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Food Hydrocolloids 37 (2014) 174e181

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Food Hydrocolloids

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Comparison of starches isolated from three different Trapa species

Hongming Gao a,1, Jinwen Cai a,1, Wenli Han b, Huyin Huai b, Yifang Chen c, Cunxu Wei a,b,*aKey Laboratories of Crop Genetics and Physiology of the Jiangsu Province and Plant Functional Genomics of the Ministry of Education, Yangzhou University,Yangzhou 225009, ChinabCollege of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, Chinac Testing Center, Yangzhou University, Yangzhou 225009, China

a r t i c l e i n f o

Article history:Received 20 September 2013Accepted 4 November 2013

Keywords:Trapa speciesStarchMorphologyCrystallineThermal propertiesHydrolysis

Abbreviations: AAG, Aspergillus niger amyloglucototal reflectance-Fourier transform infrared; DSC, diffePPA, porcine pancreatic a-amylase; SEM, scanning elepowder diffraction.* Corresponding author. College of Bioscience an

University, Yangzhou 225009, China. Tel.: þ86 514 87E-mail addresses: cxwei@yzu.edu.cn, yzuwcx@163

1 These authors contributed equally to this work.

0268-005X/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodhyd.2013.11.001

a b s t r a c t

Trapa (Trapaceae) is one of the most common aquatic plants. In order to make full use of the resource ofTrapa, we investigated two Trapa cultivated species (Trapa quadrispinosa Roxb. and Trapa bispinosa Roxb.)and one Trapa wild species (Trapa pseudoinisa Nakai) popularly found in South China. The morphology,size, weight, and contents of water, starch and soluble sugar of fruit and kernel were significantlydifferent among the three species. Starches isolated from their kernels were all oval in shape withsmooth surface and central hilum. The starch granule size was larger and amylose content was higher inthe wild species than in the two cultivated species. Starches from the three Trapa species all exhibited CA-type crystalline. T. pseudoinisa starch showed the highest swelling power and solubility. Starches fromthe three Trapa species had similar thermal properties except that T. pseudoinisa starch had higher onsettemperature and lower conclusion temperature. T. pseudoinisa starch showed similar breakdown vis-cosity and higher setback viscosity compared with starches from the other two species. Similar hydro-lysis degree by porcine pancreatic a-amylase was observed in starches isolated from the three species.However, T. bispinosa starch showed the highest hydrolysis degree and T. pseudoinisa starch showed thelowest hydrolysis degree by HCl and Aspergillus niger amyloglucosidase.

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1. Introduction

Trapa L. (Trapaceae), water caltrop, is a genus of floating-leaved aquatic plants, and is often found in shallow water field,lake, pond, creek or swampy land in tropical and sub-tropicalcountries (Chiang, Li, Huang, & Wang, 2007; Takano & Kadono,2005). The fruit (nut) is covered with a thick outer pericarp.The outer pericarp is hard, making it quite difficult to peel off toobtain the internal white kernel (Tulyathan, Boondee, &Mahawanich, 2005). The kernel of Trapa species has high con-tents of starch, soluble sugar, protein and essential minerals(Chiang et al., 2007; Takano & Kadono, 2005). Due to the sweet,tender and delicious taste, raw and cooked kernels are usuallyeaten as one of the popular starchy desserts or snacks, and are

sidase; ATR-FTIR, attenuatedrential scanning calorimetry;ctron microscope; XRD, X-ray

d Biotechnology, Yangzhou997217..com (C. Wei).

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also used as an ingredient in other foods in South China andsouthern Asian countries. Starches from Trapa species contributeto the textural properties of many foods, such as thickening ofsoups and sauces locally. Because of easy implementation andmanagement, Trapa species is a potential source of commercialstarch in Asian countries (Chiang et al., 2007; Takano & Kadono,2005). In addition, its starch may also be useful as a potentialbinder in the pharmaceutical industry (Singh, Singh, Nath, & Pani,2011).

Starch has been extensively studied and discussed in the liter-ature. Physicochemical properties of starches include morphology,size, amylose content, crystal properties, thermal properties,swelling powers and hydrolysis properties. These propertiesdetermine the application of starch in food and nonfood industries.Starches from different botanical sources have diverse physico-chemical properties. The physicochemical properties of starchesfrom some species of Trapa, such as Trapa quadrispinosa Roxb.(Chiang et al., 2007), Trapa bispinosa Roxb. (Singh et al., 2011; Tran,Lee, & Park, 2013; Tulyathan et al., 2005), Trapa natans L. (Gani, Haq,Masoodi, Broadway, & Gani, 2010; Singh, Bawa, Singh, & Saxena,2009), and Trapa taiwanensis Nakai (Wang, Chiang, Li, & Huang,2008) have been reported in previous references. However, thephysicochemical properties of starch are investigated from only

H. Gao et al. / Food Hydrocolloids 37 (2014) 174e181 175

one species in one literature. The comparison of starches has notbeen reported in different Trapa species. In addition,T. quadrispinosa, T. bispinosa, T. natans and T. taiwanensis are allwidely cultivated in China and other Asian countries. There is littleinformation on the starch from Trapa wild species.

In this study, two Trapa cultivated species (T. quadrispinosa andT. bispinosa) and one wild species (Trapa pseudoinisa Nakai), whichare commonly found in South China, were obtained from the samegrowing region and growing season. Their fruits, kernels andphysicochemical properties of starches were investigated andcompared. The aim of this study was to evaluate the physico-chemical properties of starches from three different Trapa species.

2. Materials and methods

2.1. Plant materials

Freshly harvested fruits of T. quadrispinosa and T. bispinosawereobtained from a local natural food market (Yangzhou City, China).The fruits of T. pseudoinisa were collected from creeks in the sub-urbs of Yangzhou City, China. The hard pericarp was removed withthe help of a sharp and clean stainless steel knife and the ediblekernel portion obtained was used to isolate starches.

2.2. Morphology, weight, and contents of water, starch and solublesugar of fruit and kernel

Fresh fruits and peeled kernels were photographed with aCanon digital IXUS 750 camera and weighted on an electronicanalytical balance. The water content of kernel was determinedby oven-drying method. Dry kernels were ground extensivelyand passed through a 100-mesh sieve to obtain the flour. 100 mgof flour was extracted in 10 ml 80% (v/v) ethanol for 30 min at80 �C and then centrifuged at 3000 � g for 15 min. The resultingpellet was further extracted two times with 80% ethanol. Thecombined supernatants were made up to 50 ml with 80% ethanolfor assay of soluble sugars. The ethanol-insoluble residue wasboiled in 3 ml deionized-distilled water for 20 min, cooled toroom temperature, added 3 ml of cool 9.2 M HClO4 for 10 min,and then centrifuged at 5000 � g for 15 min. The resulting pelletwas further hydrolyzed two times with 5 ml of 4.6 M HClO4 for10 min. The combined supernatants were made up to 100 mlwith deionized-distilled water for starch assay. Both superna-tants for assays of starch and soluble sugar were measured forsugar concentrations by the colorimetric method of anthrone-H2SO4 using glucose as standard (McCready, Guggolz, Silviera, &Owens, 1949).

2.3. Isolation of native starch

Native starch was isolated following a method described byWei, Qin, Zhu, et al. (2010) with a slight modification. Briefly, thekernels were homogenized with ice-cold water in a home blender.The homogenate was squeezed through four layers of cheeseclothby hand. The fibrous residue was homogenized and squeezedtwice more with ice-cold water to facilitate the release of starchgranules from the fibers. The combined extract was filtered with100, 200, 300 and 400 mesh sieves. After depositing, the super-natant was discarded and the upper non-white layer was scrapedoff. The settled starch layer was resuspended in distilled water.After six cycles of depositing and resuspending repeatedly, thewhite starch sediment was further washed with anhydrousethanol, dried at 40 �C, ground into powders, and passed througha 100-mesh sieve.

2.4. Morphology observation of starch granule

For microscopic morphology, a starch suspension (1% w/v) wasprepared with 50% glycerol. A small drop of starch suspension wasplaced on the microscope slide and covered with a coverslip. Thestarch granule shape and Maltese cross were viewed with anOlympus BX53 polarized light microscope equipped with a CCDcamera. For submicroscopic morphology, the dried starch wassuspended in anhydrous ethanol. One drop of the starch-ethanolsuspension was applied to an aluminum stub using double-sidedadhesive tape, and the starch was coated with gold beforeviewing with an environmental scanning electron microscope(SEM) (Philips XL-30).

2.5. Particle size analysis of starch

Images of iodine-stained starch granules were analyzed usingJEDA-801D morphological image analysis system (Jiangsu JEDAScience-Technology Development Co., Ltd, Nanjing, China). Morethan 2000 starch granules were analyzed per sample. Starchgranules were grouped according to the long axis length, and thenumber of starch granules in each group was counted. Plotting therelative number of starch granules against the long axis lengthproduced a starch granule size-distribution curve.

2.6. Amylose content determination

Amylose content of starch was determined using the iodinecolorimetric method of Konik-Rose et al. (2007) with some modi-fications. About 10 mg of starch was weighed (accurate to 0.1 mg)into a 10 ml screwcapped tube, then dissolved in 5 ml of ureadimethyl sulphoxide (UDMSO) solution. Dissolution was obtainedby incubating the mixture at 95 �C for 1 h with intermittent vor-texing. A 1 ml aliquot of the starch-UDMSO solution was treatedwith 1 ml of iodine solution (0.2% I2 and 2% KI, w/v) andmade up to50 ml with water. The solution was immediately mixed and placedin the darkness for 20 min. Apparent amylose content was evalu-ated from absorbance at 620 nm. The recorded values were con-verted to percent of amylose by reference to a standard curveprepared with amylose from potato (SigmaeAldrich A0512) andamylopectin from corn (SigmaeAldrich 10120).

2.7. Crystal structure analysis of starch

Crystal structure of starch was analyzed on an X-ray powderdiffraction (XRD) (D8, Bruker, Germany) spectroscope. The XRDanalysis anddetermination of the relative degreeof crystallinity (%) ofthe starcheswere carried out following themethod described byWei,Qin, Zhou, et al. (2010). Beforemeasurements, all the specimenswerestored in a desiccatorwhere a saturated solution ofNaClmaintained aconstant humidity atmosphere (relative humidity¼ 75%) for 1 week.

2.8. Structural order of starch external region

Ordered structure of starch external region was analyzed on aVarian 7000 Fourier transform infrared (FTIR) spectrometer with aDTGS detector equipped with an attenuated total reflectance (ATR)single reflectance cell containing a germanium crystal (45�

incidence-angle) (PIKE Technologies, USA) as previously describedby Wei, Xu, et al. (2010). Original spectra were corrected by sub-traction of the baseline in the region from 1200 to 800 cm�1 beforedeconvolutionwas applied using Resolutions Pro. The assumed lineshapewas Lorentzianwith a half-width of 19 cm�1 and a resolutionenhancement factor of 1.9. Intensity measurements at 1047, 1022,and 995 cm�1 were performed on the deconvoluted spectra by

Fig. 1. Morphology of fresh fruits (AeC) and kernels (aec) from T. quadrispinosa (A, a),T. bispinosa (B, b) and T. pseudoinisa (C, c).

H. Gao et al. / Food Hydrocolloids 37 (2014) 174e181176

recording the height of the absorbance bands from the baselineusing Adobe Photoshop 7.0 image software.

2.9. Swelling power and solubility of starch

The swelling power and solubility index of starch were deter-mined by heating starch-water slurries in a water bath at 95 �Caccording to the procedures of Wei et al. (2011). The values forswelling power were reported in grams per gram and that of sol-ubility index in percent.

2.10. Thermal properties of starch

Thermal properties of starch was measured using a differentialscanning calorimetry (DSC) (200-F3, NETZSCH, Germany) asdescribed previously byWei et al. (2011). Starch (w5mg, dry starchbasis) was precisely weighed and mixed with 3 times (by weight)deionized-distilled water (w15 ml). The mixture was sealed in analuminum pan overnight at 4 �C. After equilibrating for 1 h at roomtemperature, the starch sample was then heated from 25 to 110 �Cat a rate of 10 �C/min.

2.11. Pasting properties of starch

The pasting properties of starch (8% solids) was evaluated with aRapid Visco Analyzer (RVA-3D, Newport Scientific, Narrabeen,Australia). A Programmed heating and cooling cycle was used,where the samples were held at 50 �C for 1min, heated to 95 �C at arate of 12 �C/min, maintained at 95 �C for 2.5 min, cooled to 50 �C ata rate of 12 �C/min, and then held at 50 �C for 1.4 min. Parametersrecorded were peak viscosity, hot viscosity, final viscosity, break-down viscosity (peak-hot viscosity), setback viscosity (final-hotviscosity), peak time, and pasting temperature.

2.12. Hydrolysis of starch

Starch was hydrolyzed by porcine pancreatic a-amylase (EC3.2.1.1) (PPA) (SigmaeAldrich A3176), Aspergillus niger amyloglu-cosidase (EC 3.2.1.3) (AAG) (SigmaeAldrich A7095) and HCl. Thehydrolysis degrees of starch by PPA and AAG were determinedusing the method of Li, Vasanthan, Hoover, and Rossnagel (2004)with some modifications. For PPA hydrolysis, isolated nativestarch (10 mg) was suspended in 2 ml of enzyme solution (0.1 Mphosphate sodium buffer, pH 6.9, 25 mM NaCl, 5 mM CaCl2, 0.02%NaN3, 50 U PPA). For AAG hydrolysis, starch (10 mg) was suspendedin 2 ml of enzyme solution (0.05 M acetate buffer, pH 4.5, 5 U AAG).The hydrolyses of PPA and AAG were conducted in a constanttemperature shaking water bath with continuous shaking(100 rpm) at 37 and 55 �C, respectively, for 1, 2, 4, 8, 12, 24, 48, and72 h. The hydrolysis rate of starches by HCl was analyzed using themethod of Wei, Xu, et al. (2010) with minor modification. 20 mgstarch was suspended in 2 ml of 2.2 M HCl and hydrolysis wasconducted in a constant temperature shaking water bath withcontinuous shaking (100 rpm) at 35 �C for 0.5, 1, 2, 4, 6, 8, 10, 12 and14 d. After hydrolysis, starch slurries were quickly centrifuged(3000 � g) at 4 �C for 10 min. The supernatant was used for mea-surement of the solubilized carbohydrates to quantify the degree ofhydrolysis by the anthrone-H2SO4 method (McCready et al., 1949).

2.13. Statistical analysis

The data reported in all the tables were mean values and stan-dard deviation. Analysis of variance (ANOVA) by Tukey’s test(P < 0.05) was evaluated using the SPSS 16.0 Statistical SoftwareProgram.

3. Results and discussion

3.1. Morphology, weight, and contents of water, starch and solublesugar of fruit and kernel

In Trapa, the morphology of fruits offers the best diagnosticcriteria for the classification of species. Especially fruit size and thenumber of spines have been used as the most basic characters(Takano & Kadono, 2005). The mature fruits and kernels showedsignificantly different morphology and size among three differentTrapa species (Fig. 1). The mature fruit has one pair of spines in theshoulder and one pair of spines in the abdomen for T. quadrispinosa,but only one pair of spines in the shoulder for T. bispinosa. ForT. pseudoinisa, one pair of very long thin spines with terminal barbsis found in the shoulder of fruits (Fig. 1; Wan, 2000). The fruits were6.0e7.4 cm long and 3.8e4.7 cm wide for T. quadrispinosa, 5.5e6.8 cm long and 1.9e2.3 cm wide for T. bispinosa, and 2.7e3.4 cmlong and 1.0e1.3 cm wide for T. pseudoinisa. The weight and con-tents of water, starch and soluble sugar of fruit and kernel aresummarized in Table 1. For fruit and kernel, the weight ofT. quadrispinosa was the highest, and that of T. pseudoinisa was thelowest. For water content, T. bispinosa was the highest, andT. pseudoinisawas the lowest. For starch content, T. pseudoinisawasthe highest and T. bispinosa the lowest for fresh kernel, andT. quadrispinosa was the highest and T. bispinosa the lowest for drykernel. The soluble sugar content of T. pseudoinisa was the highestand that of T. bispinosa was the lowest in dry kernel.

3.2. Morphology and size distribution of starch granule

Morphologies of starch granules taken from light microscope,polarized light microscope and SEM are presented in Fig. 2. Thegranule size of T. pseudoinisa starch was significantly larger thanthat of T. quadrispinosa and T. bispinosa starches. The typical Maltese

Table 1Weights and contents of water, starch and soluble sugar of fruits and kernels from three Trapa species.a

Species Fresh fruitweight (g)

Fresh kernel Dry kernel

Weight (g) Water content (%) Starch content (%) Weight (g) Starch content (%) Soluble sugar content (%)

T. quadrispinosa 16.56 � 3.56c 7.89 � 2.30c 68.92 � 0.76b 15.71 � 0.26b 2.45 � 0.72c 50.56 � 0.85b 6.98 � 0.32bT. bispinosa 10.33 � 2.01b 5.48 � 1.16b 83.83 � 0.60c 6.61 � 0.14a 0.89 � 0.19b 40.89 � 0.89a 4.64 � 0.49aT. pseudoinisa 1.97 � 0.26a 0.99 � 0.20a 60.86 � 1.65a 16.60 � 0.53c 0.39 � 0.08a 42.41 � 1.35a 9.27 � 0.17c

a Data were means � standard deviations, n ¼ 20 for weight of fruit and kernel and 3 for the contents of water, starch and soluble sugar. Values in the same column withdifferent letters were significantly different (P < 0.05).

H. Gao et al. / Food Hydrocolloids 37 (2014) 174e181 177

crosses were in the central position of starch granules underpolarized light. Starch granules from three Trapa species were allmorphologically similar. The shapes of granules appeared to be ovaland smooth when viewed by normal light microscope and SEM,which was in agreement with the previous reports (Chiang et al.,2007; Gani et al., 2010; Singh et al., 2009; Tulyathan et al., 2005;Wang et al., 2008).

Starch granule sizes are usually measured by electrozone, imageanalysis, or laser light-scattering analysis methods. Harrigan (1997)found that using image analysis to determine starch granule sizecould yield accurate and reproducible data. Therefore, in this study,image analysis was used to determine starch granule size distri-bution. Starches from three Trapa species all showed unimodal sizedistributions (data not shown). The average particle size (the longaxis length) was 13.97, 14.11 and 17.47 mm for T. quadrispinosa,T. bispinosa and T. pseudoinisa starch, respectively (Table 2), andagreed with the results from the light and scanning electron mi-crographs (Fig. 2). In this paper, the particle size of starches fromthree Trapa species was comparable with previous results fromSEM micrographs of starch granules from Trapa species (Chianget al., 2007; Tulyathan et al., 2005; Wang et al., 2008). However,the average particle size was significantly smaller in this study thanthat (over 30 mm) determined by a laser diffraction analyzer in the

Fig. 2. Photographs of light microscope (AeC), polarized light microscope (DeF) and scaT. bispinosa (B, E, H) and T. pseudoinisa (C, F, I). Scale bar ¼ 20 mm.

previous reports (Chiang et al., 2007; Wang et al., 2008). This dif-ference between their values and ours seemed to stemmainly fromthe different assay method.

3.3. Amylose content of starch

The amylose content has a significant effect on physicochemicaland functional properties of starch. The amylose contents ofstarches from three Trapa species were determined in this study(Table 2). Amylose contents of starches showed significantlydifferent among three Trapa species. T. pseudoinisa starch had thehighest amylose content, and T. quadrispinosa starch had the lowestamylose content. T. bispinosa starch in Vietnam has amylose con-tent of 47.1% (Tran et al., 2013), that in Thailand 29.6% (Tulyathanet al., 2005). The differences between their values and oursseemed to stem possibly from the different amylose content assaymethods, plant geographical origins and growth conditions.

3.4. Crystal properties of starch

XRD has been used to reveal the presence and characteristic ofcrystalline structure of starch. According to XRD pattern, there arethree types of starch crystalline reported, known as A-, B- and C-

nning electron microscope (GeI) of starch granules from T. quadrispinosa (A, D, G),

Table 2Sizes, amylose contents, relative crystallinities and IR ratios of starches from three Trapa species.a

Species Size (mm) Amylose content (%) Relative crystallinity (%) IR ratio

1045/1022 cm�1 1022/995 cm�1

T. quadrispinosa 13.97 � 3.96a 24.68 � 0.52a 30.23 � 0.36c 0.81 � 0.01a 0.84 � 0.01aT. bispinosa 14.11 � 3.29a 26.85 � 0.42b 28.43 � 0.22b 0.86 � 0.01b 0.84 � 0.01aT. pseudoinisa 17.47 � 4.26b 31.13 � 0.83c 27.69 � 0.06a 0.85 � 0.01b 0.85 � 0.01a

a Data were means � standard deviations, n > 2000 for size and ¼3 for amylose content, relative crystallinity and IR ratio. Values in the same column with different letterswere significantly different (P < 0.05).

H. Gao et al. / Food Hydrocolloids 37 (2014) 174e181178

type. A-type crystal starch has strong diffraction peaks at about 15�

and 23� 2q, and an unresolved doublet at around 17� and 18� 2q. B-type crystal starch gives the strongest diffraction peak at around17� 2q, a few small peaks at around 15�, 20�, 22�, and 24� 2q, and acharacteristic peak at about 5.6� 2q. C-type crystal starch is amixture of both A- and B-type crystalline, and can be furtherclassified to CA-type (closer to A-type), C-type and CB-type (closerto B-type) according to the proportion of A- and B-type poly-morphs. The typical C-type crystal starch shows strong diffractionpeaks at about 17� and 23� 2q, and a few small peaks at around 5.6�

and 15� 2q. The XRD patterns of CA- and CB-type crystal starchesshow some slight differences from that of typical C-type. CA-typecrystal starch shows a shoulder peak at about 18� 2q and strongpeaks at about 15� and 23� 2q, which are indicative of the A-typepattern. CB-type crystal starch shows two shoulder peaks at about22� and 24� 2q and a weak peak at about 15� 2q, which are indic-ative of the B-type pattern. The peak at 15� 2q is strongest in A-typecrystalline and weakest in B-type crystalline (Cheetham & Tao,1998).

The XRD spectra of starches from three Trapa species are shownin Fig. 3A. Starches from three Trapa species all showed the char-acteristics of a CA-type crystalline. Both C- and A-type crystallineshad been reported for starch from Trapa species. For examples,Tulyathan et al. (2005) reported that T. bispinosa starch showed C-type XRD pattern, while Chiang et al. (2007) andWang et al. (2008)reported that T. quadrispinosa and T. taiwanensis starches showed aA-type XRD pattern. These differences suggested that the crystal-line structure of starch was easily affected by growing environ-ment, especially the temperature. The relative degrees ofcrystallinities of starches calculated from the ratio of diffractionpeak area and total diffraction area are given in Table 2.T. pseudoinisa starch showed the lowest relative degree of crys-tallinity, and T. quadrispinosa starch had the highest relative degreeof crystallinity. Usually, the crystalline degree was negativelycorrelated with the level of amylose (Cheetham & Tao, 1998),which also agreed with our study.

Fig. 3. Spectra of XRD (A) and ATR-FTIR (B) of starches from T

3.5. Ordered structure of starch external region

The development of sampling devices like ATR-FTIR combinedwith procedures for spectrum deconvolution provides opportu-nities for the study of starch external region structure (Sevenou,Hill, Farhat, & Mitchell, 2002). Though FTIR is not able to differen-tiate starch crystal type, the starches with same crystal type alwaysshow the similar FTIR spectra. The band at 1022 cm�1 is morepronounced in A-type starch than in B-type or C-type starch(Sevenou et al., 2002). The bands at 1045 and 1022 cm�1 are linkedwith order/crystalline and amorphous regions in starch, respec-tively. The ratio of absorbance 1045/1022 cm�1 is used to quantifythe degree of order in starch samples. Intensity ratios of 1045/1022and 1022/995 cm�1 are useful as a convenient index of FTIR data incomparisons with other measures of starch conformation (Sevenouet al., 2002).

The deconvoluted ATR-FTIR spectra in the region 1200e900 cm�1 of starches from three Trapa species are presented inFig. 3B. The relative intensities of FTIR bands at 1045, 1022 and995 cm�1 were recorded from the baseline to peak height, and theratios for 1045/1022 and 1022/995 cm�1 were calculated as shownin Table 2. Based on both the spectra and calculated data, starchesfrom three Trapa species showed the similar ordered structure ingranule external region and the characteristics of C-type starch,which agreed with the results of XRD.

3.6. Swelling power and solubility of starch

When aqueous suspensions of starch granules are heated, thestarch molecule hydrates and swells with a consequent leaching ofsome soluble starch into the liquid (Tulyathan et al., 2005). Theswelling power of starch indicates the degree of water absorptionof starch granules and the solubility reflects the degree of disso-lution during the starch swelling procedure (Carcea & Acquistucci,1997). Swelling power and solubility are characteristics of thestarches. The swelling power and solubility of starches from three

. quadrispinosa (a), T. bispinosa (b) and T. pseudoinisa (c).

Table 3Swelling powers, solubilities and thermal properties of starches from three Trapa species.a

Species Swelling power (g/g) Solubility (%) Thermal parameterb

To (�C) Tp (�C) Tc (�C) DT (�C) DH (J/g)

T. quadrispinosa 18.7 � 0.5a 12.6 � 1.0b 67.9 � 0.3a 73.5 � 0.3a 80.3 � 0.1b 12.3 � 0.3b 13.6 � 0.5aT. bispinosa 19.5 � 0.8ab 10.9 � 1.0a 67.9 � 0.2a 73.4 � 0.4a 80.4 � 0.2b 12.5 � 0.4b 13.5 � 0.4aT. pseudoinisa 20.0 � 0.5b 15.5 � 0.5c 69.9 � 0.1b 73.7 � 0.2a 79.3 � 0.1a 9.4 � 0.1a 13.9 � 0.1a

a Data were means � standard deviations, n ¼ 3. Values in the same column with different letters were significantly different (P < 0.05).b To: onset temperature; Tp: peak temperature; Tc: conclusion temperature; DT: gelatinization range (Tc � To); DH: enthalpy of gelatinization.

H. Gao et al. / Food Hydrocolloids 37 (2014) 174e181 179

Trapa species at 95 �C are shown in Table 3. T. pseudoinisa starchshowed the highest swelling power and solubility.

3.7. Thermal properties of starch

DSC measures and records the amount of heat involved in thestarch gelatinization. Fig. 4A presents the gelatinization thermo-grams of starches from three Trapa species, and their thermal pa-rameters are summarized in Table 3. T. quadrispinosa andT. bispinosa starches showed similar thermal properties.T. pseudoinisa starch had significantly higher gelatinization onsettemperature, lower gelatinization conclusion temperature andnarrower gelatinization range compared with T. quadrispinosa andT. bispinosa starches. Starches from three Trapa species showedsimilar gelatinization peak temperature and enthalpy. The gelati-nization properties of starch are related to a variety of factorsincluding the granule size, amylose content, proportion and kind ofcrystalline organization, and ultrastructure of the starch granules(Lindeboom, Chang, & Tyler, 2004).

3.8. Pasting properties of starch

Pasting properties of starches from three Trapa speciesmeasured by using the RVA are presented in Fig. 4B, and theirpasting parameters are given in Table 4. The peak, hot and finalviscosities of starches from three Trapa species were significantlydifferent. Peak and hot viscosity was highest in T. bispinosa starchfollowed by T. pseudoinisa and T. quadrispinosa starches. Final vis-cosity was observed to be the highest in T. pseudoinisa starch but

Fig. 4. DSC thermograms (A) and

Table 4Pasting properties of starches from three Trapa species.a

Species PV (mPa s)b HV (mPa s)b BV (mPa s)b

T. quadrispinosa 3038.3 � 12.5a 2027.3 � 9.3a 1011.0 � 4.4aT. bispinosa 3337.7 � 15.5c 2368.7 � 14.6c 969.0 � 30.1aT. pseudoinisa 3251.7 � 14.5b 2183.7 � 47.4b 1068.0 � 60.1a

a Data were means � standard deviations, n ¼ 3. Values in the same column with diffb PV, peak viscosity; HV, hot viscosity; BV, breakdown viscosity (PV � HV); FV, final vi

the lowest in T. quadrispinosa starch. T. quadrispinosa andT. bispinosa starches showed the similar setback viscosity and peaktime. The breakdown viscosity and pasting temperature did notshow significant difference among three Trapa species. The similarbreakdown viscosity of starches from three Trapa species suggestedthat they had similar resistance to heat and mechanical shear andsimilar prone to loss viscosity upon holding and shearing. Thesetback is the viscosity increase resulting from the rearrangementof amylose molecules that have leached from swollen starchgranules during cooling, and is generally used as a measure of thegelling ability or retrogradation tendency of starch (Karim, Norziah,& Seow, 2000). The pasting properties of starches have been re-ported to be influenced by size, rigidity, amylose to amylopectinratio, and swelling power of the granules (Singh, Kaur, Ezekiel, &Guraya, 2005).

3.9. Hydrolysis degree of starch

The hydrolysis degrees of starches from three Trapa species byHCl, AAG and PPA are presented in Fig. 5. The hydrolysis degreeincreased gradually with increasing hydrolysis time. Starches fromthree Trapa species showed similar hydrolysis degree by PPA.However, T. bispinosa starch showed the highest hydrolysis degreeand T. pseudoinisa starch showed the lowest hydrolysis degree byHCl and AAG.

Most uses of starch in food and nonfood (such as pharmaceutics,papers, adhesives, packaging, biofuels, etc.) applications require thedisruption of starch granules through acid, alkaline, enzyme, orhydrothermal treatments (gelatinization/melting) (Buléon &

RVA patterns (B) of starches.

FV (mPa s)b SV (mPa s)b PT (min)b PTemp (�C)b

3232.7 � 4.7a 1205.3 � 8.3a 4.3 � 0.0b 77.4 � 0.0a3538.7 � 19.3b 1170.0 � 33.9a 4.3 � 0.0b 77.1 � 0.5a3683.0 � 12.3c 1499.3 � 52.7b 4.1 � 0.0a 77.25 � 0.4a

erent letters were significantly different (P < 0.05).scosity; SV, setback viscosity (FV e HV); PT, peak time; PTemp, pasting temperature.

Fig. 5. Hydrolysis degrees of starches by HCl (A), AAG (B) and PPA (C).

H. Gao et al. / Food Hydrocolloids 37 (2014) 174e181180

Colonna, 2007; Tawil, Viksø-Nielsen, Rolland-Sabaté, Colonna, &Buléon, 2011). Acid modification is widely used in the starch in-dustry to produce thin boiling starches for use in food, paper,textile, and other industries (Rohwer & Klem, 1984). Enzyme hy-drolysis of starch is involved in many biological and industrialprocesses, such as starch metabolism in plants, digestion bymammals, malting, fermentation, glucose syrup, or bioethanolproduction. Starch is specifically hydrolyzed by amylolytic en-zymes, which can cut either one or both types of glycosidic bonds.Among these enzymes, a-amylase is the main enzyme involved inthe hydrolysis of a-1, 4-bonds (Tawil et al., 2011). The amyloglu-cosidase catalyses the hydrolysis of both a-1,4 and a-1,6 glycosidicbonds at the branching point to release b-D-glucose residues of thepolymer substrate (van der Maarel, van der Veen, Uitdehaag,Leemhuis, & Dijkhuizen, 2002). Susceptibility of starch to acidand enzyme hydrolysis is influenced by factors such as amylose toamylopectin ratio, crystalline structure, particle size and relativesurface area, granule integrity, porosity of granules, and structuralheterogeneities (Blazek & Gilbert, 2010). It is reported that theamount of native starch hydrolysis by acid or amylase is inversely

related to the amylose content (Li et al., 2004; Li, Vasanthan,Rossnagel, & Hoover, 2001). Amylase hydrolysis involves anenzyme in solution acting on a solid starch substrate. Thus, thesurface area accessible to enzyme and the efficiency of adsorptionof enzyme onto this surface are critical kinetic parameters. Largergranule has a lower ratio of surface area to volume, so it has a lowerrate of amylase hydrolysis than small granule (Bertoft & Manelius,1992). Therefore, compared with T. quadrispinosa and T. bispinosastarches, larger granule size and higher amylose content ofT. pseudoinisa starch led it to be more resistant to acid and enzymehydrolysis (Table 2, Fig. 5).

4. Conclusion

T. quadrispinosa and T. pseudoinisa had the highest and thelowest weight of fruit and kernel, respectively. Fresh kernel ofT. bispinosa had the highest water content and the lowest starchcontent, and that of T. pseudoinisa had the lowest water contentand the highest starch content. Dry kernel of T. quadrispinosa andT. pseudoinisa had the highest starch content and soluble sugarcontent, respectively. Starches from three Trapa species allexhibited central hilum granules and CA-type crystalline struc-ture. T. pseudoinisa starch had larger granule size thanT. quadrispinosa and T. bispinosa starches. T. quadrispinosa starchhad the lowest amylose content. T. pseudoinisa starch had thehighest amylose content, swelling power and solubility.T. pseudoinisa starch had lower gelatinization conclusion tem-perature and higher gelatinization onset temperature. The hy-drolysis degrees of T. pseudoinisa starch by HCl and AAG werelower than that of the others.

Acknowledgments

This studywas financially supported by grants from the NationalNatural Science Foundation of China (31170299 and 31370355), theTalent Project of Yangzhou University and the Priority AcademicProgram Development from Jiangsu Government, China.

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