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Comparative Analysis of Small-Molecule Diffusivity in Different Fat Crystal Network Xiuhang Chai, Zong Meng, Peirang Cao, Jiang Jiang, and Yuanfa Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04677 • Publication Date (Web): 05 Jan 2018 Downloaded from http://pubs.acs.org on January 5, 2018

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Journal of Agricultural and Food Chemistry

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Comparative Analysis of Small-Molecule Diffusivity in Different

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Fat Crystal Network

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Xiu hang Chai , Zong Meng , Pei rang Cao ,Jiang Jiang , Yuan fa Liu *

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State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food

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Safety and Nutrition, School of Food Science and Technology, Collaborative Innovation Center of

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Food Safety and Quality Control in Jiangsu Province, Jiangnan University, 1800 Lihu Road, Wuxi

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214122, Jiangsu, People’s Republic of China

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AUTHOR EMAIL ADDRESS: [email protected]

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*Corresponding

author

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[email protected].

[telephone

(086)510-85876799;

fax

(086)510-85876799;

e-mail

1 ACS Paragon Plus Environment


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ABSTRACT: Oil migration and fat recrystallization in fat-structured food materials can result in

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significant deterioration in food quality. Consequently, it is important to monitor and quantify the

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diffusivities of the migrants in fat crystal network. The diffusion coefficients of Nile red dye in

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liquid oils through fully hydrogenated palm kernel oil (FHPKO) / triolein (OOO) and fully

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hydrogenated soybean oil (FHSO) / triolein (OOO) systems were evaluated by the fluorescence

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recovery after photobleaching (FRAP) method. The effective diffusion coefficients (Deff) and

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mobile fraction (Mf) increased with the decrease of solid fat contents (SFC), with the changes of

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microstructure from more densely to slightly larger packed clusters for both FHPKO/OOO and

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FHSO/OOO systems. In addition, microstructural parameters of these systems were estimated by

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the image analysis. The results showed that the diffusion of dye and liquid oil was affected by the

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microstructure. The higher Deff was associated with lower fractal dimensions, lager crystal thickness,

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and larger average particle sizes. Finally, higher-permeability coefficients were calculated according

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to the Darcy’s Law and it was significantly correlated to the Deff.

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KEYWORDS: oil migration, fluorescence recovery after photobleaching, triacylglyceride crystal

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network, diffusion coefficients, fractal dimension


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INTRODUCTION

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The physical properties of fat-structured food materials, such as texture, sensory flavor and

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mouthfeel, are largely influenced by fat crystal network. The capability of a fat crystal network to

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entrap liquid oil is usually characterized by oil migration related to the interface function between the

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solid crystal and liquid oil, which leading to fat recrystallization to more stable polymorphs. Oil

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migration and fat recrystallization results in decline in food quality, which makes the products

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unacceptable 1. For example, oil migration and fat recrystallization in chocolates results in fat bloom

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on the surface 2. Chocolate is a complex mixture with solid particles distributed in liquid and solid

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triacylglyceride (TAG) phases 3. Therefore, part of these mobile components (minor lipids and

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specific TAGs) will be able to participate in transport phenomena in post-manufacture events such as

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oil diffusivity and fat recrystallization. The formulation, processing and storage conditions can

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impact the oil diffusivity and recrystallization through cocoa butter system

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reduction and sensory changes of products. Previous research has shown that TAG concentration

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gradients is one of factors leading to transport phenomena 7. Subsequently, more driving forces for

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transport phenomena have also been studied, such as structure, fat levels, solid particles 8, storage

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temperature

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dramatic breakthroughs through quantitatively analytical techniques 11, but the phenomenon of oil

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migration and fat recrystallization has not been understood well. Therefore, it is essential to make an

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in-depth understanding about the transport phenomena and influence mechanism in fat crystal

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network for industrial application.

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9, 10

4-6

, leading to quality

and so on. To date, studies on the mechanisms of oil migration have acquired some

In the recent decades, fluorescence recovery after photobleaching (FRAP) which is a



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microscopy-based technique has been widely applied in the dynamics research of various systems,

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especially in biological environments

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quantitatively analyze the translational mobility of labeled molecules with dye

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FRAP can be used to evaluate the small-molecule diffusion capacity by monitoring the fluorescence

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recovery of the bleached region. In addition, the FRAP experiment can be performed on the confocal

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scanning microscope (CLSM) for rapid analysis with high spatial resolution and minimum sample

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preparation. However, the FRAP technique has been sparingly applied to measure the diffusion in

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food system 17. Perry et al.

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new findings about structural and kinetic information of starch. Subsequently, the diffusion of mobile

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molecules in model cheeses

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also used in the dairy systems to evaluate the influence of microstructure on the diffusion of solutes

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within dairy products

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mobility of the solute molecules inside the oleogel was observed using FRAP experiment 24. Overall,

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FRAP technique has being a fast and accurate method to study the structural and kinetic information

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in food systems. In fat-contained materials, assuming that the dye and oil move in tandem, the rate of

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intensity recovery is the same as the rate of molecular diffusion. Marty et al. 25 investigated molecule

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diffusion through polycrystalline triacylglyceride networks and proved that FRAP technique was a

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powerful tool to monitor the diffusion through the complex matrices. Similarly, Nicole et al.

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analyzed the diffusion coefficients of different types of liquid oil through fat crystal networks, and

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pointed out that the differences of diffusion coefficients were ascribed to the changes of crystal

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cluster structures. However, the relationship between the diffusion and parameters of crystal

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microstructures involving in average particles size of crystal and fractal dimension in different

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12-14

. FRAP is also an important and versatile technique to 15, 16

. Consequently,

determined the diffusion coefficients in starch solution, and got some

19-21

has been studied by FRAP technique. And the FRAP method was

and the diffusion coefficient of pepsin in dairy gel

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. More recently, the

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crystal networks has not been taken into account seriously.

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Therefore, based on the FRAP technique coupled with the CSLM, the objective of the paper

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was to study the structural basis of small molecule diffusion through triacylglyceride crystal

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networks. Moreover, the combined effects of SFC and microstructures on the dye and liquid oil

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migration through the crystalline fat matrix in FHSO/OOO and FHPKO/OOO systems were also

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investigated.

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MATERIALS AND METHODS

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Sample preparation. Mixtures of triolein (OOO, Sigma-Aldrich, Shanghai, China) and fully

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hydrogenated palm kernel oil (FHPKO, Kerry specialty fats Ltd., Shanghai, China), and blends of

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OOO with fully hydrogenated soybean oil (FHSO, Kerry specialty fats Ltd., Shanghai, China) were

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prepared at the temperature of 60 °C. Blends ranged from 20 to 100% hard stock with mass ratios

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(w/w). Then, the blends were crystallized at 20 °C for 24 h for further analysis. The lipophilic

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fluorescent dye of Nile red (Sigma-Aldrich, Shanghai, China) was dissolved in the mixtures to get a

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final concentration of 1500 µmol/L 25. Mixed samples were held at 110 °C for 30 min and vortexed

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throughout to make Nile red complete dissolution. Once mixed, samples (about 15 µL) were placed

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on a preheated glass slide using a preheated capillary tube, and then a preheated cover slip glass was

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placed over the sample to produce a film with uniform thickness with no air bubbles. Finally, sample

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slides were crystallized at 20 °C in incubator for 24 h before FRAP experiments.

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Confocal imaging and FRAP. FRAP experiments were conducted on the confocal laser

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scanning microscope of Zeiss LSM510 (Zeiss Inc., Shanghai, China) equipped with an Ar/ KrAr

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laser. The Nile red fluorescence was excited with 514 nm laser (40 mW) and the fluorescence signals



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between 600 and 750 nm were recorded. A 40× objective lens with a numerical aperture (NA) of

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1.30 was applied to capture images at a resolution of 512 × 512 pixels at successive time intervals. In

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addition, the polarized light images were observed using a Zeiss Axiovert-200M inverted light

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microscope (Zeiss Inc., Shanghai, China) with a 20× (0.5 NA) objective with a Leica DFC450 video

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camera attached (Leica, Germany). All experiments were taken in a thermostated stage at 20 °C in

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air-conditioned room.

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FRAP experiments consisted of three steps: pre-bleaching, bleaching, and post-bleaching 27. In

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FRAP experiments, five reference images were acquired with the laser power intensity of 10% at the

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pre-bleaching step, then the selected areas (region of interest, ROI) with a radius r0= 22 µm were

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bleached at a 100% intensity with a bleach pulse of 30 image scans. Ultimately, the recovery images

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were taken at an interval of 20 s at 10% of the maximum laser intensity until full recovery, and the

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recovery time depended on the sample characteristics during the post-bleaching step. Three series of

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FRAP experiments were performed on each of the replicates, giving a total of nine series for each

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sample.

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FRAP analysis. Fluorescence recovery analysis in post-bleach images was carried out in Image

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J software. In order to exclude edge effects, the smaller circular area was used to determine oil

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diffusion instead of whole ROI (diameter of 7.25 µm) in order to avoid edge effects. According to

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previous reports

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effective dye diffusion coefficients can be calculated using the classical diffusion equation

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based on the Fick’s second law of diffusion.

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28-31

, during the homogenous medium and two-dimensional diffusion process, the

∁, = ∇ ∁,

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(1)

(1)


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Where, D is the lateral diffusion coefficient; ∁, is the concentration of unbleached fluorophores at position r and time t. Based on these assumptions for disk-shaped bleached 2D diffusion, data could be normalized by the mean pre-bleach intensity within the ROI. The recovery curve was written as:

=

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(2)

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Where, is fluorescence at time t, is fluorescence of the first post-bleach image (time

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0), is fluorescence at equilibrium. FRAP recovery curves were then fitted with the formula

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according to Axelrod’s theory:

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Where, K0 is the bleaching efficiency parameter, A is a fitting parameter associated with the immobile fraction, and " is the radial diffusion time. And the effective diffusion coefficients (Deff) could be determined from the exponential fits of the data (the recovery half-time t1/2) based on the FRAP recovery formula.

#$$ =

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(3)

= 1 − exp [− ] + A

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.&&×() *+/)

(4)

Where, Deff is effective diffusion coefficient, ω is bleached area radius, t1/2 is the recovery half-time. In contrast, mobile fractions (Mf) could be determined graphically according to normalization and data fitting procedures of Siggia’s model 33 as follows (Fig 1C):



=

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-

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(5)

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Where, . is the average intensity of pre-bleached images.

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Nine series FRAP experiments were done on each sample and then the data averaged. Data

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analysis and fitting were conducted by use of Origin Pro 9.0 for Windows (OriginLab, Northampton,

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MA).

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Microstructural components analysis. The fractal dimension was analyzed with the

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particle-counting method. The particle-counting fractal dimensions (Df) were measured through a

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properly thresholded and inverted pre-bleached image using the Object Image 2.01 software. A

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log-log plot of the number of crystal reflections or “particles” (Np) observed within boxes and the

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length size (R) of theses boxes gives a line with a slope equivalent to Df 34, 35.

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The particle equivalent diameters were obtained by the use of the Fovea Pro Image Processing

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Tool Kit 4.0 plug-ins (Reindeer Graphics Inc., North Carolina, USA) in Adobe Photoshop 6.0

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(Adobe System Inc., San Jose, USA). The polarized light microscopes were thresholded and inverted

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to calculate the particle equivalent diameters according to the previous report

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Supplementary Figure 1, the program assumes a circular geometry and obtains the square root of the

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quotient of the area to π. This procedure was repeated in at least 9 images for each particle size.

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. As shown in

Solid fat content (SFC). SFC was measured by pulsed nuclear magnetic resonance (pNMR) 37

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with a Bruker PC120 series NMR analyzer (Bruker, Karlsruhe, Germany)

. The water bath was

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used to cool samples rapidly and offer accurate temperature control. The instrument was

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automatically calibrated by the use of three standards (supplied by Bruker) with the solid contents of


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0, 31.3, and 74.6%, respectively. Approximately 2.5 g of each sample was placed in a glass NMR

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tube for all pNMR experiments and was kept at 110 °C for 30 min to ensure complete melting and

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destroy any crystal memory, and then stored at the temperature of 20 °C in incubator to crystalize for

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24 h prior to monitoring SFC. Then the glass NMR tubes were put into the NMR analyzer and SFC

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readings were obtained on the computer. All measurements were performed in triplicate.

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Crystal polymorphism and crystalline domain size. X-ray diffraction (XRD) data were

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collected by use of D8 Advance XRD (Bruker, Karlsrube, Germany) equipped with Cu-Kα radiation

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and Ni filter

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divergence slit of 1.0 mm, scatter slit of 1.0 mm, and receiving slit of 0.3 mm, respectively. For the

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small-angle X-ray diffraction analysis (SAXD), samples were scanned from 1 to 10 deg at a rate of

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0.02°/min. The wide-angle X-ray diffraction analysis (WAXD) was obtained through scanning the

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samples from 11 to 30 deg at the rate of 1°/min. Peak Fit software (Seasolve, Framingham, MA,

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USA) was used to analyze the obtained data in both SAXD and WAXD patterns. The thickness of the

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nanoscale crystals was calculated by the well-known Scherrer formula 39.

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. The copper lamp (λ=1.54Å for copper) was set to 30kV and 10 mA with the

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ξ = 2345678

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(6)

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Where, k is the shape factor with a value of 0.9 for crystallites of unknown shape, θ is the

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diffraction angle, FWHM is the full width at half of the maximum peak height in radians

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corresponding to the first small angle reflection reflecting the (001) plane, and λ is the XRD

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wavelength with the value of 1.54 Å for copper 40.

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Statistical analysis. All experiments were determined at least in triplicate. All data were presented as the means

±

standard deviations (SD). Significant differences between samples were



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analyzed by the use of ANOVA with Duncan’s multiple-range test in statistical analysis system

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software of SPSS. The significance of differences among mean values was identified at a level of p

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