40.Ackar_et_al

1

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

and Midhat Jai3

1Faculty of Food Technology, Kuhaeva 20, HR-31 000 Osijek, Croatia 2Institute of Public Health of the Osijek-Baranja County, F. Kreme 1, HR-31 000 Osijek, Croatia

3Faculty of Technology, Tuzla University, Univerzitetska 2, BiH-75 000 Tuzla,

Bosnia and Herzegovina

Received: May 30, 2011

Accepted: August 28, 2012

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

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: [email protected]

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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

industry application and therefore different modifications are conducted amongst which

chemical modification is most common.

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

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,

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.

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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);

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

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

(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

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

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

=

gg

gelinmatterdryofmassgelofmassSP /1/

[ ]%tssuspensioninmatterdryofmass

antupernainmatterdryofmassSOL = /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

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

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 PP-

centrifuge 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.:

[ ][ ] [ ]%100= gsampleofweighttotal

gseparatedwaterSyneresis /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.

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.:

[ ]%8.66)(

)(=

AliquotStarchTotalAbsorbanceConAAbsorbanceAmylose /4/

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

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):

)%42(4300162%Acetyl

AcetylDS

= /5/

Where: m

NVVAcetyl HClsb 100043.0)(% = /6/

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

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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

7

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

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

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),

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

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

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

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

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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

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

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

(35).

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).

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).

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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

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

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

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 dual-

modified 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

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

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

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.

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

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

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

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

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

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).

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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,

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

Sports of the Republic of Croatia.

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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 %.

Starch to / C t p /C te / C H / J/g

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

S 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 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

different than native counterparts (p

20

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

G 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

S 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

After 14 days of storage at 4 C

G 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

S 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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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

different than native counterparts (p

22

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).

Starch

Pasting

temperature/C

Peak

viscosity/BU

Viscosity

at 92 C/BU

After 20 min

at 92 C/BU

Viscosity

at 50 C/BU

After 20 min

at 50 C/BU

Breakdown*

(% breakdown)

Setback*

(% setback)

G 64.870.25d 306.335.51a 256.673.79a 240.674.16a 501.674.04a 435.004.58a 65.672.08a

(21.44)

261.01.73b

(52.03)

GGA4 63.900.14c 663.004.24c 628.502.12b 565.500.71b 968.002.83c 844.005.66c 97.504.95d

(14.71)

402.53.54c

(41.58)

GGA6 61.450.35a 653.503.54b,c 649.005.66c 579.502.12c 1034.005.66d 893.007.07d 74.001.41b

(11.32)

454.53.54d

(43.96)

GGA8 62.150.07b 650.005.66b 642.001.41c 563.007.07b 788.005.66b 726.005.66b 87.001.41c

(13.38)

225.01.41a

(28.55)

GADA4 62.650.35b 487.001.41b 476.507.78b 358.504.95b 691.509.19b 561.500.71b 128.503.54b

(26.39)

333.04.24b

(48.16)

GADA6 60.950.35a 541.002.83d 537.502.12d 397.502.12c 732.002.83c 641.502.12c 143.500.71c

(26.52)

334.54.95b

(45.70)

GADA8 60.800.14a 498.503.54c 489.004.24c 365.500.71b 737.009.90c 569.005.66b 133.002.83b

(26.68)

371.59.19c

(50.41)

S 65.600.36d 291.003.61a 272.334.16a 214.003.00a 457.673.79a 391.674.04a 77.002.65a

(26.46)

243.671.53c

(53.24)

SGA4 64.450.21c 616.004.24b 576.005.66b 540.004.24b 840.005.66d 766.005.66c 76.000.00a

(12.34)

300.01.41d

(35.71)

SGA6 63.200.42b 659.500.71c 621.002.83c 589.007.07c 765.007.07c 700.504.95b 70.506.36a

(10.69)

176.00.00b

(23.01)

SGA8 61.850.49a 667.502.12c 639.504.95d 596.005.66c 734.005.66b 694.506.36b 71.507.78a 138.00.00a

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(10.71) (18.80)

SADA4 63.500.28b 472.000.00b 465.502.12c 375.005.66c 754.005.66d 555.0011.31c 97.005.66b

(20.55)

379.00.00d

(50.27)

SADA6 61.600.14a 472.0014.14b 468.5013.44c 324.005.66b 631.001.41b 501.500.71b 148.008.49d

(31.36)

307.04.24b

(48.65)

SADA8 61.450.07a 453.504.95a 445.007.07b 322.503.54b 647.506.36c 495.002.83b 131.001.41c

(28.89)

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

24

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 %.

Swelling power (g/g) Solubility (%) %T650 nm

Starch 65 C 75 C 85 C 95 C 65 C 75 C 85 C 95 C

G 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

GADA4 9.900.15b,c 11.190.05c 15.650.11c 24.390.43b 8.340.35b 11.960.29b 19.490.68b 31.390.53a 8.450.14

GADA6 10.670.31c 14.140.08d 25.310.19d 27.500.07c 8.650.03b 14.230.17c 30.070.06d 31.310.36a 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

S 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

SGA4 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

SGA6 10.750.15c 12.030.12b 18.480.10c 22.250.25a 4.750.71a 7.010.25a 11.600.04b 12.880.17b 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 Values are means SD of triplicate. Values in the same column with different superscripts (a-d) are significantly different than native counterparts (p

25

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 %.

TS/% d. m. RS/% d. m. % amylose % Acet DS

G 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

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

significantly different (p

26

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

gel s

tren

ght /

g

Native GA4 GA6 GA8 ADA4 ADA6 ADA8

G S

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

rupt

ure

stre

ngth

/ g

Native GA4 GA6 GA8 ADA4 ADA6 ADA8

G S

0

20

40

60

80

100

120

140

160

adhe

sive

ness

/ gs

Native GA4 GA6 GA8 ADA4 ADA6 ADA8

G S

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 %.

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0

5

10

15

20

25

30

35

40

45

50

0 1 2 3 4 5 6 7number of cycles

syne

resi

s / %

G GGA4 GGA6 GGA8

GADA4 GADA6 GADA8

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7

number of cycles

syne

resi

s / %

S SGA4 SGA6 SGA8SADA4 SADA6 SADA8

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

and thawed at 30 C/2 hr.

UNEDIT

ED PRO

OF