Nienke Lindebooma
Peter R. Changb
Robert T. Tylera
a
Department of Applied
Microbiology and Food Science,
University of Saskatchewan,
Saskatoon, Canada
b
Agriculture and Agri-Food
Canada, Saskatoon, Canada
Analytical, Biochemical and Physicochemical Aspects of Starch Granule Size, with Emphasis
on Small Granule Starches: A Review
Granule size, size distribution and shape are among the most important morphologically
distinguishing factors of starches from different origins. This article provides an overview of
aspects related to starch granule size, including procedures for determining the size, the impact of
granule size on the physicochemical characteristics of starch, and biosynthetic and environmental
determinants of granule size. The focus is on small granule starches, including their isolation and
current and potential utilization.
Keywords: Starch granule; Small granule starch; Granule size determination; Starch isolation
Contents
1
Introduction............................ 89
2
Granule Size and Morphology.............. 89
2.1 Granule size............................ 89
2.2 Granule morphology..................... 90
3
Techniques for the Determination
of Granule Size.......................... 90
3.1
Microscopy.......................r..... 91
3.2 Sieving................................ 91
3.3 Electrical resistance...................... 91
3.4 Laser light scattering..................... 91
3.5 Field flow fractionation.................... 91
4
Granule Size Distribution Models........... 92
4.1 Aggregates............................. 92
4.2 Mono- and bimodal granule size
distributions............................ 92
5
Compositional, Structural and Functional Differences between Small and Large
Granules............................... 92
5.1 Amylose and amylopectin content and structure............................... 92
5.2 Gelatinization behavior.................... 93
5.3 Amylose-lipid interactions................. 93
5.4 Solubility and swelling power.............. 93
5.5 Acid and enzymatic hydrolysis............. 93
6
Determinants of Granule Size.............. 94
6.1
Environment............................ 94
6.2 Biosynthesis............................ 94
6.3 Genetics............................... 95
7
Starch Isolation......................... 95
Correspondence: Peter Chang, Research Scientist of Bioprod-ucts and Bioprocesses National
Science Program, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, S7N 0X2,
Canada. Phone: +306-956-7637, Fax: +306-956-7247, e-mail: Changp@agr.gc.ca.
7.1
Problems encountered in the isolation
of small granule starches.................. 95
7.2 Potential solutions....................... 95
8
Current and Potential Uses of Small
Granule Starch.......................... 96
8.1
Fat replacement......................... 96
8.2 Biodegradable films...................... 96
8.3 Carrier materials......................... 96
9
Conclusions............................ 97
1 Introduction
Starch is composed of two types of molecules, amylose and amylopectin, which are arranged in a
relatively water-insoluble granule of a particular size. Granule size influences the physicochemical
characteristics of starch, as well as the procedures employed in starch refining. Amylose/amylopectin ratio, molecular weight and molecular fine structure also influence the
physicochemical properties of starch. This review presents a summary of literature available in the
area of starch granule size and size determination. The focus is on starch from storage organs and
primarily the specific characteristics of small granule starches, which offer both unique
functionality and unique challenges with respect to commercial-scale refining.
2 Granule Size and Morphology
2.1 Granule size
Granule size and shape are related to the biological source from which the starch is isolated. In
general, granule size may vary from less than 1 μn to more than 100 μm. Generally, granule size
refers to the average diameter of the starch granules. For this, spherical granules
are assumed, which is seldom correct. Granule size may also be expressed as the average length of
the major and minor axes, mean maximum diameter, mean granule volume or mean surface area. In
this article, granule size is expressed as the average spherical equivalent diameter. No precise
categorization of granule size was found in the literature. For this reason, the following classes are
defined: large (>25 μm), medium (10-25 μrn), small (5-10 μm) and very small (<5 μn) granules.
This article focuses on small and very small granule starches. For convenience, the term small
granule starch will be used in reference to both. Tab. 1 is a compilation of granule size data on
small granule starches and starches exhibiting a bimodal size distribution from various sources [113]. Commercial sources of small granule starch include rice, wheat and oat. Other potential
sources of small granule starch include amaranth (Ama-ranthus cruentus L.), quinoa (Chenopodium
quinoa Willd.), taro (Alocasia macrorrhiza L), pigweed (Amaranthus ret-roflexus L), cow cockle
(Saponaria vaccaria L), canary grass (Phalaris canariensis L), cattail (Typha latifolia L), dasheen
(Colocasia esculenta L), grain tef (Eragrostis tef (Zucc.) Trotter) and dropwort (Filipendula
vulgaris Moench), all having granule sizes ranging from 0.5 to
Tab. 1. Granule sizes of starches from different origins.
10 μm. Cow cockle starch has the smallest granule size of any starch studied to date [2]. Small
granule starch may also be obtained by selective fractionation of the small granules from some
cereal starches. Starches of wheat, barley, rye, and triticale (wheat-rye hybrid) are noted for their
bimodal granule size distributions, with differentiation being made between granule fractions with
diameters smaller than and larger than 10 μn. Fractionation is achieved by air classification or
sedimentation [14]. Starch granule size distribution will be discussed in more detail later.
2.2 Granule morphology
Starch granules from different botanical origins differ in morphology. Granules of tuber and root
starches, for example, are oval, although round, spherical, polygonal, and irregular shaped granules
also exist [15]. Taro, dasheen and parsnip (Pastinaca sativa L.) starches consist of very small
granules compared to other root and tuber starches [4]. The small granule size fractions of wheat,
barley, rye and triticale have a different morphology than their larger counterparts. Granules of bean
and pea starches are characterized as thick disks with a 'cut' around the middle or at the ends and an
indentation at one end. Starch granules from fruits and nuts vary in shape. Some nut starches have
unusual granule morphology of half-spheres, although most are round in shape. Granules of small
granule starches are characterized by their very irregular, polygonal shape [4].
3 Techniques for Determination of Granule Size
Generation of granule size distribution data makes it possible to compare starches from different
botanical origins and to assess starch-processing parameters, especially the efficiency of centrifugal
operations [17]. The accuracy of the granule size distribution obtained is dependent on both the size
determination technique and the starch isolation method employed. A satisfactory size determination technique evaluates all granules without missing the small granule fraction, maintains the
granules in an unag-gregated form, and is able to distinguish between starch granules and nonstarch particles. It is also important that the technique incorporates an intrinsic allowance for
granule shape [18]. Furthermore it is a prerequisite that the granules that are analyzed are not
aggregated. Granule size analysis can be performed both in situ as well as in a recovered starch
fraction. Isolation of starch, however, may result in an incorrect granule size distribution
due to the loss of certain granule fractions, particularly the smaller granules, or to granule damage
experienced during isolation.
3.1 Microscopy
Light microscopy is suitable for use in situ by staining a small slice of sample tissue with iodine, as
well as for analysis of an isolated starch fraction [19]. However, this method is laborious and can
not be expected to provide the detailed information that can be obtained with methods such as light
scattering and field flow fractionation. The light microscope method has been developed into what
is called image analysis of optical microscopy (IA0M). Image analysis involves the processing (i.e.
the quantitative evaluation of several features) of an image of an object and enables detailed
manipulation and presentation of the data. A prerequisite to successful image analysis is that the
granules are not damaged during isolation of the starch, and samples must be homogenous [19].
Image analysis is much faster than the traditional microscopic method. However, differentiation
between starch granules and non-starch particles may be difficult. Scanning electron microscopy
(SEM) is frequently used because of the short wavelength of the electron beam, which makes it
possible to determine granule size more accurately than is the case with light microscopy. The resolution possible with SEM also provides a more detailed perspective on granule surface
characteristics and granule morphology [19].
3.2 Sieving
Granule size determination by micro-sieving is a non-microscopic technique whereby granules of
different sizes are separated in a specially-designed sieving apparatus. The relative weights of starch
granules above and below one or more aperture sizes are thereby determined. A shortcoming of this
method is that larger granules tend to be ellipsoidal in shape, hence their size tends to be
understated [20]. In addition, errors resulting from granule aggregation are of particular concern in
sieving methods. Although fractions of small granules (<10 μm) were found to be essentially free of
larger granules, the other sieved fractions were inevitably heterogeneous [20].
3.3 Electrical resistance
The electrical resistance method using the Coulter counter discriminates among particles by how
they affect the electrical resistance of an electrically conductive liquid.
The liquid, with suspended particles, is forced to flow through an orifice having an immersed
electrode on each side [18]. This procedure is not affected by either granule shape or density, and it
is claimed by many workers that the results can be more accurate than those obtained by other
techniques [18]. However, the Coulter counter was shown to be incapable of accurately measuring
granules with diameters less than 3 μrn [21]. Furthermore, the technique appears to over-size when
most of the granules are smaller than 20 μm [18].
3.4 Laser light scattering
With lower angle laser light scattering (LALLS), the light of a parallel-laser beam is deflected at an
angle dependent on the diameter and optical properties of the granules. Small granules scatter the
laser beam of electromagnetic waves at larger angles than do larger granules. This method is fast
and objective because the number of granules measured is greater than 100,000 [19]. It is possible
to evaluate granules as small as 0.1 μm in diameter [21]. A prerequisite for successful analysis
using this technique, however, is that the granules are not damaged [19]. Because most starch
granules are not truly spherical, the granule diameters determined tend to be smaller than those
measured by image analysis techniques [21].
3.5 Field flow fractionation
Field flow fractionation (FFF) techniques are a group of analytical elution methods suitable for
both size determination and fractionation of macromolecules and particles [22]. Suspended particles
are separated in a thin flow channel by the action of a gravitational or centrifugal field directed at
right angles to the channel. This process forces particles of different sizes into different streamlines
which elute at different times [22, 23]. Like LALLS, FFF techniques are able to measure large
numbers of starch granules in a relatively short period of time. The advantage of FFF over other
techniques is its ability to fractionate the sample according to granule size [19]. Furthermore, it is
fast and offers high resolution [18]. A disadvantage of sedimentation techniques is that the
assumption is made that all particles are spherical and of uniform density [18]. In addition, the
instrumentation for field flow fractionation is rather complex and expensive [19]. A prerequisite to
successful analysis using FFF techniques is that the granules are not damaged during isolation of
the starch. Field flow fractionation techniques are useful for the investigation of degradation (e.g.
enzymatic digestion) of starch granules when
digestion takes place mainly inside the starch granule. Under these circumstances, the density of the
particles is changed before any measurable change in size occurs. These density changes can be
measured by sedimentation [19].
Tab. 2 summarizes the advantages and disadvantages of the various granule size determination
methods.
Tab. 2. Advantages and disadvantages of different granule size determination techniques.
4 Granule Size Distribution Models
4.1 Aggregates
There are two aspects to the distribution of starch granule size. One is the impact of aggregation of
the granules in a starch sample on the apparent granule size distribution. The other is the actual size
distribution of individual granules. Aggregated starch granules are typical of most starch raw
materials that consist of small granules, such as quinoa, amaranth and cow cockle [24]. Large
spherical particles, 30-80 μm in diameter, can be observed in spray-dried samples, where the small
granules cluster together to minimize surface area. Clustering can also
occur in situ immediately after biosynthesis via hydropho-bic interactions among residual adhering
membrane constituents derived from the amyloplast [25], Aggregate formation complicates
obtaining an accurate perspective on the granule size distribution.
4.2 Mono- and bimodal granule size distributions
Granule size distributions are usually classified as mono-modal or bimodal. A bimodal size
distribution of large and small granules is characteristic of wheat starches as well as those from rye
and barley. The two populations are classified as A-granules (>10 μm) and B-granules (<10 μm)
and differ somewhat in their physicochemical characteristics and end-use potential [26-31]. The
proportion of small and large granules differs among genotypes [32]. Raeker et al. [21] reported that
wheat starch actually showed a trimodal rather than a bimodal granule distribution. An intermediate
(underdeveloped A-type) granule was mentioned as constituting the third group. However, most of
the time wheat endosperm is said to contain just two types of starch granules. A-type granules are
10-35 μm in diameter and account for more than 70% of the total starch weight but less than 10% of
the granules by number. B-type granules account for over 90% of the granules by number, but less
than 30% of the total starch by weight in wheat endosperm [21, 30, 33, 34].
5 Compositional, Structural and Functional Differences between Small and Large Granules
Starch composition, gelatinization and pasting properties, enzyme susceptibility, crystallinity,
swelling and solubility are all affected by granule size. However, several other factors, including
amylose/amylopectin ratio and molecular weight and granule fine structure, are also influential.
Most of the research done in this area has been on wheat and barley starches.
5.1 Amylose and amylopectin content and structure
Some reports claim that the amylose concentration is higher in the large granules of bimodal
starches [32, 34, 35], whereas others have found the same amylose concentration in both small and
large granules [36,37]. A variety of procedures have been used to determine the amylose content of
starch [38-41]. These methods all have their particular limitations, which has resulted in discrep-
ancies in the amylose contents reported for various starches. Variation in the molecular structure of
amylose and amylopectin appears to explain many of the apparent contradictions in the literature
[41, 42]. The molecular structure of the amylose and amylopectin fractions varies between granules
of different size. For barley, it was found that the number-average degree of polymerization of
amylopectin decreased with decreasing granule size, and that amylose polymerization was the same
for small and large granules [1]. Takeda [35] concluded that in barley, large granules contained
smaller, less branched amylose polymers. The branch chain length of amylopectin is also correlated
with granule size and granule size distribution. The amylopectin of large granules contained a
greater number of long amylopectin B chains and had a lower fraction infraction II ratio, one of the
structural characteristics of amylopectin, than did small granules from the same cultivar [43].
5.2 Gelatinization behavior
The gelatinization properties of starch are related to a variety of factors including the size,
proportion and kind of crystalline organization, and ultra-structure of the starch granule. Goering
and DeHaas [13] reported that small granule starch had, in general, a lower pasting temperature
than large granule starch. However, small granule size is not necessarily associated with a low
pasting temperature, as dasheen starch has a pasting temperature 20°C higher than that of most
other small granule starches. The low amylose content of dasheen and the fact that it is a tuber
starch made this gelatinization temperature totally unexpected [13]. Lorenz [24] showed that
monomodal small granule starches had higher pasting temperatures than did large granule starches.
In wheat and barley starches, the smaller B-granules paste at a higher temperature and over a wider
temperature range than do the larger A-granules [33, 37, 44, 45]. According to Eliasson and
Larsson [33], the gelatinization enthalpy of wheat starch is independent of the granule size distribution. However, others have found higher gelatinization enthalpies for A-type than for B-type starch
granules in wheat [34, 44]. This lower enthalpy value for the gelatinization of B-type granules
suggests a lower proportion of organized structures in B-granules than in A-type, or a lower
stability of the crystalline regions. Chiotelli and Le Meste [44] showed, with X-ray diffraction, that
A-type granules are indeed somewhat more crystalline than B-type granules. A difficulty in the
interpretation of such data is that differences in the apparent crystallinity of starch, as determined by
X-ray diffraction, can be attributed to differences in water content among the starch types [46, 47].
5.3 Amylose-lipid interactions
The dissociation enthalpy of the amylose-lipid complexes of small granules is higher than that of
large granules [33, 37, 44]. Soulaka and Morisson [48] and Raeker et al. [21] explained this on the
basis of the higher lipid content in B-type wheat granules. Moreover, as the gelatinization
endotherm represents essentially the difference between the endothermic energy (associated with
melting of crystallites, granule swelling and denaturation) and the exothermic energy (associated
with hydration of starch and formation of amylose-lipid complexes [49]), the greater amount of
internal lipid in B-granules (as well as better hydration) may generate a lower endothermic energy
(enthalpy underestimation) [44].
5.4 Solubility and swelling power
At similar amylose contents, small granule starches tend to have a lower pasting temperature and
more amylose leakage out of the intact granule, than do their larger granule counterparts at
temperatures of 55°C and higher [6]. According to Zheng and Sosulski [6], the swelling power of
starch is associated more with granule structure and chemical composition, particularly amylose and
lipid content, than with granule size. Higher amounts of lipid-com-plexed amylose would inhibit
swelling and gelatinization [6]. However, B-granules are associated with a higher rate of water
absorption, earlier hydration and more swelling than are A-granules [44]. The reason for this is the
less crystallized arrangement of the polysaccharide chains in B-granules (a higher proportion of
amorphous zones more accessible to water). Greater specific surface area may also contribute to the
higher water absorption of B-granules [44], though other factors like starch ageing and sorption
time might have a larger influence than surface area [50, 51]. Hellmann and Melvin [52] even concluded that the surface area of starch does not give a quantitative explanation of the water
absorption capacity of starches at 25°C from different botanical origin with varied granule sizes.
5.5 Acid and enzymatic hydrolysis
Small barley and wheat granules hydrolyze faster with acid or enzyme than do large granules [14,
53]. This again might be due to the higher surface area per unit weight of small granule starch.
Also, the pattern of enzyme digestion differs between large and small granules [41]. It has been
reported that enzymatic hydrolysis of granular starch at temperatures below the starch gelatinization
temperature results in a pitted and porous granule surface, which is readily discernible by
microscopic exami-
nation [54]. The starch granules are reduced to a sponge-like structure due to uneven hydrolysis.
For example, an enzyme like α-amylase appears to attack at discrete points on the granule surface,
forms tunnels into the granule interior, and then hydrolyzes the granule from the inside [54].
However, no pinholes were detected in small granules after incubation with a-amylase. The surface
of small granules became rough, and the granules appeared to be hydrolyzed from the outside by
surface erosion. The rough appearance of small starch granules after enzyme incubation is quite
different from the still-smooth surface of extensively degraded, large granules [54]. It is questionable, however, whether granule size is the main factor responsible for the observed differences
in the pattern of enzyme attack on small and large granules. Fannon et al. [55] proposed that the
presence of pores as an anatomical feature of some starches, and the absence of these in other
starches, affected the pattern of attack by amylases and by at least some chemical reagents. Pores
were found along the equatorial groove of large granules of wheat, rye, and barley starches, but not
in oat and rice starch, which have small granules.
6 Determinants of Granule Size
6.1 Environment
Environment seems to affect starch granule size and size distribution. Raeker et al. [21] concluded
that for wheat, the magnitude of this effect was cultivar dependent. Oli-veira et al. [56] found that
environment had a significant effect on starch traits in barley, whereas genotype/environment
interactions were not significant. In barley, temperature-induced stress reduced both the size of Aand B-type granules and the number of B-type granules [57]. This was also found in wheat [21].
When other stresses were present, the number of A-granules was reduced by high temperatures, but
proportionally to a lesser extent than was the number of B-granules [43, 57]. Different wheat
cultivars have shown considerable variation with respect to the sensitivity of the B-granule fraction
to environmental stress [58]. Significant differences in amylose content exist among cultivars,
hence the observed negative correlation between amylose content and granule size may explain, at
least in part, the relationship between cultivar and granule size [21].
6.2 Biosynthesis
Starch morphology and granule size are genetically controlled [4]. The biosynthesis of starch occurs
in the amylo-plast and/or the chloroplast. The membranous structures
and physical characteristics of plastids can impart a particular shape or morphology to starch
granules, and can affect the arrangement and association of the amylose and amylopectin molecules
within the granule [4]. Until now, research on differences in the biosynthesis of small and large
granules has focused mainly on the granules in a bimodal distribution, such as in barley and wheat
starches. Not much is known of the determinants of granule size in monomodal, small granule
starches, nor has a satisfactory explanation yet emerged from biosynthetic studies with respect to
the origin of the dual population of A- and B-granules in some cereal starches [16]. Lange-veld et
al. hypothesized that small granules are formed in vesicles budded off from out-growth of the Atype granule-containing amyloplast. This hypothesis is supported by the demonstration of the
presence of B-type granules using transmission electron microscopy and confocal laser scanning
microscopy [59]. For a general review on starch granule structure and biosynthesis, the reader is
referred to Buleon et al. [3].
The biosynthesis of cereal starches is postulated to occur by two processes. The first of these
governs the ratio and nature of the amylose and amylopectin components, whereas granule shape
and size are controlled independently, the mode depending on the cultivar [20]. In the development
of a barley kernel, the A-granules form soon after anthesis and may continue to grow throughout
grain filling, whereas B-granules are initiated some days after anthesis and remain small [58]. The
composition of B-granules differs from that of A-granules throughout development, mainly with
respect to amylose and phosphorus content. At most stages of development, the numbers of Agranules per endosperm remained constant while granule size increased, but the numbers of B-granules per endosperm increased throughout grain development [60]. Amylose content increases with
granule size and with granule maturity [61].
Takeda et al. [35] characterized the structure of amylose and amylopectin in barley, and indicated
that the genetic regulation of starch biosynthesis may differ for granules of different sizes. It was
also suggested that the regulation of biosynthesis of small granules in amyloplasts was different
from that of large granules and that of a middle fraction of granules. One group of enzymes that is
reported to influence granule size is the starch-branching enzymes [62], which are preferentially
associated with A-type starch granules in wheat endosperm. Two isoforms of a 152-kDa starchbranching enzyme [1,4-α-glucan branching enzyme] from wheat are located in the endosperm
starch granules but are not found in the endosperm soluble fraction or in pericarp starch granules
younger than 15 days post-anthesis (DPA). Small B-granules initiated
before 15 DPA incorporate the two isoforms throughout endosperm development and grow into
full-size A-gran-ules. In contrast, small starch granules harvested after 15 DPA contained only
small amounts of the branching enzyme and developed mainly into B-granules [62]. In the barley
shrunken (shx) mutant, starch synthase I was suggested as the primary mutation site [63]. The
overall size of the A-type starch granules is reduced, and the granule size-distribution is apparently
monomodal rather than bimodal as was the case in the wild type [63-65]. No mutant with altered
starch granule size distribution, analogous to the shx in barley, has been reported in other bimodal
starches.
6.3 Genetics
Little information is available with respect to the genetic control of granule size. Borem and Mather
[66] used simple interval mapping to reveal a region of chromosome 2 (2H) containing quantitative
trait loci (QTL) which affected the overall mean starch granule volume, the proportion of large Atype granules, the mean volume of A-granules, the mean maximum diameter of A-granules, and the
mean F-shape (roundness) of small B-type granules in barley starch. Composite interval mapping
revealed further QTL affecting starch granule traits on chromosomes 4 (4H; mean F-shape of Bgranules) and 7 (5H; mean maximum diameter of A-granules). Mutations in the genes affecting
amylose and amylopectin content show some effects on the granule size distribution. The presence
of the 'waxy' loci of several species results in a low amylose content of starch. Relatively low
amylopectin contents compared to non-mutant lines can occur in gene mutations that affect
amylopectin synthesis, for example at the amylose extender locus [67]. Starch from waxy barley
was reported to have a greater number of starch granules per endosperm, but the average granule
size was smaller than that of wild-type starches. In contrast, the gene that causes a relatively high
amylose content in barley starch was associated with a reduction in the mean volume of A-granules
and an increase in the mean volume of B-granules. The relative quantities (numbers) of A- and Bgranules were unaffected by either mutation [67].
7 Starch Isolation
The industrial process of starch isolation from commercial sources consists mainly of the separation
of starch from protein and fiber. Important considerations in starch isolation include avoidance of
amylolytic or mechanical damage to the starch granules during the initial isolation steps,
effective deproteinization of the starch, and minimizing the loss of small granules [68]. Commercial
production and use of small granule starch is still of relatively minor importance due to difficulties
experienced in the isolation and purification of small granule starch and the associated costs.
Processing experience on a laboratory scale has also been problematic. As a result, small granule
starch often has a high protein content and a portion of the small granule starch fraction is lost
during isolation.
7.1 Problems encountered in the isolation of small granule starches
A problem encountered in protein-starch separations is the entrapment of small granules in the
protein and fine fiber sediments generated during centrifugation [57, 68-71]. In batch
centrifugation, a dark-colored layer is generally observed on top of the white starch cake, which
contains mainly large starch granules. The interface consists of protein, non-starch polysaccharides
and smaller starch granules. When the upper layer is scraped off and discarded, which is common in
laboratory purification methods and in some industrial processes, a severe loss of small granules
occurs [72]. This affects starch yield as well as the ratio of small to large granules in starches having a bimodal starch granule size distribution.
In contemporary industrial starch refining processes, hydrocyclones are often used in lieu of
centrifuges. The advantage of hydrocyclones is that they are low in cost, contain no moving parts,
and result in a faster separation. However, important disadvantages include their relatively low
capacities and their relatively low efficiency in recovering small granules from both bimodal and
monomodal sources [73]. Granule size is a primary determinant of the efficiency of hydrocyclones
for the recovery of starch, as the sedimentation velocity of particles of similar density in a
centrifugal field is proportional to particle mass [73]. In the wheat processing industry, it is well
known that smaller granules do not sediment from aqueous suspension as rapidly as larger granules
and are partially lost in the supernatant [58].
7.2 Potential solutions
To overcome problems associated with deproteination and entrapment of small granules in the
protein layer, researchers have degraded the protein enzymatically, followed by separation of the
peptides and starch by centrifugation [74, 75]. However, the process required chroma-tography to
purify the protease so that it would not contain any amylase activity [63].
McDonald and Stark [76] suggested that instead of discarding the brown starch-protein layer, starch
in the layer should be purified separately and then added back to the white starch layer to provide a
representative starch granule preparation. Recovery of 45% of the starch from the proteinacious
brown layer was achieved by enzymatic processing of tailings with protease.
To remove β-glucans and other viscous non-starch poly-saccharides that can entrap the starch,
Wilhelm et al. [77] and Zheng and Bhatty [78] used enzyme preparations containing predominantly
β-glucanase, cellulase and arabi-noxylase activity. Subsequently, centrifugation separated the starch
slurry into well-defined phases (protein, compound starch granules, fine fiber). Wilhelm [25]
combined these non-starch-polysaccharide-degrading enzymes with proteases to effect better
purification of the starch. Another effective method for eliminating protein contaminants is the
centrifugation of the starch granules through a cesium chloride gradient. However, the cost of this
method restricts it to small-scale laboratory isolation [68], Zhao and Whistler [79] used low-gravity
centrifugation to separate a major proportion of the protein, such that when the supernatant was
centrifuged at higher gravity, starch settled with only mild contamination of the starch cake surface.
Although enhanced recovery of small granule starches is technically feasible, the extent to which
enzyme technology or alternative centrifugation regimes, for example, are adapted in commercial
practice will depend on economic factors.
8 Current and Potential Uses of Small Granule Starches
There is increasing interest in starches manufactured from novel materials for use in special
products. Starches having small granules and narrow granule size distributions can be used in fine
printing paper and plastic sheets [4, 77], as a binder with orally active ingredients, and as a carrier
material in cosmetics [80]. Microgranular starches are also suitable for one-layer-thick honeycomb
coatings and in this way can be used in the cosmetic, paper, textile and photographic industries [12].
A well-established use of small granule starch, mainly rice starch, is as a cold-water laundrystiffening agent. The small granule size affords superior penetration of starch into the fabric. The
stiffness of textiles and fabrics so treated is less affected by humidity than those treated with other
starches [81].
8.1 Fat replacement
Aqueous dispersions of small starch granules are known to produce a creamy, smooth texture that
exhibits fat-mimetic properties [4, 12, 82]. With the growing demand
for carbohydrate-based fat replacers, starch from cow cockle, for example, may find commercial
applications. In food applications, microgranular and uniform granule size starches produce a
creamy mouth feel, a desirable attribute in frozen desserts, cookies, cheesecakes and other low-fat
and fat-free food formulations. For example, rice starches, because of their uniformly small granule
size (2-8 μm, the smallest among commercially available starches), are well known to produce a
smooth creamy texture [4, 12]. Whistler [83] patented a process in which small granule starch (an
average granule size of 5 μm or less, preferably less than 3 μm, is preferred) is subjected to partial
hydrolysis with a-amylase or glucoamylase to produce a novel, granular starch composition having
an enzymatically hydrolyzed surface appearing diffuse and substantially non-porous under
microscopic examination, and exhibiting crystallinity characteristic of the corresponding native
starch granules. The partially hydrolyzed granular starch exhibited fat-like characteristics for use in
reduced-calorie processed foods.
8.2 Biodegradable films
Small granule size substantially increases the level of starch that can be incorporated into
biodegradable films while maintaining film quality [84]. Commercial application of biodegradable
films includes garbage bags, composting yard-waste bags, grocery bags and agricultural mulches.
Commercial biodegradable films are generally manufactured from low-density polyethylene
containing degradative additives such as starch and pro-oxidants. The use of starch as a
biodegradable filler satisfies thermal stability requirements and interferes minimally with the meltflow properties required of most manufacturing applications. Starch incorporation produces a
plastic film with a porous structure, which enhances the accessibility of the plastic molecules to
oxygen and microorganisms [84, 85].
8.3 Carrier materials
Small starch granules are able to combine into interesting and potentially useful porous spheres
when spray dried with small amounts of bonding agents. A variety of bonding agents can be used
including protein, gelatin, carbox-ymethyl cellulose, guar gum, locust bean gum, starch dextrin,
pectin and alginate [86]. The spherical aggregates contain open spaces in the form of
interconnecting cavities that provide extensive porosity capable of being filled and used to transport
material within the spheres. The orifice size of the spheres is dependent on the particular spray
nozzle used. A need exists in the food industry for containment of flavor essences and other
components
in a manner that will provide for oxidative protection and for controlled release over a defined
period of time [86]. Examples would be the prolonged release of chewing gum flavor or the
containment of certain components in dry mixes until placed in water or heated to a specified
temperature. The encapsulation method using spheres of bound starch granules offers a low-cost,
food-grade package that can be produced by normal processing methods [86].
9 Conclusions
A wide range of small granule starches is available in nature. However, such starches do not see
significant commercial application. A major reason for this is the difficulty experienced in
commercial-scale purification of these starches. The development of improved methods for the
refining of small granule starches could greatly facilitate the development of new applications and
new markets for such starches.
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(Received: March 28, 2003) (Revised: July 18, October 10, 2003) (Accepted: October 13, 2003)