FTB-2829

original scientific paper

Modification of Wheat Starch with Adipic Acid/Acetic Anhydride and

Glutaric Acid/Acetic Anhydride Mixtures
urica Akar 1, Drago ubari1, Jurislav Babi1, Vlasta Piliota1, Kristina Valek Lendi2

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2

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and Midhat Jai3

Faculty of Food Technology, Kuhaeva 20, HR-31 000 Osijek, Croatia

Institute of Public Health of the Osijek-Baranja County, F. Kreme 1, HR-31 000 Osijek, Croatia
3

Faculty of Technology, Tuzla University, Univerzitetska 2, BiH-75 000 Tuzla,

Bosnia and Herzegovina

Accepted: August 28, 2012

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Received: May 30, 2011

Summary

The aim of this research was to investigate the influence of modification with adipic
acid/acetic anhydride and glutaric acid/acetic anhydride mixtures (acid:anhydride ratio 1:30)
on properties of wheat starch. Starch was isolated from two wheat varieties and modified with
afore mentioned reagent mixtures in 4, 6 and 8 %. Thermophysical, pasting, physical

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properties, total and resistant starch and amylose content were determined. Gelatinisation and
pasting temperatures of both starches decreased after modification with both reagents
mixtures, with more pronounced decrease at starches modified with glutaric acid/acetic
anhydride mixture. Modified starches were less prone to retrogradation after 7 and 14 days of
storage at 4 C than native counterparts and more stable during shearing at high temperatures.
Swelling power and solubility of starches generally increased after both modifications. Gel
adhesiveness increased by modification, while stability during freeze-thaw cycles decreased.
Resistant starch content increased by modifications, with the exception of Srpanjka starch
modified with adipic acid/acetic anhydride mixtures. With increase of proportion of reagents

used for modification, degree of substitution increased.

Corresponding author; Phone: ++385 31 224 391; Fax: ++385 31 207 115; E-mail: dackar@ptfos.hr

Key words: wheat starch; adipic acid/acetic anhydride mixture; glutaric acid/acetic anhydride
mixture; thermophysical properties; pasting properties; resistant starch

Introduction
Starch is natural carbohydrate, used in industry as thickener, colloidal stabilizer, gelling,
bulking, and water retention agent in food and non-food products (1). It is produced by
isolation from its natural sources (tapioca, maize, wheat, potato, rice), with app. 33 % of

starch produced in EU from wheat. Native starch, however, does not meet all needs for

chemical modification is most common.

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industry application and therefore different modifications are conducted amongst which

By introduction of new chemical bonds (ester, ether linkages, cross-linking) starch properties,
such as gelatinisation, retrogradation and pasting properties are strongly changed.

Since mixture of adipic acid and acetic anhydride is commonly used for starch modification,
other mixtures of dicarboxylic acids and acetic anhydride have potential in usage for starch
modification as well. However, reaction between starch and these mixtures can be led in two

ways: substitution of starch or cross-linking (2). Often, mixture of cross-linked and

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substituted products is produced.

It is well established that substitution, such as acetylation, reduces gelatinisation temperature

and tendency of starch towards retrogradation (3-6). However, properties of acetylated
distarch adipate differ through literature data (7-9), probably due to different ratios of
substituted and cross-linked products obtained during modification.
Adipic acid and its salts are approved for usage in food industry under E numbers 355 357,

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while acetylated distarch adipate is approved as E 1440. Glutaric acid is recognised as good
acidulant and is approved for usage in packaging materials for food products (10). While

adipic acid rarely occurs in nature, glutaric acid is occurring in animal and plant tissues, and it
can be found in blood and urine.
The aim of this research was to investigate the influence of modification with adipic
acid/acetic anhydride and glutaric acid/acetic anhydride mixtures on thermophysical and

pasting properties of wheat starch.

Materials and Methods

Modification of starch with dicarboxylic acid/acetic anhydride mixture
Starch was isolated, as previously described, from two wheat varieties: Golubica and
Srpanjka (harvest 2008), used in previous study (11).
Adipic and glutaric acids (for syntheses) used in the research were produced by Merck,
Hohenbrunn, Germany and acetic anhydride (p. a.) was product of Kemika, Zagreb, Croatia.
Mixture of acetic anhydride and adipic or glutaric acid (30:1) was prepared by suspending:
a) 0.1290 g acid in 3.8710 g acetic anhydride for modification in 4 % (m/m);

b) 0.1935 g acid in 5.8065 g acetic anhydride for modification in 6 % (m/m);

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c) 0.2581 g acid in 7.7419 g acetic anhydride for modification in 8 % (m/m).

Starch (100 g d. m.) was suspended in distilled water (145 mL). Suspension was homogenised
by magnetic stirrer (300 rpm/30 min). pH of starch suspension was adjusted to 9.0 with 1M
NaOH and mixture of acid and anhydride was drop-wise added with maintaining pH value
close to 9 and constant stirring. After addition of mixture of acid and anhydride, starch
suspension was stirred for 30 min at room temperature. Overall reaction time was 2 hours.
Reaction was terminated by adjusting pH to 5.0 with drop-wise addition of 1M HCl.

Suspension was centrifuged (3000 rpm/5 min) and starch pellet was washed with water and

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centrifuged 3 times. Starch suspension was neutralized, centrifuged and starch pellet was air
dried. Dry matter content was determined in dried starch by oven drying (130 C/90 min).

Characterisation of modified starch

Gelatinization and retrogradation properties of native and modified starches were determined
by method described by Babi et al. (4), using differential scanning calorimeter DSC 822e

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(Mettler Toledo, Greifensee, Switzerland & Columbus, Ohio, USA) equipped with STARe
software. Starch samples were weighed into standard aluminium pan (40 L) and distilled
water was added by Hamilton microsyringe to achieve suspension containing 65 % water.
Samples were hermetically sealed and equilibrated for 24 hours at room temperature before
heat treatment in the DSC apparatus. The starch samples were heated at a rate of 10 C/min
from 25 to 95 C. After heat treatment, samples were cooled to 25 C and removed from DSC.
The starch gels were aged at 4 C and monitored for retrogradation after 7 and 14 days. The
retrogradation experiments were conducted at a heating rate of 10 C/min from 25 to 95 C.
The changes in enthalpy (H in J/g of dry starch), onset temperature (To), peak temperature
(Tp) and endset (conclusion) temperature (Te) for gelatinisation and retrogradation were
obtained from the exotherm DSC curves. Experiments were run in triplicates.
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Pasting properties of starches (7 % d.m.b., 100 g total mass) were determined using a Mycro
Visco-Analyser (Model 803202, Brabender Gmbh & Co KG, Duisburg, Germany). The starch
suspensions were heated at 7.5 C/min from 30 C to 92 C, held at 92 C for 20 min, cooled
at 7.5 C/min to 50 C, and held at 50 C for 20 min. Experiments were run in triplicates.
Swelling power (SP) and solubility (SOL) were determined by method described by Babi et
al. (3). 1 % starch suspensions (d.m.b.) were heated in shaking water bath for 30 min at 65,
75, 85 and 95 C. This temperature range is commonly used in SP and SOL measurements.
After heating, suspensions were cooled and centrifuged at 4000 rpm for 30 min. Supernatant

was decanted, and gel was weighed. Dry matter of supernatant was determined by oven

SOL were calculated from Eq.1. and 2.:

SP =

g
g

mass of gel
mass of dry matter in gel

SOL =

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drying at 105 C until constant mass was reached. Experiments were run in triplicates. SP and

mass of dry matter in supernatant

mass of dry matter in suspension

[%]

/1/
/2/

Paste clarity (in triplicates) was determined by method described by Raina et al. (2006) (12).
1% (d.w.b.) starch suspensions were heated for 30 min in boiling water bath with occasional

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shaking. After 1 hr holding at room temperature % transmittance was read at 650 nm against
distilled water as blank.

Gel texture was determined using a TA-XT Plus (Stable Microsystem). A starch suspension
(11% w/w) was prepared in a flask and then heated at 95 C for 30 min in a temperature
controlled shaking water bath with constant shaking. The cooked paste was cooled to 25 C,
weighed into plastic cans and allowed to gel in ClimaCell at 25 C and 85% r. h. The gel

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formed in the can (45 mm in height and 35 mm in diameter) was compressed at the speed of
2.0 mm/sec to the distance of 20 mm with a flat cylinder probe having 20 mm diameter. The
peak height at 20 mm compression was termed hardness, and the negative area of the curve
during retraction of the probe was termed adhesiveness (13). Five repeated measurements
were performed for each sample.
Freeze-thaw stability was measured by modified method of Lawal (2009) (14). Starch
suspension (5% w/w, d.w.b.) was heated in temperature controlled shaking water bath with
constant shaking (200 rpm) for 1 hr. The paste was weighed (10 g) in pre-weighed PPcentrifuge tubes and subjected to freeze-thaw cycles followed by centrifugation at 4000 rpm
for 30 min. Alternate freezing and thawing was performed by freezing for 22 hr at -18 C and
thawing for 2 hr at 30 C. Seven freeze-thaw cycles were performed. The weight of water
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separated after each freeze-thaw cycle was measured and extent of syneresis was calculated
following Eq. 3.:
Syneresis =

water separated [g ]
100
total weight of sample [g ]

[%]

/3/

Total starch was determined by Ewers method. Resistant starch content was determined by
AOAC 2002.02 method (15), using Megazyme enzymatic kit.
Amylose content was determined according to the method described by Gibson et al. using
Megazyme enzymatic kit (16). Starch samples were completely dispersed in dimethyl

sulphoxide (DMSO), precipitated in ethanol and dissolved in acetate/salt solution.

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Amylopectin was specifically precipitated by the addition of ConA and removed by

centrifugation. The amylose in the aliquot of the supernatant was enzymatically hydrolysed to
D-glucose, which was analysed using glucose oxidase/peroxidase reagent. Total starch in
separate aliquot of acetate/salt solution was also enzymatically hydrolysed to D-glucose,
which was analyzed using glucose oxidase/peroxidase reagent. Concentration of amylose in
starch sample was calculated from Eq.4.:

Absorbance (ConA)
66.8
Absorbance (Total Starch Aliquot )

[%]

Amylose =

/4/

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Where 66.8 was dilution factor for ConA and Total Starch extracts.
Degree of substitution (DS) was determined titrimetically, following method of Ogawa et al.
(17). Five grams of modified starch and 50 ml of distilled water were dispersed in an
Erlenmeyer flask with a stopper. Phenolphthalein was added to the suspension as an indicator,
and then 0.1 M NaOH solution was added to get red colour. After the addition of 25 ml of

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0.45 M NaOH solution, the mixture was stirred at 25 C for 30 min. The flask was corked to
prevent evaporation of produced acetate during saponification reaction. Finally, the excess
alkali in the sample mixture was titrated with 0.2 M HCl solution. A blank test was also
carried out with native starch using the same procedure. All samples were done in triplicate.
DS is defined as the average number of sites per glucose unit that possess a substituent group.
Since dicarboxylic acids were used in amounts significantly smaller than acetic anhydride, DS
is expressed through acetyl substituents and calculated as follows (Eq. 5):
DS =

% Acetyl 162
4300 (42 % Acetyl )

Where: % Acetyl =

/5/

(Vb Vs ) N HCl 0.043 100

m

Vb = ml 0.2 M HCl used to titrate the blank;

5

/6/

Vs= ml of 0.2 M HCl used to titrate the sample;

NHCl = the normality of HCl used for titration;
m = the sample mass (g).
All experimental data were analysed by analysis of variance (ANOVA) and Fishers least
significant difference (LSD) with significance defined at P<0.05. All statistical analyses were
carried out using software program STATISTICA 8 (StatSoft, Inc, USA).

Results and Discussion

Properties of native starches are discussed in detail in previous work (11).

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The DSC gelatinization parameters of starches modified with glutaric acid/acetic anhydride
and adipic acid/acetic anhydride mixtures are shown in Table 1. Onset, peak and endset
gelatinisation temperatures were decreased by both investigated modifications. Peak
gelatinisation temperature of Golubica starch was decreased from 62.68 C to 59.30 61.45
C, and at Srpanjka starch this temperature decreased from 63.72 C to 60.91 62.59 C,
depending on reagent mixture and its concentration. This, as well as broadening of
temperature range of gelatinisation (te to; results not shown), indicates that the internal

crystalline structure of modified starches was more easily destroyed at a lower temperature

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than native starch. In addition, broader temperature range indicates a greater degree of
structural heterogeneity, changes in crystal shapes and size, degree of crystal perfection and
type of starch chain intertwining (linear-linear, linear-branch, or branch-branch) (8).
Modification with mixtures of adipic acid and acetic anhydride had similar influence on both
investigated starches, while modification with mixtures of glutaric acid and acetic anhydride
had higher influence on gelatinisation temperatures of Golubica starch than of Srpanjka

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starch. Decrease of onset gelatinisation temperature of Golubica starch was proportional to

amount of glutaric acid/acetic anhydride added to starch, while this relationship wasnt
established for peak and endset temperature (tp and te of GGA8 starch were higher than of
GGA6 starch, Table 1).
Onset, peak and endset gelatinisation temperatures were also decreased by modification of
starch with glutaric acid (8), octenylsuccinic anhydride (18) and acetic anhydride (4), and by
dual modification (19). Introduction of bulky substituent groups causes steric hindrance
between starch polymer chains and therefore facilitates penetration of water molecules and
gelatinisation of starch.
Modification of both starches with glutaric acid/acetic anhydride mixture in 4 % decreased
gelatinisation enthalpy (from 7.17 J/g to 6.89 J/g for Golubica starch and from 8.47 J/g to
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7.79 J/g for Srpanjka starch), while same modification in 8 % increased it (to 8.85 J/g for
Golubica starch and 8.79 J/g for Srpanjka starch) and in 6 % had different effect, depending
on starch variety (increase to 7.48 J/g for Golubica starch and decrease to 7.96 J/g for
Srpanjka starch). Mixture of adipic acid and acetic anhydride increased gelatinisation
enthalpy of Golubica starch proportionally to amount used for modification (Table 1), which
is characteristic for cross-linked starches (20-22) due to more energy required for disruption
of intermolecular bonds.
For Srpanjka starch, gelatinisation enthalpy increased following order: SADA8 (6.97 J/g) <

SADA6 (7.51 J/g) < native starch (8.47 J/g) < SADA4 (8.61 J/g). Gelatinisation enthalpy

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gives an overall measure of crystallinity (quantity and quality) and is indicator of the loss of
molecular order within granule on gelatinisation (23). Decrease of gelatinisation enthalpy
suggests that substituent groups disrupt double helices (owing to rotation of these flexible
groups) within amorphous regions of the granules. Hence, higher the degree of substitution,
higher the decrease of gelatinisation temperatures and enthalpy (23).

Adipic and glutaric acids are dicarboxylic acids which can react as cross-linking agents.
Cross-linking at lower levels reduces the proportion of starch that can be gelatinised, thus

resulting in decrease of gelatinisation enthalpy. Gelatinisation temperatures of dual-modified

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starches (substituted and cross-linked) are generally lower than native counter parts (23).
Gelatinised starch is thermodynamically unstable system (24). Therefore, after gelatinisation,
amylose and amylopectin tend to recrystallise and form semi-cristalline gel. Rate of
retrogradation is influenced by amylose/amylopectin ratio, presence of lipids, emulsifiers and
ions, and by modification (25). Retrogradation has negative impact on textural properties of
the product (eg. bread staling (26), increase of hardness and toughness of potato chips (27),

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syneresis (28) etc.)

Retrogradation enthalpy (Table 2) of Golubica starch after 7 and 14 days of storage at 4 C

was decreased by both investigated modifications proportionally to amount of reagents

mixtures used, where enthalpy was decreased more by mixture of glutaric acid and acetic

anhydride compared to mixture of adipic acid and acetic anhydride in the same amount (eg.
1.85 J/g for GGA8 compared to 2.35 J/g for GADA8).
Retrogradation enthalpy of Srpanjka starch modified with mixture of glutaric acid and acetic
anhydride after 7 days of storage at 4 C (Table 2) decreased following order: native starch
(3.23 J/g) > SGA6 (2.15 J/g) > SGA8 (2.05 J/g) > SGA4 (2.02 J/g), while after 14 days of
storage it was proportional to increase of reagents mixture concentration. Decrease of
retrogradation enthalpy of Srpanjka starch modified with mixture of adipic acid and acetic
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anhydride after 7 and 14 days of storage was proportional to amount of reagents mixture used
(Table 2).
Decrease of retrogradation enthalpy was observed for hydroxypropylated cross-linked starch
(29), hydroxypropylated (14) and acetylated starches (4) due to bulky substituent groups
which hinder intertwining of starch polymer chains.
Starch shows unique viscosity behaviour (pasting properties) with change of temperature,
concentration and shear rate, which is very important when considering a starch as a possible
component of a food product (23).

Pasting properties of modified Golubica and Srpanjka starch are shown in Table 3. Pasting

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temperature decreased by both investigated modifications in all concentrations. Pasting occurs

after gelatinisation of starch, hence pasting temperature is higher than gelatinisation
temperature. During gelatinisation, ordered molecular structure of starch granule is disrupted,
which causes irreversible changes: granule swelling, loss of birefringence and dissolution of
starch. Pasting involves further granule swelling, leaching molecules out of granule and
complete degradation of granule. In state of gelatinisation, starch has thickening properties,
while pasting gives properties of emulsifying, gelling and taste fullness (mouth feel) (30).

Decrease of pasting temperature for Golubica starch modified with glutaric acid/acetic

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anhydride mixture followed the order: native starch (64.87 C) > GGA4 (63.90 C) > GGA8
(62.15 C) > GGA6 (61.45 C). Decrease of pasting temperature for adipic acid/acetic
anhydride modified Golubica starch was proportional to the increase of concentration of
reagents used, with no significant difference between GADA6 (60.95 C) and GADA8 (60.80
C).

Decrease of pasting temperature of Srpanjka starch was proportional to the increase of

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concentration of both reagents, with more significant influence of mixture of adipic acid and
acetic anhydride.

However, Luo et al. (9) observed increase of pasting temperature for waxy potato starch
modified with mixture of adipic acid and acetic anhydride, while Deetae et al. (31)

determined that pasting temperature could be decreased or increased by cross-linking and dual
modification, depending on reaction conditions.
Peak viscosity of both investigated starches significantly increased by both modifications.
Modification with mixture of glutaric acid and acetic anhydride doubled peak viscosity, while
modification with adipic acid/acetic anhydride mixture increased it app. 1.6 times. Maximum
viscosity reflects the ability of the granules to swell freely prior to their physical breakdown

starches that are capable to swell to higher degree are also more resistant to breakdown, but
show significant viscosity decrease after maximum viscosity is reached (32).
Viscosity at 92 C was also increased by both modifications. For Srpanjka starch modified
with mixture of glutaric acid and acetic anhydride the increase of viscosity was proportional
to amount of reagents mixture used, while for all other modified starches the increase
followed the order: native starch < starch modified with 4 % reagent < starch modified with 8
% reagent < starch modified with 6 % reagent, which corresponds to swelling power of
starches (Table 3). Hot paste viscosity increase, characteristic for cross-linked starches, was

also observed for waxy potato starch modified with mixture of adipic acid and acetic

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anhydride (9), starch modified with octenylsuccinic anhydride (33) and dual-modified starch
(31).

After 20 min holding at 92 C decrease of viscosity was observed for all investigated starches.
This is explained by the molecular dissociation that amylose and amylopectin undergo. In this
phase amylose is solubilised and liberated to aqueous phase (34).

Difference between peak viscosity and viscosity after holding at 92 C gives breakdown
value, measure of fragility of granules, their stability or resistance to the disintegration as a

result of heating and agitation (9, 34). However, simple difference does not always project the

(35).

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true stability and therefore, sometimes it is expressed as percentage of maximum viscosity

From breakdown expressed as % values (Table 3), it is visible that modification with mixture
of glutaric acid and acetic anhydride resulted in stabilisation of paste during shearing at high
temperatures for both modified starches, following order: native starch (least stable) < starch
modified in 4 % < starch modified in 8 % < starch modified in 6 % (most stable).

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Modification with adipic acid/acetic anhydride mixture resulted in increase of instability of

both starch pastes during shearing at high temperatures.
Breakdown values increased by modification of starch with octenylsuccinic anhydride (18),

while modification with adipic acid/acetic anhydride mixture (9) and hydroxypropylation
decreased these values (36).
In the cooling stage, increase of paste viscosity was observed for all investigated starches.
This is caused by molecular re-association due to formation of three-dimensional networks of
amylose (34). Increase of paste viscosity during cooling is also expressed by setback value.
Similarly to breakdown values, setback values can be expressed as % of viscosity at 50 C
(Table 3), which enables better insight in stability of paste during cooling (35).

As can be seen from results in this research, although setback values increase by modification,
% setback showed their increased stability during cooling. Most stable pastes were formed
after modification of Srpanjka starch with mixture of glutaric acid and acetic anhydride and
here stabilisation increased proportionally to modification degree. For all other investigated
modified starches this correlation wasnt established.
Lower setback values are result of restriction of tendency of starch molecules to realign after
cooling, due to steric hindrance of substituent groups (23).
Swelling power is defined as the ratio of the wet mass of the sediment gel to its dry mass.

Swelling apparently is a property of amylopectin. Crystallites within the amylopectin

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molecules determine the onset of swelling and gelatinization. The swelling of starch granules
is related to pasting behaviour and rheological properties. Swelling power has been found to
significantly correlate with a high hot-paste peak viscosity, a low pasting temperature and a
high hot-paste breakdown (37).

Swelling power of investigated starches is shown in Table 4. Swelling power of all

investigated starches increased with increase of temperature from 65 C to 95 C. As the
temperature of the medium increases, starch molecules become more thermodynamically

activated, and the resulting increase in granular mobility enhances penetration of water, which

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facilitates improved swelling capacities (38).

Modifications of both Golubica and Srpanjka starches with glutaric acid/acetic anhydride and
adipic acid/acetic anhydride mixtures resulted in increase of swelling power. The highest
increase of SP at 95 C was achieved by modification of both starches with adipic acid/acetic
anhydride mixture in 8% (from 22.23 g/g to 30.55 g/g for Golubica starch and from 23.23 g/g
to 32.88 g/g for Srpanjka starch). Introduction of bulky substituent groups into starch

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molecules by esterification leads to structural reorganisation owing to steric hindrance. This

results in repulsion between starch molecules and facilitates water percolation within
amorphous region of granules, which, in turn, results in increase of swelling power (38).
Increase of swelling power and solubility was observed for succinylated and acetylated maize
starches (38), and acetylated and dual-modified rice starches (12). However, Luo et al. (9)
reported lower swelling power of waxy potato starch due to modification with mixture of
adipic acid and acetic anhydride and Das et al. (39) reported that swelling power of dualmodified starches was lower or not significantly different from native starch.
Unlike swelling power, for wheat starch, solubility is mainly result of amylose leaching from
starch granule, since most of the amylose will be solubilised before leaking of amylopectin

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occurs (40). However, introduction of bulky substituent groups can increase solubility of
amylopectin, while cross-linking decreases it (23).
Solubility of starch generally increased after modifications with glutaric acid/acetic anhydride
and adipic acid/acetic anhydride mixtures (Table 5) with the exception of solubility at 95 C,
where decrease from 32.69% to 12.90 31.39% for Golubica starch and from 35.82% to
11.16 35.43% for Srpanjka starch was observed. However, correlation between amylose
content (Table 6) and this phenomenon was not clearly established. This may be caused by
multiple influence of modification increase of steric hindrance between amylopectin chains

due to introduction of acetyl groups, decrease of solubility due to cross-linking with mixed

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anhydrides and decrease of amylose content. Nevertheless, it can be concluded that increase
of temperature results in more apparent influence of cross-linking and amylose reduction
effects.

Paste clarity (Table 4) of Srpanjka starch after both investigated modifications, as well as
Golubica starch modified with mixture of glutaric acid and acetic anhydride decreased
significantly (eg. from 9.3%T for native Srpanjka starch to 2.47%T for SGA8), while
modification with adipic acid/acetic anhydride mixture resulted in increase of Golubica starch

paste clarity. Paste clarity is related to swelling capacity of starch, state of dispersion and the

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retrogradation tendency. It is influenced by concentration, pH, type and extent of

modification. Chemical substitution of side chains results in inhibition of ordered structure of
starch resulting in improved paste clarity (41), while cross-linking has been observed to
decrease paste clarity (23). A fairly transparent paste is desirable in fruit pie-fillings, while
opacity is desirable in salad dressings and instant desserts.
Textural properties (gel strength, rupture strength, adhesiveness) largely determine

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palatability and consumer acceptability of starch based products (42). The textural and
mechanical properties of starch gels depend mainly on the rheological properties of the
continuous amylose phase, the volume fractions of the granules, the deformability of the
granules and the interaction between the dispersed and continuous phase (20). Gel strength
and rupture strength (Fig. 1) of Srpanjka starch modified with glutaric acid/acetic anhydride
mixture decreased proportionally by modification in 4 and 6 %. However, 8 % modification
resulted in increase of these parameters in relation both to native and modified starches in 4
and 6 %. For other modified starches this relationship wasnt established.
Mali & Grossman (7) reported increase of gel strength after modification of starch with
mixture of adipic acid and acetic anhydride by extrusion, while Wattanachant et al. (36)
reported decrease of gel strength of double-modified sago starch. Rupture strength is result of
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retrogradation of starch, which causes gel firmness (39). Decreased rupture strength of
modified starches could be result of lower tendency of these starches towards retrogradation.
Adhesiveness of both investigated starches modified with glutaric acid/acetic anhydride
mixture increased significantly and proportionally to degree of modification. Modification
with adipic acid/acetic anhydride mixture caused increase of adhesion of Srpanjka starch
proportionally to modification degree. However, although Golubica starch adhesion increased
by this modification, it decreased with increase of amount of reagents mixture used. Das et
al. (39) reported that adhesion of starch gel decreased both by acetylation and double

modification.

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Stability of starch during freeze-thaw cycles was decreased after modification of both starches
(Fig. 2). However, Golubica starch modified in 6 and 8 % with both investigated reagents
mixtures didnt show increase of syneresis after 1st freeze-thaw cycle and syneresis of
Srpanjka starch modified with glutaric acid/acetic anhydride mixture was retarded. Problem
with these starches was loss of great amount of water in the 1st cycle, but not stability after it.
Van Hung & Morita (19) reported larger syneresis of cross-linked starches during first two
freeze/thaw cycles compared to native counterparts and smaller syneresis during second two

freeze/thaw cycles. Wu & Seib (43) reported that dual modified waxy barley starches

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prepared by cross-linking with POCl3 and hydroxypropylation had better freeze-thaw stability
than similar commercial starches. However, Yeh & Yeh (44) reported that resistance to
freeze-thaw cycles depended on variety of starch used. Starches with higher content of
amylopectin exhibited higher freeze-thaw stability.

Total starch content of starches isolated from wheat varieties Golubica and Srpanjka and
modified with mixtures of glutaric acid and acetic anhydride and adipic acid and acetic

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anhydride in 4, 6 and 8 % is shown in Table 5. The starch content, which is indicative of its
purity, varied from 94 % to 100 %, which is in consistence with research of Perez Sira &

Lares Amaiz (45), Li et al. (46) and Verwimp et al. (47).

Resistant starch (RS) is starch, as well as its degradation products, that is not absorbed in the
small intestine of healthy individuals. It reaches large intestine where its fermented by the
colonic microflora with the production of short chain fatty acids (mainly acetic, propionic,
butyric), CO2, H2 and, in some individuals, CH4. RS consumption is related to beneficial

implications in the management of diabetes and colonic health (48). Resistant starch (RS)
content of investigated starches is shown in Table 6. All investigated starches contained very
low proportions of resistant starch (0.21 3.06% d. m.), which is indicative of their high
digestibility.
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Modification of both starches with mixture of glutaric acid and acetic anhydride resulted in
increase of RS content, with more pronounced effect on Srpanjka starch (from 0.57% d. m.
for native to 0.69 3.06% d. m. for modified starch). RS content of Golubica starch increased
app. 130 % by modification in 4 % and app. 140 % by modification in 8 %, while 6 %
modification didnt have significant influence. For Srpanjka starch, in vitro digestibility
increased app. 530 % by modification in 4 %, app. 300 % by modification in 6 % and app.
120 % by modification in 8 %.
Although RS content in starches modified with glutaric acid/acetic anhydride was low,

increase of RS content compared to native starch was very indicative of potentiality of usage

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of this mixture for production of starch with reduced digestibility. In addition, resistant starch
content increased by modification of adley starch with glutaric acid (8).

Mixture of adipic acid and acetic anhydride increased RS content of Golubica starch, while
decreased it in Srpanjka starch. It has been reported that cross-linking decreased susceptibility
of starch to -amylase degradation (8) due to reduced availability of the inner part of starch
granule, while hydroxypropylation increased it due to increase of availability of starch chains
to enzyme attack (49).

Both reduction and increase of starch susceptibility towards enzyme degradation can be

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desirable. While starches with increased levels of RS have important role in dietotherapy of
diabetes, hyperlypidemia and colonic health (50, 51), starches with low levels of RS are
desirable materials in production of biodegradable packaging (52).
Increase of proportion of modification mixtures investigated in this research resulted in
proportional increase of degree of substitution (DS) of both starches (Table 5). DS for
starches treated with 4 % reagent varied between 0,111 and 0,115. With increase of reagent

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amount, DS increased to 0.131 0.141 (for 6 %) and 0.142 0.150 (for 8 %). Murua-Pagola

et al. (53) achieved DS 0.042 by treating starch with 4 % octenylsuccinic anhydride, while
Hui et al. (33) reported DS 0.02.
However, reaction efficiency, which can be calculated from ratio of obtained DS to theoretical
values (results not shown), decreased as level of modification increased. Reaction efficiency
for both starches modified in 4 % exceeded 70 %, modification in 6 % resulted in reaction
efficiency around 55 % and in 8 % around 45 %. This phenomenon is consistent with
previous research of Hui et al. (33).

13

Conclusions
From results shown in this research, it is visible that mixtures of glutaric or adipic acid with
acetic anhydride can be used in order to achieve decrease of gelatinisation and pasting
temperatures and tendency towards retrogradation of wheat starch during storage at 4 C. In
addition, pastes of modified starches were more stable during shearing at high temperature
and cooling than their native counterparts. However, stability during freeze/thaw cycles was
decreased by investigated modifications, and RS content was maintined at low levels.
Starches modified with glutaric acid/acetic anhydride and adipic acid/acetic anhydride

mixtures have great potential for application in systems where high stability during heating,

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cooling and storage, as well as high swelling power and solubility are required. Additional
research on reaction conditions adjustment is needed in order to achieve higher RS content.

Acknowledgement

Results shown have outcome from scientific project Development of new modified starches
and their application in food industry supported by the Ministry of Science, Education and

1.

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18

Table 1. Gelatinisation properties of starch isolated from wheat varieties Golubica (G) and
Srpanjka (S) and modified with adipic acid/acetic anhydride (ADA) and glutaric acid/acetic
anhydride (GA) mixtures in 4, 6 and 8 %.
t p /C

te / C

H / J/g

59.490.07d

62.680.03d

66.660.06d

7.170.10b

GGA4

57.620.07c

60.930.06c

64.510.08b

6.890.01a

GGA6

56.570.03a

59.900.03a

64.050.04a

7.480.07c

GGA8

56.030.11b

60.390.10b

64.880.03c

8.850.11d

GADA4

58.110.18b

61.450.15b

65.330.40b

7.210.49a

GADA6

56.030.06a

59.550.05a

63.640.03a

7.600.06a,b

GADA8

56.050.13a

59.300.53a

63.900.10a

7.850.09b

59.940.17d

63.720.02d

67.820.09d

8.470.01c

SGA4

58.060.23b

62.030.13a

66.210.17a

7.790.05a

SGA6

58.170.21b

62.590.20b

67.410.48c

7.960.04b

SGA8

57.140.14a

62.010.11a

66.830.06b

8.790.05d

SADA4

58.180.07c

62.340.13c

66.650.11c

8.610.05d

SADA6

56.360.15a

60.910.12a

65.400.06a

7.510.04b

SADA8

57.300.10b

61.760.14b

66.120.10b

6.970.03a

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to / C

Starch

to, onset temperature; tp, peak temperature; te, endset temperature; H, gelatinisation entalphy; Values are
means SD of triplicate. Values in the same column with different superscripts (a-d) are significantly

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different than native counterparts (p<0.05).

19

Table 2. Retrogradation properties of starch isolated from wheat varieties Golubica (G) and
Srpanjka (S) and modified with adipic acid/acetic anhydride (ADA) and glutaric acid/acetic
anhydride (GA) mixtures in 4, 6 and 8 % after 7 and 14 days of storage at 4 C.
Starch

to / C

tp / C

t e / C

H / J/g

After 7 days of storage at 4 C

42.570.29a

52.100.26b

60.450.05a

3.420.01d

GGA4

42.930.31a

51.600.30a

60.470.06a

2.720.01c

GGA6

42.320.33a

51.610.02a,b

60.370.25a

2.310.03b

GGA8

42.580.56a

51.870.31a

60.470.46a

1.850.05a

GADA4

42.630.35a

51.470.35a

60.200.17b

2.840.02c

GADA6

42.800.20a

51.590.17a

57.432.20a

2.450.03b

GADA8

42.330.31a

51.900.10a,b

60.630.42b

2.350.02a

43.640.42c

51.670.27b

60.440.49b

3.230.03c

SGA4

41.360.11a

51.170.30a

60.960.05b

2.020.09a

SGA6

42.200.20a.b

51.730.24b

60.520.20b

2.150.02b

SGA8

42.330.06b

51.580.19a,b

59.890.10a

2.050.05a

SADA4

42.600.10a,b

53.613.67a

60.430.11c

2.600.02c

SADA6

43.060.10b

52.510.15a

60.780.41b

2.410.01b

SADA8

42.330.21a

51.660.19a

60.480.20a

2.280.02a

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After 14 days of storage at 4 C

42.300.36a

51.400.70a

60.740.41a,b

3.810.04d

GGA4

42.230.49a

51.600.30a

61.030.35b

3.390.03c

GGA6

42.800.35a

52.730.38b

60.900.10b

3.310.02b

GGA8

42.300.36a

51.400.40a

60.300.17a

2.640.02a

GADA4

42.430.06a,b

51.300.46a

60.310.06a

3.340.01c

GADA6

42.820.29b

52.430.15b

60.890.36a

3.100.05b

GADA8

42.270.35a

51.570.15a

60.870.40a

2.870.01a

43.140.32b

51.550.24a

60.640.06a

3.700.04d

SGA4

42.470.06a

53.420.13b

60.540.39a

2.850.04c

SGA6

43.390.29b

53.870.15c

60.820.16a

2.510.01b

SGA8

43.350.43b

53.830.25c

60.610.44a

2.050.05a

SADA4

42.950.15b

53.740.24c

60.740.22a

2.810.01c

SADA6

43.380.12b

54.150.22c

61.740.26b

2.640.03b

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20

SADA8

41.980.42a

51.010.26a

60.500.17a

2.550.01a

to, onset temperature; tp, peak temperature; te, endset temperature; H, retrogradation entalphy; Values are
means SD of triplicate. Values in the same column with different superscripts (a-d) are significantly

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different than native counterparts (p<0.05).

21

Table 3. Pasting properties of starch isolated from wheat varieties Golubica (G) and Srpanjka (S) and modified with adipic acid/acetic anhydride
(ADA) and glutaric acid/acetic anhydride (GA) mixtures in 4, 6 and 8 %. Pastes contained 7 % of starch (m/m).

GGA4

Viscosity

After 20 min

Viscosity

After 20 min

temperature/C

viscosity/BU

at 92 C/BU

at 92 C/BU

at 50 C/BU

at 50 C/BU

(% breakdown)

(% setback)

64.870.25d

306.335.51a

256.673.79a

240.674.16a

501.674.04a

435.004.58a

65.672.08a

261.01.73b

(21.44)

(52.03)

63.900.14

61.450.35

663.004.24

628.502.12

Breakdown*

Peak

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Starch

Pasting

565.500.71

968.002.83

844.005.66

97.504.95

(14.71)

GGA6

653.503.54

b,c

649.005.66

642.001.41

1034.005.66

579.502.12

893.007.07

726.005.66

561.500.71

74.001.41

GGA8

62.150.07

62.650.35

650.005.66

487.001.41

563.007.07

788.005.66

87.001.41

476.507.78

358.504.95

691.509.19

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GADA4

GADA6

60.950.35a

541.002.83d

537.502.12d

397.502.12c

732.002.83c

641.502.12c

GADA8

498.503.54

489.004.24

737.009.90

291.003.61

272.334.16

457.673.79

60.800.14

65.600.36

365.500.71

214.003.00

569.005.66

391.674.04

64.450.21c

616.004.24b

576.005.66b

540.004.24b

840.005.66d

766.005.66c

SGA6

659.500.71

589.007.07

700.504.95

667.502.12

596.005.66

694.506.36

SGA8

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SGA4

63.200.42

61.850.49

621.002.83

639.504.95

765.007.07

128.503.54

734.005.66

22

333.04.24b

(26.39)

(48.16)

143.500.71c

334.54.95b

(26.52)

(45.70)

133.002.83

77.002.65

371.59.19c
(50.41)

243.671.53c

(26.46)

(53.24)

76.000.00a

300.01.41d

(12.34)

(35.71)

70.506.36

(10.69)
d

225.01.41a
(28.55)

(26.68)
a

454.53.54d
(43.96)

(13.38)
b

402.53.54c
(41.58)

(11.32)

Setback*

71.507.78

176.00.00b
(23.01)

138.00.00a

(10.71)
SADA4

63.500.28

472.000.00

465.502.12

375.005.66

754.005.66

555.0011.31

631.001.41

97.005.66

(18.80)
b

(20.55)
b

61.600.14

472.0014.14

61.450.07

468.5013.44

324.005.66

(50.27)
d

SADA6

501.500.71

148.008.49

SADA8

453.504.95

445.007.07

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(31.36)

322.503.54

647.506.36

495.002.83

131.001.41
(28.89)

379.00.00d

307.04.24b
(48.65)
325.09.90c
(50.19)

Values are means SD of triplicate. Values in the same column with different superscripts (a-d) are significantly different than native counterparts (p<0.05).

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*Breakdown= Peak viscosity-viscosity at 92 C after 20 min; Setback=viscosity at 92 C after 20 min - viscosity at 50 C before holding.

23

Table 4. Swelling power, solubility and paste clarity (%T650

nm)

of starch isolated from wheat varieties Golubica (G) and Srpanjka (S) and

modified with adipic acid/acetic anhydride (ADA) and glutaric acid/acetic anhydride (GA) mixtures in 4, 6 and 8 %.

75 C

85 C

95 C

8.600.50a

9.140.19a

11.450.04a

22.230.20a

3.810.22a

6.060.82a

11.600.02a

32.691.03a

7.800.14

GGA4

9.750.19b

11.280.12b

15.250.16b

21.980.35a

6.040.14b

8.200.10b

12.480.36b

18.600.12b

3.700.14

GGA6

11.030.25c

16.390.47d

23.951.02d

25.980.16b

7.070.09c

11.130.71c

17.940.12c

19.890.21b

4.400.00

GGA8

10.860.18c

12.880.20c

17.980.28c

26.360.64b

5.800.04b

6.890.27a,b

11.130.65a

12.900.07a

2.800.00

b,c

8.450.14

9.900.15

11.190.05

15.650.11

75 C

85 C

24.390.43

8.340.35

11.960.29

19.490.68

95 C

31.390.53

10.670.31

14.140.08

25.310.19

27.500.07

8.650.03

14.230.17

30.070.06

31.310.36

7.000.14

GADA8

9.420.36a,b

12.900.37b

18.930.05b

30.550.16d

9.990.09c

14.010.21c

24.190.43c

34.570.02b

8.100.00

8.250.12a

9.310.02a

11.960.17a

23.230.65a,b

4.530.30a

6.870.42a

13.390.04c

35.820.24d

9.300.00

10.500.18b,c

11.830.16b

18.040.25c

24.250.04b

6.540.76b

8.130.15b

13.610.33c

17.610.00c

3.500.00

SGA4

ED
ITE

GADA6

GADA4

65 C

%T650 nm

65 C

PR
OO

Starch

Solubility (%)

Swelling power (g/g)

10.750.15

12.030.12

18.480.10

22.250.25

4.750.71

7.010.25

11.600.04

12.880.17

2.470.06

SGA8

10.350.06b

12.840.01c

15.850.27b

22.350.44a

5.550.63a,b

6.390.03a

9.620.27a

11.160.38a

2.470.06

SADA4

9.970.23b

11.670.01b

17.140.38b

28.410.45b

8.260.04b

11.510.07b

22.110.08b

33.220.13a

7.000.00

SADA6

10.700.10c

12.780.03c

19.080.03c

32.570.24c

10.300.55c

14.260.07c

25.050.13c

35.430.08b

9.070.06

SADA8

10.920.16c

12.950.03d

20.070.31d

32.880.49c

12.310.82d

15.600.36d

26.770.11d

35.340.53b

8.300.10

UN

SGA6

Values are means SD of triplicate. Values in the same column with different superscripts (a-d) are significantly different than native counterparts (p<0.05).

24

Table 5. Total starch content (TS), resistant starch content (RS), % amylose and degree of
substitution (DS) of starch isolated from wheat varieties Golubica (G) and Srpanjka (S) and
modified with adipic acid/acetic anhydride (ADA) and glutaric acid/acetic anhydride (GA)
mixtures in 4, 6 and 8 %.
% amylose

% Acet

DS

96.870.16a

0.480.03a

20.270.32d

GGA4

99.200.08b

0.620.14b

14.780.31c

2.9580.000a

0.1150.000a

GGA6

99.580.38b

0.490.05a

9.340.12b

3.4140.012b

0.1330.000b

GGA8

100.300.47b

0.680.04b

6.110.33a

3.6460.000c

0.1420.000c

GADA4

96.890.14a

0.960.03c

28.501.84b

2.8900.000a

0.1120.000a

GADA6

98.780.14b

0.480.03a

27.360.47b

3.6120.000a

0.1410.000a

GADA8

99.650.28c

0.760.11b

22.810.09a

3.8360.000a

0.1500.000a

96.110.02b

0.570,11a

22.492.01b

SGA4

94.310.00a

3.060.10c

6.380.51a

2.8550.000a

0.1110.000a

SGA6

94.340.01a

1.730.05b

5.740.14a

3.3540.000a

0.1310.000a

SGA8

98.510.03c

0.690.01a

5.910.03a

3.6810.000a

0.1440.000a

SADA4

96.910.02d

0.140.01a

16.000.12a

2.9240.000a

0.1130.000a

SADA6

95.160.02b

0.210.00a

14.350.81a

3.3880.000a

0.1320.000a

SADA8

95.070.01a

0.480.00b

14.420.28a

3.6810.000a

0.1440.000a

RS/% d. m.

ED
ITE

PR
OO

TS/% d. m.

Values are means SD of triplicates. Values in the same column with different superscripts (a-d) are

UN

significantly different (p<0.05) than native counterparts.

25

4,0

gel strenght / g

3,5
3,0
2,5
2,0
1,5
1,0
0,5
0,0
GA4

GA6

GA8

ADA4

ADA6

4,0
3,5

rupture strength / g

PR
OO

ADA8

Native

3,0
2,5
2,0
1,5
1,0

0,0

GA4

GA6

GA8

ADA4

ED
ITE

Native

0,5

160

ADA6

ADA8

140

100
80
60

UN

adhesiveness / gs

120

40
20
0

Native

GA4

GA6

GA8

ADA4

ADA6

ADA8

Fig. 1. Gel texture properties of starch isolated from wheat varieties Golubica (G) and
Srpanjka (S) and modified with adipic acid/acetic anhydride (ADA) and glutaric acid/acetic
anhydride (GA) mixtures in 4, 6 and 8 %.

26

50
45

syneresis / %

40
35
30
25
20
15
10
5
0
0

GGA4

GGA6

GADA4

GADA6

GADA8

GGA8

number of cycles

PR
OO

70
60

syneresis / %

50
40
30
20
S

10
0
0

SGA4

SGA6

SADA6

SADA8

SADA4
2

SGA8

ED
ITE

number of cycles

Fig. 2. Freeze-thaw stability of starch isolated from wheat varieties Golubica (G) and
Srpanjka (S) and modified with adipic acid/acetic anhydride (ADA) and glutaric acid/acetic
anhydride (GA) mixtures in 4, 6 and 8 %. Starch pastes (5 % d.m.b.) were kept at -18 C/22 hr

UN

and thawed at 30 C/2 hr.

27