GUO Ze-Bin CHEN Bing-Yan LU Xu

ZENG Shao-Xiaoa, b, c ZHENG Bao-Donga, b, c②

a (College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China)

b (Institute of Food Science and Technology,Fujian Agriculture and Forestry University, Fuzhou 350002, China)

c (Fujian Provincial Key Laboratory of Quality Science and Processing Technology in Special Starch, Fuzhou 350002, China)

1 INTRODUCTION

Starch, which is widely used as a functional ingredient in processed foods, is a significant source of energy in human diets. Modified starches continue to outplace the unmodified (native)starches in the processed food industry due to their desirable functional properties[1]. Lotus (Nelumbo nucifera,Gaertn.)is an important plant that has been widely cultivated in China, India, Thailand, Japan, and Australia[2,3]. Lotus seed is rich in starch[4,5], which is used in the production of traditional confectionery products and food additives[6]. However,native starch has several limitations: low solubility in cold water, poor emulsification capability, easy retrogradation, and poor stability under refrigerated and processing conditions[7,8].

Ultra-high pressure (UHP)is a non-thermal process used in starch modification. Studies have reported that UHP-treated starch exhibits unique gelatinization and retrogradation properties compared to heat-gelatinized starch. Compared to heat-gelatinized starch, UHP-treated starch has intact or partially disintegrated starch granules, lower levels of leached amylose, and lower enzymatic reactivity[9-11]. The extent of gelatinization and retrogradation by UHP are highly dependent on the plant source, pressure, UHP time, starch concentration,and temperature[12,13]. Understanding the relationship between the functional and molecular properties of starch, e.g., structure and weight-averaged molecular weight (Mw), has long been a goal of food scientists[14].

Currently, there is limited information on the molecular weight distribution of UHP-treated starch and on the effect of UHP on the pasting and thermal properties of lotus seed starch. The objective of this study was to assess the effect of UHP on the structural, pasting, and thermal properties of lotus seed starch.

2 EXPERIMENTAL

2. 1 Materials and instruments

Raw lotus-seed: Green Field Fujian Food Co.,Ltd, Fujian, China; All reagents and solvents were commercially available and used without further purification. Ultra high pressure instruments: 5 L-HPP-600MPa, KeFa High Pressure Technology Co., Ltd, Baotou, China; Solid-state13C CP/MAS NMR spectrometer: AVANCE III 500, Bruker Co.,Ltd., Switzerland; HPSEC-MALLS-RI system:Wyatt Technologies, Santa Barbara, CA; Rapid viscoTManalyzer: TechMaster, Newport Scientific Pty Ltd, Australia; Differential scanning calorimeter:DSC 200 F3 Maia?, Netzsch-Ger?tebau GmbH,Germany.

2. 2 Preparation of lotus seed starch

Lotus seed starch was isolated according to the method reported by Zeng[15]. Fresh peeled raw lotus was homogenized and filtered through a 100 mesh sieve. The filtrate was collected and allowed to settle for 6 h at 4 ℃. The resulting supernatant was discarded and the sediment was washed two times with distilled water. The sediment was dried at 45 ℃ for 24 h, passed through an 80-mesh sieve,and stored in a desiccator at room temperature.

2. 3 UHP treatment of lotus seeds starch

In this experiment, 15% (w/w)of a starch-water suspension was vacuum-packaged (?100 kPa)in a polypropylene bag and subjected to 500 MPa for 10,20, 30, 40, 50, or 60 min. The UHP system was pressurized at 1 MPa/s. The UHP-treated sample was filtered, dried at 45 ℃ for 24 h, filtered through an 80 mesh sieve, and stored in a desiccator at room temperature.

2. 4 Experimental method

2. 4. 113C CP/MAS NMR spectroscopy

Solid-state13C CP/MAS NMR experiments were performed in a Bruker spectrometer with a MAS VTN 4-mm probe head. Solid-state cross-polarization/magic angle spinning (CP/MAS)spectra were obtained at a 125.7 MHz13C frequency and a 40 kHz spectral width. Other parameters consisted of an acquisition time of 50 ms, a time domain of 2 K,a transformation size of 4 K, and a line broadening of 50 Hz. A minimum of 2,400 scans was performed for each spectrum[16]. The relative degree of crystallinity was quantitatively estimated with Peakfit v4.12[17,18]using the following equation

CR= SF/ST×100%

where CRis the relative degree of crystallinity, SFis the fitting peak area of C1, and STis the total peak area of C1.

2. 4. 2 Molecular weight distribution

High performance size exclusion chromatography(HPSEC)equipped with multi-angle laser light scattering (MALLS)and refractive index (RI)detectors were used to determine the molecular weight distributions of starch. The mobile phase was DMSO with LiBr (50 mmol/L)filtered through a 0.22 μm PTFE filter and degassed by ultrasound treatment. For SEC analysis, 12.5 mg of isolated starch was dispersed in 50 mM LiBr in DMSO at 90 ℃ for 2 h during constant stirring for 24 h at room temperature[19]. Dispersed samples were centrifuged at 13,500 × g for 15 min; the resulting supernatants were analyzed by HPSEC-MALLS-RI.Diluted starch dispersions (0.02～0.2 mg/mL)were injected with a manual injector (1 mL injection loop)directly into MALLS at 0.3 mL/min. The columns consisted of Ohpak SB-G (guard column)and Ohpak SB-806 HQ, which were maintained at 35 ℃.A wavelength of 623.8 nm was used in the experiment. Mwwere calculated using the Astra V software according to the Zimm model. A secondorder Berry method was used for curve fitting. Mwcalculations were based on a mobile phase refracttive index of 1.4785 and a dn/dc value of 0.066.

2. 4. 3 Pasting properties

The pasting properties of starch were measured in a Rapid ViscoTMAnalyzer. The test method was followed by STD 1 profile (AACC method 76-21)[20]. Briefly, deionized water (25 mL)was added to starch (3.0 g, dry basis)in the RVA canister.The starch slurry was equilibrated at 50 ℃ for 1 min, heated from 50 to 95 ℃ at 12 ℃/min, held at 95 ℃ for 2.5 min, and cooled to 50 ℃ at 12 ℃/min.The speed was 960 rpm for the first 10 s and subsequently held at 160 rpm. Peak viscosity,trough viscosity, breakdown, final viscosity, setback,peak time, and pasting temperature were determined from the pasting curve. Each test was performed in triplicate.

2. 4. 4 Differential scanning calorimetry (DSC)T he thermal properties of starch were measured by DSC. In this experiment, 3.0 mg starch (dry basis)was weighed in an aluminum DSC pan and 10 μL distilled water was added. The pan was sealed, equilibrated for 1 h at room temperature, and heated from 25 to 120 ℃ in the DSC at 10 ℃/min.An empty stainless steel pan was used as reference;every measurement was performed in triplicate.Onset temperature (To), peak temperature (Tp), conclusion temperature (Tc), and enthalpy of gelatinization (ΔH)were calculated by Netzch Proteus 6.

2. 4. 5 Statistical analyses

Statistical analysis was performed using Origin-Pro 8.1, PASW Statistics 18 Statistical Analysis Software was used to transform the experimental data. Experimental data for starch characteristics and properties were analyzed by one-way analysis of variance (ANOVA). Duncan’s multiple range test was performed to assess significant differences among the experimental mean values (p ＜ 0.05).

3 RESULTS AND DISCUSSION

3. 1 13C CP/MAS NMR spectroscopy

The13C CP/MAS NMR spectra of native lotus seed starch and UHP-treated starch are shown in Fig. 1.The13C chemical shift values of each major peak are summarized in Table 1. Baik and Gidley[21,22]reported that the carbon chemical shift values of starch are at 94～105 ppm for the C(1)sites, 68～78 ppm for the C(2), C(3), and C(5)sites, 80～84 ppm for the C(4)sites, and 58～65 ppm for the C(6)sites. The signals of C(1)sites (99～102 ppm)in the NMR spectra contain information related to the crystalline and amorphous structures of starch[23].

The native lotus seed starch had multiplicity in the resonance peak of the C(1)site, with characteristics of both A- and B-type crystal structures(Fig. 1 and Table 1). Both the intensity of the major peaks and the relative crystallinity of the crystalline state gradually decreased with increasing the UHP time, suggesting that the amorphous content of the starch granules substantially increased. These results are consistent with those of a previous study[24].

Fig. 1. 13C CP/MPS NMR spectra of UHP-treated starch

Table 1. Chemical Shifts and Relative Crystallinity of Native and UHP-treated Starch

3. 2 Molecular weight distribution

The weight molar mass (Mw), number molar mass(Mn), and polydispersity index (Mw/Mn)values of native and UHP-treated lotus seed starch are shown in Table 2. Mwand Mndecreased with increasing the UHP time, suggesting that lotus seed starch was slightly degraded during UHP and formed molecular chains with a low degree of polymerization. The Mw/Mnratios of native and UHP-treated starch are 1.282, 1.208, 1.195, 1.188, 1.176, 1.167, and 1.154.The larger the value of the polydispersity index, the wider the molecular weight distribution[25].

Table 2. Molecular Weights of Native and UHP-treated Lotus Seed Starch

3. 3 Pasting properties

The viscograms of native and UHP-treated starch are shown in Fig. 2. The pasting parameters including peak viscosity (PV), trough viscosity (TV),breakdown (BD), final viscosity (FV), setback (SB),pasting temperature (PT), and peak time (PT/℃)are summarized in Table 3. Compared with native lotus seed starch, UHP-treated starch exhibited a significant (p ＜ 0.05)increase in PV, TV, FV, and PT values and a reduction in BD and SB values. BD represents the stability and resistance of starch granules to shear stress and SB reflects the rapid retrogradation of leached amylose in starch paste.Therefore, UHP-treated lotus seed starch had lower BD and SB values than the native starch, reflecting stronger starch aggregations and a lower retrogradation tendency compared to the native starch[26].These results in UHP-treated starch can be attributed to changes in starch granular structures during the transformation of the crystalline structure[27].

Fig. 2. Pasting viscosity graph of native and UHP-treated lotus seed starch

Table 3. Pasting Properties of UHP-treated Lotus Seed Starcha, b

3. 4 Differential scanning calorimetry (DSC)

The effect of UHP treatment on To (onset temperature), Tp (peak temperature), Tc (conclusion temperature), ΔT = Tc ? To (gelatinization temperature range), and ΔH (enthalpy of gelatinization)of lotus seed starch are summarized in Table 4.UHP-treated starch had a lower gelatinization temperature than native starch, which indicated that gelatinization occurred, resulting in a partial denaturation and loss of the molecular order and crystalline structure during UHP treatment. It has been reported that ΔH reflects mainly the loss of the double helical order of amylopectin crystallites[28].As shown in Table 4, ΔH decreased with increasing the pressure, indicating that UHP treatment disrupted the crystalline structure of starch granules.Therefore, native lotus seed starch samples require more energy to break the intermolecular bonds, e.g.,hydrogen bonds between water molecules of starch granules and starch crystallites[29], compared to the UHP-treated starch.

Table 4. Thermal Properties of UHP-treated Lotus Seed Starcha, b

4 CONCLUSION

The effects of UHP treatment on the structural,pasting, and thermal properties of lotus seed starch were studied. The solid-state13C CP/MAS NMR results revealed that native lotus seed starch had characteristics of C-type crystal structures and that the relative crystallinity in the crystalline state gradually decreased with increasing the UHP time.HPSEC-MALLS-RI results showed that the molecular weight (Mw)of native starch was 1.433 × 107Da; UHP-treated starch exhibited lower Mw, Mn,and polydispersity index values compared to the native starch. Pasting properties of UHP-treated starch granules changed, depending on the starch structure. The viscosity results showed that UHP-treated lotus seed starch had lower BD and SB values than the native starch, reflecting a lower retrogradation tendency. Gelatinization temperature(To, Tp, and Tc)and gelatinization enthalpy (ΔH)decreased with increasing the UHP time, indicating that UHP induced starch gelatinization.

(1)Sajilata M. G.; Singhal, R. S. Specialty starches for snack foods. Carbohydrate Polymers 2005, 59, 131–151.

(2)Kim, M. J.; Shin, H. S. Antioxidative effect of lotus seed and seedpod extracts. Food Science and Biotechnology 2012, 21, 1761–1766.

(3)Bhat, R.; Sridhar, K. R. Nutritional quality evaluation of electron beam-irradiated lotus (Nelumbo nucifera)seeds.Food Chemistry 2008, 107, 174–184.

(4)Xu, S.; Shoemaker, C. F. Gelatinization properties of Chinese water chestnut starch and lotus root starch. Journal of Food Science 1986, 51, 445–449.

(5)Zeng, S. X.; Zheng, B. D.; Lin, Y. Y.; Zhou, X. H. Granular characteristics of lotus-seed starch.Journal of the Chinese Cereals and Oils Association 2009, 62–64.

(6)Zhong, G.; Chen, Z. D.; We, Y. M. Physicochemical properties of lotus (Nelumbo nucifera Gaertn.)and kudzu (Pueraria hirsute Matsum.)starches.International Journal of Food Science & Technology 2007, 42, 1449–1455.

(7)Whistler, R. L.; Bemiller, J. N.; Paschall, E. F. Starch: chemistry and technology. Orlando: Academic Press 1984.

(8)Singh, J.; Kaur, L.; Mccarthy, O. J. Factors influencing the physico-chemical, morphological, thermal and rheological properties of some chemically modified starches for food applications—A review. Food Hydrocolloids 2007, 21, 1–22.

(9)Douzals, J. P.; Cornet, J.; Gervais, P.; Coquille, J. C. High-pressure gelatinization of wheat starch and properties of pressure-induced gels. Journal of Agricultural and Food Chemistry 1998, 46, 4824–4829.

(10)Stolt, M.; Oinonen, S.; Autio, K. Effect of high pressure on the physical properties of barley starch. Innovative Food Science & Emerging Technologies 2001, 1, 167–175.

(11)Stute, R.; Heilbronn; Klingler, R. W.; Boguslawski, S.; Eshtiaghi, M. N.; Knorr, D. Effects of high pressures treatment on starches. Starch 1996, 48, 399–408.

(12)Kawai, K.; Fukami, K.; Yamamoto, K. State diagram of potato starch–water mixtures treated with high hydrostatic pressure. Carbohydrate Polymers 2007, 67, 530–535.

(13)Oh, H. E.; Pinder, D. N.; Hemar, Y.; Anema, S. G.; Wong, M. Effect of high-pressure treatment on various starch-in-water suspensions. Food Hydrocolloids 2008, 22, 150–155.

(14)Zhong, F.; Yokoyama, W.; Wang, Q.; Shoemaker, C. F. Rice starch, amylopectin, and amylose:molecular weight and solubility in dimethyl sulfoxide-based solvents. Journal of Agricultural and Food Chemistry 2006, 54, 2320–2326.

(15)Zeng, S. X. Studies on qualitative characteristics of lotus-seed (Nelumbo nucifera Gaertn)starch and its application.Fuzhou: Fujian Agriculture and Forestry University 2007.

(16)Tan, I.; Flanagan, B. M.; Halley, P. J.; Whittaker, A. K.; Gidley, M. J. A method for estimating the nature and relative proportions of amorphous,single, and double-helical components in starch granules by13C CP/MAS NMR. Biomacromolecules 2007, 8, 885–891.

(17)Atichokudomchai, N.; Varavinit, S.; Chinachoti, P. A study of ordered structure in acid-modified tapioca starch by13C CP/MAS solid-state NMR.Carbohydrate Polymers 2004, 58, 383–389.

(18)Lin, J.; Wang, S.; Chang, Y. Effect of molecular size on gelatinization thermal properties before and after annealing of rice starch with different amylose contents. Food Hydrocolloids 2008, 22, 156–163.

(19)Pu, H. Y.; Chen, L.; Li, L.; Li, X. X. Multi-scale structural and digestion resistibility changes of high-amylose corn starch after hydrothermal-pressure treatment at different gelatinizing temperatures. Food Research International 2013, 53, 456–463.

(20)AACC. General pasting method for wheat or rye flour or starch using the rapid visco analyzer.St Paul, MN:American Association of Cereal Chemists 1999.

(21)Baik, M.; Dickinson, L. C.; Chinachoti, P. Solid-state13C CP/MAS NMR studies on aging of starch in white bread. Journal of Agricultural and Food Chemistry 2003, 51, 1242–1248.

(22)Gidley, M. J.; Bociek, S. M. Molecular organization in starches: a carbon13CP/MAS NMR study.Journal of the American Chemical Society 1985, 107, 7040–7044.

(23)Delval, F.; Crini, G.; Bertini, S.; Morin-Crini, N.; Badot, P. M.; Vebrel, J.; Torri, G. Characterization of cross-linked starch materials with spectroscopic techniques. Journal of Applied Polymer Science 2004, 93, 2650–2663.

(24)Guo, Z. B.; Liu, W. T.; Zeng, S. X.; Zheng, B. D. Effect of ultra high pressure processing on the particle characteristics of lotus-seed starch. Chin. J.of Struct. Chem. 2013, 32, 525–532.

(25)Yoo, S.; Jane, J. Molecular weights and gyration radii of amylopectins determined by high-performance size-exclusion chromatography equipped with multi-angle laser-light scattering and refractive index detectors. Carbohydrate Polymers 2002, 49, 307–314.

(26)Hu, X. T.; Xu, X. M.; Jin, Z. Y.; Tian, Y. Q.; Bai, Y. X.; Xie, Z. J. Retrogradation properties of rice starch gelatinized by heat and high hydrostatic pressure (HHP). Journal of Food Engineering 2011, 106, 262–266.

(27)Katopo, H.; Song, Y.; Jane, J. Effect and mechanism of ultrahigh hydrostatic pressure on the structure and properties of starches. Carbohydrate Polymers 2002, 47, 233–244.

(28)Mcpherson, A. E.; Jane, J. Comparison of waxy potato with other root and tuber starches. Carbohydrate Polymers 1999, 40, 57–70.

(29)Cooke, D.; Gidley, M. J. Loss of crystalline and molecular order during starch gelatinization: origin of the enthalpic transition. Carbohydrate Research 1992, 227, 103–112.