

storage at room temperature. Then, to prevent any pos-

sible overload of the measurement equipment, the exper-

imental data were continuously acquired for 12 h with

an interval of other 12 h. The overall storage period

for the starch samples was about 9 days.

The ultrasonic measurements were carried out using apulse-echo technique, in which a single transducer prop-

agated and received longitudinal waves, normally inci-

dent on the specimen surface. The typical signal

obtained is displayed in Fig. 2. In the pulse-echo mode,

an ultrasonic wave, generated by the transducer, under-

goes a first reflection at the interface between the trans-

ducer delay line and the sample, generating a first echo

of amplitude A0. If a good coupling between the trans-ducer delay line and the sample is assured by means of

fluid coupling agents, only a small part of the ultrasonic

wave is reflected back to the transducer. The remaining

part of the wave, instead, continues travelling through

the sample until it reaches the interface between the sam-

ple and the wall of the measurement cell, where a con-

siderable fraction of it returns back to the transducer.

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ARTICLE IN PRESS

The received oscillations are converted by the transducer

into an electrical pulse and displayed as a second echo of

amplitude A1 (Fig. 2).

The two echoes are relatively spaced by a time t,

called ‘‘time of flight’’, which is the time necessary for

the ultrasonic wave to travel across the sample. Thewave time of flight relates to the elastic properties of

the sample, i.e. to its ability to transmit longitudinal

waves. Moreover, the two echoes displayed in Fig. 2

have also different amplitudes because the ultrasonic

wave, during its travel inside the sample, undergoes an

attenuation depending on the material damping behav-

iour. The temporal distance between two successive ech-

oes and their amplitude ratio are important parametersfor assessing the sample acoustic behaviour through the

calculation of the velocity and attenuation of the ultra-

sonic waves.

The longitudinal velocity, c, in the pulse-echo mode,

is defined as the ratio between two times the sample

thickness, d, and the time of flight, t, of the ultrasonic

wave through the sample, calculated as the difference be-

tween the times relative to both echoes displayed in Fig.2 (t = t1 � t0) (Krautkramer & Krautkramer, 1990):

c ¼ 2dt

ð1Þ

The attenuation, a, is defined from the decrease of the

amplitude, A, of a plane wave across a thickness, d:

A ¼ A0 expð�adÞ ð2Þ

where A0 is the amplitude of the incident wave. The

attenuation a is measured in dB/mm according to

(Krautkramer & Krautkramer, 1990):

a ¼ 1

2d20 log

A0

A1

� �ð3Þ

For each velocity and attenuation measurement, ten

signal acquisitions were made and averaged. The timeof flight and the signal amplitude were computed from

the averaged signal.

It should be noted that the sound velocity in a mate-

rial depends on its density and elastic properties,

whereas the sound attenuation depends on the viscous

behaviour and homogeneity of the material. Therefore,

ultrasonic measurements of c and a would detect the

phase transformations of the material that affect itsacoustic behaviour.

The interaction between an acoustic wave and a mate-

rial can provide information on its mechanical proper-

ties. These latter can be determined from the velocity at

which the wave propagates and from themanner in which

the acoustic wave is attenuated in the sample. When the

sample dimension normal to the propagation direction

of the acoustic wave is large compared to the wavelength,the wave propagation is governed by the complex bulk

longitudinal modulus L*, related to the bulk (K*) and

shear (G*) complex moduli as follows (Ferry, 1980; Pere-

pechko, 1975):

L� ¼ K� þ 4

3G� ð4Þ

The real and imaginary components of the complex

bulk longitudinal modulus (L* = L 0 + iL00) can be calcu-

lated from the measurement of ultrasonic longitudinal

velocity and attenuation according to the following

equations (Perepechko, 1975):

L0 ¼qc2 1� ak

2p

� �2h i

1þ ak2p

� �2h i2 and L00 ¼2qc2 ak

2p

� �1þ ak

2p

� �2h i2 ð5Þ

where L 0 is the elastic or storage component of the mod-

ulus, L00 is the viscous or loss component of the modu-

lus, c is the ultrasonic velocity, a the attenuation, q the

material density and k the wavelength of propagation,

calculated as the ratio between velocity and frequency

f(k = c/f).

For the studied case, the term ak/2p remains lowerthan 0.05 over then entire ageing period, therefore the

contribution of attenuation can be neglected. Therefore,

L 0 and L00 have been calculated from the simplified

equations:

L0 ¼ qc2; L00 ¼ 2qc3ax

ð6Þ

where x is the angular frequency (x = 2pf) and q the

density of the wheat starch extrudates, which, experi-

mentally determined at room temperature by the Archi-

medes� principle, results equal to 1323 kg/m3. The

storage longitudinal modulus L 0 corresponds to the stiff-

ness of a system that is deformed, changing its dimen-

sions in one direction while, in the other twodirections, the dimensions are constraint to remain con-

stant, as it occurs in samples where two dimensions are

much larger than the third (Ferry, 1980).

2.3. Dynamic mechanical thermal analysis

Dynamic mechanical analysis (DMA) at low fre-

quency was used to monitor the changes in the mechan-ical properties occurring during retrogradation. In a

dynamic mechanical experiment, a small sinusoidal

deformation, within the linear viscoelastic range, is ap-

plied to the sample, which responds with a sinusoidal

stress shifted in phase to the input deformation if the

material is viscoelastic. The ratio of the output stress

amplitude to the input deformation amplitude defines

the complex modulus E*, which consists of a real part,the storage modulus E 0, and an imaginary part, the loss

modulus E00.

A Rheometric Scientific dynamic mechanical thermal

analyser DMTA IV operating in bending mode was

used. Rectangular strips (approximately 7 mm · 14

Fig. 3. Evolution of the ultrasonic velocity (�) and attenuation (h),

measured at 10 MHz, versus storage time for wheat starch extrudates

with 34% water (w.w.b.).

F. Lionetto et al. / Journal of Food Engineering xxx (2005) xxx–xxx 5

ARTICLE IN PRESS

mm · 2 mm) were cut from the extruded ribbons, previ-

ously stored at 25 ± 1 �C in the incubator for different

ageing times, and clamped in the single cantilever geom-

etry. The analysis was performed during heating from

�30 to 70 �C at 1 Hz with a strain amplitude of 0.1%.

A relatively low heating rate of 1 �C/min was used to en-sure adequate thermal equilibrium across the sample. In

order to alleviate potential water loss during the mea-

surement, the samples were covered with a thin film of

silicon oil. The samples were weighed before and after

the DMA experiment and no significant moisture loss

was observed.

2.4. X-ray diffraction

Wide angle X-ray diffraction (XRD) measurements

were carried out to monitor the recrystallisation of amy-

lopectin during storage. A Bruker AXS D5005 diffrac-

tometer was used. The X-ray generator was equipped

with a copper tube operating at 40 kV and 30 mA and

irradiating the sample with a monochromatic Cu Ka

radiation with a wavelength of �0.154 nm. XRD spectrawere acquired at room temperature over the 2h range of

4–38� at 0.1� intervals with a measurement time of 6 s

per 2h intervals. The angular range encompassed the

main diffraction peaks of starch crystals.

XRD diffractograms were acquired at regular storage

time intervals up to �9 days on disks (�25 mm of diam-

eter) cut from the extruded ribbons, stored at 25 ± 1 �Cin an incubator in airtight aluminium foil bags.

2.5. Differential scanning calorimetry

Starch samples of 40mg weight were sealed in high-

pressure stainless steel pans soon after the extrusion

and stored at 25 ± 1 �C in an incubator for different

times prior to analysis. The melting behaviour of the ret-

rograded starch was studied using a power compensateddifferential scanning calorimeter (DSC 7, Perkin–Elmer,

USA). An empty stainless steel pan was used as a refer-

ence. The samples were heated from 20 �C to 160 �C at

10 �C/min. Three replicas were used for each measure-

ment. The thermograms were normalized to the dry

matter weight of each sample.

3. Results and discussion

The evolution of the ultrasonic velocity and attenua-

tion at 25 �C with storage time is reported in Fig. 3 for

wheat starch extrudates containing 34% water (wet

weight basis). The ultrasonic velocity is characterized

by a non-linear increase with storage time, whereas the

ultrasonic wave attenuation shows a bell shaped curve.A first rapid increase in the ultrasonic velocity is

accompanied by a steep increase in the wave attenua-

tion. Then, a few hours before the attenuation peak,the ultrasonic velocity slows down but continues to in-

crease for very long time at a reduced rate. After 9 days

of measurement at 25 �C, the velocity increase is small

but not yet negligible, whereas the attenuation value is

settled on a smaller value than the initial one, indicating

that the aged sample presents a reduced molecular

absorption compared to that of the gelatinised sample

at the beginning of the measurement.The increase in the longitudinal velocity, shown by

starch extrudates upon storage at room temperature,

indicates that a phase transformation, with a consequent

stiffening of gelatinised starch matrix, occurred. The

stiffening of starch extrudates does not originate from

water loss, since samples, weighed before and after ultra-

sonic measurements, do not show any significant weight

variation. Therefore, the slow increase in the longitudi-nal velocity can be assigned to the retrogradation pro-

cess, mainly relating to the partial recrystallisation of

the outer chains of amylopectin, as the retrogradation

of amylose would have mostly occurred during post-

extrusion cooling and sample handling (Fredriksson

et al., 1998; Goodfellow & Wilson, 1990; Miles et al.,

1985) and would not therefore be expected contribute

significantly to the long-term starch stiffening observedby the ultrasonic experiments.

The ultrasonic attenuation may be considered as the

equivalent of a damping factor in a dynamic mechanical

experiment (Maffezzoli et al., 1999a, 1999b), represent-

ing a measure of the energy loss as the wave travels

through the retrograding starch system. The ultrasonic

attenuation can be caused both by wave scattering and

absorption. In the case of the concentrated starch sys-tem analysed in this work, the scattering contribution

is negligible, because the dimensions of the amylopectin

crystals, typically in the order of 10–100s of nm (Buleon

et al., 1998a, 1998b; Jenkins et al., 1993), are far smaller

6 F. Lionetto et al. / Journal of Food Engineering xxx (2005) xxx–xxx

ARTICLE IN PRESS

than the wavelength of the ultrasonic waves used for the

experiment at 10 MHz, which varies between 183 and

195 lm depending on the ultrasonic velocity in the

starch samples. Therefore, only wave absorption is

responsible for the wave attenuation observed in the

wheat starch extrudates. This explains why the attenua-tion is lower in fully retrograded samples rather than in

gelatinised samples tested shortly after extrusion. In

fact, the reduction of the degree of mobility, as a conse-

quence of an increased crystalline fraction, inevitably

leads to a smaller viscous dissipation of the ultrasound

energy.

Since the ultrasonic wave propagation can be consid-

ered as a high frequency dynamic mechanical analysis,the longitudinal moduli (L 0, L00) obtained from ultra-

sonic experiments at 10 MHz were compared with the

Young moduli (E 0, E00) obtained from low frequency

(1 Hz) dynamic mechanical analysis by means of a rhe-

ometer operating in bending mode, as reported in Fig. 4.

It should be kept in mind that the great difference in the

frequency of the small oscillations applied with the two

techniques affects the sample response. In general, thefaster a material is deformed, the stiffer it behaves, and

the less able it is to dissipate stresses because the molec-

ular relaxation become less likely in the increasingly

shorter experimental time scale.

The storage moduli L 0 and E 0 show a similar trend,

characterized by a continuous increase with the ageing

time, reflecting the growth of the starch elastic proper-

ties arising from the increase of the crystallinity fractionat the expenses of the amorphous phase.

Fig. 4. Comparison of the storage (a) and loss (b) components of the

complex longitudinal modulus measured using ultrasound at 10 MHz,

with the complex Young modulus measured by DMA at 1 Hz.

A great difference in the magnitude orders of L 0 and

E 0 values is expected if one accounts that, for a rubbery

material such as the starch extrudates, G 0 � K 0 (Ferry,

1980) and hence, from Eq. (4), L 0 may be considered

equal to the bulk modulus K 0, which indicates the mate-

rial compressibility under a given amount of externalpressure. The reported values of L 0 for starch extrudates

(4.44 · 109 Pa for samples which are essentially amor-

phous shortly after extrusion) are consistent with the va-

lue of water bulk modulus at 20 �C (2.2 · 109 Pa)

(Halliday, Resnick, & Walker, 1997) if one accounts

for the high water content of the studied samples (34%

by weight).

The loss moduli L00 and E00 increase in the early stagesof ageing process as the crystallinity increases. The L00

curve presents a distinct peak. Although the relaxation

originating the loss is assumed to occur mainly in the

amorphous regions, the increase in the wave energy dis-

sipation may be related to the interaction between amor-

phous and crystalline regions. The reduction of the

entity of L00 with ageing can be attributed to the concur-

rent decrease of the amorphous regions and their mobil-ity in the starch sample and to a reorganization of water

molecules inside the sample with a consequent increase

in the amount of restricted water in starch structure,

as reported in a previous paper (Lionetto, Maffezzoli,

Ottenhof, Farhat, & Mitchell, 2005).

In contrast with ultrasonic experiments, it is very dif-

ficult to follow continuously the retrogradation process

using mechanical (DMA) measurements over severaldays. Conventional dynamic mechanical analysers, de-

signed for studying synthetic polymers, are not fully sui-

ted for biosystems such as starch gels because of risks of

significant loss of water (the most effective plasticizer for

this class of materials) during the test as it is not easy to

control the relative humidity surrounding the sample

while continuously increasing the temperature. Coating

the sample with an inorganic oil film can be adoptedas a solution, but the efficiency of such approach is

uncertain and, in addition, the oil may have a plasticiz-

ing effect on some biopolymers. Moreover, on ageing,

the surface of the sample does not remain perfectly flat,

leading to a slight error in the modulus calculation. The

experimental difficulties in using DMA experiments to

follow starch retrogradation, particularly in situ, explain

the relatively limited reports of experimental data of E 0

and E00 during retrogradation shown in Fig. 4.

The experimental set-up developed in this study for

the ultrasonic tests overcomes all these problems. The

retrogradation of the same starch sample can be moni-

tored continuously for many days in situ, without water

loss. Finally, a constant pressure is applied on the sam-

ple, assuring a flat surface absolutely necessary for a

good transmission of the ultrasonic waves from thetransducer to the sample and vice versa. This leads to

a strong reduction of the experimental errors enabling

F. Lionetto et al. / Journal of Food Engineering xxx (2005) xxx–xxx 7

ARTICLE IN PRESS

to obtain reliable measurements. Therefore, the ultra-

sonic dynamic mechanical analysis can be a valid alter-

native to the low frequency dynamic mechanical analysis

for studying the growth of the viscoelastic properties

during starch retrogradation.

The ultrasonic results have been also compared withthose of ‘‘more established’’ techniques in this research

area, wide-angle X-ray diffraction (XRD) and differen-

tial scanning calorimetry (DSC), which monitor starch

retrogradation through the growth of the diffraction

peaks of recrystallised starch and the increase of its

melting enthalpy, respectively. In Fig. 5 the extent of ret-

rogradation, as monitored by different techniques, has

been plotted as a function of the storage time. The stor-age longitudinal modulus (L 0), the loss modulus (L00),

the DSC melting enthalpy and the X-ray crystallinity in-

dex values reported in Fig. 5 were divided by their indi-

vidual maximum value. XRD and DSC data were

collected discretely at intervals of several hours.

The progress of retrogradation, monitored by the dif-

ferent techniques, is in good agreement. The loss modu-

lus L00 presents a peak slightly before the plateau valuereached by the crystalline index, calculated from XRD

data, and by melting enthalpy, measured by DSC. On

further storage, a strong decrease in L00 and a continu-

ous increase in the L 0 curve is observed from ultrasonic

experiments, whereas XRD and DSC data indicate a

sluggish growth of crystalline fraction inside the sample.

The more likely explanation of this behaviour is that,

after three days of storage at room temperature, a reor-ganization of crystallites inside starch sample occurs.

Such crystal perfection phenomena have been demon-

strated also by an increase of melting temperature ob-

served by DSC measurements reported in a previous

study (Lionetto et al., 2005).

Fig. 5. Comparison of the extent of retrogradation at 25 �C of wheat

starch extrudates (34% water w.w.b.) as sensed by different techniques.

The consequent more homogeneous distribution of

the crystallites leads to a reduction of L00 and a further

slight stiffening of the starch sample, monitored by the

continuous and gradual increase of L 0.

The close correlation among the acoustic properties

of starch extrudates and other physical properties, mea-surable by DSC and XRD, is an indication of the valid-

ity of the ultrasonic wave propagation in monitoring the

retrogradation process of starch based systems.

4. Conclusions

In this work, the propagation of ultrasonic longitudi-nal waves was applied to monitor non-destructively the

retrogradation process of extruded wheat starch. A spe-

cifically developed experimental set-up, consisting of an

ultrasonic probe in contact with the starch sample and a

purpose built sample-holder, enabled a continuous mea-

surement of ultrasonic velocity and attenuation during a

several days lasting experiment without sample

dehydration.Ultrasonic wave propagation was able to monitor the

recrystallisation of amylopectin molecules because the

acoustic behaviour of the material is greatly affected

by the changes in its viscoelastic properties during

retrogradation.

An increase in the ultrasonic velocity with storage

time was a good indication of the growth of the elastic

properties arising from the increased crystalline fractionin starch extrudates. The bell-shaped behaviour of the

ultrasonic wave attenuation suggested a high degree of

viscous dissipation of the ultrasonic energy in corre-

spondence of the attenuation peak and, consequently,

a more homogeneous reorganization of crystallites in

starch samples.

The potential of the ultrasonic wave propagation to

be used as a high frequency DMA technique for a con-tinuous monitoring of retrogradation was assessed by

comparing ultrasonic results with those obtained from

low frequency dynamic mechanical experiments.

The results of the ultrasonic monitoring of starch ret-

rogradation were in good agreement with those ob-

tained from X-ray diffraction and differential scanning

calorimetry. The close correlation among the acoustic

properties of starch extrudates and other physical prop-erties, measurable by DSC and XRD, was a further indi-

cation of the validity of the ultrasonic wave propagation

in monitoring the retrogradation process of starch based

systems.

Acknowledgements

The present work was carried out with the support of

the Marie Curie Fellowship financed by the European

8 F. Lionetto et al. / Journal of Food Engineering xxx (2005) xxx–xxx

ARTICLE IN PRESS

Community (EC Contract Number: HPMT-CT-2001-

00404). The authors would like to thank Mrs V. Street

for assistance in the extrusion of the samples.

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