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Effects of chain length and degree of unsaturation of fatty acids on structure and in vitro digestibility of starch-protein-fatty acid complexes Mengge Zheng, Chen Chao, Jinglin Yu, Les Copeland, Shuo Wang, and Shujun Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04779 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018

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Journal of Agricultural and Food Chemistry

Effects of chain length and degree of unsaturation of fatty acids on structure and in vitro digestibility of starch-protein-fatty acid complexes

Mengge Zhengab, Chen Chaoab, Jinglin Yua, Les Copelandc, Shuo Wanga*, Shujun Wangab*

a

State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, China

b

College of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China

c

The University of Sydney, Sydney Institute of Agriculture, School of Life and Environmental Sciences, NSW Australia 2006

* Corresponding authors: Dr. Shuo Wang or Dr. Shujun Wang Tel: 86-22-60912486; Fax: 86-22-60912489; E-mail: [email protected] or [email protected]

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ACS Paragon Plus Environment


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Abstract: The effects of chain length and degree of unsaturation of fatty acids (FAs)

2

on structure and in vitro digestibility of starch-protein-FA complexes were

3

investigated in model systems. Studies with the Rapid Visco Analyser (RVA) showed

4

that the formation of ternary complex resulted in higher viscosities than those of

5

binary complex during the cooling and holding stages. The results of Differential

6

Scanning Calorimetry (DSC), Raman and X-ray diffraction (XRD) showed that the

7

structural differences for ternary complexes were much less than those for binary

8

complexes. Starch-protein-FA complexes presented lower in vitro enzymatic

9

digestibility compared with starch-FAs complexes. We conclude that shorter chain

10

and lower unsaturation FAs favor the formation of ternary complexes, but decrease

11

the thermal stability of these complexes. FAs had a smaller effect on the ordered

12

structures of ternary complexes than on those of binary complexes, and little effect on

13

enzymatic digestibility of both binary and ternary complexes.

14 15 16

Keywords: starch-protein-fatty acid complexes; starch-fatty acid complexes; fatty

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acid chain length; fatty acid unsaturation; in vitro digestibility

18 19 20 21 22 2


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INTRODUCTION

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Many cereal-based foodstuffs are composed of starch, proteins and lipids. Interactions

25

among these macronutrients during processing influence significantly quality

26

characteristics of finished foods, such as flavor, texture, mouth-feel and digestibility.

27

Amylose can form helical inclusion complexes with various types of lipids, naturally

28

or during food processing.1-4 As has been shown in many studies, the formation of

29

these complexes reduces the solubility and swelling power of starch in water, alters

30

the rheological properties of starch pastes, increases gelatinization temperature,

31

reduces gel rigidity, retards retrogradation and reduces the susceptibility to enzymic

32

hydrolysis.5-7 Both chain length and degree of unsaturation of fatty acids have been

33

shown to affect the structure and functional properties of starch-fatty acid

34

complexes.8-14

35 36

Ternary interactions between starch, lipids and proteins have been demonstrated using

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Rapid Visco Analyzer (RVA) and high performance size exclusion chromatography

38

(HPSEC) studies.15-18 The functional properties of the starch in the complexes were

39

shown to be altered.19 Studies with laser confocal micro-Raman (LCM-Raman)

40

spectroscopy, Fourier transform infrared spectroscopy (FTIR) and X-ray Diffraction

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(XRD) showed that starch, β-lactoglobulin and fatty acids formed a more ordered

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V-type crystalline structure compared with the respective binary starch-fatty acid

43

complexes.20 The digestibility of starch, as the most important glycemic carbohydrate

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in foods, is of considerable interest nutritionally in relation to the increasing incidence 3



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of obesity and diet-related chronic diseases. However, the digestibility of starch in

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processed foods is not well understood, especially the susceptibility of complexes

47

between starch, lipids and proteins to enzymic breakdown, and potential implications

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for human nutrition. Starch digestibility in processed foods is affected by interactions

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with other constituents,19 but does not seem to be correlated closely with the degree of

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starch gelatinization.21 Hence, there is a need for a greater understanding of

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interactions between starch, lipids and proteins, and of the properties of resulting

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complexes that may form. In particular, there is little information on the effect of

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chain length and degree of unsaturation of fatty acids on the formation of ternary

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complexes involving starch, lipids and proteins.

55 56

Hence, the objectives of the present study were to use a model system to investigate

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the effects of structural attributes of FAs on the formation, structure and in vitro

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enzymatic digestibility of model starch-protein-fatty acids complexes, and to compare

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the properties of these complexes with the corresponding starch-FA complexes. This

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information is essential to developing an understanding of the interactions between

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macronutrients during food processing and of the bioavailability of these

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macronutrients for human nutrition.

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MATERIALS AND METHODS

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Materials

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Maize starch (MS, 10.2% moisture and 22.7% amylose content), β-lactoglobulin (βLG, 4


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from bovine milk, ≥90%), octanoic acid (C8:0, OcA), decanoic acid (C10:0, DA),

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lauric acid (C12:0, LA), myristic acid (C14:0, MA), palmitic acid (C16:0, PA), stearic

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acid (C18:0, SA), oleic acid (C18:1, OA) and linoleic acid (C18:2, LiA), porcine

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pancreatic α-amylase (PPA, A3176, EC 3.2.1.1, type VI-B from porcine pancreas, 16

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units/mg) were purchased from Sigma Chemical Co. (St. Louis, Mo., U.S.A.).

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Amyloglucosidase

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oxidase/peroxide, GOPOD) were purchased from Megazyme International Ireland Ltd.

74

(Bray Co., Wicklow, Ireland). All other chemical reagents were of analytical grade

75

and were from Sigma-Aldrich Chemical Corporation (Shanghai, China).

(AMG,

3260

U/mL),

D-Glucose

Assay

Kit

(glucose

76 77

Rapid Viscosity Analysis (RVA)

78

The pasting profiles of MS-FA and MS-βLG-FA mixtures were determined using a

79

Rapid Visco Analyzer (RVA-4) (Perten Instruments Australia, Macquarie Park, NSW,

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Australia) according to the method described elsewhere.20 Maize starch (2.0 g, wet

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weight), FAs (100 mg) and βLG (200 mg) were weighed accurately into a RVA

82

canister and deionised water was added to make a total weight of 28.0 g.

83

The pasting profiles of the mixtures were monitored according to the STD 1 protocol

84

supplied with the instrument. Briefly, starch slurries were held at 50 °C for 1 min

85

before heating at a rate of 12°C /min to 95 °C, holding at 95 °C for 3 min, and then

86

cooling at a rate of 12 °C /min to 50 °C and held at 50°C for 4 min. The speed of the

87

mixing paddle was 960 rpm for the first 10 s, then 160 rpm for the remainder of the

88

experiment. Pastes of MS were prepared by the same protocol and used as the 5



89

reference. The RVA pastes were frozen in liquid nitrogen immediately after the

90

conclusion of the protocol, freeze-dried, ground into powder using a mortar and pestle,

91

and passed through a 150 µm sieve. The resulting powders were stored in sealed

92

containers at 4 °C for further structural characterization and in vitro enzymatic

93

digestibility analysis.

94 95

Differential Scanning Calorimetry (DSC)

96

Thermal properties of MS-FA and MS-βLG-FA mixtures were examined using a

97

differential scanning calorimeter (200 F3, Netzsch, Germany) equipped with a

98

thermal data analysis station. The sample preparation was as described in our previous

99

study.20 Samples (3 mg) were weighed accurately into 40 µL aluminum pans, and

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deionized water or the appropriate amount of βLG solution was added to give a

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samples/water (or βLG solution) ratio of 1:3 (w/w) and to obtain a MS/βLG/FA ratio

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of 20:2:1 (w/w/w) or a MS/FA ratio of 20:1 (w/w). The pans were sealed, equilibrated

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overnight at room temperature, and heated from 20 to 120 °C at a rate of 10 °C/min.

104

After heating, the gelatinized samples were cooled to 20 °C at 5 °C/min, and the pans

105

were reheated without delay to 120 °C at a rate of 10 °C/min. An empty pan was used

106

as the reference. The thermal transition parameters (onset (To), peak (Tp), conclusion

107

(Tc) temperatures and enthalpy change (∆H)) for MS, MS-FA and MS-βLG-FA

108

mixtures were determined from the data recording software.

109 110

Laser Confocal Micro-Raman (LCM-Raman) Spectroscopy 6


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The short-range molecular order of MS-FA and MS-βLG-FA complexes obtained

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from the RVA was determined using a Renishaw Invia Raman microscope system

113

(Renishaw, Gloucestershire, UK) equipped with a Leica microscope (Leica

114

Biosystems, Wetzlar, Germany); a 785 nm green diode laser source was used. The

115

spectra from 3200 to 100 cm-1 were collected from at least five different positions on

116

each sample with a resolution of approximately 7 cm-1. The full width at half

117

maximum (FWHM) of the band at 480 cm-1 was obtained using the WiRE 2.0

118

software to characterize the short-range molecular order of complex samples.22

119 120

Fourier Transform Infrared (FT-IR) Spectroscopy

121

The FTIR spectra of complexes obtained from the RVA were obtained using a Tensor

122

27 FTIR spectrometer (Bruker, Germany) equipped with a KBr beam splitter and a

123

DLaTGS detector. The samples were ground with KBr powder at a ratio of 1:150

124

(w/w), and the fine powders were pressed into transparent pellets and examined by

125

the transmission method. The spectra were scanned in the range of 4000 to 400 cm−1

126

at room temperature, with an accumulation of 64 scans and at a resolution of 4 cm-1.23

127 128

X-ray Diffraction (XRD)

129

The crystallinity of complexes obtained from the RVA was determined using a

130

D/max-2500vk/pc X-ray diffractometer (Rigaku Corporation, Tokyo, Japan) with a

131

Cu-Kα source (λ = 0.154 nm) operating at 40 kV and 40 mA. The samples were

132

equilibrated over a saturated NaCl solution at room temperature for one week before 7



133

measurement. The moisture-equilibrated samples were packed tightly in round glass

134

cells and examined over the range of 5o to 30o (2θ) at a scanning rate of 2o/min and a

135

step size of 0.02°.

136 137

In Vitro Enzymatic Digestibility

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The in vitro enzymatic digestibility of MS-FA and MS-βLG-FA complexes was

139

analyzed based on Englyst’s method24 and according to the procedure of Wang et al25

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with minor modifications as follows. The mixture of hydrolytic enzymes consisted of

141

porcine pancreatic α-amylase (PPA) and amyloglucosidase (AMG). According to the

142

manufacturer’s instructions, PPA solution was prepared by suspending 1.22 g of PPA

143

(16 U/mg) in 11.9 mL deionized water at 37 oC with magnetic stirring for 10 min. The

144

mixture was centrifuged at 3000 g for 15 min and 0.1 mL of AMG was added to 8.0

145

mL of the supernatant. Samples of the complexes (100 mg, dry weight basis) were

146

dispersed in 25 mL sodium acetate buffer (0.2 M, pH 6) containing 6.67 mmol/L

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CaCl2, and 5 mL of enzyme mixture containing 8228 U PPA and 204 U AMG was

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added. The complex-enzyme mixtures were incubated at 37 °C with stirring at 260

149

rpm for 2 h. Aliquots (0.2 mL) of the hydrolysate were withdrawn at specific time

150

points (from 0 to 120 min), and mixed with 0.8 mL of absolute ethanol to stop

151

enzyme reactions. After centrifuging at 13000 rpm for 3 min, the supernatant was

152

used to determine the glucose content by the Megazyme GOPOD kit. The hydrolysis

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percentages of starch samples were calculated from the amount of glucose produced

154

(multiplied by 0.9 to convert to the anhydro-glucosyl form) divided by the initial 8


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amount of glucose equivalents in the starch.

156 157

Statistical Analysis

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All results were measured at least in triplicate (three samples were used), and the

159

results are reported as the mean values and standard deviations. In the case of XRD,

160

only one measurement was performed. One way analysis of variance (ANOVA)

161

followed by post-hoc Duncan’s multiple range tests (p 0.05). ND, not detected

28


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Table3. FWHM of the Raman Band at 480 cm-1 of MS-FA and MS-βLG-FA Complexes. FWHM at 480 cm-1

Types of FAs MS-FA

MS-βLG-FA

OcA

17.09±0.08b

16.26±0.24a

DA

16.55±0.16a

16.17±0.29a

LA

16.44±0.37a

16.15±0.17a

MA

16.59±0.15a

16.51±0.44ab

PA

18.63±0.10c

16.30±0.23a

SA

20.27±0.13d

16.78±0.31b

OA

20.06±0.26d

17.32±0.30c

LiA

18.53±0.10c

16.35±0.21a

Values are means ± SD. Means with similar letters in a column do not differ significantly (p > 0.05).

29



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Figure 1

100

MS MS-OcA MS-DA MS-LA MS-MA MS-PA MS-SA MS-OA MS-LiA

2500

Viscosity(cP)

2000 1500

A 90

80

70

1000 60 500

Temperature(oC)

3000

50

0

40

-500 -1

0

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16

Time(min)

4000 3500

Viscosity(cP)

100

MS MS-βLG-OcA MS-βLG-DA MS-βLG-LA MS-βLG-MA MS-βLG-PA MS-βLG-SA MS-βLG-OA MS-βLG-LiA

3000 2500

B 90

80

2000

70

1500 60

1000 500

50

0 -500

-1

0

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16

Time(min)

30


40

Temperature(oC)

4500

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Figure 2

9

A

MS-LiA

-1.4

B

MS-LiA

MS-OA

MS-OA

8

7

MS-PA 6

MS-MA

MS-SA

-1.6

DSC flow

DSC flow

MS-SA

MS-PA -1.8

MS-MA MS-LA

MS-LA

5

-2.0

MS-DA

MS-DA

4

MS-OcA

MS-OcA

3 30

40

50

60

70

80

90

100

110

120

-2.2 20

130

30

40

60

70

80

C

MS-βLG-LiA

-1.0

100

D

MS-βLG-LiA MS-βLG-OA

MS-βLG-OA

8

-1.2

MS-βLG-SA

MS-βLG-SA 7

-1.4

MS-βLG-PA 6

MS-βLG-MA MS-βLG-LA

5

MS-βLG-DA 4

DSC flow

DSC flow

90

Temperature( C)

Temperature( C)

9

50

o

o

MS-βLG-PA -1.6

MS-βLG-MA -1.8

MS-βLG-LA MS-βLG-DA

-2.0

MS-βLG-OcA

MS-βLG-OcA -2.2

3 30

40

50

60

70

80

90

100

110

120

130

20

o

30

40

50

60

70 o

Temperature( C)

Temperature( C)

31


80

90

100


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Figure 3

A MS-LiA

Relative Intensity

100000

MS-OA MS-SA MS-PA

50000

MS-MA MS-LA 0

MS-DA MS-OcA -50000 3000

2500

2000

1500

1000

500

0

500

0

-1 Wavenumbers(cm )

B MS-βLG-LiA 100000

Relative Intensity

MS-βLG-OA MS-βLG-SA MS-βLG-PA

50000

MS-βLG-MA MS-βLG-LA MS-βLG-DA

0

MS-βLG-OcA 3000

2500

2000

1500

1000

-1 Wavenumbers(cm )

32


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Figure 4

150

A

MS-LiA MS-OA

Transmittrance intensity

100

MS-SA MS-PA

50

MS-MA MS-LA

0

MS-DA MS-OcA

-50 -1

1710cm

-1

2857cm -100 3500

3000

2500

2000

1500

1000

500

0

-1 Wavenumbers(cm )

250

B

MS-βLG-LiA

Transmittrance intensity

MS-βLG-OA 200

MS-βLG-SA MS-βLG-PA

150

MS-βLG-MA MS-βLG-LA

100

MS-βLG-DA MS-βLG-OcA

50 -1

-1

0 3500

1540cm

2846cm 3000

2500

2000

1500

1000

-1 Wavenumbers(cm )

33


500

0


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Figure 5

Diffraction intensity (cps)

7000 6000

LiA

5000

OA

4000

SA

3000

PA

2000

MA

1000

LA DA

0

OcA

-1000 5

10

15

20

Diffraction angle (2θ)

34


25

30

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Figure 6

100

Hydrolysis percentage(%)

A 80

60

MS MS-OcA MS-DA MS-LA MS-MA MS-PA MS-SA MS-OA MS-LiA

40

20

0 0

20

40

60

80

100

120

Time(min)

100


B 80

60

MS MS-β LG-OcA MS-β LG-DA MS-β LG-LA MS-β LG-MA MS-β LG-PA MS-β LG-SA MS-β LG-OA MS-β LG-LiA

40

20

0 0

20

40

60

80

Time(min)

35


100

120


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Table of Contents Graphic

7000

MS

MS

FAs

MS-βLG-LiA MS-LiA(C18:2) MS-βLG-OA MS-OA(C18:1) MS-βLG-SA MS-SA(C18:0) MS-βLG-PA MS-PA(C16:0)

6000

Diffraction intensity (cps)

Binary complex

FAs

5000 4000 3000 2000

MS-βLG-MA 1000

MS-MA(C14:0) MS-βLG-LA MS-LA(C12:0)

0 -1000 5

10

15

20

25

30

Diffraction angle (2θ)

MS

β-LG

β-LG

FAs

Ternary complex


100

80

60

40

MS MS-MA(C14:0) MS-β LG-MA

20

0 0

20

40

60

80

Time(min)

36


100

120

Effects of Chain Length and Degree of Unsaturation of Fatty Acids on

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