Preparation and Characterization of Debranched-Starch

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Preparation and Characterization of Debranched-Starch/ Phosphatidylcholine Inclusion Complexes Weiwei Cheng, Zhigang Luo,* Lin Li, and Xiong Fu* Carbohydrate Lab, College of Light Industry & Food Sciences, South China University of Technology, Guangzhou 510640, China ABSTRACT: In this study, debranched-starch/phosphatidylcholine inclusion complexes were prepared. The effect of reaction parameters such as reaction temperature, reaction time, and addition amount of phosphatidylcholine on the phosphatidylcholine payload and inclusion rate was investigated. The phosphatidylcholine payload and inclusion rate prepared under the optimal conditions were 106 mg/g and 84.8%, respectively. The formation of debranched-starch/phosphatidylcholine inclusion complexes was confirmed by the results of XRD and FT-IR. Furthermore, the molecular, cluster, and fractal structures of the complexes were investigated using 13C CP/MAS NMR and SAXS. The results indicated that the inclusion complexes were formed by hydrophobic interactions between alkyl chain of phosphatidylcholine and debranched-starch helix cavity. The complexes possessed a mass fractal structure, and a semicrystalline structure with a Bragg distance of 19.04 nm formed. After complexation, the stability of phosphatidylcholine was significantly improved, and phosphatidylcholine of the complexes can be gradually released with pancreatin treatment. This study revealed that debranched-starch can be used as an effective carrier of phosphatidylcholine for the purpose of improving its stability. KEYWORDS: debranched-starch, phosphatidylcholine, complexes, preparation

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alcohol precipitation.14 However, amylose obtained by these methods is often costly because of the complicated production process and the consumption of organic compounds such as alcohol, n-butyl, and pentanol. In this study, to reduce the cost and achieve industrialization, amylose was obtained by hydrolysis of native potato starch with pullulanase, and the obtained debranched-starch (amylose) was used for the first time as wall material to encapsulate phosphatidylcholine by its single helical to improve the stability of phosphatidylcholine. The effect of reaction temperature, reaction time, and addition amount of phosphatidylcholine on the phosphatidylcholine payload and inclusion rate was investigated. In addition, the obtained debranched-starch/ phosphatidylcholine complexes were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), 13C cross-polarization/magic angle spinning nuclear magnetic resonance (13C CP/MAS NMR), small-angle X-ray scattering (SAXS), and scanning electron microscopy (SEM), and the stability and digestibility properties of the complexes were also studied.

INTRODUCTION Phosphatidylcholine is a naturally occurring mixture with diverse fatty acid side chains such as stearic, oleic, and palmitic acids.1,2 It is the best known kind of phospholipids.2 Because of its outstanding physiological functionalities, such as slowing aging, enhancing memory, reducing blood fat, and preventing diabetes, and its surface activity, phosphatidylcholine has been widely used in food, pharmaceutical, and cosmetic industries.3−5 However, due to its rich content of unsaturated fatty acids, phosphatidylcholine is sensitive to heat and light, and very easy to oxidize during manufacture, storage, and consumption, which restricts its applications in industries. Up to now, there are few reports on improving the stability of phosphatidylcholine. Amylose, one of the two main fractions of starch, is a predominantly linear polymer, which is composed of α-Dglucose mainly linked by α-1,4 linkages. In the presence of ligands such as alcohols,6 aroma compounds,7 and lipids,8 amylose can undergo a conformation change resulting in a single, left-handed helix that has a hydrophilic surface and hydrophobic inside helical channel. Hence, amylose can be used as a wall material to encapsulate hydrophobic guest molecules to form inclusion complexes, and meanwhile amylose single helices were arranged to form a V-type crystalline structure known as V-amylose.9 Although the ability of amylose to form inclusion complexes with a wide variety of molecules has been known for several decades, only recently has amylose been systematically studied as a possible vehicle for bioactive compounds.10−12 In view of the degradability and availability of amylose, the application of amylose as a foodgrade delivery material has become a research hotspot in recent years.13 Conventionally, amylose is obtained by separation of the two components of starch, mainly by methods of aqueous dispersion, aqueous leaching, selective retrogradation, or © 2015 American Chemical Society

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MATERIALS AND METHODS

Materials. Commercial food grade potato starch (amylose content 19.42%) was obtained from Qinghai Weston Co. Ltd. (Qinghai, China). Pullulanase (E.C. 3.2.1.41., activity 1000 ASPU/g) was supplied by Genencor (China) Bio-Products Co. Ltd. (Wuxi, China). One unit of pullulanase activity was defined as the amount of enzyme that liberates one glucose reducing equivalent from pullulan per minute at pH 4.5 at 60 °C. Phosphatidylcholine (phosphatidylcholine >95%, Epikuron 200) was obtained from Lucas Meyer (Hamburg, Received: Revised: Accepted: Published: 634

August 28, 2014 January 3, 2015 January 4, 2015 January 4, 2015 DOI: 10.1021/jf504133c J. Agric. Food Chem. 2015, 63, 634−641

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Journal of Agricultural and Food Chemistry Germany) and used without further purification. Pancreatin (8 × USP, from porcine pancreas) was purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals used were of analytical grade. Preparation of Debranched-Starch. Starch (50 g, dry basis) was mixed with 450 g of water in a 500 mL three necked flask. The slurry was adjusted to pH 4.5 with 0.5 mol/L HCl and gelatinized in a thermostatic water bath at 98 °C with stirring for 1 h. After gelatinization, the mixture was cooled to 60 °C and the debranching reaction was started by adding pullulanase (10 ASPU/g). To completely debranch starch, the solution was incubated at 60 °C, pH 4.5 for 4 h. The thoroughness of the debranched reaction was confirmed by 1H NMR, which can distinguish α-1,6 from α-1,4 linkages of starch according to chemical shifts of 4.7−5.0 ppm for α1,6 linkages and 5.1−5.4 ppm for α-1,4 linkages.15,16 According to previous literature,15,16 when the α-1,6 peak located at 4.7−5.0 ppm disappears altogether, all of the α-1,6 branch linkages are suggested to be completely hydrolyzed. In this study, when potato starch was hydrolyzed by pullulanase at the above reaction conditions, the α-1,6 peak located at 4.7−5.0 ppm disappears altogether, indicating that potato starch has been completely debranched. The hydrolysis percent and blue value of the debranched-starch were determined using the methods of Bruner17 and Gilbert and Spragg,18 respectively. Hydrolysis rate and blue value of the debranched-starch made under the above conditions were 15.11% and 1.12, respectively. The debranched-starch solution obtained was stored at 60 °C for further use. Preparation of Debranched-Starch/Phosphatidylcholine Inclusion Complexes. Different amounts of tetrahydrofuran (10, 15, 20, 25, 30 g) containing 20 wt % phosphatidylcholine were added to the debranched-starch solutions, and after reacting in a thermostatic water bath at a certain temperature (40, 50, 60, 70, 80 °C) for 0.5, 1, 2, 3, and 4 h, respectively, the mixture was slowly cooled to ambient temperature and then stored at 4 °C for 18 h. Samples were centrifuged, washed to remove all tetrahydrofuran with deionized water, and dried in an oven at 40 °C overnight. The recovery of debranched-starch/phosphatidylcholine inclusion complexes is in the range of 35−40 g. The products prepared under the optimal conditions were used for further analysis. As control, a debranchedstarch solution sample without phosphatidylcholine was slowly cooled to ambient temperature and then stored at 4 °C for 18 h. The mixture was centrifuged, washed with deionized water, and dried in an oven at 40 °C overnight. The obtained uncomplexed retrograded debranchedstarch (URDS) product was used for further analysis. Determination of the Phosphatidylcholine Payload and Inclusion Rate. The determination of the phosphatidylcholine payload and inclusion rate was done by phosphous molybdenum blue spectrophotometric method.19 Debranched-starch/phosphatidylcholine complexes (0.5 g) and URDS (0.5 g) were accurately weighed and mineralized by HNO3 and HClO4, respectively. The mineralized liquid was transferred to a 50 mL flask and diluted with distilled water to volume. After being diluted to an appropriate concentration, the phosphous content of the complexes was determined by the phosphous molybdenum blue spectrophotometric method with URDS as blank, and the phosphatidylcholine payload was obtained by multiplying the phosphous content by a specific conversion coefficient. The phosphatidylcholine payload (X, mg/g) was calculated by the following equation:

X = (C × N × V1 × 25)/m0

where m1 (g) is the weight of the debranched-starch/phosphatidylcholine complexes obtained, X (mg/g) is the phosphatidylcholine payload of the sample, and m2 (g) is the weight of phosphatidylcholine added. Characterization of Debranched-Starch/Phosphatidylcholine Complexes. XRD analysis was performed on a D8 ADVANCE X-ray diffractometer (Bruker, Karlsruhe, Germany) operating at 40 mA and 40 kV by using Cu Kα radiation (λ = 0.1542 nm). The scattering angle (2θ) was varied from 5° to 60° with a scanning step of 0.04°. FT-IR spectra of samples were acquired on a VECTOR 33 FT-IR spectrophotometer (Bruker, Frankfurt, Germany) using KBr disk technique. The samples were mixed with anhydrous KBr and then compressed into thin disk-shaped pellets. The spectra were obtained with a resolution of 2 cm−1 in a wavenumber range of 400−4000 cm−1. 13 C CP/MAS NMR experiments were performed on a Bruker AVANCE AV 400 spectrometer (Bruker, Rheinstetten, Germany) equipped with a 4 mm MAS probe. Samples (ca. 100 mg) were packed in 4 mm zirconia rotor and spun at 6000 r/min. The spectra were obtained at ambient temperature with a sampling time of 17 ms and a relaxation delay time of 2 s. All chemical shifts were reported in parts per million (ppm). SAXS measurements were performed using a SAXSess small-angle X-ray scattering system (Anton Paar, Graz, Austria), operated at an acceleration voltage of 40 kV and current of 50 mA. A Cu Kα radiation with a wavelength of 0.1542 nm was used as X-ray source. Prior to analysis, all samples used for the experiments were prepared by mixing the starch with distilled water and equilibrated at ambient temperature for 24 h; the total moisture content of each sample was 60%. Each sample then was filled into a capillary of 0.01 mm wall thickness, the sample-to-detecter distance was 261.2 mm, and the temperature was kept at 26 °C during the measurement. The data were collected by the IP reader softwware and read out by a Perkin-Elmer storage phosphor system. The background and smeared intensity were removed by applying the data to the SAXSquant 3.0 software. The average repeat distance (i.e., thickness of semicrystalline lamellae) of the amorphous and crystalline lamellar of each sample can be calculated as d = 2π /q

where d (nm) is the lamellar repeat distance and q (nm−1) is the scattering vector;20 the relationship between q and θ can be calculated as q = (4π sin θ )/λ

(4)

where λ (nm) is the wavelength of the X-ray source and 2θ is the scattering angle. SEM observations were performed using a model 1530VP scanning electron microscope (LEO, Oberkochen, Germany). The accelerating voltage was 20 kV. The samples were mounted on an aluminum stub with a double sticky tape, and coated with gold in a vacuum before examination. Stability of the debranched-starch/phosphatidylcholine inclusion complexes and URDS/phosphatidylcholine physical mixture (phosphatidylcholine payload106 mg/g) was evaluated by measuring peroxide value (POV) after the samples had been stored at 50 °C for 0, 3, 7, 13, and 20 days. The measurement of POV was according to ISO 3960:2001. Digestion tests in simulated stomach condition and by enzymatic hydrolysis were performed according to previous literature12 with little modification. The release of phosphatidylcholine from the complexes in simulated stomach conditions was tested by incubating the debranched-starch/phosphatidylcholine complexes (30 mg) with 2 mL of HCl (pH = 2) for 2 h at 37 °C under continuous stirring. The release of phosphatidylcholine from the complexes in simulated small intestine conditions was tested by incubating the debranched-starch/ phosphatidylcholine complexes (30 mg) with 2 mL of pancreatin solutions for 24 h at 37 °C. The amount of phosphatidylcholine released following the simulated stomach and simulated small intestine conditions was measured by extracting the reaction substrate (after freeze-drying) with n-hexane, and the phosphatidylcholine payload was

(1)

where C (mg/mL) is the phosphous content of the mineralized liquid (after being diluted) determined from the phosphous standard curve, N is the dilution ratio of the mineralized liquid, V1 (mL) is the total volume of the mineralized liquid, 25 is the conversion coefficient from phosphous to phosphatidylcholine, and m0 (g) is the amount of the sample weighed. The inclusion rate (Y) was calculated by the following equation:

Y = (m1 × X × 100%)/(m2 × 1000)

(3)

(2) 635

DOI: 10.1021/jf504133c J. Agric. Food Chem. 2015, 63, 634−641

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Journal of Agricultural and Food Chemistry

Figure 1. Effect of reaction parameters on soybean lecithin payload and inclusion rate. (a) Effect of reaction temperature (reaction time, 1 h; addition amount of soybean lecithin solution, 25 g). (b) Effect of reaction time (reaction temperature, 60 °C; addition amount of soybean lecithin solution, 25 g). (c) Effect of addition amount of soybean lecithin solution (reaction temperature, 60 °C; reaction time, 2 h). Values in the same curve with different superscript letters are significantly different (P < 0.05). measured by ultraviolet spectrophotometry according to our previous method.4 The release rate (R) of phosphatidylcholine was calculated as

R = (m1/m0) × 100%

Effect of Reaction Time. As presented in Figure 1b, at the starting hours, the phosphatidylcholine payload and inclusion rate increased steadily with time increasing. However, when the reaction lasted for more than 2 h, an apparent reduction of phosphatidylcholine and inclusion rate was observed (P < 0.05). One reason for this phenomenon is probably the oxidative degradation of phosphatidylcholine after reacting at 60 °C for more than 2 h. Another reason might be that, similar to the complexation of cyclodextrin with guest molecules, the complexation of debranched-starch with phosphatidylcholine is also a dynamic process.21 At the starting hours, phosphatidylcholine entered the hydrophobic cavity of debranched-starch by hydrophobic interactions. However, when the reaction time was too long, part of the phosphatidylcholine was released from the cavity. Effect of Addition Amount of Phosphatidylcholine. The influence of addition amount of phosphatidylcholine on the phosphatidylcholine payload and inclusion rate was investigated, and the result is shown in Figure 1c. The phosphatidylcholine payload and inclusion rate increased with the increase in the addition amount of phosphatidylcholine at the first stage. As the addition amount of phosphatidylcholine solution increased from 10 to 20 g, the phosphatidylcholine payload increased rapidly from 43 to 86 mg/g, and the inclusion rate almost kept constant (P < 0.05). When the addition amount of phosphatidylcholine solution was further increased to 25 g, the phosphatidylcholine payload reached its maximum of 106 mg/g, while the inclusion rate slightly reduced. Therefore, the optimal addition amount of phosphatidylcholine solution was 25 g; that is, the mass ratio of phosphatidylcholine to native starch was 1:10.

(5)

where m1 (g) is the quantity of phosphatidylcholine released calculated according to the ultraviolet spectrophotometric method, and m0 (g) is the phosphatidylcholine payload of the complexes calculated according to the phosphous molybdenum blue spectrophotometric method. Statistical Analysis. All determinations were replicated three times, and mean values and standard deviations were reported. Analyses of variance (anova) were performed, and the mean separations were performed by Tukey’s HSD test (P < 0.05) using SigmaStat version 2.0 (Jandel Scientific/SPSS Science, Chicago, IL).

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RESULTS AND DISCUSSION The complexation of phosphatidylcholine with debranchedstarch is influenced by many factors. The effect of reaction parameters (reaction temperature, reaction time, and addition amount of phosphatidylcholine) on the phosphatidylcholine payload and inclusion rate was studied, and the results are shown in Figure 1. Effect of Reaction Temperature. Reaction temperature had a great influence on the phosphatidylcholine payload and inclusion rate. As shown in Figure 1a, as the reaction temperature increased from 40 to 60 °C, the phosphatidylcholine payload and inclusion rate increased rapidly from 22 to 81 mg/g and 17.6% to 64.8%, respectively. However, when the reaction temperature was higher than 60 °C, a decrease in phosphatidylcholine payload and inclusion rate was observed. The reason for this phenomenon is probably that phosphatidylcholine is prone to deterioration when the reaction temperature is too high.4 Thus, the optimal reaction temperature of 60 °C was selected for the following runs. 636

DOI: 10.1021/jf504133c J. Agric. Food Chem. 2015, 63, 634−641

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Journal of Agricultural and Food Chemistry XRD Analysis. X-ray diffraction was used to verify the formation of debranched-starch/phosphatidylcholine inclusion complexes. As shown in Figure 2a, the diffraction pattern of

vibration, respectively. Three characteristic peaks at 1155, 1080, and 1019 cm−1 were assigned to the C−O stretching vibrations. The peak at 1644 cm−1 was attributed to the δ(OH) bending vibration. 27 As shown in Figure 3b, the spectrum of phosphatidylcholine was characterized by the absorption at 1738 cm−1, which was attributed to the CO stretching vibration.4 The FT-IR spectrum of the URDS/phosphatidylcholine physical mixture showed approximate superimposition of the individual spectrum of URDS and phosphatidylcholine. Most of the absorption bands of phosphatidylcholine were covered by that of URDS, except the CO stretching vibration located at 1738 cm−1. The FT-IR spectrum of the debranchedstarch/phosphatidylcholine complexes was almost the same as that of URDS except the shoulder peak (1736 cm−1) corresponding to the CO stretching vibration. However, as compared to the physical mixture, the CO stretching vibration peak of phosphatidylcholine was apparently weakened; the reason is probably that part of the CO bonds were covered by debranched-starch after the complexation of debranched-starch with phosphatidylcholine. Similar results were found by Yang et al.25 who investigated the formation of inclusion complexes of amylose and conjugated linoleic acid. From the FT-IR spectra, the authors suggested that the driving force for self-assembly between amylose and conjugated linoleic acid is the hydrophobic interaction. 13 C CP/MAS NMR Analysis. The 13C NMR spectrum and the peak data of (a) URDS, (b) URDS/phosphatidylcholine physical mixture (phosphatidylcholine payload 106 mg/g), and (c) debranched-starch/phosphatidylcholine inclusion complexes are presented in Figure 4 and Table 1. The serial

Figure 2. X-ray diffraction spectra. (a) URDS, (b) soybean lecithin, (c) URDS/soybean lecithin physical mixture, and (d) debranchedstarch/soybean lecithin inclusion complexes.

URDS was a typical B-type crystalline structure with main diffraction peaks at 5.7°, 17.2°, 22.3°, and 24.2°.22 The diffractogram of phosphatidylcholine (Figure 2b) exhibited a big and broad peak at around 20.3°, which indicated the amorphous characteristics of phosphatidylcholine.23 As presented in Figure 2c, the diffractogram of URDS/phosphatidylcholine physical mixture (phosphatidylcholine payload 106 mg/g) was a simple superimposition of URDS and phosphatidylcholine, which remained the main diffraction peaks of URDS and showed the characterictics of phosphatidylcholine. As compared to URDS/phosphatidylcholine physical mixture, the diffractogram of debranched-starch/ phosphatidylcholine inclusion complexes (Figure 2d) confirmed the formation of inclusion complexes, as inferred from the diffraction peaks at 7.6°, 12.5°, and 19.9°, which was a typical diffraction pattern of V-type amylose inclusion complexes.10,24 FT-IR Analysis. As a common analysis tool, FT-IR spectrum has been widely applied to confirm the formation of inclusion complexes.25,26 Figure 3 illustrates the FT-IR spectra of URDS (Figure 3a), phosphatidylcholine (Figure 3b), URDS/phosphatidylcholine physical mixture (Figure 3c, phosphatidylcholine payload 106 mg/g), and debranched-starch/phosphatidylcholine inclusion complexes (Figure 3d). In the spectrum of URDS, the extremely broad band at 3384 cm−1 and the peak at 2930 cm−1 corresponded to the O−H and C−H stretching

Figure 4. 13C CP/MAS NMR spectra. (a) URDS, (b) URDS/soybean lecithin physical mixture, and (c) debranched-starch/soybean lecithin inclusion complexes.

number of the peaks in Figure 4 and Table 1 corresponds to the carbon atoms of the molecular formula presented in Figure 4. In the spectrum of URDS, the peaks were assigned as follows: 66.59 ppm, C6; 77.44 ppm, C2, C3, C5; 86.53 ppm, C4; and 106.46 ppm, C1.28 The 13C NMR spectrum of URDS/ phosphatidylcholine physical mixture was almost the same as URDS, except two new peaks located at 37.09 and 176.89 ppm, which were attributed to the alkyl carbon and carbonyl carbon atoms, respectively.29,30 In comparison with URDS and URDS/ phosphatidylcholine physical mixture, the spectrum of debranched-starch/phosphatidylcholine inclusion complexes displayed typical characteristics of inclusion complexes, of which the C-4 peak at 86.46 ppm was better resolved from the combined C-2, C-3, C-5 peak and moved downfield, the C-3 peak was partially resolved from the C-2, C-5 peak, and the peak of C-1 was sharper and moved downfield.31,32 The

Figure 3. FT-IR spectra. (a) URDS, (b) soybean lecithin, (c) URDS/ soybean lecithin physical mixture, and (d) debranched-starch/soybean lecithin inclusion complexes. 637

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Journal of Agricultural and Food Chemistry Table 1. Characteristics of 13C CP/MAS NMR Spectra ppm samples

C-7

C-6

C-3,5

URDS physical mixture inclusion complexes

37.09 35.92

66.59 66.44 66.61

77.44 76.85 77.29

C-2

C-4

C-1

C-8

80.19

86.53 86.46 87.42

106.46 106.42 108.13

176.89 176.95

the complexes. In previous literature, similar results were found by Zabar et al.37 who investigated the nano characteristics of the amylose/fatty acid complexes. In this study, the researchers suggested that interactions between fatty acid and amylose tend to form lamella of distinct nanometric dimensions. The structure of the debranched-starch/phosphatidylcholine complexes may be described as the crystalline regions of folded debranched-starch chains were separated alternately by regions of imperfections, chain ends, and probably residues of phosphatidylcholine, which lay outside the helix cavity of debranched-starch. It was worth noting that the semicrystalline structure in the complexes that was formed by crystallization of linear polymers from solution was different from that of native starch, of which the concentric regions of alternating amorphous and crystalline structures known as growth rings are formed by the radial orientation of the amylopectin molecules.29 In previous literature, SAXS data have also been used to analyze the fractal structure of native and modified starch.19,36 The fractal structures can be characterized by the fractal dimension D, which can be obtained from the scattering power law equation:

chemical shifts of C-1 and C-4 were more remarkable because α-1,4 linkages are more sensitive to conformational changes.33 Morever, the peaks attributed to the alkyl carbon (35.92 ppm) and carbonyl carbon (176.95 ppm) atoms were also observed in the spectrum of the complexes. However, as compared to that of the physical mixture, the peak (35.92 ppm) corresponding to the alkyl carbon atoms of the complexes shifted upfield by 1.17 ppm. The chemical shift was caused by the combined trans and gauche conformation of the fatty acid chain of the lipids upon complexation with debranched-starch. Similar results were found by Lebail et al.34 who investigated the complexation of amylose with fatty acids. Furthermore, according to Snape et al.31 who demonstrated that all polar groups larger than carboxyl must lie outside the helix cavity of amylose, we can conclude that the debranched-starch/phosphatidylcholine inclusion complexes were formed by the hydrophobic interactions between the alkyl chain of phosphatidylcholine and the helix cavity of debranched-starch, with the rest of the parts of phosphatidylcholine lying outside the helix cavity. SAXS Analysis. Characterization of the cluster and fractal structure of (a) native potato starch, (b) URDS, and (c) debranched-starch/phosphatidylcholine inclusion complexes was performed using SAXS, and the scattering patterns are presented in Figure 5. As shown in Figure 5a, native starch

I ∝ qα

(6)

where I is the scattering intensity, and α is an exponent that can take the range from −1 to −4 and can be obtained from the slope between log I and log q.20 In case of −4 < α < −3, the scattering can be judged as reflection from the surface, and the objects can be seen as possessing a surface fractal structure with the fractal dimension Ds = α + 6, which indicates the irregularity of the surface. When the scattering surface is smooth, Ds becomes 2.20 In case of −3 < α < −1, the scattering objects can be seen as possessing a mass fractal structure (which means that the density profile of the scattering object has a self-similar nature) with the fractal dimension Dm = −α, which indicates the degree of compactness of the physical arrangement of the mass.20 As can be seen from Figure 5a, native starch had an obvious surface fractal structure with α = −3.92 and Ds = 2.08, indicating that native starch had a surface fractal structure and the surface of the granules was smooth. However, the slope of the scattering patterns of URDS and debranched-starch/ phosphatidylcholine complexes was −2.02 and −1.82, respectively, indicating that both URDS and debranchedstarch/phosphatidylcholine complexes possessed a mass fractal structure with Dm = 2.02 and 1.82, respectively. The Dm value of URDS was slightly higher than that of the complexes, which suggested that the molecules of URDS were arranged more tightly than that of the complexes. SEM Analysis. Scanning electron microscopy was used to investigate the morphology of URDS and debranched-starch/ phosphatidylcholine complexes at different magnification. The surface of URDS appeared to be smooth with no distinct features (Figure 6a,b). However, as shown in Figure 6c,d, after complexation with phosphatidylcholine, the complexes dis-

Figure 5. SAXS curves. (a) Native potato starch, (b) URDS, and (c) debranched-starch/soybean lecithin inclusion complexes.

showed one characteristic peak at about q = 0.66 nm−1, which corresponds to a Bragg distance (d) of about 9.52 nm. This peak is thought to arise from the long periodicity occurring from the semicrystalline structure (i.e., alternating crystalline and amorphous lamellae structure) of the starch granules.35,36 As can be seen from Figure 5b and c, after the gelatinization, enzymatic hydrolysis, and retrogradation process, the crystalline structure of native starch was completely destroyed, and the peak located at about 0.66 nm−1 disappeared. The SAXS pattern of URDS was smooth, and no distinct characteristic peak was observed within the testing range. However, after complexation with phosphatidylcholine, a new shoulder peak was observed in the SAXS curve of the complexes at about q = 0.33 nm−1. This shoulder indicated that a semicrystalline structure with a Bragg distance (d) of about 19.04 nm existed in 638

DOI: 10.1021/jf504133c J. Agric. Food Chem. 2015, 63, 634−641

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Journal of Agricultural and Food Chemistry

Figure 6. SEM images. (a) URDS ×2000, (b) URDS ×10 000, (c) inclusion complexes ×2000, and (d) inclusion complexes ×10 000.

played a rough appearance with thin lamellar structures embedded in the matrix. This phenomenon is probably due to the fact that the regular arrangement of debranched-starch was disrupted by the phosphatidylcholine molecules with alkyl chain encapsulated in the helix cavity of debranched-starch and the rest of the molecule parts of phosphatidylcholine lying outside the helix cavity. In previous literature, similar results were found in the amylose/fatty acid inclusion complexes.29,37 Stability Analysis. The determination of POV is a method that is widely used to evaluate the deterioration of lipids.13 The higher is the value of POV, the severer is the deterioration of lipids. As can be seen from Figure 7, the POV of both the debranched-starch/phosphatidylcholine inclusion complexes

and the URDS/phosphatidylcholine physical mixture increased with storage time increasing. However, the POV of the complexes was significantly lower than that of the physical mixture at any storage time. This result indicated that after complexation with debranched-starch, the stability of phosphatidylcholine was significantly improved. This result was in good agreement with other studies using amylose as embedding material of lipids.12,13 Therefore, not only amylose extracted from native starch, but also debranched-starch can be used as wall material to improve the stability of lipids. Digestibility Analysis. The percentage of phosphatidylcholine released after the complexes were incubated under the simulated stomach condition and hydrolyzed by pancreatin is shown in Figure 8. As can be seen from Figure 8a, the complexes were relatively stable under stomach conditions, and only 5% of phosphatidylcholine was released after incubating for 2 h at the simulated stomach conditions. However, after being hydrolyzed by pancreatin for 24 h (Figure 8b), nearly all of the phosphatidylcholine was gradually released, which indicated that the debranched-starch/phosphatidylcholine inclusion complexes could be effectively hydrolyzed by pancreatin. Similar results were found by Lalush et al.12 who studied the enzymatic digestion behaviors of amyloseconjugated linoleic acid complexes. In conclusion, this Article described an exploratory study on the synthesis of debranched-starch/phosphatidylcholine inclusion complexes, which is a facile method to improve the stability of phosphatidylcholine. The optimum conditions for preparing the debranched-starch/phosphatidylcholine complexes were concluded as follows: reaction temperature, 60 °C; reaction time, 2 h; and the mass ratio of phosphatidylcho-

Figure 7. Stability of debranched-starch/soybean lecithin inclusion complexes and URDS/soybean lecithin physical mixture stored at 50 °C for 0, 3, 7, 13, and 20 days. Values in the same curve with different superscript letters are significantly different (p < 0.05). 639

DOI: 10.1021/jf504133c J. Agric. Food Chem. 2015, 63, 634−641

Article

Journal of Agricultural and Food Chemistry

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line to native starch (dry basis), 1:10. The phosphatidylcholine payload and inclusion rate measured under optimum conditions were 106 mg/g and 84.8%, respectively. The formation of debranched-starch/phosphatidylcholine inclusion complexes was confirmed by XRD and FT-IR. The debranched-starch/phosphatidylcholine inclusion complexes were formed mainly by the hydrophobic interactions between the alkyl chain of phosphatidylcholine and the helix cavity of debranched-starch, with the rest of the molecule parts of phosphatidylcholine lying outside the helix cavity. Moreover, a new semicrystalline structure with a Bragg distance of about 19.04 nm was formed in the complexes. The complexes possessed a mass fractal structure and displayed a rough appearance with thin lamellar structures embedded in the matrix. After complexation with debranched-starch, the stability of phosphatidylcholine was significantly improved, and the phosphatidylcholine encapsulated can be gradually released upon pancreatin treatment.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-20-87113845. Fax: +86-20-87113848. E-mail: [email protected]. *Tel.: +86-20-87113845. Fax: +86-20-87113848. E-mail: [email protected]. Funding

This research was supported by the National Natural Science Foundation of China (31130042, 21376097), the program for New Century Excellent Talents in University (NCET-130212), the Guangdong Natural Science Foundation (S2013010012318), the Key Project of Science and Technolo g y o f G ua n g d o n g P r o v i n c e ( 2 0 1 2 B 0 9 1 1 00 4 4 3 , 2012B091100047), and the Fundamental Research Funds for the Central Universities, SCUT (2013ZZ0070). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED URDS, uncomplexed retrograded debranched-starch; XRD, Xray diffraction; FT-IR, Fourier transform infrared spectroscopy; 13 C CP/MAS NMR, 13C cross-polarization/magic angle spinning nuclear magnetic resonance; SAXS, small-angle Xray scattering; SEM, scanning electron microscopy

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REFERENCES

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DOI: 10.1021/jf504133c J. Agric. Food Chem. 2015, 63, 634−641

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DOI: 10.1021/jf504133c J. Agric. Food Chem. 2015, 63, 634−641