Comparative Analysis of Small-Molecule Diffusivity in Different Fat

Subscriber access provided by READING UNIV

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

1

Comparative Analysis of Small-Molecule Diffusivity in Different

2

Fat Crystal Network

3

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

4

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

5

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

6

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

7

214122, Jiangsu, People’s Republic of China

8

9

AUTHOR EMAIL ADDRESS: [email protected]

10

11

12

13

14

15

16

17

*Corresponding

author

18

[email protected].

[telephone

(086)510-85876799;

fax

(086)510-85876799;

e-mail

1 ACS Paragon Plus Environment


Page 2 of 30

19

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

20

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

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32 33 34

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

35

network, diffusion coefficients, fractal dimension


Page 3 of 30

36


INTRODUCTION

37

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

38

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

39

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

40

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

41

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

42

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

43

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

44

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

45

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

46

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

47

impact the oil diffusivity and recrystallization through cocoa butter system

48

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

49

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

50

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

51

temperature

52

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

53

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

54

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

55

network for industrial application.

56

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



Page 4 of 30

57

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

58

especially in biological environments

59

quantitatively analyze the translational mobility of labeled molecules with dye

60

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

61

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

62

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

63

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

64

food system 17. Perry et al.

65

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

66

molecules in model cheeses

67

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

68

within dairy products

69

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

70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

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

22

18

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

23

. More recently, the

26


Page 5 of 30

79


crystal networks has not been taken into account seriously.

80

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

81

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

82

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

83

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

84

investigated.

85

MATERIALS AND METHODS

86

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

87

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

88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

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



Page 6 of 30

100

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

101

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

102

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

103

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

104

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

105

air-conditioned room.

106

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

107

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

108

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

109

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

110

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

111

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

112

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

113

sample.

114

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

115

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

116

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

117

previous reports

118

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

119

based on the Fick’s second law of diffusion.

120

28-31

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

∁, = ∇ ∁,

32

(1)

(1)


Page 7 of 30

121 122

123 124


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:

=

125

(2)

126

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

127

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

128

according to Axelrod’s theory:

130 131

132 133

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.

#$$ =

134

135 136

137 138

(3)

= 1 − exp [− ] + A

129

.&&×() *+/)

(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):



=

139

-

Page 8 of 30

(5)

140

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

141

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

142

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

143

MA).

144

Microstructural components analysis. The fractal dimension was analyzed with the

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

to calculate the particle equivalent diameters according to the previous report

153

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

154

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

155

36

. As shown in

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

156

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

. The water bath was

157

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

158

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


Page 9 of 30


159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

and Ni filter

167

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

168

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

169

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

170

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

171

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

172

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

38

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

01

ξ = 2345678

173

(6)

174

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

175

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

176

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

177

wavelength with the value of 1.54 Å for copper 40.

178 179

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



Page 10 of 30

180

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

181

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

Comparative Analysis of Small-Molecule Diffusivity in Different Fat

Recommend Documents