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Insights into the formation and structures of starch-protein-lipid complexes Shujun Wang, Mengge Zheng, Jinglin Yu, Shuo Wang, and Les Copeland J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05772 • Publication Date (Web): 15 Feb 2017 Downloaded from http://pubs.acs.org on February 17, 2017
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Journal of Agricultural and Food Chemistry
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Insights into the Formation and Structures of Starch-Protein-Lipid
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Complexes
3 Shujun Wanga*, Mengge Zhenga, Jinglin Yub, Shuo Wangac*, Les Copelandd
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a
Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science & Technology, Tianjin 300457, China
7 8 b
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Research Centre of Modern Analytical Technique, Tianjin University of Science & Technology, Tianjin 300457, China
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Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing
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Technology & Business University, Beijing 100048, China
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d
School of Life and Environmental Sciences, University of Sydney, NSW Australia 2006
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* Corresponding authors: Dr. Shujun Wang or Dr. Shuo Wang
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Mailing address: No 29, 13th Avenue, Tianjin Economic and Developmental Area (TEDA), Tianjin
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300457, China
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Phone: 86-22-60912486
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E-mail address:
[email protected] or
[email protected] 22 23 1
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ABSTRACT: The aim of the present study was to characterize the multi-scale
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structures of ternary complexes of a model system of starch, fatty acid (FA) and
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β-lactoglobulin (βLG) prepared using a Rapid Visco Analyser (RVA). The addition of
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βLG to starch-lauric acid or starch-oleic acid RVA pastes resulted in the increased
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intensity or occurrence of a new viscosity peak during cooling when the RVA protocol
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was repeated. The viscosity peak was attributed to the formation of starch-βLG-FA
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complexes. Differential scanning calorimetry (DSC) results showed clearly that
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starch-βLG-FAs complex was formed as gelatinized starch was cooled in the presence
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of βLG and FAs. The results of Raman, FTIR and X-ray diffraction analyses showed
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that starch can interact with βLG and FAs to form a ternary V-type crystalline
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complex, which had a greater short-range molecular order and higher relative
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crystallinity compared with binary starch-FA complex. The present study provided
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insights into the structure of a model starch-protein-fatty acid complex, as an example
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of what might occur during food processing.
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Keywords: ternary complex; binary complex; V-type crystalline complex; Raman
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spectroscopy; X-ray diffraction
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INTRODUCTION
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Starch, lipids and proteins are macronutrient constituents of many foods and major
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sources of calories in the human diet. During food processing, the changes that these
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macronutrients undergo, and the often complex interactions between them, determine
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the quality, nutritional and organoleptic properties of finished food products.1,2
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Complexes between starch and lipids (predominantly fatty acids or monoglycerides)
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may be present naturally in native starch or they may form during food processing or
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storage. While amylose is well-known to form complexes with fatty acids, 3-6 there is
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some experimental evidence which indicates that amylopectin may also interact with
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fatty acids.7-10 The formation of amylose-lipid complexes reduces swelling power and
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starch solubility, increases gelatinization temperature, retards the retrogradation of
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starch and decreases the susceptibility of starch to enzymatic digestion.2,11,12 Proteins
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can also form complexes with lipids through electrostatic attractions or hydrophobic
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interactions with lipophilic/hydrophobic regions of the proteins.13 Under appropriate
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conditions, starch can form conjugates with protein, which can alter the
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physiochemical properties of both the starch and protein components.14
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Binary interactions between starch and lipids have been studied extensively, but
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knowledge of the interactions between starch, lipids and proteins, and the
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microstructure of the complexes is still very limited. The formation of ternary
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complexes between starch, proteins and fatty acids (FAs) were described initially
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when the pasting properties of sorghum flour were examined.15 Subsequently, 3
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interactions between starch, proteins and lipids have been studied by high
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performance size exclusion chromatography (HPSEC), multi angle laser scattering
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(MALLS), X-ray diffraction (XRD) and differential scanning calorimetry (DSC).16-18
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Interactions between the three components alter starch pasting, rheological and
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gelatinization properties, and the rate and extent of digestion of the three
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macronutrient components.1 A self-assembled ternary complex was able to carry
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sparingly soluble small molecules in the lumen of the amylose helix, which has some
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potential applications in the delivery and release of hydrophobic drugs and
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nutritional health products.19
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To better understand the formation and structures of starch-protein-lipid complex,
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changes in pasting and gelatinization properties of a model system of starch in the
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presence of FAs and/or β-lactoglobulin (βLG) were monitored. Meanwhile, the
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multi-scale structures of the ternary complexes prepared from RVA pastes were
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characterized by a combination of XRD, Fourier Transform infrared spectroscopy
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(FTIR) and Raman spectroscopy. The results obtained will help to better understand
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the mechanism of formation of starch-protein-fatty acid complexes, which may form
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between these food macroconstituents during food processing.
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MATERIALS AND METHODS
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Materials. Maize starch (MS, 10.2% moisture and 22.7% amylose content),
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β-lactoglobulin (βLG, from bovine milk, ≥90%), octanoic acid (C8:0, OcA), decanoic 4
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acid (C10:0, DA), lauric acid (C12:0, LA), myristic acid (C14:0, MA), palmitic acid
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(C16:0, PA), stearic acid (C18:0, SA), oleic acid (C18:1, OA) and linoleic acid (C18:2,
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LiA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other
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chemicals were of analytical grade and were from Sigma-Aldrich Chemical
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Corporation (Shanghai, China).
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Pasting Viscosity Analysis. The pasting profiles of MS-βLG-FA mixtures were
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investigated using a Rapid Visco Analyser (RVA-4) (Perten Instruments Australia,
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Macquarie Park, NSW, Australia) according to the method of Zhang et al (2004) with
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minor modifications as follows. Exactly 2.0 g (10% moisture) of MS was added into
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the RVA canisters, followed by the addition of 100 mg of the appropriate FA and/or
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200 mg βLG, as described subsequently. Deionised water was added to make a total
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weight of 28.0 g. The pasting profiles of the mixtures were determined according to
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STD 1 protocol provided with the RVA instrument. Briefly, the mixtures were held in
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the RVA at 50 oC for 1 min, heated from 50 to 95 oC at a rate of 12 oC/min, held at 95
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o
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min. The heating process was accompanied by a constant shear at 960 rpm for the first
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10 s followed by a constant shear at 160 rpm until the end of the analysis. The effect
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on the pasting profiles of adding components separately or in different sequences was
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determined by repeating the STD 1 RVA protocol after the respective addition was
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made to pasted samples. The RVA pastes were frozen quickly in liquid nitrogen,
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freeze-dried, ground using a mortar and pestle, and passed through a 150 µm sieve.
C for 2.5 min, cooled from 95 to 50 oC at a rate of 12 oC/min, and held at 50 oC for 2
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The resulting powders were stored in sealed containers at 4 oC until further structural
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analysis.
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The pastes obtained from the RVA protocols will be referred to by a short-hand
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nomenclature, in which S, P and F represent the starch, βLG and FA components
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respectively, and a subscripted number indicates the RVA run in which the component
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was added. For example, S1P2F3 will describe a sample in which the RVA STD 1
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protocol was performed three times, firstly with starch alone, then after adding βLG,
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and then again after adding the FA.
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An unpasted mixture of the three components was used as the reference. This mixture
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was prepared by firstly dissolving 100 mg of the fatty acid in ethanol, after which 2 g
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of starch was added and the mixture stirred thoroughly. After evaporation of ethanol
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in a fume hood, the necessary amount of βLG solution was added to obtain a
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starch:βLG:FA ratio of 20:2:1 (w/w/w). The resulting mixture, which is referred to
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subsequently as the three-component mixture, was freeze-dried and ground into a
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powder.
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Differential Scanning Calorimetry. Thermal properties of MS and mixtures of MS
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with βLG, FA or βLG and FA were examined using a differential scanning calorimeter
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(200 F3, Netzsch, Germany) equipped with a thermal analysis data station. For all the
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DSC measurements, samples (3 mg) prepared as described subsequently were 6
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weighed accurately into 40 µL aluminum pans and deionized water was added to give
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a ratio of 3:1 (w/w) water:starch (or starch mixtures). Mixtures of MS and LA (20:1,
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w/w) were prepared according to the method described in Wang et al (2016). Mixtures
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of MS:βLG (20:2, w/w) and MS:βLG:LA (20:2:1, w/w/w) were prepared by a βLG
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from a solution in deionized water. The pans were sealed, equilibrated overnight at
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room temperature, and heated from 20 to 120 oC at a rate of 10 oC /min. After cooling
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to 20 oC at 5 oC/min, the pans were reheated to 120 oC at a rate of 10 oC /min. An
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empty pan was used as the reference.
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Laser Confocal Micro-Raman Spectroscopy. The short-range molecular order of
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samples obtained from the RVA was determined using a Renishaw Invia Raman
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microscope system (Renishaw, Gloucestershire, United Kingdom) equipped with a
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Leica microscope (Leica Biosystems, Wetzlar, Germany). A 785 nm green diode laser
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source was used. Spectra in the range of 3200 to 100 cm-1 were taken from at least
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five different spots on each sample with a resolution of approximately 7 cm-1. The full
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width at half maximum (FWHM) of the band at 480 cm-1 was obtained to characterize
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the short-range molecular order of starch samples using the software of WiRE 2.0.20
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Fourier Transform Infrared (FTIR) Spectroscopy. The FTIR spectra of samples
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obtained from the RVA were obtained using a Tensor 27 FTIR spectrometer (Bruker,
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Germany) equipped with a KBr beam splitter and a DLaTGS detector. The samples
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were mixed with KBr powder at a ratio of 1:150 (w/w). After mixing and grinding, the 7
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fine powders were pressed into the transparent pellets and examined by the
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transmission method. The spectra were scanned from 4000 to 400 cm−1, with an
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accumulation of 64 scans and at a resolution of 4 cm-1.
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X-ray Diffraction (XRD). The relative crystallinity in the samples obtained from the
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RVA was determined using a D/max-2500vk/pc X-ray diffractometer (Rigaku
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Corporation, Tokyo, Japan) operating at 40 kV and 40 mA. The freeze-dried samples
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were equilibrated over a saturated NaCl solution at room temperature for one week
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before analysis. The XRD spectra were obtained from 5o to 30o (2θ) at a scanning rate
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of 2o/min.
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Statistical Analysis. Results were reported as the mean values and standard
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deviations of at least duplicate measurements. In the case of X-ray diffraction (XRD),
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only one measurement was performed. One way analysis of variance (ANOVA)
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followed by post-hoc Duncan’s multiple range tests (p 0.05). ND, not detected
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Table 2. FWHM of the band at 480 cm-1 determined by Raman of MS, MS paste, the three-component mixture, binary and ternary complex samples. FWHM at 480 cm-1
Samples
683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716
Maize starch（MS）
15.70±0.13a
Three component mixture
15.87±0.19a
MS-βLG-LA
16.18±0.22b
MS-LA
16.37±0.18bc
MS-βLG
21.41±0.45d
MS paste
22.31±0.19e
Values are means ± SD. Means with similar letters in a column do not differ significantly (p > 0.05).
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Table of Contents Graphic
717 718
FA
MS
β-LG
ng ati He
Cooli ng
Repeating
MS FA
β-LG Emulsifying action
Starch-Protein-Lipid complex
719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735
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