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Chemical and rheological properties of a starch-rich fraction from the pulp of the fruit cupuassu (Theobroma grandiflorum)
Materials Science and Engineering C 29 (2009) 651–656
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m s e c
Chemical and rheological properties of a starch-rich fraction from the pulp of the fruit cupuassu (Theobroma grandiflorum) Lúcia C. Vriesmann, Joana L.M. Silveira, Carmen L. de O. Petkowicz ⁎ Universidade Federal do Paraná, Departamento de Bioquímica e Biologia Molecular, CP 19046, CEP 81531-990, Curitiba-PR, Brazil
a r t i c l e
i n f o
Article history: Received 8 May 2008 Received in revised form 28 November 2008 Accepted 9 December 2008 Available online 16 December 2008 Keywords: Theobroma grandiflorum Cupuassu Starch Amylose Gel Rheology
a b s t r a c t The pulp obtained from the fruit of cupuassu (Theobroma grandiflorum) was extracted with hot aqueous 0.1% citric acid to give fraction 0.1CA-2 in 15% yield. This was the predominant component polysaccharide, 91% of which was composed of starch, by an iodine test and monosaccharide composition, and its 13C NMR spectrum was consistent with that of a high amylose starch. The content of amylose found in fraction 0.1CA-2 was 71%. This value is higher than those of common starches of cereal grains, tubers, roots, and other fruits. The fraction was submitted to rheological examination, gels being prepared on heating with concentrations of 4 to 7% (w/w). A non-Newtonian behavior was observed, and gel viscosity and strength depended on the concentration. The presence of starch, as well as the presence of previously investigated pectin, conferred the high viscosity and gelling capability of the pulp. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Starch is the main reserve polysaccharide of many plants. It is an important renewable and a low-cost polymer, occurring as granules, which determine its physical properties. Due its thickening and gelling properties, it is used in the food, pharmaceutical, and chemical industry [1–3]. Cereal grains, legume seeds, tubers, and certain fruits contain from 30 to 85% starch on a dry-weight basis [3]. Commercial starches are obtained mainly from yellow corn, although potato, wheat, tapioca, rice and sorghum also are significant sources [2,3]. Many reports have described the characterization of starch from cereals, roots and tubers [4–7]. The fruit starches have also been investigated, mainly those from banana [8,9], mango [8,10], apple [11], squash [12], cherimoya [13] and Kamo Kamo [14]. Starch consists of a mixture of two polymers, amylose and amylopectin. Whereas amylose is an essentially linear molecule, consisting of (1→4)-linked α-D-glucopyranosyl units, amylopectin is a highly branched molecule with substitution of these at O-6 by α-Dglucopyranosyl branches. These are composed of (1→4)-linked α-Dglucopyranosyl units with various lengths [1–3]. It has been demonstrated that the pulp of the fruit of Theobroma grandiflorum, Schumann (family Sterculiaceae), growing in the Brazilian Amazon, known locally as cupuassu, contains a considerable amount of starch as well as pectin polysaccharides [15,16]. The pulp is ⁎ Corresponding author. Tel.: +55 41 3361 1661; fax: +55 41 3266 2042. E-mail address: [email protected] (C.L.O. Petkowicz). 0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.12.011
greatly appreciated for its pleasant acidic taste, being consumed fresh or processed, mainly as juice, ice cream, candy and jellies [17]. It has an intense fragrance [17], whose volatile compounds have been extensively analyzed [18], as well as its content of vitamin C [19]. As chemical and rheological characterization of native starch can lead to improvements for predicting desirable functional properties of starchy products, we now study some chemical and rheological features of the native starch-rich fraction obtained from the pulp of cupuassu fruit [15]. 2. Experimental 2.1. Extraction of fraction 0.1CA-2 After enzyme-inactivation with methanol–H2O (4:1, v/v) under reflux for 20 min, the grounded pulp fruit was defatted with ptoluene-ethanol (2:1, v/v) in a Soxhlet and dried. The residue was submitted to sequential extractions with water (25 °C and 60 °C), citric acid (0.1%, 0.5%, 1%, 2.5% and 5%, using temperatures of 50 °C and 100 °C at each concentration) and NaOH (2 mol L– 1 and 4 mol L– 1 NaOH; 25 °C) [15,16]. Fraction 0.1CA-2 was obtained with 0.1% citric acid at 100 °C for 60 min. The extract was concentrated and treated with ethanol (2:1 v/v) in order to obtain precipitated polysaccharide, which was then washed three times with ethanol and dried under vacuum. The fraction was identified according to the conditions applied on extraction: 0.1 displays the concentration of citric acid (CA) and “−2” was used for the second hot extraction (100 °C).
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2.2. Chemical characterization of fraction 0.1CA-2 Total carbohydrate was determined by the phenol–sulfuric acid colorimetric method [20], employing glucose as standard. Uronic acid was estimated by the sulfamate/3-phenylphenol colorimetric method [21], using galacturonic acid as standard. Protein was measured by the Hartree method [22], with BSA as standard. Amylose content was determined by the Chrastil method [23] with a modification, using 80% amylose and waxy amylopectin (0% amylose) (Sigma) as standards. Intermediate amylose proportions were obtained by mixing appropriate amounts of each. A straight line plot was used to determine the content of amylose in the sample. Fraction 0.1CA-2 was solubilized in distilled water at 100 °C during 10 min, then alkalinized with 1 mol L– 1 NaOH and kept for 5 min at 100 °C. After cooling at 25 °C, it was submitted to the procedure for amylose determination. The monosaccharide composition was determined after total acid hydrolysis with 2 mol L− 1 trifluoroacetic acid (5 h, 100 °C). The monosaccharides, obtained on evaporation to dryness, were reduced with NaBH4 and then acetylated with pyridine-acetic anhydride (1:1 v/v, 16 h, at 25 °C). The resulting alditol acetates were analyzed by gas–liquid chromatography (GLC) using a model 5890 S II HewlettPackard gas chromatograph at 220 °C (flame ionization detector and injector temperature, 250 °C) with a DB-210 capillary column (0.25 mm internal diameter × 30 m, film thickness 0.25 µm), the carrier gas being nitrogen at 2.0 mL min− 1 [16]. For high pressure size exclusion chromatography (HPSEC) analyses, suspension of the sample in 0.1 mol L− 1 NaNO2 solution containing NaN3 (0.5 g L− 1) was heated to 80 °C during 2 h under magnetic stirring. HPSEC was carried out on polysaccharide solution at 25 °C, using a multidetection equipment in with a Waters 2410 differential refractometer (RI) and a Wyatt Technology Dawn F multiangle laser light scattering (MALLS) detector were adapted on-line. Four Waters Ultrahydrogel 2000/500/250/120 columns were connected in series and coupled to the multidetection equipment. The mobile phase consisted of a 0.1 mol L− 1 NaNO2 solution containing 0.5 g L− 1 NaN3 (vacuum-filtered through a 0.22 µm membrane filter) at a flow rate of 0.6 mL min− 1 and pressure 830 psi. The sample, previously filtered (0.22 µm; Millipore), was injected (100 µL loop) at a 1.5 mg mL− 1. HPSEC data were collected and analyzed by a Wyatt Technology ASTRA program. 13 C NMR spectra of fraction 0.1CA-2 were obtained using a Bruker DRX 400 Avance spectrometer incorporating Fourier transform (FT) on a solution in D2O at 70 °C. Chemical shifts are expressed as δ (ppm), using the resonances of CH3 groups of acetone as internal standard (δ 30.2). 2.3. Rheological analysis of fraction 0.1CA-2 Solutions of fraction 0.1CA-2 at concentrations of 4, 5, 6 and 7% (w/w) were prepared by stirring in water for 16 h at 25 °C. They were then heated at 92 °C with continuous stirring for 15 min and cooled at room temperature. The samples were stored at 5 °C prior to rheological examination. These were carried out using a model RS 75 Haake Rheometer, coupled with a DC5 heating circulator, with a C60/2° (cone and plate geometry) or PP20 sensor (plate and plate geometry). Mechanical responses of the samples were determined by subjecting them to a frequency sweep (0.1–10 Hz) at 25 °C at tension (τ) between 1 and 4 Pa. These values refer to the viscoelastic-linear region, where the gel structure was preserved. The temperature sweeps, heating (5–95 °C) and subsequent cooling (95–5 °C), were performed at a rate of 1 °C min− 1, at a frequency of 1 Hz. Before starting the experiments, the exposed sample edge was covered with a thin layer of low viscosity mineral oil to minimize evaporation losses during measurements. Continuous flow ramps in the CR mode (controlled rate) were performed at 25 °C. The sensor was programmed to increase the shear
rate from 0.1 to 100 s− 1 (up curve) in 100 s followed immediately by a reduction from 100 to 0.1 s− 1 in 100 s (down curve). The shear stress (τ) was then measured as a function of shear rate. As done by Techawipharat, Suphantharika and BeMiller (2008) [24], data from the down curve of the shear cycle were used to characterize the flow behavior of the cupuassu starch samples. The experimental data were evaluated and fitted according to the rheological models of Herschel– Bulkley (τ = τ0 + Kγn), Power-law (τ = Kγn), and Bingham (τ = τ0 + ηpγ), where τ is the shear stress (Pa), γ is the shear rate (s− 1), K is the consistency coefficient (Pa s n), n is the flow behavior index (dimensionless), τ0 is the yield stress (Pa), and ηp is the Bingham plastic viscosity (Pa s). The software RheoWin 3 Data Manager was employed to obtain the rheological and statistical parameters. The temperature of all analyses was controlled with a Peltier system. 3. Results and discussion The pulp of cupuassu, which sums ∼40% of the total weight of the fresh fruit, was used to the isolation and characterization of its polysaccharides [15,16]. Fraction 0.1CA-2, obtained by extraction with hot 0.1% citric acid, was the main fraction, yielding 15% on an enzymeinactivated, defatted, dry-weight basis. Due to its high yield, it was selected for detailed chemical and rheological analysis. 3.1. Characterization of fraction 0.1CA-2 3.1.1. Chemical composition The monosaccharide composition of fraction 0.1CA-2 showed it to contain 91% glucose (Table 1) and this and a blue coloration with iodine test suggested the presence of starch. The glucose content was close those reported by Freitas et al. [7] for starches of yam (Dioscorea alata: 88%) and cassava (Manihot utilisssima: 87.4%). The monosaccharides Rha, Ara, Gal, and uronic acid were detected, typical of pectic polysaccharides, in small quantities of 0.6, 0.4, 1, and 6.6%, respectively. The protein content of fraction 0.1CA-2 was negligible at 1%. The amylose content of fraction 0.1CA-2 determined by colorimetric method [23] was 71% (R2 = 0.9993). According to Chrastil [23], the same color intensity is obtained after sample solubilization in NaOH, Me2SO, or urea-Me2SO, and the choice depends only on the solubility of the sample. Fraction 0.1CA-2 was found to be more soluble in aqueous NaOH than in urea-Me2SO or Me2SO. When only Glc content of fraction 0.1CA-2 (Table 1) is take into account, the content of amylose increases to 77%. Thus, the amylose content of starch present in fraction 0.1CA-2 from cupuassu is larger than that reported for starch from roots and tubers (10–38%) [6,7,25] and also of normal corn (21.4–32.5%) [25,26], wheat (18–30%) and rice (5–28.4%) starches [26]. Comparing with other fruits, this fraction from cupuassu pulp presented a native high amylose starch, differing of apple (26.0–29.3% of amylose) [11], cherimoya (15.4–16.3%) [13], squash (12.9–18.2%) [12] and Kamo Kamo fruit starches (17.2%) [14]. Amylose level greatly affects the properties of starch and highamylose starch has attracted attention because of their beneficial properties in many food and non-food applications [27]. It has been reported that long-term intake of dietary amylose may be valuable in decreasing insulin response while maintaining proper control of glucose tolerance and low levels of blood lipids [28]. Therefore,
Table 1 Monosaccharidea composition of fraction 0.1CA-2 obtained from the pulp of cupuassu Fraction
Rha
Ara
Xyl
Man
Gal
Glc
Uronic acidb
1.0
90.8
6.6
mol% 0.1CA-2 a b
0.6
0.4
0.4
0.2
Determined by GLC of derived alditol acetates. Determined by colorimetric method, Filisetti-Cozzi and Carpita [21].
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Fig. 1. 13C NMR spectra of fraction 0.1CA-2 in D2O at 70 °C (A) and DEPT-135 (B).
consumption of cupuassu pulp which contains high-amylose starch could have some health advantages in relation to the other fruits known to contain starch. 3.1.2. 13C-NMR analysis As expected, the 13C-NMR spectrum of fraction 0.1CA-2 contained 6 main signals (Fig. 1A), typical of starch with a high proportion of amylose and minor ones of amylopectin [29]. Present were signals of (1→4)-linked units of glucopyranose (C-1; 99.5 ppm), O-substituted C-4 (77.4 ppm). The resonance at 73.2, 71.5 and 71.2 ppm corresponded to C-3, C-2 and C-5 respectively. The signal at 60.6 ppm arose from unsubstituted C-6, confirmed by a DEPT-135 spectrum (Fig. 1B), which contained an inverted signal. Minor signals were present near to the C-1, C-4 and C-5 signals, which should arise from amylopectin. Similar 13C-NMR assignments for a starch fraction of mango pulp and for a standard corn starch with a high amylose content have been reported by Iagher et al. and Freitas, respectively [10,30]. 3.1.3. HPSEC-MALLS analysis Fraction 0.1CA-2 was analyzed by HPSEC using MALLS and RI detectors (Fig. 2). The RI gives a signal proportional to concentration
Fig. 2. Elution profile of fraction 0.1CA-2 obtained by HPSEC-MALLS/RI.
whereas the MALLS response depends on both concentration and molar mass. Although the alkaline media (pH13–14) has been considered good solvent for amylose, according to the literature the unperturbed dimensions are adopted at pH 7 and 25 °C, whatever the ionic strength [31]. Milles et al. [32] isolated amylose from starch and determined the molecular weight by light scattering using aqueous solutions of amylose. In the present work, for HPSEC analysis, fraction 0.1CA-2 (71% amylose) was solubilized in 0.1 mol L− 1 NaNO2 solution containing NaN3 (0.5 g L− 1) at 80 °C. Fig. 2 showed fraction 0.1CA-2 to be heterogeneous. A broad RI peak, representing the proportion of the molecules present, appeared between 45 and 58 min and is characteristic of molecules with lower molecular weight, with another being eluted at ∼ 38 min with higher molecular weight. With MALLS, this appeared overlapping with a minor RI peak of higher molecular weight. The profile of fraction 0.1CA-2 with both detectors is similar to that obtained by SEC-MALLS for a low molecular weight polydisperse amylose sample [33]. Its RI response showed a small shoulder eluted before a large peak and whose LS profile had a large peak eluted and then with a shoulder. The authors interpreted these responses as indicating that amylose solutions of low molecular weight are heterogeneous with respect to molecular weight distribution, containing a high molecular weight population at a low elution volume and the majority with lower molecular weight at higher elution volumes. The RI profile of fraction 0.1CA-2 is similar as those of debranched amylopectins of corn, sorghum, barley, wheat, and rice, measured by a intermediate SEC pressure [34], or by flow field-flow fractionation analysis [35], exhibiting in the same way, a multi-modal chain distribution and also it is close to that of the hot-water soluble starch of Bolivar rice cultivar [36]. The authors suggest that the amylopectin molecules of Bolivar are bigger, are more capable of inter- and intramolecular interactions, and may be more difficult to dissolve in hot water, and thus the hot-water soluble fraction of Bolivar starch predominantly consisted of amylose. The results suggest that the peak eluted at ∼38 min could be a high-molecular amylopectin, while the peaks eluted later correspond to an intermediate-molecular weight and low-molecular weight amylose fractions.
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Fig. 3. Frequency sweeps at 25 °C of fraction 0.1CA-2 at 4, 5, 6 and 7% (w/w) at tensions of 1.5, 4.0, 1.0 and 3.0 Pa, respectively.
According to Cheetam and Tao [37], the amylose content in corn starches has a strong correlation with the molecular size of its amylose, the molecular size of amylose decreasing with the increase in amylose content. If that was true for other species, amylose from cupuassu should be a low molecular weight polymer and consequently more soluble. The general results are in agreement with reports that consider starch to be a polymolecular and polydisperse polysaccharide [2,3]. 3.2. Rheological analysis Starch is a biopolymer largely employed by industry [3]. Highamylose starches have the ability to form strong gels and films. In the food industry, the high gelling strength of these starches makes them especially useful for producing sweets. The film-forming ability of these starches prevents moisture loss in fried products crispy and reduces their fat uptake upon cooking. High-amylose starches can be processed into ‘resistant starch’, which has nutritional benefits, and also may be used for the creation of biodegradable packing materials and adhesives [27,38]. As discussed by Prokopowich and Biliaderis [39], gelation of amylose (concentration b 10%) involves rapid formation of double helical structures from an amorphous sol upon cooling, which acts as junction zones among polymer chains, leading to the establishment of a three-dimensional hydrated gel network. On the other hand, gelation of amylopectin (concentration b 10%) is a much slower process involving recrystallization of the outer branches. As fraction 0.1CA-2 was the main polysaccharide fraction obtained from cupuassu pulp and was composed mainly by starch with a high amylose content (71%), it was submitted to rheological analysis. Solutions were prepared at concentrations of 4 to 7% (w/w). 3.2.1. Dynamic rheological properties of fraction 0.1CA-2 at different concentrations Frequency sweeps for gelatinized starch-rich fraction, at different concentrations, are shown in Fig. 3. The elastic moduli (G′) at all concentrations were relatively frequency-independent, while viscous moduli (G″) were only slightly dependent on frequency. There was a gradual increase of G″ with an increase of frequency for all tested concentrations.
Rosalina and Bhattacharya [40] and Khondkar et al. [41] obtained similar rheological characteristics for some corn and waxy corn starches, respectively. They proposed that starch samples give rise to a weak-gel behavior or a high viscosity gel respectively. Dynamic oscillatory results showed a gel-like behavior for all concentrations of fraction 0.1CA-2 with G′ being significantly larger than G″ over studied frequencies (0.1–10 Hz). These data, together with G′ being independent of frequency, characterize the presence of a network structure [42]. Furthermore, G′ of the sample with a higher concentration (7%) was about ten times higher than G″, characterizing a stronger gel [43]. The G′ of fraction 0.1CA-2 gels as a function of concentration measured at three different frequencies can be seen in Fig. 4. The magnitude of G′ increased with increasing sample concentration. The variation of the elastic modulus with concentration for 0.1CA-2 gels followed the relation G′ ∝ C5. Iturriaga et al. [44] evaluated starch gels from seven novel argentine rice genotypes and the dependence of storage modulus on concentration followed the Power-law, G′ ∝ Cn, with n = 2.9–3.2 for the non-waxy genotypes and n = 1.1 for waxy starch. Other authors have reported that G′ varies as C2.6–3.2 for non-waxy starches [45,46]. Ring (1983) cited by Miles et al. [47] have reported that G′ ∝ C7 for amylose gels in the concentration range 1.5–7%, which is similar to the results for high-amylose starch from cupuassu (fraction 0.1CA-2). However, it has been pointed out by the same author that the values of G′ are sensitive to thermal history of the amylose sample. Also, the differences in the origin, molecular weight of the samples of amylose could affect the values of G′ as well as the methods of preparation of the gels. The value of C0 has been used as an estimate of the lowest concentration where gelation is possible and it is obtained by the extrapolation of the values of G′ to zero [31]. A C0 of 0.9% was observed for amylose gels in 0.2 and 0.5 M KCl. Although we have not enough data to determine C0, when the concentration of 0.9% was used in the relation obtained for 0.1CA-2, the value determined for G′ was 0.018 Pa. Ortega-Ojeda et al. [48,49] reports that at the same concentration, the moduli values for potato starch (20.4% amylose) were not as high as for amylose. A lower minimum concentration (1.7%) was required for amylose to observe solid-like behavior [48]. On the other hand, potato starch showed G′ N G″ only when concentration was approximately higher than 4%.
Fig. 4. G′ as a function of concentration for 0.1CA-2 gels at different frequencies (25 °C).
L.C. Vriesmann et al. / Materials Science and Engineering C 29 (2009) 651–656
Fig. 5. Temperature dependence of elastic modulus (G′) of fraction 0.1CA-2 from cupuassu fruit pulp (4–7%, w/w) during heating trace at 1 Hz.
Samples of gelatinized fraction 0.1 CA-2 also were submitted to temperature sweeps, by cooling and heating curves from 5 to 95 °C and 95 to 5 °C, respectively. Fig. 5 shows the temperature dependence of the elastic modulus (G′) at a heating rate of 1 °C min− 1. Two-step mechanisms were observed at all concentrations of the fraction, namely a slow G′ decreases from 5 to 40 °C, then a sharp decrease of the G′ modulus between 65 to 70 °C. The profiles revealed losses in the gel strength above this temperature suggesting that structural changes occurred in the three dimensional network after the thermal cycle of the sample. This is similar to those described for starches from rice [50]. A temperature sweep of the 5% fraction 0.1CA-2 during the complete thermal cycle (heating followed by cooling) is shown in Fig. 6. The G′ underwent a decrease of about ten times during heating, approximately twice that of G″, and during cooling, both moduli underwent a gradual increase, but more pronounced for G″, so much so that reaching 5 °C, its value was greater than that of the start of the analysis. Thus, after thermal variation, the sample had a lower solid character than that of the start of the experiment. According to Yu and Christie [51], the thermal behavior of starches is complex. Several physicochemical changes may occur during heating, involving gelatinization, crystallization, volume expansion, molecular degradation and motion of water, among others. 3.2.2. Flow behavior of fraction 0.1CA-2 at different concentrations The viscosity of gelatinized starch is influenced by the extent of swelling of the granules prior to their rupture, as well as the expansion and dispersion of resistant starch granules after the rupture of the
Fig. 6. Variation of G′ and G″ (1 Hz, 4 Pa) during initial heating from 5 to 95 °C and subsequent cooling from 95 to 5 °C for a 5% (w/w) 0.1 CA-2 gel.
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granular structure. When the viscosity of starch pastes is determined, the volume of swollen granules and their inherent deformability are important [52]. Fig. 7 shows viscosity curves for fraction 0.1CA-2 at different concentrations (4 to 7 w/w %) at 25 °C. A decrease in the viscosity with shear rate increase was observed, characterizing a non-Newtonian behavior under the examined conditions. The non-Newtonian behavior of starch solution was reported by many other researches [24,53–55]. The viscosity value increased with the increase in the fraction 0.1CA-2 concentration, as expected for polysaccharides in solution. For example, at concentrations of 4, 5, 6 and 7% the absolute viscosity — η was 14120; 37230; 63960 and 90410 mPa s respectively, with all measurements determined at 1.2 s− 1 (low shear rate). These results showed that the viscosity of fraction 0.1CA-2 in aqueous solution is strongly dependent on starch concentration. A similar behavior was observed by Chaudemanche and Budtova [56] for pregelatinized corn starch (70% amylose) at concentrations higher than 1% at 60 °C. Under high shear, the gelatinized starch granule structure was broken down, which caused a drop in paste viscosity [57]. The experimental data for the cupuassu starch (fraction 0.1CA-2) were evaluated according to different rheological models. The parameters of the Herschel–Buckley, Power-law and Bingham models obtained from regression analysis between the shear stress and the shear rate from 4 to 7% (w/w) samples are summarized in Table 2. The Bingham model is a special case of Herschel–Bulkley model when n is equal to unity [53]. For the values of n in Herschel–Bulkley, which were far from unity, the samples of this study can not be appropriately described by the Bingham model. Considering the R2 values, Herschel–Buckley (R2 ≥ 0.98) was found to be the most adequate model to describe the flow behavior of the sample in this study. Gelatinized starch dispersions are described as non-Newtonian fluids which may also exhibit a yield stress at low shear [53,58], which seems the case of starch of this study at concentration of 4–7%. According to Lagarrigue and Alvarez [58], the yield stress value depends on concentration, mass fraction of swollen granules, granule mean diameter and gelatinization procedure. Although other models have been used, gelatinized starch dispersions are usually represented by the Power-law or the Herschel–Bulkley model in the range 1– 1500 s− 1. The consistency index (K) and the flow behavior index (n) depend on the kind of starch, its concentration and temperature [58]. As can be seem from Table 2, K increases with the increasing of concentration of 0.1CA-2, showing increasing viscosities. Similar
Fig. 7. Influence of shear rate on the absolute viscosity of gels of fraction 0.1CA-2 at 25 °C.
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Table 2 Comparison of different models for the flow behavior of fraction 0.1CA-2 samples obtained from the pulp of cupuassu Concentration (%, w/w)
Model
K (Pa sn)
τ0 (Pa)
n
ηp (Pa s)
R2
4
Herschel–Bulkley Power-law Bingham Herschel–Bulkley Power-law Bingham Herschel–Bulkley Power-law Bingham Herschel–Bulkley Power-law Bingham
1.52 5.22 – 10.22 21.94 – 11.52 23.66 – 12.16 24.72 –
10.34 – 15.08 38.40 – 107.1 40.43 – 117.8 44.45 – 120.1
0.70 0.51 – 0.55 0.45 – 0.55 0.45 – 0.55 0.44 –
– – 0.2630 – – 0.5416 – – 0.5992 – – 0.5998
0.980 0.946 0.951 0.989 0.985 0.960 0.996 0.993 0.964 0.997 0.992 0.960
5
6
7
observations were reported for cassava starch [53], sago starch pastes [55] and corn starch [59]. 4. Conclusions For the first time, a starch-rich fraction from the pulp of cupuassu fruit (T. grandiflorum) is described with a yield of 15% related to defatted-dried material. The fraction was composed predominantly by a high amylose (71%) starch, characterized by a blue coloration with iodine solution, monosaccharide composition and 13C NMR spectroscopy. This fraction contained a minor proportion of pectin that may contribute to its gel-like behavior. Analysis of the rheological properties of the fraction showed a non-Newtonian behavior at concentrations from 4 to 7% (w/w). Considering the R2 values, Herschel–Buckley was found to be the most adequate model to describe the rheological comportment of the samples. Dynamic oscillatory experiments indicated a highly elastic behavior with G′ significantly larger than G″, being independent of the range of examined frequencies (0.1–10 Hz), confirming gel characters for all concentrations. A gel-like behavior was demonstrated for fraction 0.1CA-2, a native high amylose starch, suggesting a potential as a gelling additive in pharmaceutical, cosmetic and food applications. Acknowledgements The authors thank the Brazilian agencies, CNPq and Fundação Araucária-PRONEX for financial support, and Dr. Philip A. J. Gorin for help with the English language. References [1] P. Van Hung, T. Maeda, N. Morita, Trends in Food Science & Technology 17 (2006) 448–456. [2] H.F. Zobel, A.M. Stephen, in: A.M. Stephen (Ed.), Food Polysaccharides and Their Applications, Marcel Dekker, 1995, pp. 19–66. [3] J.N. BeMiller, in: R.L. Whistler, J.N. BeMiller (Eds.), Industrial Gums: Polysaccharides and Their Derivatives, 3rd, Academic Press, san Diego, 1993, pp. 579–600. [4] G.E. Vandeputte, V. Derycke, J. Geeroms, J.A. Delcour, Journal of Cereal Science 38 (2003) 53–59. [5] N. Singh, N. Isono, S. Srichuwong, T. Noda, K. Nishinari, Food Hydrocolloids 22 (2008) 979–988. [6] R. Hoover, Carbohydrate Polymers 45 (2001) 253–267. [7] R.A. Freitas, R.C. Paula, J.P.A. Feitosa, S. Rocha, M.-R. Sierakowski, Carbohydrate Polymers 55 (2004) 3–8. [8] C.E. Millán-Testa, M.G. Méndez-Montealvo, M.A. Ottenhof, I.A. Farhat, L.A. BelloPérez, Journal of Agricultural and Food Chemistry 53 (2005) 495–501. [9] L.A. Bello-Pérez, E. Agama-Acevedo, L. Sánchez-Hernández, O. Paredes-López, Journal of Agricultural and Food Chemistry 47 (1999) 854–857.
[10] F. Iagher, F. Reicher, J.L.M. Ganter, International Journal of Biological Macromolecules 31 (2002) 9–17. [11] D.G. Stevenson, P.A. Domoto, J. Jane, Carbohydrate Polymers 63 (2006) 432–441. [12] D.G. Stevenson, S. Yoo, P.L. Hurst, J. Jane, Carbohydrate Polymers 59 (2005) 153–163. [13] O. Goňi, M.I. Escribano, C. Merodio, LWT — Food Science and Technology 41 (2008) 303–310. [14] J. Singh, O.J. McCarthy, H. Singh, P.J. Moughan, L. Kaur, Carbohydrate Polymers 67 (2007) 233–244. [15] L.C. Vriesmann, Extração, caracterização e aspectos reológicos de polissacarídeos da polpa dos frutos de Theobroma grandiflorum (cupuaçu) (2008). MSc. Thesis. Universidade Federal do Paraná, Curitiba, Brasil. Available on-line on http://hdl. handle.net/1884/13859. [16] L.C. Vriesmann, C.L.O. Petkowicz, Carbohydrate Polymers (in press). doi:10.1016/j. carbpol.2008.12.007. [17] P.B. Cavalcante, Frutas Comestíveis da Amazônia, 5th ed., CEJUP: CNPq: Museu Paraense Emílio Goeldi, Belém, 1991, pp. 90–91. [18] C.E. Quijano, J.A. Pino, Food Chemistry 104 (2007) 1123–1126. [19] M.C. Vieira, A.A. Teixeira, C.L.M. Silva, Journal of Food Engineering 43 (2000) 1–7. [20] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Analytical Biochemistry 28 (1956) 350–356. [21] T.M.C.C. Filisetti-Cozzi, N.C. Carpita, Analytical Biochemistry 197 (1991) 157–162. [22] E.F. Hartree, Analytical Biochemistry 48 (1972) 422–427. [23] J. Chrastil, Carbohydrate Research 159 (1987) 154–158. [24] J. Techawipharat, M. Suphantharika, J.N. BeMiller, Carbohydrate Polymers 73 (2008) 417–426. [25] J. Jane, Y.Y. Chen, L.F. Lee, A.E. McPherson, K.S. Wong, M. Radosavljevic, T. Kasemsuwan, Cereal Chemistry 76 (1999) 629–637. [26] N. Singh, J. Singh, L. Kaur, N.S. Sodhi, B.S. Gill, Food Chemistry 81 (2003) 219–231. [27] N.W. Polaske, A.L. Wood, M.R. Campbell, M.C. Nagan, L.M. Pollak, Starch/Stärke 57 (2005) 118–123. [28] K. Behall, J. Hallfrisch, European Journal of Clinical Nutrition 56 (2002) 913–920. [29] P.A.J. Gorin, Advances in Carbohydrate Chemistry and Biochemistry 38 (1981) 13–104. [30] R.A. Freitas, Estrutura e interações entre galactoxiloglucanas e amidos (2003). Ph D. Thesis, UFPR, Curitiba, Brasil. [31] J.-L. Doublier, L. Choplin, Carbohydrate Research, 193 (1989) 215–226. [32] M.J. Milles, V.J. Morris, S.G. Ring, Carbohydrate Research 135 (1985) 257–269. [33] P. Roger, V. Tran, J. Lesec, P. Collona, Journal of Cereal Science 24 (1996) 247–262. [34] J.-H. Chung, J.-A. Han, B. Yoo, P.A. Seib, S.-T. Lim, Carbohydrate Polymers 71 (2008) 365–371. [35] P. Roger, B. Baud, P. Colonna, Journal of Chromatography A 917 (2001) 179–185. [36] J.A. Patindol, B.C. Gonzalez, Y.-J. Wang, A.M. McClung, Journal of Cereal Science 45 (2007) 209–218. [37] N.W.H. Cheetam, L. Tao, Carbohydrate Polymers 33 (1997) 251–261. [38] S. Jobling, Current Opinion in Plant Biology 7 (2004) 210–218. [39] D.J. Prokopowich, C.G. Biliaderis, Food Chemistry 52 (1995) 255–262. [40] I. Rosalina, M. Bhattacharya, Carbohydrate Polymers 48 (2002) 191–202. [41] D. Khondkar, R.F. Tester, N. Hudson, J. Karkalas, J. Morrow, Food Hydrocolloids 21 (2007) 1296–1301. [42] A.H. Clark, S.B. Ross-Murphy, Advanced Polymer Science 83 (1987) 57–192. [43] E.R. Morris, in: A.M. Stephen (Ed.), Food Polysaccharides and Their Applications, Marcel Dekker, New York, 1995, pp. 517–546. [44] L.B. Iturriaga, B.L. Mishima, M.C. Añon, Food Research International 39 (2006) 660–666. [45] C.G. Biliaderis, B.O. Juliano, Food Chemistry 48 (1993) 243–250. [46] V.M.F. Lai, M.-C. Shen, A.-I. Yeh, B.O. Juliano, C.-y. Lii, Cereal Chemistry 78 (2001) 596–602. [47] M.J. Miles, V.J. Morris, P.D. Orford, S.G. Ring, Carbohydrate Research 135 (1985) 271–281. [48] F.E. Ortega-Ojeda, H. Larsson, A.-C. Eliasson, Carbohydrate Polymers 57 (2004) 55–66. [49] F.E. Ortega-Ojeda, H. Larsson, A.-C. Eliasson, Carbohydrate Polymers 56 (2004) 505–514. [50] M. Tako, S. Hizukuri, Journal of Carbohydrate Chemistry 18 (1999) 573–584. [51] L. Yu, G. Christie, Carbohydrate Polymers 46 (2001) 179–184. [52] X.-Z. Han, O.H. Campanella, H. Guan, P.L. Keeling, B.R. Hamaker, Carbohydrate Polymers 49 (2002) 315–321. [53] L.-m. Che, D. Li, L.-j. Wang, N. Özkan, X.D. Chen, Z.-H. Maoa, Carbohydrate Polymers 74 (2008) 385–389. [54] M.A. Rao, P.E. Okechukwu, P.M.S. da Silva, J.C. Oliveira, Carbohydrate Polymers 33 (1997) 273–283. [55] I.M. Nurul, B.M.N.M. Azemi, D.M.A. Manan, Food Chemistry 64 (1999) 501–505. [56] C. Chaudemanche, T. Budtova, Carbohydrate Polymers 72 (2008) 579–589. [57] X.-Z. Han, O.H. Campanella, H. Guan, P.L. Keeling, B.R. Hamaker, Carbohydrate Polymers 49 (2002) 315–321. [58] S. Lagarrigue, G. Alvarez, Journal of Food Engineering 50 (2001) 189–202. [59] P.N. Bhandari, R.S. Singhal, D.D. Kale, Carbohydrate Polymers 47 (2002) 365–371.
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m s e c
Chemical and rheological properties of a starch-rich fraction from the pulp of the fruit cupuassu (Theobroma grandiflorum) Lúcia C. Vriesmann, Joana L.M. Silveira, Carmen L. de O. Petkowicz ⁎ Universidade Federal do Paraná, Departamento de Bioquímica e Biologia Molecular, CP 19046, CEP 81531-990, Curitiba-PR, Brazil
a r t i c l e
i n f o
Article history: Received 8 May 2008 Received in revised form 28 November 2008 Accepted 9 December 2008 Available online 16 December 2008 Keywords: Theobroma grandiflorum Cupuassu Starch Amylose Gel Rheology
a b s t r a c t The pulp obtained from the fruit of cupuassu (Theobroma grandiflorum) was extracted with hot aqueous 0.1% citric acid to give fraction 0.1CA-2 in 15% yield. This was the predominant component polysaccharide, 91% of which was composed of starch, by an iodine test and monosaccharide composition, and its 13C NMR spectrum was consistent with that of a high amylose starch. The content of amylose found in fraction 0.1CA-2 was 71%. This value is higher than those of common starches of cereal grains, tubers, roots, and other fruits. The fraction was submitted to rheological examination, gels being prepared on heating with concentrations of 4 to 7% (w/w). A non-Newtonian behavior was observed, and gel viscosity and strength depended on the concentration. The presence of starch, as well as the presence of previously investigated pectin, conferred the high viscosity and gelling capability of the pulp. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Starch is the main reserve polysaccharide of many plants. It is an important renewable and a low-cost polymer, occurring as granules, which determine its physical properties. Due its thickening and gelling properties, it is used in the food, pharmaceutical, and chemical industry [1–3]. Cereal grains, legume seeds, tubers, and certain fruits contain from 30 to 85% starch on a dry-weight basis [3]. Commercial starches are obtained mainly from yellow corn, although potato, wheat, tapioca, rice and sorghum also are significant sources [2,3]. Many reports have described the characterization of starch from cereals, roots and tubers [4–7]. The fruit starches have also been investigated, mainly those from banana [8,9], mango [8,10], apple [11], squash [12], cherimoya [13] and Kamo Kamo [14]. Starch consists of a mixture of two polymers, amylose and amylopectin. Whereas amylose is an essentially linear molecule, consisting of (1→4)-linked α-D-glucopyranosyl units, amylopectin is a highly branched molecule with substitution of these at O-6 by α-Dglucopyranosyl branches. These are composed of (1→4)-linked α-Dglucopyranosyl units with various lengths [1–3]. It has been demonstrated that the pulp of the fruit of Theobroma grandiflorum, Schumann (family Sterculiaceae), growing in the Brazilian Amazon, known locally as cupuassu, contains a considerable amount of starch as well as pectin polysaccharides [15,16]. The pulp is ⁎ Corresponding author. Tel.: +55 41 3361 1661; fax: +55 41 3266 2042. E-mail address: [email protected] (C.L.O. Petkowicz). 0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.12.011
greatly appreciated for its pleasant acidic taste, being consumed fresh or processed, mainly as juice, ice cream, candy and jellies [17]. It has an intense fragrance [17], whose volatile compounds have been extensively analyzed [18], as well as its content of vitamin C [19]. As chemical and rheological characterization of native starch can lead to improvements for predicting desirable functional properties of starchy products, we now study some chemical and rheological features of the native starch-rich fraction obtained from the pulp of cupuassu fruit [15]. 2. Experimental 2.1. Extraction of fraction 0.1CA-2 After enzyme-inactivation with methanol–H2O (4:1, v/v) under reflux for 20 min, the grounded pulp fruit was defatted with ptoluene-ethanol (2:1, v/v) in a Soxhlet and dried. The residue was submitted to sequential extractions with water (25 °C and 60 °C), citric acid (0.1%, 0.5%, 1%, 2.5% and 5%, using temperatures of 50 °C and 100 °C at each concentration) and NaOH (2 mol L– 1 and 4 mol L– 1 NaOH; 25 °C) [15,16]. Fraction 0.1CA-2 was obtained with 0.1% citric acid at 100 °C for 60 min. The extract was concentrated and treated with ethanol (2:1 v/v) in order to obtain precipitated polysaccharide, which was then washed three times with ethanol and dried under vacuum. The fraction was identified according to the conditions applied on extraction: 0.1 displays the concentration of citric acid (CA) and “−2” was used for the second hot extraction (100 °C).
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2.2. Chemical characterization of fraction 0.1CA-2 Total carbohydrate was determined by the phenol–sulfuric acid colorimetric method [20], employing glucose as standard. Uronic acid was estimated by the sulfamate/3-phenylphenol colorimetric method [21], using galacturonic acid as standard. Protein was measured by the Hartree method [22], with BSA as standard. Amylose content was determined by the Chrastil method [23] with a modification, using 80% amylose and waxy amylopectin (0% amylose) (Sigma) as standards. Intermediate amylose proportions were obtained by mixing appropriate amounts of each. A straight line plot was used to determine the content of amylose in the sample. Fraction 0.1CA-2 was solubilized in distilled water at 100 °C during 10 min, then alkalinized with 1 mol L– 1 NaOH and kept for 5 min at 100 °C. After cooling at 25 °C, it was submitted to the procedure for amylose determination. The monosaccharide composition was determined after total acid hydrolysis with 2 mol L− 1 trifluoroacetic acid (5 h, 100 °C). The monosaccharides, obtained on evaporation to dryness, were reduced with NaBH4 and then acetylated with pyridine-acetic anhydride (1:1 v/v, 16 h, at 25 °C). The resulting alditol acetates were analyzed by gas–liquid chromatography (GLC) using a model 5890 S II HewlettPackard gas chromatograph at 220 °C (flame ionization detector and injector temperature, 250 °C) with a DB-210 capillary column (0.25 mm internal diameter × 30 m, film thickness 0.25 µm), the carrier gas being nitrogen at 2.0 mL min− 1 [16]. For high pressure size exclusion chromatography (HPSEC) analyses, suspension of the sample in 0.1 mol L− 1 NaNO2 solution containing NaN3 (0.5 g L− 1) was heated to 80 °C during 2 h under magnetic stirring. HPSEC was carried out on polysaccharide solution at 25 °C, using a multidetection equipment in with a Waters 2410 differential refractometer (RI) and a Wyatt Technology Dawn F multiangle laser light scattering (MALLS) detector were adapted on-line. Four Waters Ultrahydrogel 2000/500/250/120 columns were connected in series and coupled to the multidetection equipment. The mobile phase consisted of a 0.1 mol L− 1 NaNO2 solution containing 0.5 g L− 1 NaN3 (vacuum-filtered through a 0.22 µm membrane filter) at a flow rate of 0.6 mL min− 1 and pressure 830 psi. The sample, previously filtered (0.22 µm; Millipore), was injected (100 µL loop) at a 1.5 mg mL− 1. HPSEC data were collected and analyzed by a Wyatt Technology ASTRA program. 13 C NMR spectra of fraction 0.1CA-2 were obtained using a Bruker DRX 400 Avance spectrometer incorporating Fourier transform (FT) on a solution in D2O at 70 °C. Chemical shifts are expressed as δ (ppm), using the resonances of CH3 groups of acetone as internal standard (δ 30.2). 2.3. Rheological analysis of fraction 0.1CA-2 Solutions of fraction 0.1CA-2 at concentrations of 4, 5, 6 and 7% (w/w) were prepared by stirring in water for 16 h at 25 °C. They were then heated at 92 °C with continuous stirring for 15 min and cooled at room temperature. The samples were stored at 5 °C prior to rheological examination. These were carried out using a model RS 75 Haake Rheometer, coupled with a DC5 heating circulator, with a C60/2° (cone and plate geometry) or PP20 sensor (plate and plate geometry). Mechanical responses of the samples were determined by subjecting them to a frequency sweep (0.1–10 Hz) at 25 °C at tension (τ) between 1 and 4 Pa. These values refer to the viscoelastic-linear region, where the gel structure was preserved. The temperature sweeps, heating (5–95 °C) and subsequent cooling (95–5 °C), were performed at a rate of 1 °C min− 1, at a frequency of 1 Hz. Before starting the experiments, the exposed sample edge was covered with a thin layer of low viscosity mineral oil to minimize evaporation losses during measurements. Continuous flow ramps in the CR mode (controlled rate) were performed at 25 °C. The sensor was programmed to increase the shear
rate from 0.1 to 100 s− 1 (up curve) in 100 s followed immediately by a reduction from 100 to 0.1 s− 1 in 100 s (down curve). The shear stress (τ) was then measured as a function of shear rate. As done by Techawipharat, Suphantharika and BeMiller (2008) [24], data from the down curve of the shear cycle were used to characterize the flow behavior of the cupuassu starch samples. The experimental data were evaluated and fitted according to the rheological models of Herschel– Bulkley (τ = τ0 + Kγn), Power-law (τ = Kγn), and Bingham (τ = τ0 + ηpγ), where τ is the shear stress (Pa), γ is the shear rate (s− 1), K is the consistency coefficient (Pa s n), n is the flow behavior index (dimensionless), τ0 is the yield stress (Pa), and ηp is the Bingham plastic viscosity (Pa s). The software RheoWin 3 Data Manager was employed to obtain the rheological and statistical parameters. The temperature of all analyses was controlled with a Peltier system. 3. Results and discussion The pulp of cupuassu, which sums ∼40% of the total weight of the fresh fruit, was used to the isolation and characterization of its polysaccharides [15,16]. Fraction 0.1CA-2, obtained by extraction with hot 0.1% citric acid, was the main fraction, yielding 15% on an enzymeinactivated, defatted, dry-weight basis. Due to its high yield, it was selected for detailed chemical and rheological analysis. 3.1. Characterization of fraction 0.1CA-2 3.1.1. Chemical composition The monosaccharide composition of fraction 0.1CA-2 showed it to contain 91% glucose (Table 1) and this and a blue coloration with iodine test suggested the presence of starch. The glucose content was close those reported by Freitas et al. [7] for starches of yam (Dioscorea alata: 88%) and cassava (Manihot utilisssima: 87.4%). The monosaccharides Rha, Ara, Gal, and uronic acid were detected, typical of pectic polysaccharides, in small quantities of 0.6, 0.4, 1, and 6.6%, respectively. The protein content of fraction 0.1CA-2 was negligible at 1%. The amylose content of fraction 0.1CA-2 determined by colorimetric method [23] was 71% (R2 = 0.9993). According to Chrastil [23], the same color intensity is obtained after sample solubilization in NaOH, Me2SO, or urea-Me2SO, and the choice depends only on the solubility of the sample. Fraction 0.1CA-2 was found to be more soluble in aqueous NaOH than in urea-Me2SO or Me2SO. When only Glc content of fraction 0.1CA-2 (Table 1) is take into account, the content of amylose increases to 77%. Thus, the amylose content of starch present in fraction 0.1CA-2 from cupuassu is larger than that reported for starch from roots and tubers (10–38%) [6,7,25] and also of normal corn (21.4–32.5%) [25,26], wheat (18–30%) and rice (5–28.4%) starches [26]. Comparing with other fruits, this fraction from cupuassu pulp presented a native high amylose starch, differing of apple (26.0–29.3% of amylose) [11], cherimoya (15.4–16.3%) [13], squash (12.9–18.2%) [12] and Kamo Kamo fruit starches (17.2%) [14]. Amylose level greatly affects the properties of starch and highamylose starch has attracted attention because of their beneficial properties in many food and non-food applications [27]. It has been reported that long-term intake of dietary amylose may be valuable in decreasing insulin response while maintaining proper control of glucose tolerance and low levels of blood lipids [28]. Therefore,
Table 1 Monosaccharidea composition of fraction 0.1CA-2 obtained from the pulp of cupuassu Fraction
Rha
Ara
Xyl
Man
Gal
Glc
Uronic acidb
1.0
90.8
6.6
mol% 0.1CA-2 a b
0.6
0.4
0.4
0.2
Determined by GLC of derived alditol acetates. Determined by colorimetric method, Filisetti-Cozzi and Carpita [21].
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Fig. 1. 13C NMR spectra of fraction 0.1CA-2 in D2O at 70 °C (A) and DEPT-135 (B).
consumption of cupuassu pulp which contains high-amylose starch could have some health advantages in relation to the other fruits known to contain starch. 3.1.2. 13C-NMR analysis As expected, the 13C-NMR spectrum of fraction 0.1CA-2 contained 6 main signals (Fig. 1A), typical of starch with a high proportion of amylose and minor ones of amylopectin [29]. Present were signals of (1→4)-linked units of glucopyranose (C-1; 99.5 ppm), O-substituted C-4 (77.4 ppm). The resonance at 73.2, 71.5 and 71.2 ppm corresponded to C-3, C-2 and C-5 respectively. The signal at 60.6 ppm arose from unsubstituted C-6, confirmed by a DEPT-135 spectrum (Fig. 1B), which contained an inverted signal. Minor signals were present near to the C-1, C-4 and C-5 signals, which should arise from amylopectin. Similar 13C-NMR assignments for a starch fraction of mango pulp and for a standard corn starch with a high amylose content have been reported by Iagher et al. and Freitas, respectively [10,30]. 3.1.3. HPSEC-MALLS analysis Fraction 0.1CA-2 was analyzed by HPSEC using MALLS and RI detectors (Fig. 2). The RI gives a signal proportional to concentration
Fig. 2. Elution profile of fraction 0.1CA-2 obtained by HPSEC-MALLS/RI.
whereas the MALLS response depends on both concentration and molar mass. Although the alkaline media (pH13–14) has been considered good solvent for amylose, according to the literature the unperturbed dimensions are adopted at pH 7 and 25 °C, whatever the ionic strength [31]. Milles et al. [32] isolated amylose from starch and determined the molecular weight by light scattering using aqueous solutions of amylose. In the present work, for HPSEC analysis, fraction 0.1CA-2 (71% amylose) was solubilized in 0.1 mol L− 1 NaNO2 solution containing NaN3 (0.5 g L− 1) at 80 °C. Fig. 2 showed fraction 0.1CA-2 to be heterogeneous. A broad RI peak, representing the proportion of the molecules present, appeared between 45 and 58 min and is characteristic of molecules with lower molecular weight, with another being eluted at ∼ 38 min with higher molecular weight. With MALLS, this appeared overlapping with a minor RI peak of higher molecular weight. The profile of fraction 0.1CA-2 with both detectors is similar to that obtained by SEC-MALLS for a low molecular weight polydisperse amylose sample [33]. Its RI response showed a small shoulder eluted before a large peak and whose LS profile had a large peak eluted and then with a shoulder. The authors interpreted these responses as indicating that amylose solutions of low molecular weight are heterogeneous with respect to molecular weight distribution, containing a high molecular weight population at a low elution volume and the majority with lower molecular weight at higher elution volumes. The RI profile of fraction 0.1CA-2 is similar as those of debranched amylopectins of corn, sorghum, barley, wheat, and rice, measured by a intermediate SEC pressure [34], or by flow field-flow fractionation analysis [35], exhibiting in the same way, a multi-modal chain distribution and also it is close to that of the hot-water soluble starch of Bolivar rice cultivar [36]. The authors suggest that the amylopectin molecules of Bolivar are bigger, are more capable of inter- and intramolecular interactions, and may be more difficult to dissolve in hot water, and thus the hot-water soluble fraction of Bolivar starch predominantly consisted of amylose. The results suggest that the peak eluted at ∼38 min could be a high-molecular amylopectin, while the peaks eluted later correspond to an intermediate-molecular weight and low-molecular weight amylose fractions.
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Fig. 3. Frequency sweeps at 25 °C of fraction 0.1CA-2 at 4, 5, 6 and 7% (w/w) at tensions of 1.5, 4.0, 1.0 and 3.0 Pa, respectively.
According to Cheetam and Tao [37], the amylose content in corn starches has a strong correlation with the molecular size of its amylose, the molecular size of amylose decreasing with the increase in amylose content. If that was true for other species, amylose from cupuassu should be a low molecular weight polymer and consequently more soluble. The general results are in agreement with reports that consider starch to be a polymolecular and polydisperse polysaccharide [2,3]. 3.2. Rheological analysis Starch is a biopolymer largely employed by industry [3]. Highamylose starches have the ability to form strong gels and films. In the food industry, the high gelling strength of these starches makes them especially useful for producing sweets. The film-forming ability of these starches prevents moisture loss in fried products crispy and reduces their fat uptake upon cooking. High-amylose starches can be processed into ‘resistant starch’, which has nutritional benefits, and also may be used for the creation of biodegradable packing materials and adhesives [27,38]. As discussed by Prokopowich and Biliaderis [39], gelation of amylose (concentration b 10%) involves rapid formation of double helical structures from an amorphous sol upon cooling, which acts as junction zones among polymer chains, leading to the establishment of a three-dimensional hydrated gel network. On the other hand, gelation of amylopectin (concentration b 10%) is a much slower process involving recrystallization of the outer branches. As fraction 0.1CA-2 was the main polysaccharide fraction obtained from cupuassu pulp and was composed mainly by starch with a high amylose content (71%), it was submitted to rheological analysis. Solutions were prepared at concentrations of 4 to 7% (w/w). 3.2.1. Dynamic rheological properties of fraction 0.1CA-2 at different concentrations Frequency sweeps for gelatinized starch-rich fraction, at different concentrations, are shown in Fig. 3. The elastic moduli (G′) at all concentrations were relatively frequency-independent, while viscous moduli (G″) were only slightly dependent on frequency. There was a gradual increase of G″ with an increase of frequency for all tested concentrations.
Rosalina and Bhattacharya [40] and Khondkar et al. [41] obtained similar rheological characteristics for some corn and waxy corn starches, respectively. They proposed that starch samples give rise to a weak-gel behavior or a high viscosity gel respectively. Dynamic oscillatory results showed a gel-like behavior for all concentrations of fraction 0.1CA-2 with G′ being significantly larger than G″ over studied frequencies (0.1–10 Hz). These data, together with G′ being independent of frequency, characterize the presence of a network structure [42]. Furthermore, G′ of the sample with a higher concentration (7%) was about ten times higher than G″, characterizing a stronger gel [43]. The G′ of fraction 0.1CA-2 gels as a function of concentration measured at three different frequencies can be seen in Fig. 4. The magnitude of G′ increased with increasing sample concentration. The variation of the elastic modulus with concentration for 0.1CA-2 gels followed the relation G′ ∝ C5. Iturriaga et al. [44] evaluated starch gels from seven novel argentine rice genotypes and the dependence of storage modulus on concentration followed the Power-law, G′ ∝ Cn, with n = 2.9–3.2 for the non-waxy genotypes and n = 1.1 for waxy starch. Other authors have reported that G′ varies as C2.6–3.2 for non-waxy starches [45,46]. Ring (1983) cited by Miles et al. [47] have reported that G′ ∝ C7 for amylose gels in the concentration range 1.5–7%, which is similar to the results for high-amylose starch from cupuassu (fraction 0.1CA-2). However, it has been pointed out by the same author that the values of G′ are sensitive to thermal history of the amylose sample. Also, the differences in the origin, molecular weight of the samples of amylose could affect the values of G′ as well as the methods of preparation of the gels. The value of C0 has been used as an estimate of the lowest concentration where gelation is possible and it is obtained by the extrapolation of the values of G′ to zero [31]. A C0 of 0.9% was observed for amylose gels in 0.2 and 0.5 M KCl. Although we have not enough data to determine C0, when the concentration of 0.9% was used in the relation obtained for 0.1CA-2, the value determined for G′ was 0.018 Pa. Ortega-Ojeda et al. [48,49] reports that at the same concentration, the moduli values for potato starch (20.4% amylose) were not as high as for amylose. A lower minimum concentration (1.7%) was required for amylose to observe solid-like behavior [48]. On the other hand, potato starch showed G′ N G″ only when concentration was approximately higher than 4%.
Fig. 4. G′ as a function of concentration for 0.1CA-2 gels at different frequencies (25 °C).
L.C. Vriesmann et al. / Materials Science and Engineering C 29 (2009) 651–656
Fig. 5. Temperature dependence of elastic modulus (G′) of fraction 0.1CA-2 from cupuassu fruit pulp (4–7%, w/w) during heating trace at 1 Hz.
Samples of gelatinized fraction 0.1 CA-2 also were submitted to temperature sweeps, by cooling and heating curves from 5 to 95 °C and 95 to 5 °C, respectively. Fig. 5 shows the temperature dependence of the elastic modulus (G′) at a heating rate of 1 °C min− 1. Two-step mechanisms were observed at all concentrations of the fraction, namely a slow G′ decreases from 5 to 40 °C, then a sharp decrease of the G′ modulus between 65 to 70 °C. The profiles revealed losses in the gel strength above this temperature suggesting that structural changes occurred in the three dimensional network after the thermal cycle of the sample. This is similar to those described for starches from rice [50]. A temperature sweep of the 5% fraction 0.1CA-2 during the complete thermal cycle (heating followed by cooling) is shown in Fig. 6. The G′ underwent a decrease of about ten times during heating, approximately twice that of G″, and during cooling, both moduli underwent a gradual increase, but more pronounced for G″, so much so that reaching 5 °C, its value was greater than that of the start of the analysis. Thus, after thermal variation, the sample had a lower solid character than that of the start of the experiment. According to Yu and Christie [51], the thermal behavior of starches is complex. Several physicochemical changes may occur during heating, involving gelatinization, crystallization, volume expansion, molecular degradation and motion of water, among others. 3.2.2. Flow behavior of fraction 0.1CA-2 at different concentrations The viscosity of gelatinized starch is influenced by the extent of swelling of the granules prior to their rupture, as well as the expansion and dispersion of resistant starch granules after the rupture of the
Fig. 6. Variation of G′ and G″ (1 Hz, 4 Pa) during initial heating from 5 to 95 °C and subsequent cooling from 95 to 5 °C for a 5% (w/w) 0.1 CA-2 gel.
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granular structure. When the viscosity of starch pastes is determined, the volume of swollen granules and their inherent deformability are important [52]. Fig. 7 shows viscosity curves for fraction 0.1CA-2 at different concentrations (4 to 7 w/w %) at 25 °C. A decrease in the viscosity with shear rate increase was observed, characterizing a non-Newtonian behavior under the examined conditions. The non-Newtonian behavior of starch solution was reported by many other researches [24,53–55]. The viscosity value increased with the increase in the fraction 0.1CA-2 concentration, as expected for polysaccharides in solution. For example, at concentrations of 4, 5, 6 and 7% the absolute viscosity — η was 14120; 37230; 63960 and 90410 mPa s respectively, with all measurements determined at 1.2 s− 1 (low shear rate). These results showed that the viscosity of fraction 0.1CA-2 in aqueous solution is strongly dependent on starch concentration. A similar behavior was observed by Chaudemanche and Budtova [56] for pregelatinized corn starch (70% amylose) at concentrations higher than 1% at 60 °C. Under high shear, the gelatinized starch granule structure was broken down, which caused a drop in paste viscosity [57]. The experimental data for the cupuassu starch (fraction 0.1CA-2) were evaluated according to different rheological models. The parameters of the Herschel–Buckley, Power-law and Bingham models obtained from regression analysis between the shear stress and the shear rate from 4 to 7% (w/w) samples are summarized in Table 2. The Bingham model is a special case of Herschel–Bulkley model when n is equal to unity [53]. For the values of n in Herschel–Bulkley, which were far from unity, the samples of this study can not be appropriately described by the Bingham model. Considering the R2 values, Herschel–Buckley (R2 ≥ 0.98) was found to be the most adequate model to describe the flow behavior of the sample in this study. Gelatinized starch dispersions are described as non-Newtonian fluids which may also exhibit a yield stress at low shear [53,58], which seems the case of starch of this study at concentration of 4–7%. According to Lagarrigue and Alvarez [58], the yield stress value depends on concentration, mass fraction of swollen granules, granule mean diameter and gelatinization procedure. Although other models have been used, gelatinized starch dispersions are usually represented by the Power-law or the Herschel–Bulkley model in the range 1– 1500 s− 1. The consistency index (K) and the flow behavior index (n) depend on the kind of starch, its concentration and temperature [58]. As can be seem from Table 2, K increases with the increasing of concentration of 0.1CA-2, showing increasing viscosities. Similar
Fig. 7. Influence of shear rate on the absolute viscosity of gels of fraction 0.1CA-2 at 25 °C.
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Table 2 Comparison of different models for the flow behavior of fraction 0.1CA-2 samples obtained from the pulp of cupuassu Concentration (%, w/w)
Model
K (Pa sn)
τ0 (Pa)
n
ηp (Pa s)
R2
4
Herschel–Bulkley Power-law Bingham Herschel–Bulkley Power-law Bingham Herschel–Bulkley Power-law Bingham Herschel–Bulkley Power-law Bingham
1.52 5.22 – 10.22 21.94 – 11.52 23.66 – 12.16 24.72 –
10.34 – 15.08 38.40 – 107.1 40.43 – 117.8 44.45 – 120.1
0.70 0.51 – 0.55 0.45 – 0.55 0.45 – 0.55 0.44 –
– – 0.2630 – – 0.5416 – – 0.5992 – – 0.5998
0.980 0.946 0.951 0.989 0.985 0.960 0.996 0.993 0.964 0.997 0.992 0.960
5
6
7
observations were reported for cassava starch [53], sago starch pastes [55] and corn starch [59]. 4. Conclusions For the first time, a starch-rich fraction from the pulp of cupuassu fruit (T. grandiflorum) is described with a yield of 15% related to defatted-dried material. The fraction was composed predominantly by a high amylose (71%) starch, characterized by a blue coloration with iodine solution, monosaccharide composition and 13C NMR spectroscopy. This fraction contained a minor proportion of pectin that may contribute to its gel-like behavior. Analysis of the rheological properties of the fraction showed a non-Newtonian behavior at concentrations from 4 to 7% (w/w). Considering the R2 values, Herschel–Buckley was found to be the most adequate model to describe the rheological comportment of the samples. Dynamic oscillatory experiments indicated a highly elastic behavior with G′ significantly larger than G″, being independent of the range of examined frequencies (0.1–10 Hz), confirming gel characters for all concentrations. A gel-like behavior was demonstrated for fraction 0.1CA-2, a native high amylose starch, suggesting a potential as a gelling additive in pharmaceutical, cosmetic and food applications. Acknowledgements The authors thank the Brazilian agencies, CNPq and Fundação Araucária-PRONEX for financial support, and Dr. Philip A. J. Gorin for help with the English language. References [1] P. Van Hung, T. Maeda, N. Morita, Trends in Food Science & Technology 17 (2006) 448–456. [2] H.F. Zobel, A.M. Stephen, in: A.M. Stephen (Ed.), Food Polysaccharides and Their Applications, Marcel Dekker, 1995, pp. 19–66. [3] J.N. BeMiller, in: R.L. Whistler, J.N. BeMiller (Eds.), Industrial Gums: Polysaccharides and Their Derivatives, 3rd, Academic Press, san Diego, 1993, pp. 579–600. [4] G.E. Vandeputte, V. Derycke, J. Geeroms, J.A. Delcour, Journal of Cereal Science 38 (2003) 53–59. [5] N. Singh, N. Isono, S. Srichuwong, T. Noda, K. Nishinari, Food Hydrocolloids 22 (2008) 979–988. [6] R. Hoover, Carbohydrate Polymers 45 (2001) 253–267. [7] R.A. Freitas, R.C. Paula, J.P.A. Feitosa, S. Rocha, M.-R. Sierakowski, Carbohydrate Polymers 55 (2004) 3–8. [8] C.E. Millán-Testa, M.G. Méndez-Montealvo, M.A. Ottenhof, I.A. Farhat, L.A. BelloPérez, Journal of Agricultural and Food Chemistry 53 (2005) 495–501. [9] L.A. Bello-Pérez, E. Agama-Acevedo, L. Sánchez-Hernández, O. Paredes-López, Journal of Agricultural and Food Chemistry 47 (1999) 854–857.
[10] F. Iagher, F. Reicher, J.L.M. Ganter, International Journal of Biological Macromolecules 31 (2002) 9–17. [11] D.G. Stevenson, P.A. Domoto, J. Jane, Carbohydrate Polymers 63 (2006) 432–441. [12] D.G. Stevenson, S. Yoo, P.L. Hurst, J. Jane, Carbohydrate Polymers 59 (2005) 153–163. [13] O. Goňi, M.I. Escribano, C. Merodio, LWT — Food Science and Technology 41 (2008) 303–310. [14] J. Singh, O.J. McCarthy, H. Singh, P.J. Moughan, L. Kaur, Carbohydrate Polymers 67 (2007) 233–244. [15] L.C. Vriesmann, Extração, caracterização e aspectos reológicos de polissacarídeos da polpa dos frutos de Theobroma grandiflorum (cupuaçu) (2008). MSc. Thesis. Universidade Federal do Paraná, Curitiba, Brasil. Available on-line on http://hdl. handle.net/1884/13859. [16] L.C. Vriesmann, C.L.O. Petkowicz, Carbohydrate Polymers (in press). doi:10.1016/j. carbpol.2008.12.007. [17] P.B. Cavalcante, Frutas Comestíveis da Amazônia, 5th ed., CEJUP: CNPq: Museu Paraense Emílio Goeldi, Belém, 1991, pp. 90–91. [18] C.E. Quijano, J.A. Pino, Food Chemistry 104 (2007) 1123–1126. [19] M.C. Vieira, A.A. Teixeira, C.L.M. Silva, Journal of Food Engineering 43 (2000) 1–7. [20] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Analytical Biochemistry 28 (1956) 350–356. [21] T.M.C.C. Filisetti-Cozzi, N.C. Carpita, Analytical Biochemistry 197 (1991) 157–162. [22] E.F. Hartree, Analytical Biochemistry 48 (1972) 422–427. [23] J. Chrastil, Carbohydrate Research 159 (1987) 154–158. [24] J. Techawipharat, M. Suphantharika, J.N. BeMiller, Carbohydrate Polymers 73 (2008) 417–426. [25] J. Jane, Y.Y. Chen, L.F. Lee, A.E. McPherson, K.S. Wong, M. Radosavljevic, T. Kasemsuwan, Cereal Chemistry 76 (1999) 629–637. [26] N. Singh, J. Singh, L. Kaur, N.S. Sodhi, B.S. Gill, Food Chemistry 81 (2003) 219–231. [27] N.W. Polaske, A.L. Wood, M.R. Campbell, M.C. Nagan, L.M. Pollak, Starch/Stärke 57 (2005) 118–123. [28] K. Behall, J. Hallfrisch, European Journal of Clinical Nutrition 56 (2002) 913–920. [29] P.A.J. Gorin, Advances in Carbohydrate Chemistry and Biochemistry 38 (1981) 13–104. [30] R.A. Freitas, Estrutura e interações entre galactoxiloglucanas e amidos (2003). Ph D. Thesis, UFPR, Curitiba, Brasil. [31] J.-L. Doublier, L. Choplin, Carbohydrate Research, 193 (1989) 215–226. [32] M.J. Milles, V.J. Morris, S.G. Ring, Carbohydrate Research 135 (1985) 257–269. [33] P. Roger, V. Tran, J. Lesec, P. Collona, Journal of Cereal Science 24 (1996) 247–262. [34] J.-H. Chung, J.-A. Han, B. Yoo, P.A. Seib, S.-T. Lim, Carbohydrate Polymers 71 (2008) 365–371. [35] P. Roger, B. Baud, P. Colonna, Journal of Chromatography A 917 (2001) 179–185. [36] J.A. Patindol, B.C. Gonzalez, Y.-J. Wang, A.M. McClung, Journal of Cereal Science 45 (2007) 209–218. [37] N.W.H. Cheetam, L. Tao, Carbohydrate Polymers 33 (1997) 251–261. [38] S. Jobling, Current Opinion in Plant Biology 7 (2004) 210–218. [39] D.J. Prokopowich, C.G. Biliaderis, Food Chemistry 52 (1995) 255–262. [40] I. Rosalina, M. Bhattacharya, Carbohydrate Polymers 48 (2002) 191–202. [41] D. Khondkar, R.F. Tester, N. Hudson, J. Karkalas, J. Morrow, Food Hydrocolloids 21 (2007) 1296–1301. [42] A.H. Clark, S.B. Ross-Murphy, Advanced Polymer Science 83 (1987) 57–192. [43] E.R. Morris, in: A.M. Stephen (Ed.), Food Polysaccharides and Their Applications, Marcel Dekker, New York, 1995, pp. 517–546. [44] L.B. Iturriaga, B.L. Mishima, M.C. Añon, Food Research International 39 (2006) 660–666. [45] C.G. Biliaderis, B.O. Juliano, Food Chemistry 48 (1993) 243–250. [46] V.M.F. Lai, M.-C. Shen, A.-I. Yeh, B.O. Juliano, C.-y. Lii, Cereal Chemistry 78 (2001) 596–602. [47] M.J. Miles, V.J. Morris, P.D. Orford, S.G. Ring, Carbohydrate Research 135 (1985) 271–281. [48] F.E. Ortega-Ojeda, H. Larsson, A.-C. Eliasson, Carbohydrate Polymers 57 (2004) 55–66. [49] F.E. Ortega-Ojeda, H. Larsson, A.-C. Eliasson, Carbohydrate Polymers 56 (2004) 505–514. [50] M. Tako, S. Hizukuri, Journal of Carbohydrate Chemistry 18 (1999) 573–584. [51] L. Yu, G. Christie, Carbohydrate Polymers 46 (2001) 179–184. [52] X.-Z. Han, O.H. Campanella, H. Guan, P.L. Keeling, B.R. Hamaker, Carbohydrate Polymers 49 (2002) 315–321. [53] L.-m. Che, D. Li, L.-j. Wang, N. Özkan, X.D. Chen, Z.-H. Maoa, Carbohydrate Polymers 74 (2008) 385–389. [54] M.A. Rao, P.E. Okechukwu, P.M.S. da Silva, J.C. Oliveira, Carbohydrate Polymers 33 (1997) 273–283. [55] I.M. Nurul, B.M.N.M. Azemi, D.M.A. Manan, Food Chemistry 64 (1999) 501–505. [56] C. Chaudemanche, T. Budtova, Carbohydrate Polymers 72 (2008) 579–589. [57] X.-Z. Han, O.H. Campanella, H. Guan, P.L. Keeling, B.R. Hamaker, Carbohydrate Polymers 49 (2002) 315–321. [58] S. Lagarrigue, G. Alvarez, Journal of Food Engineering 50 (2001) 189–202. [59] P.N. Bhandari, R.S. Singhal, D.D. Kale, Carbohydrate Polymers 47 (2002) 365–371.