Food Chemistry 190 (2016) 50–59

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Development of an analytical method to measure insoluble and soluble

starch in sugarcane and sweet sorghum products
Marsha R. Cole a,⇑, Gillian Eggleston a, Audrey Gilbert b, Yoo Jin Chung a
a
Southern Regional Research Center, Agricultural Research Services, U.S. Department of Agriculture, 1100 Robert E. Lee Blvd., New Orleans, LA, USA
b
Department of Food Science, Agroparistech, Paris, France

a r t i c l e i n f o a b s t r a c t

Article history: A rapid research method using microwave-assisted probe ultrasonication was developed to quantify
Received 15 July 2014 total, insoluble, and soluble starch in various sugar crop products. Several variables affecting starch sol-
Received in revised form 11 December 2014 ubilisation were evaluated, (1) heating method, (2) boiling time, (3) probe ultrasonication time, (4) water
Accepted 13 May 2015
loss, (5) concentration, (6) sample colour, and (7) sample. The optimised method solubilises <40,000 ppm
Available online 14 May 2015
insoluble starch with microwave-assisted sonication in 6 min, has acceptable precision (<6% CV), accu-
racy (P95%), uses a corn starch reference, and incorporates a colour blank to remove contribution from
Keywords:
natural colourants found in industrial samples. This method was validated using factory samples and
Starch solubilisation
Insoluble starch
found applicable to sugarcane and sweet sorghum bagasse (3% CV), mixed juices (2%), massecuites
Soluble starch (4%), molasses (7%), and raw sugars (12%), 100% satisfactory performance z-scores were also obtained.
Sugarcane Total starch values obtained with this method were significantly higher than those measured using other
Sweet sorghum methods presently accepted by the sugar industry.
Probe ultrasonication Published by Elsevier Ltd.
Sugar products
Microwave

1. Introduction processing problems for sugarcane factories and refineries (Cole

et al., 2013). In particular, insoluble starch impedes the action of
Starch is a polysaccharide composed of a chain formed of a-amylases to control starch in factories and refineries. If insoluble
a-D-(1,4)-glucopyranosyl repeating units that is extracted into fac- starch is not totally solubilised it can persist in downstream sugar-
tory juices by milling or diffusing sugar-rich crops like sugarcane cane products like syrups, massecuites, molasses, and raw sugars
and sweet sorghum. Starch occurs as granules of complex structure (Cole, Rose, Chung, & Eggleston, 2014). Sweet sorghum is well
and consists of amylose (linear glucopolymer) and amylopectin known to contain considerably more starch than sugarcane
(branched glucopolymer), which are collectively responsible for (Lingle, 2010), which can also interfere with processing but has
its rigidity and difficult solubilisation (Chen & Chou, 1993). the added potential to increase ethanol yields during sweet sor-
However, starch granules lose their crystallinity when boiled in ghum juice fermentation (Eggleston, Cole, & Andrzejewski, 2013).
the presence of excess water, which occurs during the Understanding the total concentration of starch in sweet sorghum
heat-intensive processes in sugar or syrup manufacture of sugar- juice and bagasse can facilitate the application of a-amylases for
cane or sweet sorghum, respectively. This causes both amylose subsequent saccharification and fermentation steps.
and amylopectin to leach from the semi-crystalline starch granule Unfortunately for the world-wide sugarcane industry, starch con-
as it becomes increasingly more soluble (Chen & Chou, 1993). centrations have been increasing in recent years mostly because
Until recently, it was assumed that starch was mostly solu- of green mechanical cane harvesting, newer cultivars, and environ-
bilised by the end of the heat intensive clarification and evapora- mental and seasonal influences (Zhou et al., 2008). Furthermore,
tion stages in the sugarcane factory (Godshall, Clarke, & Dooley, U.S. carbonatation refineries have implemented a penalty on
1991). However, Eggleston, Cole, Gilbert, and Rose (2013) and >250 ppm soluble starch in raw sugars. This problem is being exac-
Cole et al. (2013) have reported that this is not always true, espe- erbated by the current starch methods used in the sugar industry
cially when high starch sugarcane is delivered to the factory. High that mostly measure soluble starch and have very little impact
concentrations of insoluble, swollen, and soluble starch often cause on insoluble starch (Eggleston et al., 2013). Thus, a method that
measures soluble and insoluble starch in products across entire
⇑ Corresponding author. factory and refinery processes is urgently needed (1) to mitigate
E-mail address: Marsha.Cole@ars.usda.gov (M.R. Cole). the processing and financial damages incurred to sugarcane

http://dx.doi.org/10.1016/j.foodchem.2015.05.049
0308-8146/Published by Elsevier Ltd.
 M.R. Cole et al. / Food Chemistry 190 (2016) 50–59 51

factories and (2) to better assess potential ethanol profits of 2. Experimental

untapped fermentable glucose from sweet sorghum starch.
Current starch methods in the sugar industry were founded on 2.1. Chemicals and sugar samples
the pioneering work of Balch (1953) and Charles (1968) who used
alcohol-acid induced precipitation or heat-acid induced solubilisa- Corn starch, potato starch, sucrose (99.95%), and other chemi-
tion, respectively, to solubilise starch in raw juices and sugars. cals were analytical grade from Sigma Aldrich Company (St.
Soluble starch concentrations are then treated with iodine to form Louis, MO). Sugarcane and sweet sorghum products, including
a starch-triiodide (I
3 ) complex (blue colour) that is measured with juices, syrups, raw sugars, and bagasse were kindly provided by
either a spectrophotometer or colorimeter and quantified against a multiple Louisiana sugarcane factories and a Florida sweet sor-
potato starch reference (Smith & Cheng, 1978). Examples of exist- ghum biorefinery.
ing iodometric starch methods include the ICUMSA (International
Commission for Uniform Methods of Sugar Analysis) Methods GS 2.2. Brix (% dissolved refractometric solids)
1-16 and Methods GS 1-17 (2013) and South African Sugar
Technologists’ Association (SASTA, Anonymous, 1985). In these The soluble solids content of the simulated juices were deter-
current methods, sugar products are generally boiled in water or mined using degrees Brix in triplicate with a
acidified CaCl2 solution between 5 and 15 min to ‘‘solubilise’’ temperature-controlled refractometer (model TCR 15-30; Index
starch; but Eggleston et al. (2013) reported that not all the starch Instruments, FL) to an accuracy of ±0.02 Brix. Brix is the
was solubilised and therefore these methods often ‘‘underestimate weight-weight percent of dissolved solids in 100 g of solution. All
the total amount of starch in the sugar product.’’ Even conductively Brix measurements were completed at room temperature and per-
boiling a sugarcane factory syrup for up to 80 min did not solu- formed on simulated juices before adding starch.
bilise all the starch (Eggleston, Cole, Gilbert et al., 2013) and more
than 8 h was needed to solubilise starch in raw sweet sorghum 2.3. Digital microscopy
juices (data not published). Research methods also exist that utilise
a-amylases and a-amyloglucosidases to convert total starch to The presence of insoluble (granular) starch was determined by
quantifiable glucose (AACC International, 2000; McCleary, Gibson, adding one drop of filtered iodine solution (filtered twice through a
& Mugford, 1997; Sakar & Day, 1994), which is typically useful 0.22 lm syringe filter) to two drops of the sugar product under
for the sweet sorghum saccharification and fermentation biorefin- investigation and observing if blue starch granules were present
ing processes. Enzymatic starch hydrolysis methods are not gener- under a digital microscope. Digital micrographs were taken with
ally used at the sugarcane factory or refinery because they are an Olympus MIC-D™ digital microscope (Center Valley, USA). At
expensive, require large enzyme dosages, and are too labour and least five random sub-samples of each stained sample were
time intensive for their high-throughput needs (Cole et al., 2013). photographed.
Thus, other treatments need to be investigated that totally solu-
bilise insoluble starch in sugar products.
2.4. Effect of microwave irradiation time on the solubilisation of
The purpose of this study was to develop a research method to
insoluble corn starch
measure insoluble and soluble starch in sugar products and the
principle behind the development of this new method is illustrated
Dispersions of commercial corn starch (910–40,000 ppm) were
in Fig. 1. For this research method to be successful, the following
made in 15 Brix sucrose solutions to obtain final starch concen-
five questions needed to be answered: (1) Is all the starch solu-
trations of 6,066–267,000 ppm/Brix. Known weights of starch sam-
bilised? (2) Have any unwanted reactions occurred? (3) Does the
ples (10 mL) placed in 125 mL volumetric flasks (KimaxÒ,
iodometric reaction still work in this new solubilised starch solu-
Cole-Palmer, IL, USA) were vortexed (5 s, 3,000 rpm) and heated
tion? (4) Is all the soluble starch still intact, i.e., has unwanted
at full power in a commercial microwave oven (Kenmore,
breakdown of starch occurred? (5) Can it quickly and reproducibly
Sears-Roebuck and Company, IL, USA) with a maximum output of
measure samples with high and low starch concentrations? To
1,100 W at 2.45 GHZ in 30 s intervals from 30 to 180 s. A watch
address these questions, we explored various physical and chemi-
glass (110 mm O.D.; Sigma Aldrich, MO, USA) was used to cover
cal treatments (Cole, Eggleston, Gilbert, & Chung, 2014). The final,
the flask during microwaving. After microwaving, the samples
validated method was based on microwave-assisted probe
were rehydrated to the original weights to ensure starch concen-
ultrasonication.
trations did not change due to microwave-induced evaporation.

Fig. 1. General principle to calculate total, soluble, and insoluble starch in factory samples. Adapted from Eggleston et al. (2013).
52 M.R. Cole et al. / Food Chemistry 190 (2016) 50–59

The amount of gelatinised (swollen) and totally solubilised 2.8. Effect of probe ultrasonication on microwave-assisted
starches were differentiated by passing the sample through an solubilisation of simulated mixed juice
Acrodisc syringe filter (25 mm, 0.45 lm GHP GxF prefilter, VWR,
PA, USA). Thus, filtered samples indicated only soluble starch and Probe ultrasonication amplitude (60%) and 20 kHz frequency
non-filtered samples indicated both swollen and soluble starches. was studied on 10 mL simulated 15 Brix juices containing
After cooling, an 800 lL sample was treated with 200 lL of 900 ppm insoluble corn starch and 10 ppm solubilised corn starch.
0.25 M HCl and 1,000 lL iodometric reagents (1 mM KIO3 and After microwaving each sample for 60 s and reconstituting it with
5 mM KI) before measuring absorbance at 600 nm (UVmini-1240, water, probe ultrasonication was applied for 5 min. Each sample
Shimadzu, MD, USA). An iodometric blank (Ib) consisting of was reconstituted gravimetrically with water before filtering and
800 lL water, 200 lL of 0.25 M HCl, and 1,000 lL of iodometric reacting with iodometric reagents and HCl. Absorbance was mea-
reagents and an acidified colour blank (Cb) consisting of 800 lL sured at 600 nm. All of the sample preparation and treatments
sample (or appropriate dilution), 200 lL of 0.25 M HCl, and were carried out in duplicate and were corrected for Cb and Ib.
1,000 lL of water were subtracted from all sample measurements. Energy output (J) was recorded after each sample was processed.
Initial concentrations of soluble starch (untreated sample) were
determined by filtering non-microwaved dispersions through a 2.9. Final USDA method to measure total starch in sugar products
syringe filter to separate the insoluble granules from the soluble
starch in the sample. All measurements were completed in quadru- Factory samples, i.e., mixed juice, syrup, massecuites, and
plicate. Absorbance contribution from particle scattering was molasses, were adjusted to 615 Brix with deionised water and kept
determined by: on ice until use. Fresh bagasse (25% w/w) was pulverised in water
   for 2 min using a high-speed commercial blender fixed with a
Cbnot filtered cross-blade (Magic Bullet MBR-1701, Homeland Housewares,
% Scattering ¼ 1  1  100%
Cbfiltered LLC., CA, USA). Massecuites and molasses were preheated slowly
to 40 °C and stirred manually for 5 min to completely homogenise
Absorbance contribution from colourants was determined by: sample. All factory and bagasse samples were filtered through a
437 lm polyethylene macrofilter (SpectrameshTM; Spectrum
  
Cbfiltered Medical Industries, CA, USA) to remove large impurities before
% Colour ¼ 1  1  100%
Absfiltered;sample use. Ten millilitres of the 15 Brix simulated juice sample (i.e.,
1,000–40,000 ppm insoluble corn starch) or factory sample filtrate
was vortexed for 5 s at 3,000 rpm and added to 125 mL volumetric
flasks. Each sample was weighed and recorded before covering it
2.5. Effect microwave time has on temperature and water loss
with a watch glass and microwaving it for 60 s at 100% power.
After microwaving, the sample was vortexed and reconstituted
Temperature was monitored digitally after each microwave
with water to the original weight. Probe ultrasonication at 60%
interval to distinguish whether the 15 Brix samples containing
intensity was applied for 5 min to the 10 mL sample. Each soni-
1,000–20,000 ppm insoluble starch were boiled at the same rate.
cated sample was filtered through a 0.45 lm syringe filter. The fil-
Differences in weights before and after microwave boiling were
tered sample (800 lL) was then treated with HCl and iodometric
recorded as water loss.
reagents. Absorbance was measured at 600 nm using a UV–vis
spectrophotometer.
2.6. Effect of conductive boiling time in water on the solubilisation of To determine the concentration of naturally soluble starch,
insoluble corn starch untreated juice was also filtered through a 0.45 lm syringe filter
and reacted similarly with iodometric reagents and HCl, and then
For comparison purposes, a traditional starch dissolution the absorbance at 600 nm was measured. An Ib and a Cb, respec-
method was also conducted as a standard reference. Corn starch tive to untreated and treated samples, were both subtracted from
(1,000–20,000 ppm) was suspended in 10 mL of a 15 Brix sucrose all sample measurements. All measurements were undertaken in
solution. Each sample was weighed and vortexed before conduc- duplicate. After extrapolating concentrations from the corn starch
tively boiling it for 1–31 min in a rolling water bath. After boiling, calibration curve, insoluble starch was calculated by subtracting
the starch sample was vortexed and reconstituted with water to the soluble starch concentration (untreated sample) from the total
correct starch concentrations. Starch values were determined as starch concentration (treated sample) as illustrated in Fig. 1.
outlined in Section 2.4 and completed in quadruplicate. The calibration curve was made using 5,000 ppm insoluble corn
starch dispersed in a 15 Brix sucrose solution. A 10 mL aliquot of
the dispersion was prepared similarly as outlined above. Ten serial
2.7. Effect of probe ultrasonication time and amplitude on the dilutions (1:1) to a concentration range (5–5,000 ppm) were made
solubilisation on simulated mixed juice in water before reacting with iodometric reagents and HCl and
measuring absorbance at 600 nm.
A Sonics & Materials Inc. (Newtown, CT, USA) ultrasonic proces-
sor VCX 750 model, operating at 20 kHz, and equipped with a stan- 2.10. ICUMSA GS1-17 method for starch quantitation
dard probe (6 mm diam.) was used to treat simulated 15 Brix juices
containing 1,000–40,000 ppm insoluble corn starch. Probe ultra- Starch in sugarcane and sweet sorghum products were also
sonication of a pre-weighed simulated juice sample (10 mL) was measured using the ICUMSA GS1-17 method for comparison and
subjected to 20–60% amplitude and 20 kHz frequency for 4– accuracy purposes. A summary of the method is briefly described
31 min. After probe ultrasonication, the samples were weighed, herein. Three aliquots (3 mL) of juice or 15 Brix sugar solutions
reconstituted with water, filtered through 0.45 lm membrane, (1 colour blank control and 2 juice samples measured in duplicate
and determined in quadruplicate as outlined in Section 2.4. after gently defrosting to room temperature) were prepared for
Energy output (J) was recorded after each sample was processed. analysis in corresponding 15 mL glass test-tubes. A colour blank
 M.R. Cole et al. / Food Chemistry 190 (2016) 50–59 53

control was incorporated in this method to determine the percent at the molecular level when absorbing energy at high frequencies,
contribution of sample colourants to the absorbance values. Juices i.e., 300–300,000 MHz (Bilbao-Sainz, Butler, Weaver, & Bent, 2007;
were maintained on ice until analysis. Each juice sample was Roy & Gupta, 2003). It has been reported that microwave irradia-
boiled individually for 5 min (raw sugar) or 10 min (juice) and tion significantly mobilises starch protons and increases moisture
placed on ice to chill to room-temperature. Once cooled, the iodo- intake, which results in losses of crystallinity much faster than
metric reagents consisting of 1.2 mL of 2 M CH3COOH, 0.25 mL of when boiled on top of a hot surface (conductively boiled samples).
602 mM or 10% w/w KI, and 2.5 mL of 1.66 mM KIO3 were added Moreover, microwave irradiation is less affected by starch type,
to the juice sample. Low-shear mixing the juice, i.e., by inverting amylose-to-amylopectin ratios, concentration, size, or
five times, and the iodometric reagents yielded either an amber phosphate-ester content (Bilbao-Sainz et al., 2007; Lewandowicz,
(low starch content) or dark blue (high starch content) solution Jankowski, & Fornal, 2000; Palav & Seetharaman, 2006).
with coloured precipitate. The sample was then clarified with Time–temperature profiles revealed that microwave irradiated
5 min centrifugation at 3,000 rpm before measuring absorbance samples reached boiling temperatures (see Supplementary
of the supernatants at 600 nm using a UV–vis spectrophotometer. Information, Fig. S1A) much quicker than those conductively boiled
The Ib was similarly prepared following ICUMSA GS1-17 by substi- (data not shown) which agrees with the finding previously
tuting juice with water. The method does not include a Cb; how- reported by Bilbao-Sainz et al. (2007). Initial starch concentrations
ever, one was prepared by mixing 3 mL of juice with 1.2 mL of did not limit the efficiency of microwave boiling as observed when
2 M CH3COOH and 2.75 mL of deionised water in a 15 mL glass conductively boiled. A rapid rise in temperature was found for all
test-tube. Both Cb and Ib were subtracted from the absorbance. starch concentrations studied, with boiling temperatures reached
Then, the starch concentrations were calculated from a potato after the first 30 s of microwave heating, which were subsequently
starch calibration curve using the equation of the curve. maintained throughout the course of microwaving (Fig. S1A). A
strong quadratic correlation was observed between water loss
2.11. Precision of the USDA starch method to determine starch in and the microwave heating time (Fig. S1B). As microwave heating
industrial sugar products and simulated juices time continued, the rate of water loss was 1.86 g/s (R2 = 0.9824).
Samples no longer contained visible liquid after 180 s microwaving
The USDA research method was validated following ICUMSA and remnants were either viscous or crystallized to the bottom of
protocols. Accuracy and precision were obtained by analysing the glassware (Fig. S1B).
known starch concentrations in 15 Brix simulated juices as well Simulated juices that were microwave boiled had higher yields
as factory samples in duplicate in eight successive runs. Each of swollen and soluble starch as compared to samples conductively
duplicate analysis was an independent execution of the method boiled for equal time lengths (Fig. 2A). Approximately 30% of the
on the simulated juice. The mean of the data set (Xmean) and stan- conductively boiled insoluble starch (65,000 ppm) was gelatinised
dard deviation (SD) were calculated, as well as the relative stan- and solubilised after 60 s. Longer periods of conductive boiling did
dard deviation (RSD) or coefficient of variation (CV) (Thompson, improve starch gelatinisation and solubilisation and was affected
Ellison, & Wood, 2002): largely by concentration, but was far less effective than when
  microwave boiled (Fig. 3). After microwave boiling <5,000 ppm
SD
RSD ¼  100 insoluble starch, 90% and 60% of the dispersion became gela-
X mean
tinised and solubilised, respectively (Fig. 2A) with greater solubil-
Percent (%) recovery was used to determine the accuracy of the isation occurring on starch dispersions containing low amounts
method on starch samples of known concentration, in which Xmean of insoluble starch (<1,000 ppm). Fig. 2B illustrates the changes
was divided by the theoretical starch concentration (Xtheoretical) and observed when 20,000 ppm insoluble starch was microwave or
multiplied by100: conductively boiled over time. Quantifiable amounts of gelatinised
and solubilised starch did not occur until 4 min of conductive boil-
X mean
% Recovery ¼  100 ing. As conductive boiling time progressed, small increases in
X theoretical
starch gelatinisation and solubilisation occurred (Fig. 2B). In com-
parison, microwave boiling required approximately 10-times less
2.12. Statistics time and solubilised 5-times more starch than conductive boiling
(Fig. 2B). The differences between microwave and conductive boil-
Statistics were conducted using Microsoft Excel™ version 2007 ing performance may be attributable to three key explanations.
with SP3 MSO. Performance z scores were calculated using the fol- First, moisture migration is limited from the exterior to interior
lowing formula (Thompson, Ellison, & Wood, 2006): of the starch granule during conductive boiling whereas, micro-
wave irradiation uses both internal and external water to induce
xX
z¼ gelatinisation. Secondly, heat conduction is very slow compared
SDPA
to microwave irradiation (Xue, Sakai, & Fukuoka, 2008). Lastly,
where x is the data value, X is the assigned value, and SDPA is the the lack of continuous agitation in the test-tube during conductive
standard deviation for proficiency assessment. jzj scores <2.00 are boiling may have limited gelatinisation and even more so, solubil-
satisfactory, jzj scores between 2.00 and 3.00 are questionable, isation (Sakonidou, Karapantsios, & Raphaelides, 2003; Xue et al.,
and jzj scores >3.00 are unsatisfactory (Thompson et al., 2006). 2008; Zylema, Grider, Gordon, & Davis, 1985). Since the
20,000 ppm starch dispersion appeared heterogenous and still con-
3. Results and discussion tained insoluble, precipitated granules at the bottom of the
test-tube after either microwaving or conductively boiling
3.1. Heating effects of microwave and conventional boiling (Fig. 3), it was assumed that the limiting factor affecting its solubil-
isation was the lack of agitation to evenly disperse the granules
The successful application of microwave irradiation in food pro- and make them available to the surrounding aqueous environ-
cessing to gelatinise starch is based on the rapid and even heating ment. Therefore, the effect of agitation before conductive boiling
that occurs when water molecules in food orient themselves with or microwave irradiation of insoluble corn starch was studied with
the alternating electromagnetic field. As a result, heat is generated quantitative chemistry and visual evaluation (see next Section 3.2).
54 M.R. Cole et al. / Food Chemistry 190 (2016) 50–59

Fig. 2. (A) Percent yield of swollen and soluble starch from 1,000 ppm, 5,000 ppm, or 20,000 ppm insoluble corn starch after boiling with conductive or microwave irradiation
for 60 and 120 s. Asterisks (⁄) indicate no swollen or soluble starch detected. (B) The effect of microwave or conductive boiling on the solubilisation of 20,000 ppm insoluble
corn starch over time.

3.2. Vortexing effect on samples: quantitative and visual evaluations (Zylema et al., 1985). Starch yields obtained for non-vortexed sam-
ples after 60 s microwaving decreased quadratically (R2 = 0.962)
Samples containing <1,000 ppm insoluble starch were less with increased insoluble starch concentrations.
dependent upon vortexing than higher concentrations (data not Fig. 3 also compares starch yields (swollen and soluble)
shown). In some instances with low-shear mixed (non-vortexed) acquired after microwaving already vortex-mixed insoluble starch
samples, an irreproducible, semi-transparent paste was formed at samples. Overall, pre-vortexing insoluble starch dispersions before
the bottom of the test-tube. When starch concentrations were microwave heating, improved the gelatinisation of broad starch
increased to between 1,000 and 5,000 ppm, a small amount of concentrations. Increased gelatinisation and solubilisation of lower
granular starch remained unchanged or formed an opaque paste concentrations of insoluble starch (65,000 ppm) was observed;
with uneven consistency in the middle of a low viscosity gel. In however, vortex mixing became increasingly more critical with
non-vortexed samples with starch concentrations >5,000 ppm, increasing starch concentrations >10,000 ppm (Fig. 3). More pas-
rapid settling and subsequent buildup of dense, granular starch te/gel precipitation was seen at the bottom of the test-tubes after
occurred at the bottom of the test-tube when conductively boiled 60 s microwaving which did not become more homogeneous with
(Fig. 4). This indicates that high starch-to-water ratios in raw sugar longer heating times. Insoluble corn starch dispersions ranging
solutions or mixed juices may not become adequately gelatinised between 20,000 and 40,000 ppm were increasingly viscous, opa-
using the protocol detailed in the ICUMSA GS1-17 method que, and consisted of only one even phase after 60 s microwaving.
 M.R. Cole et al. / Food Chemistry 190 (2016) 50–59 55

10,000 ppm Insoluble Corn Starch

Microwave Heang (60 s) Conducve Heang (30 min)

Swollen
and
Soluble
Layer
Insoluble
Layer

Without Vortexing With Vortexing With Vortexing

120 Microwave Heang (60 s) Microwave Heang (120 s)

% Starch Yield (swollen + soluble)

100

80

60

40

20

Insoluble Corn Starch (ppm)

Not Vortex Vortex
Fig. 3. Effect of vortex-mixing 1,000 ppm, 5,000 ppm, or 10,000 ppm insoluble corn starch in 15 Brix solutions before microwave (60 s) or conductive boiling (30 min).

Fig. 4. Percent yield of starch solubilisation from 20,000 ppm insoluble corn starch as a function of probe ultrasonication time and energy (10,000 J) in the presence and
absence of 60 s microwave heat.

Overall, large quantifiable decreases in starch yields were found swollen, except 20,000 ppm and 40,000 ppm, which were 89%
with increased starch concentrations. Greater homogeneity was and 47% swollen, respectively.
observed in samples that were vortex mixed and microwave boiled Excessive differences in starch-to-water ratios can limit the
than those samples agitated and boiled with conductive heating. heating process and limit starch’s susceptibility to heat.
After vortex-mixing, all starch dispersions were at least 95% Sakonidou et al. (2003) and Zylema et al. (1985) reported that
56 M.R. Cole et al. / Food Chemistry 190 (2016) 50–59

the lack of appropriate mixing of a starch sample during boiling literature (Czechowska-Biskup, Rokita, Lotfy, Ulanski, & Rosiak,
may not only result in heterogenous samples but also limit the 2005; Price & Lenz, 1993) Because of this, we investigated the
water absorption capacity needed to effectively swell and/or solu- impact that 60 s microwave irradiation and 5 min probe ultrason-
bilise starch granules. This is because granule swelling and neces- ication (60% amplitude) had on (i) 1,000 ppm amylose and
sary heat transfer becomes much more limited as starch-to-water 1,000 ppm amylopectin and (ii) a simulated sugarcane mixed juice
ratios increase. This may be overcome, however, by mixing the containing 10 ppm soluble corn starch and 900 ppm insoluble corn
sample and improving convective heat transfer (Zylema et al., starch. Overall, minor losses (±5% loss) in binding affinity were
1985). As shown in Fig. 3, sufficient agitation of starch dispersions observed between amylose or amylopectin and iodine despite
before microwave irradiation provided an optimal balance obtaining 99.7% yield of total solubilised starch in the simulated
between starch settling rate and heat transfer that was unmatched mixed juice. Slight degradation or losses in starch-I 3 complexes
by conductive boiling methods. Also, the combination improved may have occurred as a result of high sono-mechanical activity
the percentage of starch that became gelatinised and solubilised which has been reported to reduce viscosity and subsequently
during the short heating period. fragment starch. Starch fragmentation also becomes more evident
after it has been gelatinised and processed for long periods (Iida
3.3. Probe Ultrasonication of Starch Samples et al., 2008). Iida et al. (2008) also concluded that probe ultrason-
ication helped to increase the solubilisation of swollen starch, but
As our previous results showed that vortex mixing and at the expense of a one magnitude loss in MW distribution of sol-
microwaving alone do not fully solubilise starch in simulated sugar uble starch (debranching of amylopectin chains). Severe debranch-
solutions, we investigated an additional technique to solubilise ing of amylopectin would cause a minimal reduction in starch-I 3
starch after it has been gelatinised by microwaving. Specifically, absorbance values given that amylopectin has a lower binding
we investigated the use of probe ultrasonication, which is a type affinity to iodine than amylose and debranced amylopectin would
of high power acoustic energy (16–20 kHz) that promotes mixing lead to better iodine binding (White, 2001). It must also be men-
and enhances the even distribution of difficult-to-solubilise mate- tioned that only hours of continuous probe ultrasonication and
rials into the surrounding liquid medium (Chandrapala, Oliver, percent amplitudes can alter or fragment the chemical structure
Kentish, & Ashokkumar, 2012; Iida, Tuziuti, Yasui, Towata, & of soluble starch. Therefore, minimal losses in amylose or amy-
Kozuka, 2008; Jambraka et al., 2010). Probe ultrasonication is also lopectin starch-I 3 absorbance were more likely due to the forma-
known to reduce sample-processing times, consume less energy, tion of trace amounts of oligosaccharides that were too short to
increase sample recovery, and reduce thermal degradation effects. complex with iodine (Iida et al., 2008; Mecozzi, Amici,
The majority of studies on the use of probe ultrasonication in foods Pietrantonio, & Acquistucci, 1999).
have been focused entirely on either (1) fundamental studies
between probe ultrasonication and starch or (2) its ability to min- 3.4. Incorporation of a colour blank and filtering step
imise the viscosity of fully pasted starch dispersions, since probe
ultrasonication alone does not solubilise granular starch Since the natural colourants and small particles present in sugar
(Jambraka et al., 2010; Szent-Gyorgyi, 1933; Zuo, Hébraud, products can overlap with the starch-I 3 absorbance, it was impor-
Hemar, & Ashokkumar, 2012; Zuo, Knoerzer, Mawson, Kentish, & tant to remove these interferences and correct starch values. This is
Ashokkumar, 2009). To the best of our knowledge, this is the first critical since sample colourants and particles can easily mask
report that suggests the use of microwave-assisted probe ultrason- relatively low starch quantities and cause an overestimation of
ication to totally solubilise the insoluble starch content in all agri- starch values, especially in raw sugars. Comparisons between
cultural and sugar products across the whole manufacturing untreated and treated (either conductively boiled or with
process. microwave-assisted probe ultrasonication) samples in the
Probe ultrasonication with and without heat was first studied ICUMSA GS1-17 and USDA research methods showed that the
on 1,000–40,000 ppm insoluble corn starch in 15 Brix sucrose solu- absorbance contributed from sample colourants to total starch
tions (Fig. 4). In the absence of any heat, low starch concentrations was considerable and varied by sample type. Particle scattering
such as those found in raw sugars, required less time and energy accounted for 29 ± 6% and 47 ± 11% of raw sugar and mixed
input to become soluble; yet, higher concentrations such as those juice absorbance values analysed by the ICUMSA GS1-17 method,
in mixed juices and bagasse required greater amplitudes (>50%) i.e., after 5 min centrifugation at 3,000 rpm. Colourants in treated
to solubilise the large amounts of insoluble starch and longer pro- samples contributed 8 ± 2% and 27 ± 3% colour in raw sugars
cessing times (data not shown). At 60% probe ultrasonication and mixed juices, respectively, as measured by the ICUMSA
amplitude, only 40% of a 20,000 ppm insoluble corn starch dis- GS1-17.
persion became solubilised and longer processing times did not For the USDA research method, colourants in the starch absor-
impart improved yields (Fig. 4). bance values of filtered and untreated samples (not
In the presence of heat, separate experiments showed that microwave-assisted probe ultrasonicated) accounted for 42% of
pre-boiling either conductively or by microwave irradiation before mixed juices, 31% of massecuites, 105% of molasses values,
probe ultrasonication equally solubilised <5,000 ppm insoluble 49% of bagasse extract, and 22% of raw sugar. It was found that
starch. However, microwaving was able to improve the probe the high colour of molasses and some massecuites were reduced
ultrasonication efficiency and solubilisation of higher insoluble when samples were diluted to 5 to 10 Brix before processing,
starch concentrations nearly 20–60% (Fig. 4) and was much quicker which did not affect total starch quantification on a ppm/Brix scale
than conductively boiled samples of the same concentration. (data not shown). For unfiltered and untreated mixed juices and
Maximum solubilisation of 620,000 ppm insoluble starch occurred bagasse extract, 86% of the colour values were due to suspended
with 60 s microwaving and 5 min probe ultrasonication (or 6 min particles scattering light. The percent colour contribution to
total processing time) at 60% amplitude, since starch yields starch-I 3 absorbance in microwave-assisted probe-ultrasonicated
decreased quadratically (R2 = 0.999) with longer probe ultrasonica- samples was reduced to 18% in mixed juices, 22% in masse-
tion time (Fig. 4). cuites, 33% in molasses, 9% in bagasse extract, and 12% in
Many authors have hypothesised that ultrasound may facilitate raw sugars. By filtering the untreated and treated samples instead
the decomposition of polymeric species in aqueous solutions, but of centrifuging them, the USDA research method removed 100% of
this was not evident in this study and there are mixed reviews in particle scattering, markedly reduced colourant contribution to the
 M.R. Cole et al. / Food Chemistry 190 (2016) 50–59 57

starch-I
3 absorbance values, and increased accuracy of the method. Jane et al., 1999; Swanston et al., 2001; Takeda, Hizukuri, &
Less than 2% of the starch-I
3 absorbance studied was from particle Juliano, 1987). As a result, overall starch-I
3 values differ signifi-
scattering in refined sugars with no observable contribution from cantly. Because sweet sorghum and sugarcane starch are better
its natural colourants (Cole, Eggleston et al. (2014)). represented by corn starch rather than potato starch, a calibration
curve with corn starch will provide more accurate starch quantities
in factory by-products.
3.5. Linearity
3.6. Precision of USDA method to quantify starch in simulated and
Current sugar industry starch methods measure sugarcane factory mixed juices
starch calibrated to potato starch, mostly because potato starch
is easy to solubilise in boiling water. Godshall, Triche, and Moore The starch values of five simulated mixed juices and factory
(2004) compared the production of calibration curves from potato, sugar products measured with the final USDA method are listed
rice, and corn starches that were boiled in water (conductive heat- in Table 1 with performance statistics. The best performance statis-
ing) for 5 min. Godshall et al. (2004) advocated the need to use tics were observed for sucrose solutions containing P10,000 ppm
potato starch as a reference because it can be completely solu- or 67,000 ppm/Brix insoluble starch with a CV 6 4%. Coefficients
bilised, whereas corn starch could not be used because it was not of variance increased as insoluble starch decreased, but still with
fully solubilised after 5 min boiling. Thus, potato starch has been acceptable precision 67%. For simulated mixed juice (15 Brix) con-
a common starch reference used across the sugar industry, despite taining 1% soluble corn starch and 99% insoluble corn starch, 99.3%
it being unrelated in composition or chemical properties to sugar- yield was obtained with an excellent CV% of 1%. No unsatisfactory
cane or sweet sorghum starch. In strong contrast, corn starch, is performance z scores or questionable data were obtained for
considerably more similar to sugarcane and sweet sorghum starch simulated juices containing 910–40,000 ppm or 6,066–
than potato starch (Eggleston, Cole, Gilbert et al., 2013; 267,000 ppm/Brix starch content (Thompson et al., 2006).
Figueira, Carvalho, & Sato, 2011), even having comparable As expected, mean starch values for sweet sorghum bagasse
amylose-to-amylopectin ratios (Chen & Chou, 1993). This is not were the highest at 13,385,118 ppm/Brix (115,379 ppm).
surprising since sugarcane, sweet sorghum, and corn are all grasses Sugarcane factory mixed juice, massecuites, molasses, and raw
(Poaceae). Calibration curves prepared with either solubilised sugar samples contained 2,607, 1,071, 673, and 1,164 ppm/Brix
potato (ICUMSA GS1-17) or solubilised cornstarch (USDA method) total starch, respectively. Although the mixed juice, massecuites,
are illustrated in Fig. 5. and molasses had CVs 6 7%, the CV% for the raw sugar sample
Acceptable linearity for both corn and potato starches was was the highest (i.e., CV = 12%). The low starch content in the
achieved with excellent R2 values >0.99. The linear range of the raw sugar and difficult repetitive sampling is a contributing factor
corn starch calibration curve in Fig. 5 was 0–1,275 ppm. The limit to lower precision observed in raw sugars since the mean starch
of detection was 39 ppm for corn starch and 92 ppm for potato concentration and standard deviation were both very small num-
starch. However, higher starch quantities can be measured by bers. When the mean concentration value is a small number, than
diluting the starch sample and then staining with iodometric the CV values can become quite large simply because a small (but
reagents. acceptable) standard deviation is being compared with another
When comparing calibration curves acquired for potato and small number (Coleman & Vanatta, 2012). No unsatisfactory per-
corn starches, it was seen that potato starch absorbed 2-times formance z scores or questionable data were obtained for any fac-
more than corn starch at equal concentrations (Fig. 5). A greater tory sample studied (Thompson et al., 2006).
divergence in absorbance occurred as concentrations exceeded
900 ppm. After determining that there was no difference between 3.7. Comparison of USDA method to sugar industry method to quantify
the iodometric reagents, it was concluded that the 2-fold differ- starch in factory samples
ence in slopes was due to the starch source. Godshall et al.
(2004) similarly reported that corn starch produced a lower slope Total starch in the mixed juice measured with the ICUMSA
than potato starch because less colour developed during iodine GS1-17 method was dramatically lower (3,226 ppm/Brix) than
complexation. Figueira et al. (2011) also reported that equal con- when determined by the USDA research method
centrations of potato starch-I3 complexes were twice more absorb- (10,467 ppm/Brix) (Fig. S2). Furthermore, the added benefit of
ing than iodine complexes with sugarcane and corn starches under the USDA method is that it was able to measure the ratio of soluble
similar conditions. This is because the binding affinities between to insoluble starch in each sample (Table 1). The differences in
iodine and starch as a granule, as well as to amylose and starch concentration observed between soluble (USDA) and total
amylopectin, differ with botanical source (Jane & Shen, 1993; starch (ICUMSA GS1-17) content, was attributed to the probable
swelling of some insoluble starch in the juice after the advocated
15 min boiling in water (ICUMSA GS1-17) which is still capable
of contributing to absorbance. By filtering the sample and extrap-
olating the results to a corn starch calibration curve, it was found
that much of the absorbance from the ICUMSA GS1-17 treated
sample was due to scattered starch (swollen and suspended) and
natural juice colourants; and, once removed, the total starch was
not much different from the initial soluble starch concentration
(data not shown).
Total starch values in raw sugar were 700 ppm/Brix and
1,112 ppm/Brix for ICUMSA GS1-17 and USDA methods, respec-
tively. Using the USDA method, the overall starch composition in
this raw sugar was found to be 51.7% soluble and 48.3% insoluble.
Cole, Eggleston et al. (2014) also reported that approximately 90%
Fig. 5. Standard curves generated using ICUMSA GS1-17 method (potato starch) or of the raw sugars analysed from various factories in Louisiana con-
USDA Research method (corn starch). tained 2–3 more total starch than that determined using the
58 M.R. Cole et al. / Food Chemistry 190 (2016) 50–59

Table 1
Data and performance statistics for the USDA starch solubilisation research method in various simulates and actual factory products.

Data statistics
Artificial 15 Brix sucrose solutions with insoluble corn starch Factory samples
Theoretical starch sample 6,066a 6,700 33,000 67,000 133,000 200,000 267,000 Mixed Bagasse Raw Massecuites Molasses
(ppm/Brix) Juice Sugar
Number of results 8 8 8 8 8 8 8 8 8 18 8 8
Number of excluded results 0 0 0 0 0 0 0 0 0 0 0 0
Mean starch concentration 6,026 6,761 33,693 67,947 133,508 200,274 253,603 2,607 13,385,118 1,164 1,071 673
(ppm/Brix)
% Recovery 99.3 100.9 102.1 101.4 100.4 100.1 94.9 n/ac n/a n/a n/a n/a
Median starch concentration 6,050 6,854 33,542 68,871 134,376 201,359 252,316 2,593 13,403,069 1,132 1,060 654
(ppm/Brix)
Standard deviation (ppm/Brix) 84 374 1,629 2,862 2,915 3,462 6,735 64 426,205 145 43 44
CV% 1 6 5 4 2 2 3 2 3 12 4 7
Result range (ppm/Brix) ±218 ±949 ±4462 ±6873 ±8973 ±10,519 ±21,039 ±222 ±1.3  106 ±463 ±134 ±121
Performance statistics
Assigned value (ppm/Brix) 6,050 6,854 33,542 68,871 134,376 201,359 252,316 2,593 13,403,069 1,131 1,060 654
SDPAb (ppm/Brix) 6,957 1,028 5,031 10,331 20,156 30,204 37,847 389 2,010,460 170 159 98
Satisfactory z scores% 100 100 100 100 100 100 100 100 100 100 100 100
Questionable z scores% 0 0 0 0 0 0 0 0 0 0 0 0
Unsatisfactory z scores% 0 0 0 0 0 0 0 0 0 0 0 0
a
Sample contains 1% or 66 ppm/Brix soluble corn starch and 99% insoluble corn starch or 6000 ppm/Brix in a 15 Brix sucrose solution.
b
SDPA is Standard Deviation for Proficiency Assessment.
c
n/a, data is not applicable or evaluated in this study.

ICUMSA GS1-17 method in which 15–40% was from insoluble method and compared to current starch methods used in the sugar
starch. Much of the starch found in the refined sugars analysed industry.
in that that study were also severely underestimated although
>80% of the starch was soluble. This is an important result for Acknowledgements
the sugar industry, as insoluble rather than soluble starch may
be the cause of difficult filtration in carbonatation refineries, yet Mention of trade names, commercial products, or methods in
current starch methods do not detect insoluble starch and under- this article is solely for the purpose of providing specific informa-
estimate total starch. tion and does not imply recommendation or endorsement by the
U.S. Department of Agriculture (USDA). USDA is an equal opportu-
4. Conclusions nity provider and employer.

A rapid and precise research method has been developed to Appendix A. Supplementary data
measure the total amount of starch in a variety of industrial sugar
products. Furthermore, the method is capable of measuring the Supplementary data associated with this article can be found, in
percent contribution from soluble and insoluble starch in these the online version, at http://dx.doi.org/10.1016/j.foodchem.2015.
products. The method is sensitive, highly selective to starch and, 05.049.
compared to current sugar industry starch methods, it is more
accurate, flexible and can rapidly analyse all samples with broad
References
starch concentration ranges (i.e., bagasse, juices, syrups, molasses,
massecuites and raw sugars) and wide Brix values. AACC International. Approved methods of analysis (10th ed.). Total starch assay
Utilising microwave irradiation, insoluble starch granules up to procedure (Megazyme amyloglucosidase/alpha-amylase method). Approved
a concentration limit of 40,000 ppm were evenly gelatinised in AACC International, St. Paul, MN, USA. http://dx.doi.org/10.1094/
AACCIntMethod-76-13.01.
60 s. The application of probe ultrasonication (60% amplitude; Anonymous. (1985). Laboratory manual for South African sugar factories, including
5 min) afterwards allowed for total solubilisation of the starch, official methods. In Proceedings of the South African Sugar Technologists
which could not be achieved with conductive boiling. Because total Association (3rd ed.) (pp. 263, 325, 326).
Balch, R. T. (1953). Further notes on starch in Louisiana cane juices and raw sugars.
starch solubilisation can be achieved, a standard curve could be Sugar Journal, 15(8), 11–15.
generated from corn starch which is a much better representative Bilbao-Sainz, C., Butler, M., Weaver, T., & Bent, J. (2007). Wheat starch gelatinisation
of sugarcane and sweet sorghum starches than potato starch, as under microwave irradiation and conduction heating. Carbohydrate Polymers,
69(2), 224–232.
they are all grass (Poaceae) crops. The lower limit of detection Chandrapala, J., Oliver, C., Kentish, S., & Ashokkumar, M. (2012). Ultrasonics in food
for this research method was found to be 39 ppm. Accuracy as processing – Food quality assurance and food safety. Trends in Food Science &
determined by percent recoveries were >99% or greater for Technology, 26(2), 88–98.
Charles, D. F. (1968). Quick starch method of analysis on raw sugars. In Proc. 1968
620,000 ppm insoluble corn starch and 95% for the 40,000 ppm Tech. session on cane sugar refining research (pp. 75–81).
insoluble corn starch sample. Precision generally improved with Chen, J. C. P., & Chou, C. C. (1993). Chemicals used as sugar processing aids. In Cane
increased starch contents and coefficients of variance (CV) for Sugar Handbook: A Manual for Cane Sugar Manufacturers and Their Chemists,
12th ed.): John Wiley & Sons Inc.
mixed juice, bagasse, molasses, massecuites, and raw sugar were
Cole, M., Eggleston, G., Gilbert, A., & Chung, Y. J. (2014). Development of a research
2%, 3%, 7%, 4%, and 12%, respectively with no unsatisfactory or method to measure insoluble and soluble starch in factory and refinery
questionable performance z scores. Future research is now needed products. In Proceedings of the 2014 sugar industry technologists (Vol. LXXIII,
to develop a method that could be used at a factory, refinery, or pp. 212–229).
Cole, M., Eggleston, G., Gilbert, A., Rose, I., Andrzejewski, B., St. Cyr, E., et al. (2013).
biorefinery using existing or simple equipment. The USDA research The presence and implication of soluble, swollen, and insoluble starch at the
method will be used to underpin the development of a factory sugarcane factory and refinery. International Sugar Journal, 115(1380), 844–851.
 M.R. Cole et al. / Food Chemistry 190 (2016) 50–59 59

Cole, M., Rose, I., Chung, Y. J., & Eggleston, G. (2014). A structured approach to target McCleary, B. V., Gibson, T. S., & Mugford, D. C. (1997). Measurement of total starch in
starch solubilisation and hydrolysis for the sugarcane industry. Food Chemistry. cereal products by amyloglucosidase-a-amylase method: Collaborative study.
http://dx.doi.org/10.1016/j.foodchem.2014.05.151. Journal of AOAC International, 80, 571–579.
Coleman, D., & Vanatta, L. (2012). Statistics in analytical chemistry: Part 48— Mecozzi, M., Amici, M., Pietrantonio, E., & Acquistucci, R. (1999). Ultrasound-
Relative standard deviations (RSDs). American Laboratory, 30–33. assisted analysis of total carbohydrates in environmental and food samples.
Czechowska-Biskup, R., Rokita, B., Lotfy, S., Ulanski, P., & Rosiak, J. M. (2005). Ultrasonics Sonochemistry, 6(3), 133–139.
Degradation of chitosan and starch by 360-kHz ultrasound. Carbohydrate Palav, T., & Seetharaman, K. (2006). Mechanism of starch gelatinisation and polymer
Polymers, 60(2), 175–184. leaching during microwave heating. Carbohydrate Polymers, 65(3), 364–370.
Eggleston, G., Cole, M., & Andrzejewski, B. (2013). New commercially viable Price, G. J., & Lenz, E. J. (1993). The use of dosimeters to measure radical production
processing technologies for the production of sugar feedstocks from sweet in aqueous sonochemical systems. Ultrasonics, 31(6), 451–456.
sorghum (Sorghum bicolor L. Moench) for manufacture of biofuels and Roy, I., & Gupta, M. (2003). Applications of microwaves in biological sciences.
bioproducts. Sugar Tech, 15(3), 232–249. Current Science, 85, 1685–1693.
Eggleston, G., Cole, M., Gilbert, A., & Rose, I. (2013). A new look at starch in the Sakar, D., & Day, D. (1994). A simple assay for starch in sugar mills process streams.
sugarcane factory and refinery: The presence of soluble and insoluble starch. In Proceedings of the 1994 American Society of sugarcane technologists, 14 (pp. 58–
Sugar Industry Technologists, Inc., 72(1066), 326–339. 62).
Eggleston, G., Viator, R. P., Gateuil, A., Fenger, J., White, P., Jackson, W., et al. (2013). Sakonidou, E. P., Karapantsios, T. D., & Raphaelides, S. N. (2003). Mass transfer
Effects of seasonal variations of sugarcane stalk and extraneous matter quantity limitations during starch gelatinisation. Carbohydrate Polymers, 53(1), 53–61.
and quality as they affect recoverable sugar, starch, and fiber: Part 1. Smith, W. T., & Cheng, C. (1978). Use of 3,5-dinitrosalicylate reagent for glucose
International Sugar Journal, 115(1375), 477–487. determination in mixed solvents. Analytical Letters, 11(2), 191–194.
Figueira, J. D. A., Carvalho, P. H., & Sato, H. H. (2011). Sugarcane starch: Quantitative Swanston, J. S., Ellis, R. P., Morrison, I. M., Mackay, G. R., Dale, M. F. B., Cooper, A.,
determination and characterization. Food Science and Technology (Campinas), et al. (2001). Combining high-amylose and waxy starch mutations in barley.
31(3). Journal of the Science of Food and Agriculture, 81(6), 594–603.
Godshall, M. A., Clarke, M. A., & Dooley, C. D. (1991). Starch: Process problems and Szent-Gyorgyi, A. (1933). Chemical & biological effects of ultra-sonic radiation.
analytical developments. IN Proceedings of the 1990 sugar processing research Nature, 131. 278-278.
conference (pp. 244–264). Takeda, Y., Hizukuri, S., & Juliano, B. O. (1987). Structures of rice amylopectins with
Godshall, M. A, Triche, R., & Moore, S. J. (2004). A rapid starch test for use in cane low and high affinities for iodine. Carbohydrate Research, 168, 79–88.
mills. Thompson, M., Ellison, S. L. R., & Wood, R. (2002). Harmonized guidelines for single
ICUMSA. Method GS 1–16. The determination of starch in raw sugar by a modified laboratory validation of methods of analysis (IUPAC Technical Report). Pure
BSES Method – Official. Approved Bartens, Berlin, Germany. Applied Chemistry, 74, 835–855.
ICUMSA. Method GS 1–17. The determination of starch in raw sugar by the spri Thompson, M., Ellison, S. L. R., & Wood, R. (2006). The international harmonized
rapid starch test – Tentative. Approved Bartens, Berlin, Germany. protocol for the proficiency testing of analytical chemistry laboratories. Pure
Iida, Y., Tuziuti, T., Yasui, K., Towata, A., & Kozuka, T. (2008). Control of viscosity in Applied Chemistry, 78, 145–196.
starch and polysaccharide solutions with ultrasound after gelatinisation. White, P. J. (2001). Properties of corn starch. In A. R. Hallauer (Ed.), Specialty corns
Innovative Food Science & Emerging Technologies, 9(2), 140–146. (pp. 33–57). Boca Raton, FL: CRC Publishers.
Jambraka, A. R., Hercega, Z., Šubaric, D., Babić, J., Brnčić, M., Brnčić, S. R., et al. (2010). Xue, C., Sakai, N., & Fukuoka, M. (2008). Use of microwave heating to control the
Ultrasound effect on physical properties of corn starch. Carbohydrate Polymers, degree of starch gelatinisation in noodles. Journal of Food Engineering, 87(3),
79, 91–100. 357–362.
Jane, J.-L., Chen, Y. Y., Lee, L. F., McPherson, A. E., Wong, K. S., Radosavljevic, M., et al. Zhou, M. M., Kimbeng, C. A., Eggleston, G., Viator, R. P., Hale, A. L., & Gravois, K. A.
(1999). Effects of amylopectin branch chain length and amylose content on the (2008). Issues of starch in sugarcane processing and prospects of breeding for
gelatinisation and pasting properties of starch. Cereal Chemistry Journal, 76(5), low starch content in sugarcane. Sugar Cane International, 26(3), 3–13.
629–637. Zuo, Y. Y. J., Hébraud, P., Hemar, Y., & Ashokkumar, M. (2012). Quantification of
Jane, J.-L., & Shen, J. J. (1993). Internal structure of the potato starch granule high-power ultrasound induced damage on potato starch granules using light
revealed by chemical gelatinisation. Carbohydrate Research, 247, 279–290. microscopy. Ultrasonics Sonochemistry, 19(3), 421–426.
Lewandowicz, G., Jankowski, T., & Fornal, J. (2000). Effect of microwave radiation on Zuo, J. Y., Knoerzer, K., Mawson, R., Kentish, S., & Ashokkumar, M. (2009). The
physico-chemical properties and structure of cereal starches. Carbohydrate pasting properties of sonicated waxy rice starch suspensions. Ultrasonics
Polymers, 42(2), 193–199. Sonochemistry, 16(4), 462–468.
Lingle, S. (2010). Opportunities and challenges of sweet sorghum as a feedstock for Zylema, B. J., Grider, J. A., Gordon, J., & Davis, E. A. (1985). Model wheat starch
biofuel. In G. Eggleston (Ed.). Sustainability of the sugar and sugarethanol systems heated by microwave irradiation and conduction with equalized
industries (Vol. 1058, pp. 177–188). Washington, DC: American Chemical heating times. Cereal Chemistry, 62, 447–453.
Society.