Interaction of granular maize starch with lysophosphatidylcholine evaluated by calorimetry, mechanical and microscopy analysis

Interaction of granular maize starch with lysophosphatidylcholine

evaluated by calorimetry, mechanical and microscopy analysis

Jorge F. Toro-Vazqueza,*, Carlos A. Gomez-Aldapab, Antonio Aragon-Pinaa,Edmundo Brito-de la Fuentea, Elena Dibildox-Alvaradoa, Miriam Charo-Alonsoa

aUniversidad Autonoma de San Luis Potosı, Facultad de Ciencias Quımicas-CIEP, Av. Dr Manuel Nava 6, Zona Universitaria 78210,

San Luis Potosı, Mexico City, MexicobUniversidad Autonoma de Queretaro, Facultad de Quımica-PROPAC, Cerrro de las Campanas SN, Queretaro, Qro, Mexico

Received 10 April 2002; revised 24 February 2003; accepted 3 March 2003

Abstract

In this study we evaluated the thermo-mechanical properties of maize starch pastes (80% wt/wt) under the effect of exogenous

lysophosphatidylcholine (LPC) using differential scanning calorimetry (DSC), dynamic mechanical spectrometry (DMS), and scanning

electron microscopy (SEM). Particular attention was paid to the development of the amylose-LPC inclusion complex. Results from SEM and

DSC showed that with no exogenous LPC, granular maize starch developed the amylose network structure for starch gelling at 80–95 8C. In

comparison, at 1.86 and 3.35% of LPC, heating up to 130 8C was needed to develop the three-dimensional network required for starch

gelling. Results showed that at these LPC concentrations LPC interacted mainly with amylose within the starch granule. At concentrations

$8.26% the LPC interacted with amylose both inside the granule and on the granule’s surface. At such LPC concentrations heating to 130 8C

did not fully develop the starch network structure for gelling. These results suggested that a higher thermal stability was achieved by starch

granules because of LPC inclusion complex formation. DSC or DMS did not detect the development of this complex, probably because its

formation took place below the onset of gelatinization under conditions of limited molecular mobility. Subsequently, a lower level of

organization (i.e. complex in form I) was achieved than in the complex developed at high temperature and water excess (i.e. complex in form

II). On the other hand, the changes in the starch granule structure observed by SEM as a function of the time–temperature variable were well

described by the phase shift angle ðdÞ rheograms for starch pastes with and without addition of LPC.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: Lysophosphatidylcholine; Maize; Gelatinization; Starch granule; Rheology

1. Introduction

Three events occur during conventional time–tempera-

ture processing of starch, namely gelatinization, gelation,

and retrogradation (Biliaderis, 1991). All these events are

metastable processes resulting from the starch–water

interaction. However, the extent to which each process

occurs depends on the starch type (Jane et al., 1999; Boltz

and Thompson, 1999) (i.e. waxy and high amylose

starches), water to starch ratio, and presence of solutes

such as monosaccharides (Salde and Levine, 1991;

Eliasson, 1992; Yuan and Thompson, 1998), salts

(Paredes-Lopez and Hernandez-Lopez, 1991), lipids

(Godet et al., 1995; Villwock et al., 1999; Kim et al.,

1992), and phospholipids (Jovanovich et al., 1992). Thus,

0733-5210/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/S0733-5210(03)00026-2

Journal of Cereal Science 38 (2003) 269–279

www.elsevier.com/locate/jnlabr/yjcrs

* Corresponding author. Tel.: þ52-444-8262450; fax: þ52-444-

8262371/72.

E-mail address: [email protected] (J.F. Toro-Vazquez).

Abbreviations: LPC, lysophosphatidylcholine; DSC, differential

scanning calorimetry; DMS, dynamic mechanical spectrometry; SEM,

scanning electron microscopy; To, onset temperature of DSC transition

peak; Tp, peak temperature of DSC transition peak; Te, ending temperature

of DSC transition peak; DH, enthalpy for the transition peak; LVR, linear

viscoelastic region for rheological measurements; s.d., standard deviation;

w.b., wet basis; d.b., dry basis; DHG, transition energy for starch

gelatinization; DH1M, transition energy for inclusion complex melting

during the initial heating; DH2M, transition energy for inclusion complex

melting during reheating; DHC, transition energy for complex

crystallization.

amylose, and possibly the longest linear branches of

amylopectin, develop inclusion complexes with fatty

acids, monoglycerides, and lysophosphatidyl choline

(LPC). Formation of amylose–lipid inclusion complexes

produces a transition in the amylose molecular structure

from a disordered coil to a helix structure (i.e. helical

inclusion complex) (Hoover, 1998; Biliaderis and Tono-

gai, 1991). This process increases the molecular structural

order of amylose, a process commonly associated to a

crystallization process responsible for the V-type X-ray

diffraction pattern found in some high amylose starches

(Hoover, 1998; Biliaderis and Tonogai, 1991) (e.g.

amylomaize, wrinkled pea starch). Nevertheless, the

crystalline nature of the complex is in debate since most

native cereal starches fail to yield the characteristic V-type

crystallization diffraction pattern. However, the lack of V-

type X-ray diffraction does not necessarily establish the

absence of amylose–lipid complex. It just indicates the

absence of amylose helices organized in crystalline three-

dimensional structures, yet the presence of the amylose–

lipid complex might be as an amorphous structure

(Hoover, 1998; Biladieris, 1992). In fact, the presence of

amylose complexed with monoacylglycerides and lyso-

phospholipids in native maize starch has been established

by solid state NMR (Morrison et al., 1993). On the other

hand, a study by Le Bail et al. (1999) using simultaneous

thermal and synchrotron X-ray diffraction measurements,

showed that crystallinity by the amylose–lipid complex

occurs in native maize starch at water contents above 50%

(w.b.) only after complete gelatinization of starch. Results

from different investigations (Hoover, 1998; Biladieris,

1992; Le Bail et al., 1999) show that the amorphous (i.e.

form I) or crystalline (form II) structure accomplished by

the amylose–lipid complex depends on the interactions

between heating temperature, water content in the starch–

lipid system, and extent of amylose leached out from the

starch granule.

In food systems, lipids interact with starch in the

granular state. Nevertheless, most of the research done has

used extracted amylose, and interaction with lipids is

achieved at temperatures well above gelatinization tem-

perature for granular starch (Godet et al., 1995; Liu et al.,

1997). It is essential to understand the amylose–lipid

complex formation with granular starch under temperature

conditions used in food processing. The aim of this study

was to investigate the interaction between granular maize

starch and exogenous LPC under time– temperature

conditions where swelling, gelatinization, and gelation of

starch occur. LPC is a main component of polar lipids in

cereal starches and particularly in maize starch (Morrison,

1988). The methodologies used in this study included

differential scanning calorimetry (DSC), dynamic mechan-

ical spectrometry (DMS), and scanning electron

microscopy (SEM).

2. Materials and methods

2.1. Starch samples

Dent maize was sampled from storage silos (MASECA,

Guadalajara, Jal., Mexico). The grains were hammer-milled

(Pulvex 200, 0.8 mm aperture sieve; Pulvex, Mexico) and

maize flour was obtained. Wet milling was carried out at

5 8C to avoid annealing at sub-gelatinization temperatures

(Krueger et al., 1987). Native starch isolation was carried out

following the procedure previously described (Toro-Vaz-

quez and Gomez-Aldapa, 2001). The chemical analysis

indicated a starch purity of 95.6% (^2.8%, d.b.), 22.4%

(^1.1% d.b.) amylose, no detectable protein, and 6.8%

(^0.1%, w.b.) of moisture. The methods of analysis are

reported elsewhere (Toro-Vazquez and Gomez-Aldapa,

2001).

2.2. Differential scanning calorimetry

Previous to the DSC analysis L-a-lysophosphatidyl-

choline (approx. 99% purity, Sigma Chemicals, St Louis,

Missouri, USA) solutions were prepared at 0.46, 0.93, 2.33,

4.66, and 9.32% (wt/wt) using deionized water. In stainless

steel pans (Perkin Elmer, Norwalk, CT, USA) 20 mg of LPC

solution (i.e. 0.46–9.32% LPC) or deionized water (i.e. 0%

LPC) was added with a micro syringe to 5 mg of granular

starch. With this model system a constant starch to LPC

solution (or water) ratio of 1:4 was obtained with LPC

concentrations of 0, 1.86, 3.35, 8.26, 16, and 30.4%

(%wt/wt starch). Samples were analysed in a TA Instrument

Model 2920 (TA Instrument Inc. New Castle, DE, USA)

previously calibrated with Indium (melting

temperature ¼ 156.6 8C, melting heat ¼ 28.45 J/g). The

baseline from 20 to 140 8C was obtained with both reference

and sample cells empty. The temperature program used

involved heating from 30 to 130 8C (i.e. first heating stage)

and maintaining the system at this temperature for 1 min.

Then, cooling from 130 to 30 8C (i.e. cooling stage) and

maintaining this temperature for 2 min. Finally, the system

was reheated from 30 to 130 8C (i.e. reheating stage). In all

cases the heating/cooling rate used was 5 8C/min. The onset

ðToÞ, peak ðTpÞ, and ending ðTeÞ temperature for the different

transitions were determined using the first derivative of the

heat capacity of the sample calculated with the DSC

software library. Thus, To and Te were established as the

temperatures where the first derivative of the heat capacity

of the sample initially departed from or returned to the

baseline, respectively (Fig. 1). In contrast, Tp was

established as the temperature where the first derivative of

the heat capacity of the sample crossed the baseline (i.e. the

inflexion point of the transition curve, Fig. 1). In the same

way, enthalpy (DH, J/g of starch d.b.) for the different

transitions was calculated by integration of the correspond-

ing endothermal or exothermal peak. Integration was done

between the respective To and Te with the DSC software. To

J.F. Toro-Vazquez et al. / Journal of Cereal Science 38 (2003) 269–279270

compensate for the difference in heat capacity associated

with the excess of water (80% wt/wt) in the starch paste, a

sealed pan with 20 mg of water was used in the reference

cell. At least duplicate thermograms were done.

2.3. Scanning electron microscopy

Using the procedure described above, additional starch

samples were prepared. The system was heated in the DSC

(5 8C/min) starting at 30 8C and after each 5 8C interval the

sample pan was quench cooled (200 8C/min) to 30 8C, taken

out of the DSC, and the starch removed from the pan,

transferred to a test tube, and frozen rapidly by putting it in a

freezer (<25 8C/min; Ultralow temperature Ultima, Revco)

to 220 8C. After 3 h at this temperature the sample was

freeze-dried (240 8C, 50 mbar for 3.5 h). The lyophilized

sample was placed onto an aluminum slide using elec-

trically conductive tape (Bal-Tec, Furstentum Liechten-

stein), and sputter coated with gold at 10 mbar for 90 s

(Polaron SC-7610, Fisson Instruments, CA, USA). The

same procedure was repeated to cover the whole 35–130 8C

interval. Samples were examined and photographed with a

Leica Stereoscan S420i (Cambridge, England).

2.4. Dynamic mechanical spectroscopy analysis

All measurements were done in a dynamic mechanical

spectrometer UDS 200 Parr Physica (Anton Para, Austria)

in the oscillatory mode. A 50 mm-diameter parallel-plate

geometry (MP 31, Parr Physica) was used with a 0.5 mm

gap. Temperature control of the sample was attained with a

Peltier system located in the base of the parallel plate

geometry. The angular velocity was 10 s21, and the

heating/cooling rate was 5 8C/min. An environmental

chamber saturated to 100% humidity was used to limit

water evaporation from the sample during measurements.

Starch pastes (1:4 starch to LPC solution) were prepared in

assay tubes at LPC concentrations of 0, 1.86 and 16%

(%wt/wt). The linear viscoelastic region (LVR) of the starch

pastes was determined for the conditions of temperature and

strain percentages shown in Table 1. Then, the storage, loss

modulus (G0 and G00, respectively) and phase shift angle ðdÞ

of the samples were measured within the LVR during starch

swelling, gelatinization, and gelation according to the time–

temperature conditions shown in Table 1. Data capture and

analysis were performed with the UDS 200 software (v1.90,

Physica Mebtechnic Gmbh, Stuttgart, Germany).

Fig. 1. Representative thermogram for maize starch with 1.86% of LPC showing the determination of the onset ðToÞ, peak ðTpÞ, and end ðTeÞ temperatures for

starch gelatinization with the use of the baseline and first derivate of the heat flow.

Table 1

Interval of strain applied to starch as a function of temperature to establish

the linear viscoelastic region (LVR) in the measurement of the rheological

parameters during starch heating and cooling. Heating/cooling rate used

5 8C/min

Stage Condition during the

establishment of the

LVR

Temperature and strain program

applied to the sample

T8

(8C)

Strain

(%)

T8

(8C)

Strain in the LVR

(%)

Heating 50 0.1–100 25–50 75

60 0.1–100 50.1–60 65

65 0.01–10 60.1–65 2.5

70 0.01–10 65.1–70 1.7

80 0.01–10 70.1–80 1.5

90 0.01–10 80.1–90 0.95

95 0.01–10 90.1–95 0.66

Isothermal 1 min to 95 0.01–10 1 min to 95 0.45

Cooling 90 0.01–10 95–90 0.4

80 0.01–10 89.9–80 0.3

25 0.01–10 79.9–25 0.25

J.F. Toro-Vazquez et al. / Journal of Cereal Science 38 (2003) 269–279 271

3. Results and discussion

Fig. 2 shows the maize starch thermograms at 0, 1.86,

and 16% of LPC concentrations. As previously indicated,

results from different investigations (Hoover, 1998; Bila-

dieris, 1992; Le Bail et al., 1999) show that the amorphous

(i.e. form I) or crystalline (form II) structure developed by

the amylose–lipid complex depends on the interactions

between heating temperature, water content in the starch–

lipid system, and extent of amylose released from the starch

granule. Then, given the water content (80% wt/wt) and

the DSC temperature program used the starch and starch-

LPC transitions evaluated in the first heating stage were

gelatinization and inclusion complex melting (Fig. 2(A)),

during the cooling stage the inclusion complex crystal-

lization (Fig. 2(B)), and in the reheating stage the second

melting of the complex (Fig. 2(C)) (Biladieris, 1992; Le

Bail et al., 1999). The second endotherm observed during

the first heating stage was considered, based in the results of

Le Bail et al. (1999), a melting endotherm of crystalline

structures and not just a decomplexing endotherm of

the amorphous complexes. Such results showed that

Fig. 2. Thermograms of granular maize starch with 0% (A), 1.86% (B), and 16% (C) of LPC.

J.F. Toro-Vazquez et al. / Journal of Cereal Science 38 (2003) 269–279272

crystallinity by the amylose–lipid complex, measured

simultaneously by thermal and synchrotron X-ray diffrac-

tion, occurs in normal maize starch at water contents above

50% (w.b.) only after complete gelatinization of starch

(Le Bail et al., 1999). For normal maize starch at 80% water

(w.b.), as the one used in this investigation, such crystal-

lization was observed at 80–82 8C (Le Bail et al., 1999).

Consequently, the following discussion of results was done

within this framework.

During the first heating stage the addition of LPC

increased the endotherm present at temperatures above the

Te of the corresponding endotherm for maize starch

gelatinization (Fig. 2(B) and (C)). This second endotherm,

associated with the melting of the LPC inclusion complex,

had To in the range between 91.3 and 94.3 8C and Te

between 106.8 and 109.2 8C (Fig. 2(B) and (C)). This

endotherm, although present (To < 93 8C, Te < 105.6 8C,

and DH ¼ 0:59 J=g; s.d. ¼ 0.026; Fig. 2(A)) in the absence

of exogenous LPC, was less apparent. Based on previous

results (Toro-Vazquez and Gomez-Aldapa, 2001), the

native phosphorous concentration (125.4 ^ 0.4 mg/g

starch) present in the maize starch used in this investigation

indicated a tentative maximum LPC concentration of

206 mg/100 g starch. Such concentration (0.206% wt/wt)

is lower than the smallest concentration of added LPC used

in this study (1.86% wt/wt). Then, the limited amount of

native LPC present in the maize starch might be the reason

for the difficult in observing the second endotherm during

the first heating. On the other hand, in the present study no

exothermic contribution, associated with the development

of crystallinity during the formation of the inclusion

complex (Hoover, 1998; Biliaderis and Tonogai, 1991)

was apparent between the starch gelatinization endotherm

and the second endotherm (i.e. amylose-LPC melting)

(Fig. 2(A)), even upon addition of LPC (Fig. 2(B) and (C)).

The exotherm for the inclusion complex crystallization was

just observed during the cooling stage, after the heating

stage (e.g. 30–130 8C; Fig. 2). Upon reheating, the complex

melted again as made evident by an endotherm with To

between 88.7 and 99.1 8C and Te between 107 and 113.7 8C

(Fig. 2). Results from other investigations (Biladieris, 1992;

Le Bail et al., 1999) suggest the use of temperatures up to

150–160 8C and heating rates lower that 5 8C/min (i.e. 1 or

3 8C/min) to observe, during the first heating, the exotherm

corresponding to the crystallization of amylose– lipid

complexes. Such conditions would allow the separation of

the melting endotherm of poorly crystalline complexes from

the crystallization exotherm of amylose–lipid complexes,

which upon further heating melt at higher temperatures

(Biladieris, 1992; Le Bail et al., 1999). However, such

conditions were not used in this investigation. On the other

hand, it is important to point out that To, Tp, and Te for the

gelatinization endotherm was not significantly affected by

the LPC concentration (data not shown).

The effect of exogenous LPC on the transition energy for

starch gelatinization ðDHGÞ, inclusion complex melting

during heating ðDH1MÞ and reheating ðDH2MÞ stages, and for

complex crystallization ðDHCÞ is shown in Fig. 3. It is

apparent that DHG increased above the native values (8.83 J/

g, s.d. ¼ 0.68) when 1.86% (10.71 J/g, s.d. ¼ 0.13), 3.35%

(10.48 J/g, s.d. ¼ 0.64), and 8.26% (9.86 J/g, s.d. ¼ 0.08) of

LPC were present in the system ðP , 0:05Þ. Above 8.26%

of LPC the DHG decreased even to values below the ones

observed in native starch (i.e. at 30.4% of LPC,

DHG ¼ 6:80 J=g; s.d. ¼ 0.15; P , 0:05). After cooling and

further reheating DH2M showed a linear increment as

exogenous LPC concentration increased from 0% (0.5 J/g,

s.d. ¼ 0.02), 1.86% (3.26 J/g, s.d. ¼ 0.41) to 3.35% (5.65 J/

g, s.d. ¼ 0.73), attaining a plateau at 8.26% (6.96 J/g,

s.d. ¼ 0.29) and 16% (7.12 J/g, s.d. ¼ 0.15) of LPC, and

then decreasing at 30.4% of LPC (4.88 J/g, s.d. ¼ 0.05). It is

important to point out that DH2M was statistically the same

as DH1M up to an LPC concentration of 3.35% ðP , 0:05Þ.

Nevertheless, DH2M was significantly higher than DH1M in

the starch systems at exogenous LPC concentrations greater

than 8.26% ðP , 0:005Þ (Fig. 3). This showed that upon

addition of exogenous LPC $ 3.35%, linear starch com-

ponents (i.e. amylose and the longest amylopectin branches)

within the granules did not fully interact with LPC after

starch gelatinization during the first heating stage. At such

exogenous LPC concentrations, additional formation of

complex was developed during heating to 130 8C (i.e. above

Te of gelatinization). This additional complex formation

was measured during the cooling stage by the exothermic

heat for development (i.e. crystallization) of the inclusion

complex ðDHCÞ. Thus, for the whole interval of LPC

concentration investigated DHC did not observe a significant

relationship with DH1M. In contrast, a significant

negative linear relationship was observed between DHC

and DH2M ½DHC ¼ 20:817ðDH2MÞ2 1:173; R20:9730;

P0:005�. This confirmed that during the first heating stage

amylose and the longest amylopectin branches within starch

granules did not fully interact with LPC after starch

gelatinization, particularly at LPC $ 3.35%.

The above results were additionally interpreted with

photomicrographs obtained for starch and starch-LPC

systems as a function of temperature (Figs. 4–6). Addition-

ally, it was considered that at the water content used

molecular diffusion (i.e. mobility) was enough to allow

accessibility of amylose by LPC diffusion inside the granule

and also amylose leaching outside the granule during starch

swelling (Le Bail et al., 1999). In consequence, both

processes, the molecular interaction inside and outside the

starch granule, were involved in the amylose-LPC complex

formation during heating of maize starch. Obviously, the

first process occurred under conditions of lower water

availability than in the second process. The reason for this is

the constant change of moisture content occurring within the

starch as water diffuses from the outside (i.e. 100% water)

toward inside the granule (i.e. 6.8%) during the DSC

determination (Biliaderis, 1991; Toro-Vazquez and Gome-

z-Aldapa, 2001). Overall, it was evident that when

J.F. Toro-Vazquez et al. / Journal of Cereal Science 38 (2003) 269–279 273

Fig. 4. Micrographs of maize starch with 0% exogenous LPC at 65 8C (A), 80 8C (B), 95 8C (C), and 130 8C (D). Magnification 1500 £ .

Fig. 3. Changes in DH for starch gelatinization ðDHGÞ, dissociation ðDH1M and DH2M), and formation of the amylose—LPC complex ðDHCÞ as a function of

LPC concentration. X, starch gelatinization; A, melting of amylose—LPC complex, first heating; S, complex formation during cooling; O, melting of

amylose—LPC complex during reheating.

J.F. Toro-Vazquez et al. / Journal of Cereal Science 38 (2003) 269–279274

Fig. 5. Micrographs of maize starch with 1.86% exogenous LPC at 65 8C (A), 80 8C (B), 95 8C (C), and 130 8C (D). Magnification 1500 £ .

Fig. 6. Micrographs of maize starch with 16% exogenous LPC at 65 8C (A), 80 8C (B), 95 8C (C), and 130 8C (D). Magnification 1500 £ .

J.F. Toro-Vazquez et al. / Journal of Cereal Science 38 (2003) 269–279 275

compared to starch with no added LPC (Fig. 4), in the

presence of exogenous LPC (Fig. 5) some starch retained its

granular structure at temperatures above gelatinization (i.e.

80 and 95 8C). We hypothesize that during the initial heating

at LPC concentrations of 1.86 and 3.35%, the phospholipid

diffusing along with water toward the inside of the granule

during starch swelling, interacted with the more accessible

amylose, i.e. amylose in the amorphous regions. It has been

demonstrated that complexes formed at low temperatures

(i.e. below 70–80 8C) are amorphous in nature (e.g. form I)

with very low or no crystallographic register (Hoover, 1998;

Biladieris, 1992). Nevertheless, these complexes produce a

melting endotherm (Biladieris, 1992), and the higher DHG

observed at LPC concentrations of 1.86 and 3.35% might be

the result of the concomitant events of starch gelatinization

and decomplexing of the amorphous complexes (Fig. 3).

Additionally, as observed in Fig. 5 at 1.86% of LPC, heating

to 130 8C was needed to develop the three-dimensional

network required for starch gelling. In comparison, with no

added LPC granular maize starch at temperatures of

80–95 8C developed the network structure for starch gelling

(Fig. 4). On the other hand, at exogenous LPC $8.26%

(Fig. 6), it was apparent that a film was present on the

granular surface, which was quite evident with 16% LPC at

95 8C (Fig. 6C). We suggest that at LPC .8.26%, the

phospholipid interacted with amylose inside the granule, but

as LPC concentration increased it interacted also with

amylose on the granule surface. As a result of the

LPC-amylose interaction on the granule surface, starch

achieved higher thermal stability and some granular

structure was retained after the first heating to 130 8C

(Fig. 6(D)). Under these LPC conditions heating to 130 8C

did not fully develop the starch network structure required

for gelling. These observations suggested that the decrease

in DHG at LPC $8.26% (Fig. 3) was the result of partial

starch gelatinization.

Then, the results presented here suggest that LPC

diffused to the interior of the starch granule before

gelatinization takes place. Consequently, the initial

interaction between LPC with linear components (i.e.

amylose and long branches of amylopectin) of starch took

place below the onset of gelatinization ðToÞ under

conditions of limited molecular mobility. Therefore, the

inclusion complex initially developed within the starch

granule would have a different level of organization (i.e.

complex in form I) than the complex developed further on

with amylose leached out of the granule under conditions

of high temperature and water excess (i.e. complex in

form II).

On the other hand, we used the regression equation

developed by Sievert and Holm (1993) to quantify

amylose in granular starch. This technique is based on

the development of the amylose-LPC inclusion complex

with exogenous LPC, which is quantified by DSC through

DH2M. Thus, with DH2M data determined in the present

investigation the Sievert and Holm equation (Sievert and

Holm, 1993) showed that upon addition of 1.86% LPC,

the amylose participating in the inclusion complex was

11.07% (d.b.). In the same way, for 3.35, 8.26, 16, and

30.4% of exogenous LPC, the corresponding amylose

concentrations were 19.16, 23.60, 24.14, and 16.55%,

respectively. Thus, given the actual concentration of

amylose in the granular maize starch used in the present

research (22.4% d.b.), an LPC concentration lower than

8.26% was not enough to interact with all amylose present

in the starch granules. In contrast, at 8.26 and 16% LPC

there is a tendency to overestimate amylose probably

because DH2M also included the melting of the inclusion

complex of LPC with the long linear regions of

amylopectin. At 30.4% LPC, DH2M underestimates

amylose since some granular starch structure was retained

even after reheating at 130 8C, limiting the release of

amylose and the subsequent development of the inclusion

complex. Therefore, determination of amylose by DSC

through DH2M measurements must be considered with

reservations since the correct LPC-amylose ratio must be

used in the determination. Unfortunately, such LPC-

amylose ratio is not known in advance.

The rheograms for granular maize starch at different

exogenous LPC concentration are shown in Fig. 7. The

use of d in viscoelastic systems is based on the

measurements of G0 and G00 modulus. Thus, in a purely

viscous system (i.e. water) d is 908, and subsequently

G0 ¼ 0 and G00 ¼ G* ; where G* is the complex modulus.

However, during granular starch swelling a decrease in d

should accompany the water restriction in the system with

the corresponding increase in the storage modulus (i.e.

G0 . 0). Eventually, if the system becomes gelled d! 08

or when fully crystallized (i.e. purely elastic) d ¼ 08; and

subsequently G0 ¼ G* and G00 ¼ 0. Such changes occur

along with a d peak associated with the heat released by

the molecular association during gelling or crystallization.

Then, changes in the viscoelastic properties of starch–

water dispersions might be established by the relative

proportions between G0 and G00 profiles. The d profile

encompasses both parameters evaluating, in a quite

sensitive way, viscoelastic changes of complex systems

such as the one here studied. Then, the d profile of the

starch-LPC-water was evaluated applying the time–

temperature program shown in Table 1 (Fig. 7).

Thus, below the temperature where reversible starch

swelling occurred ðT8 , 55 8CÞ, no significant d changes

were observed. When a temperature between 60 and 65 8C

was achieved, a dramatic decrease in d occurred (Fig. 7).

This decrease in d was associated with the water

restriction in the system because of starch swelling.

Thus, at 0% LPC most starch granules were swollen at

65 8C (Fig. 4(A)) in comparison to starch granules under

no temperature effect (Fig. 8(C)). The decrease in d

occurred at higher temperature in the presence of LPC

(Fig. 7, 1.86 and 16% of exogenous LPC), because of the

formation of the amylose-LPC complex both within

J.F. Toro-Vazquez et al. / Journal of Cereal Science 38 (2003) 269–279276

Fig. 8. Micrographs of granular maize starch after heating to 95 8C and cooling (5 8C/min) to 40 8C. Starch with 0% exogenous LPC (A), 16% exogenous LPC

(B) and control (starch without thermal treatment) (C).

Fig. 7. Phase shift angle ðdÞ for granular maize starch as a function of the time-temperature variable. Rheograms for 0, 1.86, and 16% exogenous LPC are

shown.

J.F. Toro-Vazquez et al. / Journal of Cereal Science 38 (2003) 269–279 277

the granule and on the granule surface. These events

limited water uptake from the granules and consequently

their swelling (Figs. 5 and 6). In fact, the higher the LPC

concentration the higher the temperature at which a

decrease in d was observed (Fig. 7). Such a relationship

between LPC concentration and starch swelling was not

detected through DSC.

After the corresponding Te for starch gelatinization with

no added LPC, the rheogram showed a d peak around 95 8C

(Fig. 7), which corresponded to the starch gel structure

development through a three-dimensional network as

observed by the SEM photomicrographs (Figs. 4(C) and

8(A)). However, in the presence of exogenous LPC, such a d

peak was smaller (i.e. 1.86% LPC, Fig. 7) and less evident

(i.e. 16% LPC, Fig. 7) with the subsequent lack of

development of the three-dimensional network (Figs. 5(C),

6(C) and 8(B)). Under these conditions, the granular

structure of starch was still evident, although the granules

were swollen and deformed (Fig. 8(B)). As a result, during

cooling and in the absence of exogenous LPC, a second

major d peak was observed around 55–60 8C (Fig. 7). Such

a d peak, associated with amylose gelling (Fig. 8(A)), was

almost imperceptible in the presence of LPC (Fig. 7, 1.86%

and 16% LPC; Fig. 8(B)). This last phenomenon was

associated with the limited extent of amylose leached out

the granule, due to the inclusion complex formation within

and on the starch granule.

Thus, the thermal behavior of granular starch is much

more complicated than that exhibited by conventional

thermoplastic materials. This is because of the physico-

chemical and structural changes that occur in starch during

heating and cooling cycles. Such a situation is further

complicated by the molecular interactions of starch

components with compounds naturally present in the

granule (i.e. native LPC) or intentionally added to the

system. More than one analytical technique, as shown in this

investigation, is needed to study the starch behavior under

time–temperature conditions and its implications in texture,

stability and overall food quality. Unfortunately, the

processes used in the food industry for heat treatment of

starch-based products are quite different from the situation

in a rheometer chamber or a DSC pan. Conditions such as

stirring, heating rate, and pressure must affect the develop-

ment of the inclusion complex. The results presented here

where the complex is developed between starch components

within the granule, are quite different to the situation where

both amylose and LPC in solution interact in solution and

then the resulting complex is precipitated to be studied.

Under the former conditions the complex formation and its

effect on granular starch thermal behavior depends on the

amount of lipids naturally present or added. In contrast, with

a precipitated complex the starch structure is already lost

and thermal behavior of the complex is independent of the

amount of lipids added. In food systems, most of the time

lipids interact with starch in the granular state.

Acknowledgements

The present work was supported by CONACYT through

the grant # 485100-5-3939PB and the Universidad Auton-

oma de San Luis Potosi through the grant C01-FAI-10-

19.78.

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