Food Research International 75 (2015) 41–49

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Chemical composition, digestibility and emulsification properties of

octenyl succinic esters of various starches
Senay Simsek a,⁎, Maribel Ovando-Martinez a, Ali Marefati b, Malin Sj b,c
, Marilyn Rayner b
a
North Dakota State University, Department of Plant Sciences, PO Box 6050, Dept. 7670, Fargo, ND 58108-6050, USA
b
Lund University, Department of Food Technology, Engineering, and Nutrition, P.O. Box 124, Lund SE-22100, Sweden
c
Speximo AB, Medicon Village SE, 223 81 Lund, Sweden

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

Article history: Octenyl succinate starches are commonly used as emulsifiers and texturizing agents in many food-systems. Rice,
Received 13 January 2015 tapioca, corn, wheat and potato starches were modified with octenyl succinic anhydride (OSA) at 3% level. Struc-
Received in revised form 12 May 2015 tural characterization, molecular weight, starch digestibility and physical properties of starch granule stabilized
Accepted 14 May 2015
emulsions were studied for modified starches. Modified potato (0.022) and wheat (0.018) starches had the
Available online 16 May 2015
highest and lowest degrees of OSA substitution, respectively. For all starches, amylose and amylopectin molecular
Keywords:
mass was significantly (P b 0.05) lower for OSA starches. OSA modification may have hydrolyzed the small am-
Octenyl succinate starch ylose and amylopectin chains, or caused rearrangement of the starch molecules. Although the starch modification
NMR improved emulsification properties, botanical source showed more influence on this parameter. Overall, botan-
FTIR ical source had more influence on functional properties than degree of substitution. Further studies on OSA group
HPSEC–MALS distribution and fine molecular structure of amylopectin and relationship with functional properties will be
Digestibility important.
Pickering emulsions Published by Elsevier Ltd.

1. Introduction
Starch in its native form often has limited use as an ingredient in
food-systems. For this reason, starch may be modified to improve its
functional properties, especially to enhance emulsification and nutri-
tional properties. Such modification alters the physical and chemical
characteristics of the native starch (Shih & Daigle, 2003). Chemical mod-
ification with octenyl succinic anhydride (OSA) is one type of starch
modification used in the food industry (Ai, Nelson, Birt, & Jane, 2013).
OSA esterification enhances emulsification properties of starch by incor-
porating hydrophobic alkenyl groups from OSA into the hydrophilic
starch molecule. The incorporation of the hydrophobic groups results
in surface active properties which are useful in stabilizing emulsions
(Shogren, Viswanathan, Felker, & Gross, 2000; Song, He, Ruan, & Chen,
2006). OSA esterified starches have been widely used as emulsion stabi-
lizers in molecular form (Nilsson & Bergenståhl, 2006), as surface active
hydrocolloids and more recently in the form of intact granules produc-
ing Pickering type emulsions (Rayner et al., 2014; Timgren, Rayner,
Dejmek, Marku, & Sjöö, 2013). Pickering emulsions are stabilized by
solid particles in the size range of tens of nm to tens of μm. Adsorption
of the oil water interface occurs at the surface of these particles due to
⁎ Corresponding author.
E-mail address: senay.simsek@ndsu.edu (S. Simsek).
their partial dual wettability for aqueous and non-aqueous phases.
After adsorption of the oil water interface at the particle surface, the par-
ticles are essentially trapped at the oil water interface creating a thick
interfacial barrier (Aveyard, Binks, & Clint, 2003; Dickinson, 2006).
Pickering emulsions in the context of food products have received
increasing research interest due to the high degree of stability provided
by the particle layer at the oil water interface, which prevents coales-
cence, even for large droplets. Other research has shown that Pickering
emulsions may also act to reduce Ostwald ripening (Yusoff & Murray,
2011), improve barrier properties and freeze thaw stability (Marefati,
Rayner, Timgren, Dejmek, & Sjöö, 2013; Matos, Timgren, Sjöö, Dejmek,
& Rayner, 2013; Rayner et al., 2014), and in some cases decrease the
rate of oxidation (Kargar, Fayazmanesh, Alavi, Spyropoulos, & Norton,
2012). The use of starch to create Pickering emulsions is very attractive
as there is large natural variation with respect to granule size, shape
and functional properties among various botanical sources (Jane,
Kasemsuwan, Leas, Zobel, & Robyt, 1994). Furthermore, OSA esteri-
fied starch has been reported to have distinctive digestibility prop-
erties. The rate and extent of the starch digestion of OSA esterified
starches are reduced when compared to native starches, resulting
in high levels of slowly digestible starch and moderately resistant
starch content (Ai et al., 2013; Han & BeMiller, 2007). The capacity
of the OSA esterified starch to stabilize emulsions and its resistant
starch content make it an ideal material for the encapsulation of bio-
active compounds in targeted delivery systems (Li et al., 2012).

http://dx.doi.org/10.1016/j.foodres.2015.05.034
0963-9969/Published by Elsevier Ltd.
42 S. Simsek et al. / Food Research International 75 (2015) 41–49
The properties of OSA esterified starch depend on the level of OSA
substitution (degree of substitution, DS) (Ai et al., 2013; Bai, Shi,
Herrera, & Prakash, 2011) and the hydrophobic and hydrophilic charac-
ter of the OSA groups (Bao, Xing, Phillips, & Corke, 2003). However, OSA
esterified starch properties are greatly dependent on the botanical
source of the starch. The amylose and amylopectin from starches of dif-
ferent botanical sources vary substantially in molecular weight and fine
structure. The OSA starch properties will be affected by amylose/amylo-
pectin ratio, crystallinity and molecular packing (Bertoft, 2013). OSA
substitution has previously been determined to occur mostly in the
amorphous region without affecting the crystallinity and, that OSA
groups were found primarily in the periphery of the starch granule
in maize and potato starches (Shogren et al., 2000; Wang et al., 2013).
At the molecular level, it has been suggested that OSA substitution oc-
curs close to the branching points of the amylopectin (Bai, Kaufman,
Wilson, & Shi, 2014; Bai et al., 2011; Shogren et al., 2000). Therefore,
because there are differences in fine structure of starches of different
botanical sources, it will be of interest to study OSA esterified starches
from several botanical sources which receive the same level of modifica-
tion under the same modification conditions and the effect on the
chemical and emulsification properties. In this study OSA starches
were prepared using rice, corn, wheat, tapioca and potato starches,
representing the top sources of starch around the world and their
structure, digestibility and emulsification properties after OSA mod-
ification was studied.
2. Materials and methods
2.1. Material
Native wheat, corn, rice and potato starches were obtained from
Sigma-Aldrich and tapioca starch was acquired as a gift from Ingredion.
Octenyl succinic anhydride (OSA) was purchased from Dixie Chemical
Company. Medium chain triglyceride oil (Miglyol 812) was purchased
from Sasol AG, Germany.
2.2. Esterification with octenyl succinic anhydride
Starches (100 g, dry basis, db) were dispersed in water (225 mL)
with stirring. The pH of the slurry (at ~25 °C) was adjusted to 8.5–9.0
with 1 M NaOH. Octenyl succinic anhydride (OSA, 3% of the weight of
the starch) was added during continuous stirring at room temperature
(~ 25 °C), while maintaining the pH at 8.5. A burette was used to add
the OSA in a slow steady stream of approximately 0.1 ml/min. After
6 h, the starch slurry was neutralized to pH 7.0 with 1 M HCl. The modi-
fied starch was centrifuged (2500 rpm, 15 min). The residue was washed
three times with water and once with acetone, and air-dried (40 °C, 24 h)
(Han & BeMiller, 2007).
2.3. 1H nuclear magnetic resonance spectroscopy
Before conducting the nuclear magnetic resonance (NMR) spectros-
copy experiments, the starch samples were purged with deuterium
oxide (D2O) three times, lyophilizing between each purge. The samples
were dissolved in D2O (0.6 ml, 85 °C, 2 h) and placed in NMR tubes (8 in.,
5 mm, thin wall). 1H spectra were taken using a Bruker 400 MHz NMR
(Billerica, USA). The analysis was conducted at 25 °C for 64 scans with a
delay time of 1 s. The degree of substitution (DS) was calculated
according to the method reported by Shih and Daigle (2003). The in-
ternal standard was considered to be the equatorial proton of the
anhydroglucose unit (AGU) of starch (5.2–5.4 ppm). The extent of OSA
substitution was determined by integration of the methyl protons of the
OSA (0.8–0.9 ppm). Thus, the DS = A0.8–0.9 / (3 × A5.10–5.26), where A is
the integral value of the peak assigned.
2.4. Fourier transform-infrared spectroscopy (FT-IR)
During the esterification, hydroxyl groups of starch molecules were
substituted by carbonyl groups of OSA which can be confirmed by FTIR
(Wang et al., 2013). A Fourier transform infrared spectrometer (FTIR,
Nicolet 8700 Thermo Scientific) was used to obtain the IR spectrum of
native and OSA starches. Approximately 1.5 mg of sample was ground
with potassium bromide (KBr) and pressed to form a pellet disc. The
disc was placed in the sample compartment before the spectra was ob-
tained. The samples were scanned over the wavelength range from 400
to 4000 cm cm−1.
2.5. Molecular weight of amylose and amylopectin
For the determination of starch molecular mass and apparent
amylose content, the starch was prepared according to the method
of Grant, Ostenson, and Rayas-Duarte (2002). The starch was dis-
solved in a 1:10 (v/v) solution of 6 M urea and 1 M KOH and heated
for 90 min at 100 °C. The samples were then neutralized using 1 M
HCl and filtered through 0.45 μm nylon syringe filters before analy-
sis by high performance size exclusion chromatography (Simsek,
Whitney, & Ohm, 2013) (HPSEC, Agilent Technologies, USA) with
multi-angle light scattering (MALS, Wyatt Technology, USA). The
dn/dc value for calculation of the starch molecular mass was 0.146
(You & Lim, 2000). The Debye model with a fit degree of one was
used for calculation of the molar mass. The results were fitted to a
first order polynomial model.
2.6. Starch digestibility
In vitro starch digestibility of native and OSA starches was analyzed
using the method described by Englyst, Kingman, and Cummings
(1992). The samples (0.3 g) with 0.1 M sodium acetate buffer (20 mL,
pH 5.2) were gelatinized in boiling water for 30 min and put in a water
bath (37 °C) with agitation (100 strokes/min). Guar gum (50 mg) and 5
glass beads were added to each tube. One blank and glucose standard
tubes were prepared. Five milliliters of enzyme solution was added to
each tube at 1 min intervals. The enzyme solution was prepared as fol-
lows: amyloglucosidase solution (70 U/mg, 24 mg in 12 mL of deionized
water), invertase solution (≥300 U/mg, 60 mg in 8 mL of deionized
water), pancreatin solution (3 g in 20 mL of deionized water, stirred for
10 min at 4 °C and centrifuged). The pancreatin solution (108 ml) was
mixed with the invertase and amyloglucosidase solutions, and was fresh-
ly prepared for the digestion analysis. Aliquots (0.5 mL) of the samples
were taken at 20 min intervals over a total of 180 min and were mixed
with 5 mL of absolute ethanol and centrifuged. The glucose released
was measured at 510 nm in parallel with a standard curve of glucose
using the glucose oxidase assay (Megazyme International Ireland). The
rapidly digestible starch (RDS), slowly digestible starch (SDS) and resis-
tant starch (RS) were determined as expressed in percentage (%). RDS is
considered the portion of the starch hydrolyzed from 0 to 20 min, SDS
is the starch hydrolyzed between 20 and 120 min and the RS is the starch
remaining after 120 min of digestion (Englyst et al., 1992). The hydrolysis
index (HI) was obtained by dividing the area under the hydrolysis curve
of the sample by the area obtained for white bread (hydrolysis curve
0 min to 180 min). The estimated glycemic index (eGI) of the samples
was calculated using the equation described by Granfeldt, Bjorck,
Drews, and Tovar (1992):
eGI ¼ 8:198 þ 0:862  HI:
2.7. Emulsion preparation
Emulsions were prepared in glass tubes using starch granules as
stabilizers. Medium chain triglyceride oil as dispersed phase and
 S. Simsek et al. / Food Research International 75 (2015) 41–49 43

phosphate buffer (5 mM, pH 7, 0.2 M NaCl) as continuous phase. Oil where d is the measured diameter of a drop and n is the total num-
(7% v/v), buffer (93% v/v), and starch (214 mg/mL oil) in a total vol- ber counted. This is calculated from the light scattering data obtain-
ume of 7 mL were emulsified first with a vortex mixer (10 s) and ed by Malvern Mastersizer with the analysis software (Ver. 5.60).
then homogenized by high-shear mixing in an Ystral mixer (Ystral
GmbH, Germany) at 22,000 rpm for 30 s (Timgren et al., 2013).
2.9. Statistical analysis
The emulsions were stored at 5 °C for 1, 3, 6 and 15 days to observe
the emulsion stability based on particle size.
The modifications and analyses were done in duplicate. The statisti-
cal software package SAS 9.3 was used to perform analysis of variance
2.8. Microstructure and particle size distribution of starch granules and
(ANOVA) with completely random design (CRD). The mean separation
emulsions
was done using least significance difference (LSD) with α = 0.05.
The microstructure of the starch granules and the initial emulsions
was characterized by light microscopy (Olympus BX50, Japan) and 3. Results and discussions
using a digital camera (DFK 41AF02 Imaging source, Germany) 1 day
after emulsification. The images were processed using the Java image pro- 3.1. Nuclear magnetic resonance spectroscopy of native and OSA starches
cessing software ImageJ (NIH, Version 1.42 m). A laser diffraction particle
size analyzer (Mastersizer 2000 Ver. 5.60, Malvern, Worcestershire, UK) The use of 1H-NMR provides evidence of OSA esterification of the
was used in order to determine the particle size distribution of the starch starch molecules. The peaks observed in 1H-NMR spectra of native
granules and the starch granule stabilized emulsion oil drops. The sample starch, arising from the glucose in starch were assigned according to lit-
was added to the flow system containing milliQ-water and was pumped erature. The 1H-NMR spectra of the native and OSA esterified tapioca
through the optical chamber at a pump velocity of 2000 rpm. The refrac- starch were shown in Fig. 1. The peaks at 4.10–3.18, 4.64, 4.96, and
tive index (RI) of the sample was set to 1.54 (starch) since the oil droplets 5.38 ppm represent hydrogen at: positions 2, 3, 4, and 5, reducing end
were covered by starch granules and therefore could be considered to β-form, position 6, α-1-6 hydrogen and the internal hydrogen at position
scatter light as a large granule (Bromley & Hopkinson, 2002; Rayner, 1 of the glucose units, respectively (Bai & Shi, 2011; Bai et al., 2011; Shih
Timgren, Sjöö, & Dejmek, 2012). The RI of the continuous phase was set & Daigle, 2003). In OSA starches, the appearance of peaks between 0.80
to 1.33 (water) and the obscuration was between 10 and 20%. The volume and 2.7 ppm was seen; which is associated with OSA esterification of
mean diameter, d[43] and the surface mean drop diameter, d[32] were the starch molecules. The peak at 0.8 ppm corresponded to the methyl
reported to describe particle size distributions, and calculated using protons of the OSA. The intensity of the peak at 0.8 ppm varied among
equations: botanical sources, reflecting differences in the DS of each starch. A peak
was also observed at 0.90 ppm. According to Bai et al. (2011), this peak
is indicative of the terminal methyl protons in some substituted octenyl
Xn Xn
di
3
di
4 succinate groups, which are hydrophobic. These hydrophobic octenyl
d32 ¼ Xi¼1
n 2
d43 ¼ Xi¼1
n 3 succinate groups might aggregate in aqueous media and cause shifting
d
i¼1 i
d
i¼1 i of the methyl peak. Peaks observed around 1.2–2.7 ppm, in OSA starches,

Fig. 1. 1H nuclear magnetic resonance spectroscopy of native and OSA esterified tapioca starches. N: native and M: modified. T: tapioca.
44 S. Simsek et al. / Food Research International 75 (2015) 41–49

were from the protons of the methylene groups of the succinic anhydride to the C–O of the C–O–C in the polysaccharide; the peaks close to 1081
(Cizova, Koschella, Heinze, Ebringerova, & Srokova, 2007). and 1160 cm−1 are characteristics of the anhydroglucose ring C–O
The DS of OSA esterified starch has been determined using the stretch; the peak near to 930 cm−1 was assigned to the skeletal mode vi-
1
H-NMR by comparing the intensities of the methyl protons of OSA bration of α-(1–4) glycosidic linkage (Miao et al., 2014; Wang, Su, &
substituted in the glucose units of the starch molecule (Bai et al., Wang, 2010); while the peak near to 860 cm−1 corresponds to the C–H
2011; Shih & Daigle, 2003). The DS of the OSA esterified starches and CH2 deformations (Miao et al., 2014).
in this study is shown in Table 1 and the DS was in the following Compared to native starches, the spectrum of OSA esterified starches
order potato = rice N corn N tapioca = wheat. The percentage of OSA showed two additional peaks around 1750 and 1570 cm−1. The peak at
used for modification was chosen based on the level approved by the 1750 cm−1 is attributed to the characteristic IR stretching vibration of
Food and Drug Administration for food-use purpose. The starches stud- C = O, suggesting the formation of ester carbonyl groups; while the
ied were from cereal (rice, corn, and wheat), tuber (potato), and root peak at 1570 cm−1 is ascribed to the asymmetric stretching vibration
(tapioca) sources. Potato and rice starch presented the highest DS, of carboxylate RCOO− (Song et al., 2006). The values observed for
while tapioca and wheat showed the lowest values. It was observed these two peaks in OSA esterified starches were similar to those report-
that the DS varied significantly (P b 0.05) and is dependent on the ed in literature. These results indicate that the hydroxyl groups in the
botanical source. This could be due to differences in the phosphorous, starch were substituted by ester carbonyl and carboxyl groups of OSA.
protein and lipid content, granular surface and structure of the starch The 1H NMR and FT-IR analyses show that the esterification of starches
among the different botanical sources (Sweedman, Tizzotti, Schäfer, & with OSA was successful and that the starch botanical source signifi-
Gilbert, 2013). cantly (P b 0.05) effected the level of esterification, measured by DS.
Cereal starches present A-type crystalline patterns, while potato and However, it is important to determine how the OSA esterification is af-
tapioca present B type crystalline patterns. The principal structural fecting the chemical and functional properties of starches from the dif-
differences between these two types of crystals are found in the ferent botanical sources.
amorphous backbone (Bertoft, 2013). Starches with higher degree
of branching of amylopectin, have lower DS; due to stearic factors 3.3. Amylopectin and apparent amylose contents and molecular weights
(Yusoff & Murray, 2011). The differences observed in the DS among bo-
tanical sources could be affected by the amorphous structure of the starch, Alteration of amylopectin and amylose will change the functionality
causing differences in the distribution of OSA esterification onto the starch of the OSA esterified starches. The apparent amylose and amylopectin
structure (Sweedman et al., 2013). Another factor that can affect the content of native and modified starches, and their respective molecular
introduction of OSA groups into the starch structure could be the OSA sol- weights were determined by HPSEC–MALS–RI (Table 1). The amy-
ubility in the starch slurry during the modification, affecting its diffusion lopectin content varied from 69.85% to 75.53%. Among native
into the starch granule (Shogren et al., 2000; Wang et al., 2013). More- starches, tapioca, rice and wheat starches did not have significant
over, the presence of channels and pores on the starch granular surface differences (P b 0.05) in amylopectin content. Native corn starch
can affect the OSA diffusion through the starch granule (Wang et al., showed the lowest amylopectin content (71.06%), which was signif-
2013). The structural diversity of starch and the OSA solubility could affect icantly (P b 0.05) lower than the native starches from other botani-
the level of DS for each botanical source even though the same level of cal sources. The results for the apparent amylose content followed a
OSA is used. similar trend as that seen for amylopectin content. Among the native
starches, corn had significantly (P b 0.05) higher apparent amylose con-
3.2. Fourier transform infrared spectroscopy of native and OSA starches tent than the other native starches. OSA esterified corn starch had signif-
icantly (P b 0.05) higher apparent amylose content than the other OSA
The FT-IR spectra of both native and OSA starch have similar profiles. esterified starches, while OSA esterified tapioca starch had the lowest ap-
Fig. 2 shows the FT-IR spectra of native and OSA esterified tapioca starch parent amylose content.
(shown as representative spectra). Broad peaks appeared approximately There were some significant (P b 0.05) differences seen when com-
at 3440 cm−1, indicating the presence of hydroxyl groups (O–H). Peaks paring the amylopectin and amylose contents of the OSA esterified
at 2931 cm−1 and 1650 cm−1 representing the C–H stretching vibration starches to their native counter parts. OSA esterified corn starch had sig-
and bound water present in the starch, respectively, were also seen in the nificantly (P b 0.05) higher apparent amylose content than native corn
FT-IR spectra (Miao et al., 2014). The fingerprint region of the starch spec- starch. The apparent amylose content of OSA esterified tapioca starch
trum has five characteristics peaks between 800 and 1200 cm−1, attribut- was significantly (P b 0.05) lower than the native tapioca starch. It is
ed to the C–O bond stretching. The peak around 1015 cm−1 is attributed clear that the differences in botanical source of the starches have
some effect on changes to the amount of apparent amylose after
Table 1 OSA esterification. Amylose has been determined to greatly affect
Degree substitution, amylose and amylopectin content and molecular weight of native the starch functionality and even small differences in amylose con-
and modified starchesa.
tent can change the functional properties and digestibility of the
Sample DS Molecular weight starch (J. Jane et al., 1999).
Amylopectin Amylose Amylopectin Amylose The starches of the various botanical sources respond differently to
the treatment during the OSA esterification procedure because of their
(%) (Da)
differences in starch granular structure, crystallinity and starch fine
Native corn ND 71.06 d 28.94 b 1.41 × 107 e 2.82 × 106 a structure. OSA substitution has been found to occur in the interior,
Modified corn 0.022 b 69.85 e 30.15 a 1.34 × 107 f 1.31 × 106 e
amorphous region of amylopectin without changing the crystallinity
Native tapioca ND 74.16 b,c 25.84 d,c 1.74 × 107 d 1.39 × 106 d
Modified tapioca 0.018 c 75.53 a 24.47 e 1.22 × 107 g 8.63 × 105 g (at 3% OSA level), as well on the surface of starch granules (Bai et al.,
Native rice ND 74.56 b,a 25.44 d,e 1.88 × 107 a 1.64 × 106 b 2014; Shogren et al., 2000). The changes in apparent amylose content
Modified rice 0.026 a 74.59 b,a 25.41 d,e 1.83 × 107 b 1.01 × 106 f may be partially due to OSA esterification or to the mild alkaline treat-
Native potato ND 73.42 c 26.58 c 7.32 × 106 h 8.11 × 105 h
ment during the modification procedure. The introduction of OSA
Modified potato 0.026 a 73.88 b,c 26.12 d,c 7.16 × 106 h 6.25 × 105 i
Native wheat ND 74.75 b,a 25.25 d,e 1.78 × 107 c 1.44 × 106 c groups into the starch structure, whether at branch points of amylopec-
Modified wheat 0.018 c 74.88 b,a 25.12 d,e 1.43 × 107 e 1.32 × 106 e tin and/or along the amylose molecule, may increase the hydrodynamic
DS: degree substitution; Da: Daltons.
volume of these molecules; as a consequence increasing the fraction of
a
Values in the same column with the same letter are not significantly different (P b 0.05, starch which eluted first during HPSEC separation. The mild alkaline
n = 2). treatment could also result in leaching of small amounts of amylose
 S. Simsek et al. / Food Research International 75 (2015) 41–49 45

Fig. 2. Fourier transform infrared spectroscopy of native and OSA esterified tapioca starches. N: native and M: modified. T: tapioca.

molecules which may be close to the surface of the starch granules potato starches. Esterification with OSA has been proposed as a method
(BeMiller & Whistler, 2009). of preparation of slowly digestible starch (Han & BeMiller, 2007), and
The molecular weight of amylopectin and amylose from native and our results show that most of the OSA esterified starches had signifi-
modified starches is presented in Table 1. The results showed that cantly (P b 0.05) higher SDS content than their native starch counter-
after modification with OSA, the molecular weights of amylopectin parts. OSA esterified rice starch was the only type to have lower SDS
and amylose decreased significantly (P b 0.05), although the decrease than the native starch counterpart. However, OSA esterified rice, along
is relatively small. The molecular weight decrease suggests a low degree with all the other OSA esterified starches had significantly (P b 0.05)
of hydrolysis of the small amylose and amylopectin chains or rearrange- higher resistant starch contents than the native starches, regardless of
ments of the structure, size or compactness of the starch molecules ac- botanical source. Taking into account the unique digestibility of OSA
cording to the botanical source and the conditions (alkaline system and starches, plus the amphiphilic properties of the OSA esterified starches
agitation) used during the starch modification. During OSA modifica- makes these modified starches preferential material as a delivery carrier
tion; the small chains of amylose and amylopectin in the amorphous material for bioactive compounds in colon-targeted delivery systems (Li
zone may be hydrolyzed, allowing the clusters to remain intact (crystal- et al., 2012).
line zone of amylopectin), which are reordered in a compact molecule. The hydrolysis index (HI) of a food is a measurement of the rate at
During such rearrangement of the structure and the esterification of which the starch is hydrolyzed by the digestive enzymes in the gut.
OSA groups into the starch structure, the molecules lose flexibility and This parameter can be used to determine the estimate glycemic index
increase in hydrodynamic volume; which is evident by the changes in (eGI). The eGI of a food product is the primary term used to represent
amylopectin and amylose contents and molecular weights. the physiological properties of dietary carbohydrates (Granfeldt et al.,
1992). The HI and eGI of native and modified starches are presented
3.4. Digestibility properties of OSA starches in Table 2. The HI values of the starches ranged from 84.88 to 94.89
and the eGI of the starches ranged from 81.37 to 89.99. The HI of OSA es-
The in vitro starch digestibility of native and OSA esterified starches terified starches decreased significantly (P b 0.05) in comparison with
is shown in Table 2. RDS values have previously been seen to decrease the HI of native starches from the same botanical source. The same
with OSA esterification at 3% in starches of different botanical sources trend was observed in the eGI of the samples. The decrease in rate of di-
(Han & BeMiller, 2007). The results in this study also show a decrease gestion may be caused by physical interference or the hydrophobicity
of RDS content for OSA esterified starches, although the decrease is caused by the bulky OSA group attached to the starch. The OSA groups
not as large as was determined by Han and BeMiller (2007). The lowest attached to the starch have been shown to act as an uncompetitive in-
RDS contents were seen in native (62.13%) and OSA esterified (55.85%) hibitor to α-amylase and amyloglucosidase. During hydrolysis the re-
lease of the enzyme is delayed by the presence of the OSA molecule,
effectively reducing enzyme activity and hydrolysis of the starch (He,
Table 2
Starch digestibility of different starch sources modified with OSAa. Liu, & Zhang, 2008). In this study, samples with higher DS did not nec-
essarily have lower eGI. This means that the DS may not be the only in-
Sample RDS SDS RS HI eGI
fluence on the digestibility of the starches. Because of differences in
(%, DWB) starch granule morphology, amylose content, degree of branching and
Native corn 79.29 b 15.35 e 5.36 f 91.47 b,c 87.05 b,c crystalline structure, the botanical source of the starch can also play a
Modified corn 70.57 d 20.03 d 9.40 b 84.88 e 81.37 e role in the rate of starch digestion (Singh, Dartois, & Kaur, 2010). The
Native tapioca 80.62 a 15.36 e 4.02 g 92.58 a,b 88.00 a,b starches from the different botanical sources have different granular
Modified tapioca 63.49 e 22.58 c 13.93 a 87.93 d 84.00 d
morphologies and crystalline structure as well as slight differences in
Native rice 81.33 a 16.32 e 2.35 h 90.86 b,c 86.52 b,c
Modified rice 76.83 c 14.13 f 9.04 b 88.12 d 84.16 d amylose content. These characteristics of the starches from different bo-
Native potato 62.13 f 30.55 b 7.32 d 92.37 b 87.82 b tanical sources will influence the digestibility (Singh et al., 2010). For a
Modified potato 55.85 g 36.02 a 8.13 c 87.76 d 83.85 d branched polymer such as starch, its starch backbone provides a range
Native wheat 80.92 a 12.96 g 6.11 e 94.89 a 89.99 a
of substrates with controlled macromolecular architectures which
Modified wheat 71.53 d 19.14 d 9.33 b 89.60 c,d 85.44 c,d
can affect the structural parameters (Tizzotti, Sweedman, Schäfer, &
OSA: Octenyl succinic anhydride; DWB: dry weight basis; RDS: rapidly digestible starch; Gilbert, 2013) and in turn affect the digestibility properties. However,
SDS: slowly digestible starch; RS: resistant starch; HI: hydrolysis index; eGI: estimated glyce-
mic index.
we are unable to determine conclusively if the degree of OSA esterifica-
a
Values in the same column with the same letter are not significantly different (P b tion or botanical source plays a larger role in the digestibility of the
0.05, n = 2). starches in this study.
46 S. Simsek et al. / Food Research International 75 (2015) 41–49

3.5. Characterization of the starch granule stabilized emulsions
The use of OSA esterified intact starch granules as a novel powder
material stabilizing Pickering oil in water emulsions was studied. The
microstructure of both native and modified starches was observed
with light microscopy. The granular morphology was as is considered
typical for each botanical source (Jane et al., 1994). Corn starch present-
ed spherical and polygonal granules and potato starch had large oval
and spherical granules. The rice starch showed polyhedral compound
granules, tapioca presented unique granular morphology having trun-
cated spherical granules and wheat starch showed the typical lenticular
and spherical granules of bimodal size (Jane et al., 1994). The particle

Fig. 3. Particle size distribution of starch granules and emulsion drops for native and modified starches during different days. N: native and M: modified. C: corn; T: tapioca; R: rice;
P: potato; W: wheat.
 S. Simsek et al. / Food Research International 75 (2015) 41–49 47

Table 3 Table 4
Size distribution of native and modified starch of different botanical source in phosphate Size distribution of droplets stabilized by different starch sources modified with OSA.a.
buffera.
Sample Day 1 Day 15
Sample d[43] d[32]
d[43] d[32] d[43] d[32]
(μm)
(μm) (μm)
Native corn 14.75 d 7.806 d
Modified corn 103.8 c 35.83 c 96.61 c 30.16 c
Modified corn 15.00 d 8.018 d
Modified tapioca 148.8 a 108.2 a 128.5 a 89.85 a
Native tapioca 14.52 d 7.590 d
Native rice 69.95 d 7.517 d 38.99 e 5.794 e
Modified tapioca 15.23 d 7.921 d
Modified rice 47.79 e 15.85 d 44.66 e 14.56 d,e
Native rice 4.898 f 3.218 f
Modified potato 75.09 d 31.90 c 53.99 d 23.11 d,c
Modified rice 7.231 e 3.658 e
Modified wheat 127.2 b 92.57 b 119.1 b 70.18 b
Native potato 49.97 a 21.58 a
Modified potato 48.80 b 22.00 a d[43]: volume mean diameter; d[32]: surface mean diameter; μm: micrometers.
a
Native wheat 20.19 c 10.25 c Values in the same column with the same letter are not significantly different (P b
Modified wheat 21.05 c 10.74 b 0.05, n = 2).
d[43]: volume mean diameter; d[32]: surface mean diameter; μm: micrometers.
a
Values in the same column with the same letter are not significantly different (P b aqueous dispersion it would be more energetically favorable for them
0.05, n = 2). to aggregate.
The oil-in-water emulsion using native and OSA starch as a particle
size distribution of the starches dispersed in phosphate buffer analyzed stabilizer was prepared and the formation and stability of droplets
by light scattering is shown in Fig. 3 (dark blue line). The granule size were analyzed. Among native starches, rice starch was the only
followed this order from smallest to largest: rice b tapioca b corn b one that stabilized the oil droplets in an emulsion. Due to the non-
wheat b potato in both native and modified starches (Table 3). In the hydrophobic nature of the native starch, the oil–water interface should
case of modified starches, a very slight increase of the mean diameter not be able to adsorb at the surface of the native starch granules and
size (d[43] and d[32]) can be detected for some of the starches (Table 3). stabilize an emulsion (Timgren et al., 2013). Timgren et al. (2013) con-
This increase in mean particle size OSA starch could be explained by ducted a study of emulsification properties of different starches showing
aggregation, since the more hydrophobic OSA starch granules are in an that the size of the granules had a large impact on the emulsion stability.

Fig. 4. Light microscope micrographs of the emulsions stabilized by native rice starch and OSA modified starch in the first day (20×). N: native and M: modified. C: corn; T: tapioca; R: rice;
P: potato; W: wheat. Length of scale bar = 25 μm.
48 S. Simsek et al. / Food Research International 75 (2015) 41–49
They demonstrated that small granule sizes have the best emulsification
properties. Among the starches used in this study, rice presented the low-
est granule size, which could be one of the reasons why this starch formed
an emulsion in its native form.
The oil droplets formed in the emulsion stabilized by OSA starch dur-
ing the first day are shown in Fig. 4. Starch granules formed a densely
packed layer at the oil–water interface, indicating the stabilization of
the emulsions by the OSA starches, due to their amphiphilic nature.
The droplet size and size distribution of the oil droplets (Table 4 and
Fig. 3) were examined for emulsions stabilized by OSA starches. Both
modified and native rice starch showed the smallest mean volume
and surface diameter (d[43] and d[32]) compared with the rest of the
starches (Table 4). There was no statistically significant increase in
emulsion drop size after 15 days of storage, indicating that once droplets
are formed they are highly stable even at large (N 100 μm) droplet diam-
eters. Droplets in Pickering emulsions will be more stable when uni-
formly covered with the starch granules, which may be the reason for
their high stability at larger droplet diameters (Kuipers, 2009) Less of
the stabilizer (OSA esterified starch) dropped to the bottom of the
tube for emulsions prepared with OSA esterified rice, tapioca and
wheat starches, which can be related to the size of the starch granules
from the different botanical sources. According to Rayner, Sjöö,
Timgren, and Dejmek (2012), the main peak in the particle size dis-
tribution of starch stabilizing Pickering emulsions represents the
starch granule stabilized emulsion drops and the small peak (on
the left side of the main peak) represents the free starch granules.
Taking this into account, modified rice, tapioca and wheat starches
presented small peaks for the material representing free starch granules
(around 1–10 μm for rice, and 10–30 μm for tapioca and wheat)
(Table 3), supporting that these starches are more appropriate for
use in Pickering emulsions due to the fact that they release a small
amount of free starch into the continuous phase during storage time.
There is a clear difference between size distribution for native and OSA es-
terified starches. The size distribution graphs (Fig. 3) of the native starch
emulsions show a larger frequency of free starch granules while stabilized
droplets are the predominant feature for the OSA esterified starch emul-
sions. However, native rice starch has nearly equal portions of free gran-
ules and stabilized droplets. The OSA esterified corn starch emulsion has
the largest proportion of free granules among the OSA esterified starches.
Also, the large size of the potato starch granules makes it difficult to distin-
guish between the free granules and emulsion droplets.
OSA esterification improves the emulsion capacity of the starch
compared to native starches; but this does not mean that the OSA ester-
ification is the only influence on the ability of starch to stabilize the
emulsion. Rather, emulsion properties also depend on the starch gran-
ule size distribution and the stability of the oil droplets formed. Even
so, the branch structure of the starch backbone plays an important
role in the stability of emulsions, as a higher degree of branching implies
more rigid macromolecules, which may affect OSA distribution after
modification. Differences in distribution of OSA molecules on the starch
granules may result in a thicker adsorbed layer around the droplet,
suggesting more stability (Sweedman et al., 2013). Further study of
the fine structure of the starch backbone will be important to determine
how this parameter influences emulsions stabilized by OSA esterified
starches.
4. Conclusions
In conclusion, the structural characterization of OSA starches from dif-
ferent botanical sources indicated that the OSA esterification proceeded to
varied degrees for each starch source. This was evident because of the
identification of the methyl and methylene groups with 1H NMR, and
peaks observed in the FT-IR spectra corresponding to the carboxyl and
ester carbonyl of OSA into the starch structure. OSA esterified starches
had increased SDS and RS, as well as, lower HI and eGI. The esterification
with OSA resulted in unique digestibility properties, which may lend their
application to colon targeted delivery systems for drugs or bioactive
compounds. Although OSA esterification improves the emulsification
properties, the size of the starch granule showed a strong influence on
this parameter. Overall, the botanical source was determined to play an
important role in the functionality of the OSA esterified starches. Howev-
er, it may be of interest to conduct further investigation on the use of dif-
ferent starch types from a single botanical source (example, rice starches
with different amylose contents). According to these results, we suggest
that OSA esterified rice, tapioca and wheat starches may be useful in
targeted delivery systems due to RS content and emulsification proper-
ties. An important parameter that may affect the functional properties
of OSA starches is the fine structure of starch. So, further investigation is
necessary to determine where the OSA esterification occurs in the fine
structure of the starch components, and how will the starch structure
affect such substitution? It will be important to analyze the OSA group
distribution into the granule and structure of starch as well as on the sur-
face, and determine their effects on the functional properties for future
studies.
Acknowledgment
The Swedish research group is partially funded by the Antidiabetic
Food Centre (Grant Number 2009/27), a VINNOVA VINN Excellence Cen-
ter at Lund University.
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