Bulletin UASVM Agriculture, 69(2)/2012
Print ISSN 1843-5246; Electronic ISSN 1843-5386
Influence of Physical Treatments on the Potato Starch Granules Micro- and
Ultrastructure
Maria Iasmina MOZA1), Monica MIRONESCU 2), Adrian FLOREA3)
1
2
Faculty of Sciences, “Lucian Blaga” University of Sibiu, Romania; [email protected]
Faculty of Agricultural Sciences, Food Industry and Environmental Protection, “Lucian Blaga”
University of Sibiu, Romania; [email protected]
3
Faculty of Medicine, "Iuliu Haţieganu" University of Medicine and Pharmacy Cluj-Napoca,
Romania; [email protected]
Abstract: In this research, potato starch was subjected to physical treatments in order to analyse the
changes at the micro- and ultrastructural levels and finally to identify the optimal conditions for
disintegrating the starch granules. For the analysis, 10% aqueous solutions of potato starch were
treated with a) microwaves; b) heat and c) heat combined with ultrasounds. For each treatment type,
the duration was 5 minutes, excepting the microwave treatment, where the time was 1 and 5 minutes;
heat power varied from 180W to 900W at the tests with microwave, temperature varied between 65oC
and 100oC in the thermal experiments and at the thermal analysis combined with ultrasounds
temperature had values between 35oC and 85oC. The effect of these treatments was analysed using
microscopic techniques (optical and scanning electron microscopy) in order to describe the
morphological and micro- and ultrastructural modifications of the starch granules. Results indicated
that the morphological and microstructural differences mostly depend on the chosen treatment and on
the working conditions. The microwaves treatment was shown to be more effective on disintegrating
the starch granule than the thermal action combined with ultrasounds or the thermal treatment alone.
Starch granule was cracked at the heat power of 180W even after 1 minute of treatment; after 5
minutes, the gel-like structure was clearly formed for all the granules. Ultrasounds also modified the
granule structure by cracking. High temperature (minimal 85oC) was essential for the propagation of
ultrasounds or for the thermal treatment in order to assure the efficacy of the physical treatments on
the granule.
Keywords: potato starch, microwave, ultrasounds, thermal, microstructure, SEM, OM
INTRODUCTION
Starch is the most common used polysaccharide in human nutrition and the most
abundant reserve carbohydrate stored in different plant organs including seeds, fruits, tubers
and roots. Starch is included in many foods because it contributes greatly to their textural
properties - having therefore multiple industrial applications (Herceg et al., 2010), and
because is one of the most common easy to recover biopolymer (Tomasik, 2003).
Starch granule has a complex structure composed of amorphous regions containing
especially amylose (AMY) and crystalline regions with amylopectin (AMP) as major
compound (Donald et al., 2001). When heated, in the presence of excess water, starch
granules lose their crystallinity, absorb large amounts of water, leach out AMY followed by
AMP fusion and loss of semi-crystalline structure; finally, a gel or a pasty mass is formed.
This process is called gelatinization and has as main effect the starch granule destabilization
(Atkin et al., 1999; Waigh et al., 2000; Che et al., 2007a, b).
Classical, starch gelatinises through the controlled application of heat and moisture.
The thermal treatment is a procedure known from ancient times and extensively studied, in
which a number of irreversible processes take place resulting in losing the ordered structure
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and even the identity of the starch granules and forming a gel-like substance (Molina-García,
2007; Chung et al., 2009). This treatment changes various properties of starch, such as
swellability, solubility, pasting properties, gel texture and crystallinity, and promotes its
increased thermal stability and reduced retrogradation (Adebowale et al., 2005). The effect of
the heat treatment is attributed to hydrogen bonds formed between water and molecular chains
within starch granules (Singh et al., 2009) and is dependent on the starch concentration and
temperature (Mironescu et al., 2011).
There is an increasing trend toward the use of microwave applications in food
processing due to the fact that microwave energy is more efficient than the traditional heating
process since it ensures homogenous operation in the whole volume of substance, greater
penetrating depth, and selective absorption (Rajko et al., 1997). Some previous studies have
shown that the microwave heating influences the process of swelling and gelatinization of
starch granules and thus could be very efficient in destroying the starch crystalline
arrangement and obtaining a soft gel (Palav and Seetharaman, 2006; 2007). Anderson and
Guraya (2006) studied the effects of the microwave on digestibility, pasting, and
morphological properties of the heated starches in order to improve the bioconversion and
bioethanol production from raw sago starch. Nikolić et al. (2008) also investigated the
microwave-assisted liquefaction as a pretreatment for the bioethanol production by SSF of
corn meal with Saccharomyces cerevisiae var. ellipsoideus. Some previous studies have
shown that application of microwave irradiation pretreatment may significantly increase the
conversion of starch materials to glucose (Zhu et al., 2006; Palav and Seetharaman, 2007).
Another physical treatment is represented by the ultrasounds. The ultrasonic process
has been confirmed to be applicable to many kinds of starches: corn, potato, tapioca, and
sweet potato (Iida et al., 2008). Ultrasound treatment caused disrupting of starch granules by
cavitational forces and made the granule more permeable to water during the heating step.
When applying more powerful ultrasound, starch granules become much more mechanically
damaged mainly in its amorphous region (Herceg et al., 2010). It has been demonstrated that
at the applied ultrasound frequency, degradation of the starch molecule is caused both by OH
radicals and by mechanochemical effects (Czechowska-Biskup et al., 2005).
The objective of this study was to find a preliminary treatment for disrupting the
compact structure of potato starch granule, in order to facilitate the enzymatic hydrolysis,
using tree physical treatment involving microwave, heat and heat combined with ultrasounds
and to select the most effective one with the optimal parameters.
MATERIALS AND METHODS
Potato starch was kindly provided by Boromir, Sibiu, Romania. For the structural
analyses, 10% aqueous solutions were used. Experiments consisted in treating the starch
solutions with microwaves, heat and heat combined with ultrasounds. For each treatment
several working parameters were selected. For heating, three temperatures were selected:
65°C, 85°C and 100°C. The treatment with ultrasounds was conducted at 45 kHz and at three
temperatures: 35°C, 65°C and 85°C. The treatment with microwaves was performed at three
powers: 180, 540 and 900W. The exposure time was 5 minutes for all the three sets of
experiments, excepting the experiments with microwaves, where the treatment time was 1 and
5 minutes.
The effect of treatments on the micro- and ultrastructural modifications of the starch
granules was analysed using microscopic techniques. Optical microscopy (OM) was realised
with the Axiostar Plus microscope (Carl Zeiss, Göttingen, Germany) at 100× and 1000×. At
the OM analysis, iodine was added for better visualization of starch transformations. Iodine as
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Lugol solution (iodine and potassium iodide in water) is used as indicator of gelatinisation
since it colours very well AMY in dark blue and AMP in red-brownish; the native starch
granules are coloured only at the surface because the dye cannot penetrate the granule,
whereas the iodine enters very easy in the destructed granules and colours them (Belitz and
Grosch, 1999).
In order to study their ultrastructural aspects, the starch granules were processed for
scanning electron microscopy (SEM) according to the usual protocol (Flegler et al., 1993).
The different samples containing starch granules were placed on glass cover slips mounted on
aluminium stubs, and dried at the room temperature. Then, the samples were dehydrated and
coated with a thin layer of gold atoms in a Polaron E–5100 plasma-magnetron sputter coater
(Polaron Equipment Ltd., Watford, U.K.). The samples were examined with a JEOL JSM–25
Scanning Microscope (Jeol Ltd. Tokyo, Japan), at 25 KV acceleration voltage and 700× and
1500× magnification. The images were captured with a Deben Pixie–3000 image processor
(Deben U.K. Ltd., Debenham, U.K.).
RESULTS AND DISCUSSION
OM and SEM analysis of the untreated starch granules evidenced the form of the
native potato granules, which were mostly oval and some of them round. The granules had
different sizes; they were compact and insoluble in water (Fig. 1 and 2). The growth rings
were well visualised (Fig. 1B). All granules were coloured in violet by iodine (Fig. 1A)
indicating that they were integral.
A- ×100, B- ×1000
Fig. 1. Untreated potato starch granules in 10%
aqueous solution observed by OM
A- ×700, B- ×1500
Fig. 2. Untreated potato starch granules in 10%
aqueous solution observed by SEM
Fig. 3 and 4 evidence the modifications induced in the starch granule micro- and
ultrastructure by heating in excess of water. Most of the granules became bigger due to
soaking and swelling. Theoretical, potato starch forms already gel at 65oC (Belitz and Grosch,
1999). As Fig. 3A1 shows, most of the granules lost their initial granular structure and
became differently coloured with iodine, but some granules still preserved their shape at this
temperature as observed in Fig. 3A and 4A. When the heating temperature increased at 85oC,
the percent of granules which were destabilised was near 100% (Fig. 3B1). The SEM analysis
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in the sample treated at 65oC evidenced the AMY loss by the starch granule (amylose
leaching) and the formation of junction zones intermediated by AMY, characteristic for a gel
structure as showed also in the literature (Atkin et al., 1999) (Fig. 4A).
Fig. 3. Potato starch granule in 10% aqueous solution at the heat treatment for 5 min visualised by
OM: A: 65 °C, B: 85 °, C: 100 °C / 1- ×100, 2- × 1000
Fig. 4. Potato starch granule in 10% aqueous solution at the heat treatment for 5 min visualised by
SEM: A: 65 °C, B: 85 °, C: 100 °C / 1- ×700, 2- ×1500
When temperature increased to higher values (85oC) the granule modified its shape,
became wrinkled and lost the initial shape (Fig. 3B and 4B). Some pores, visible at SEM,
were observed (Fig. 3B2), being probably formed during AMY leaching.
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The typical gel-like structure was clearly visible at 100oC when the starch granule
was entirely deformed and its contents were melted in a mass substance of AMY and AMP
(Fig. 3C and 4C). After cooling it formed a dense colourless gel.
When ultrasounds were applied at various temperatures, microstructure was also
modified but not entirely: the granule showed some cracks (Fig. 5). The cracks appeared only
when temperature was quite high (85oC) (Fig. 5C), demonstrating that high temperature is
essential for the propagation of ultrasound and for the efficacy of this treatment on the starch
granules. No other significant changes were observed under the temperature of 85oC. The
result shows the bigger importance of heat on the starch gelatinisation, compared with
ultrasounds.
Fig.5. Potato starch granule in 10% aqueous solution at the treatment with ultrasound combined with
heat for 5 min visualised by OM: A: 35 °C , B: 65 °C, C-: 85 °C, / 1- ×100, 2- ×1000
The results obtained at the treatment with microwaves are presented in Fig. 6 and 7.
The granule morphology was strongly modified, appearing wrinkled and dehydrated, even at
the lowest microwave power, 180W and a very short treatment (1 min), as observed in Fig.
6.A1. and 7.A1. Time seems to be very important. When the same treatment was applied for a
longer time (5 min), the damages induced by microwaves were more obvious, the gel
appeared slightly violet at the staining with iodine, sign of its total disorganization (Fig.
6.B1). The action of microwaves with the heat power 180W for 5 min. was similar with that
of temperature 100oC (Fig. 4.C2), whereas the short treatment allowed the granule to preserve
its shape even if irreversible changes occurred, as the SEM image shows (Fig. 7.A1). The
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granular shape was maintained even when the heat powers of 540W and 900W were used
(Fig. 7.A2 and 7.A3).
1
2
3
Fig. 6. Potato starch granule in 10% aqueous solution at the treatment with microwave for 1 min (A)
and 5 min (B) visualised by OM: 1= 180 W, 2= 540 W, 3= 900 W/ a ×100, b ×1000
1
2
3
Fig. 7. Potato starch granule in 10% aqueous solution at the treatment with microwave for 1 min (A)
and 5 min (B) visualised by SEM: 1= 180 W, 2= 540 W, 3= 900 W/ a ×700, b ×1500
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The treatments at the heat powers of 540W and 900W for 5 minutes gave a gel-like
structure (Fig. 7.B2 and 7.B3). These two treatments had as result the water release and starch
cooking; they were very aggressive for the starch granule and not necessary since the action
of microwave with 180W gave starch gelatinisation.
CONCLUSIONS
Three preliminary treatment involving microwave, heat and heat combined with
ultrasound were applied for the disintegration of the potato starch granules in 10% aqueous
solution. The effects of the different treatments were studied using the usual protocols of OM
and SEM.
All the treatments used changed the morphology and microstructure of the potato
starch granules. The thermal treatment had an increased action on the starch granule with the
increase of the temperature from 65oC to 100oC, as OM analysis showed. The SEM images
revealed the stages of gel formation, with the AMY leaching and AMP fusion – processes
very well observed beginning with the temperature 65oC. Several pores, probably remained
from the sites where amylose was released from the granule, were visible in the samples
treated at the lowest temperature. The temperatures of 85oC and 100oC resulted in gels with a
quasi-continuous appearance.
The ultrasounds (45 kHz) combined with heat at temperatures between 35°C to 85°C
didn’t have a significant influence on the potato starch granule. Small ruptures were identified
in the samples treated at 85°C.
When treated with microwaves, all potato granule content was expulsed and formed
a gel mass. Starch granule disintegrated after the exposure at the minimal heat power 180 W
for very short time (1 minute) but still preserved its shape. When the treatment time was
increased to 5 minutes, a gel-like structure was formed. The use of higher powers (540W or
900W) gave products which were dried and cooked.
In comparison with the two other treatments (heat and ultrasounds), the treatment
with microwave was the most effective. This preliminary treatment was selected for a future
research, to be the solution used before the enzymatic hydrolysis.
REFERENCES
1. Adebowale, K. O., Afolabi, T. A. and Olu-Owolabi, B. I. (2005). Hydrothermal treatments of
Finger millet (Eleusine coracana) starch. Food Hydrocolloids 19: 974–983.
2. Anderson, A. K. and Guraya, H. S. (2006). Effects of microwave heat-moisture treatment on
properties of waxy and non-waxy rice starches in Food Chemistry 97 : 318–323.
3. Atkin N., Cheung S., Abeysekera R. and Robards A. (1999). Localisation of amylose and
amylopectin in starch granules using enzyme-gold labelling, Starch/Stärke, 51: 163–172.
4. Belitz, H.D. and Grosch, W. (1999). Food chemistry, Springer Verlag Berlin Heidelberg
5. Che, L. M., Li, D., Wang, L. J., Chen, X. D. and Mao, Z. H. (2007a). Micronization and
hydrophobic modification of cassava starch. Int. J. Food Prop., 10: 527–536.
6. Che, L. M., Li, D., Wang, L. J., Özkan, N., Chen, X. D. and Mao, Z. H. (2007b). Effect of
high-pressure homogenization on the structure of cassava starch. Int. J. Food Prop., 10(4): 911–922.
7. Chung, H. J., Liu, Q. and Hoover, R..(2009). Impact of annealing and heat-moisture treatment
on rapidly digestible, slowly digestible and resistant starch levels in native and gelatinized corn, pea
and lentil starches. Carbohydr. Polym., 75: 436–447.
8. Czechowska-Biskup, R., Rokita, B., Lotfy, S., Ulanski, P. and Rosiak, J. M. (2005).
Degradation of chitosan and starch by 360-kHz ultrasound. Carbohydr. Polym., 60(2): 175–184.
9. Donald, A. M., Kato, K. L., Perry, P. A. and Waigh, T. A. (2001). Scattering studies of the
internal structure of starch granules. Starch/Sta¨rke, 53: 504–512.
310
10. Flegler, S. L., Heckman, I. W. Jr. and Komparens, K. L. (1993). Scanning and transmission
electron microscopy: an introduction. W. H. Freeman & Co., New York.
11. Iida, Y., Tuziuti, T., Yasui, K., Towata, A. and Kozuka, T. (2008). Control of viscosity in
starch and polysaccharide solutions with ultrasound after gelatinization. Innovat. Food Sci. Emerg.
Tech., 9: 140–146.
12. Herceg, I. L., Jambrak, A. R.., Šubarić, D., Brnčić, M., Brnčić, S. R., Badanjak, M., Tripalo,
B., Ježek, D., Novotni, D. and Herceg, Z. (2010).Texture and Pasting Properties of Ultrasonically
Treated Corn Starch. Czech J. Food Sci. 28(2): 83–89.
13. Mironescu, M., Mironescu, I.D. and Oprean, L. (2011). Steady and oscillatory shear
behaviour of semi-concentrated starch suspensions. Procedia Food Sci., 1: 322-327.
14. Molina-García, A. D., Horridge1, E., Sanz1, P. D. and Martino, M. N. (2007). Nanostructure
of starch high-pressure treated granules discovered by low temperature scanning electron microscopy,
in Modern Research and Educational Topics in Microscopy.
15. Nikolić, S., Mojović, L., Rakin, M., Pejin, D. and Savić, D. (2008). A microwave-assisted
liquefaction as a pretreatment for the bioethanol production by the simultaneous saccharification and
fermentation of corn meal. Chem. Ind. Chem. Eng. Q., 14 (4): 231−234.
16. Palav, T. and Seetharaman, K. (2006). Mechanism of starch gelatinization and polymer
leaching during microwave heating. Carbohydr. Polym., 65: 364−370.
17. Palav, T. and Seetharaman, K. (2007). Impact of microwave heating on the physicochemical properties of a starch-water model system in Carbohydr. Polym., 67: 596−604 .
18. Rajko, R., Szabo, G., Vidal-Valverde, C. and Kovacs, E. (1997). Designed experiments for
reducing antinutritive agents in soybean by microwave energy. J. Agri. Food Chem., 45: 3565–3569.
19. Singh, G. D., Bawa, A. S., Riar, C. S. and Saxena, D. C. (2009). Influence of heat-moisture
treatment and acid modifications on physicochemical, rheological, thermal and morphological
characteristics of indian water chestnut (Trapa natans) starch and its application in biodegradable
films. Starch/Sta¨rke 61: 503–513.
20. Tomasik, P., (2003) Chemical and Functional Properties of Food Saccharides. 1st Ed., CRC,
Boca Raton.
21. Waigh, T. A., Gidley, M. J., Komanshek, B. U. and Donald, A. M. (2000). The phase
transformations in starch during gelatinisation: A liquid crystalline approach. Carbohyd. Res., 328 (2):
165−176.
22. Zhu, S., Y. Wu, Z. Y. and C. Wang (2006). Comparison of three microvawe/chemical
pretreatment processes for enzymatic hydrolysis of rice straw. Biosyst. Eng., 93: 279−283.
311