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Exploring the relationship between structural and airwater interfacial properties of wheat (Triticum aestivum L.) gluten hydrolysates in a food system relevant pH range Arno G.B. Wouters, Ellen Fierens, Ine Rombouts, Kristof Brijs, Iris J. Joye, and Jan A. Delcour J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05062 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 28, 2017

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Journal of Agricultural and Food Chemistry

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Exploring the relationship between structural and air-water interfacial

2

properties of wheat (Triticum aestivum L.) gluten hydrolysates in a

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food system relevant pH range

4

Arno G.B. Woutersa*, Ellen Fierensa, Ine Romboutsa, Kristof Brijsa, Iris J. Joyea,b Jan A. Delcoura

5 6 7

a

8

Research Center (LFoRCe), KU Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium.

9

b

10

Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition

Food Science Department, University of Guelph, 50 Stone Road East Guelph, Ontario, N1G 2W1,

Canada.

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*Corresponding author.

13

Tel.: +32 (0) 16 372035

14

E-mail address: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

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The relationship between structural and foaming properties of two tryptic and two peptic wheat

17

gluten hydrolysates was studied at different pH conditions. The impact of pH on foam stability

18

(FS) of the samples heavily depended on the peptidase used and the degree of hydrolysis

19

reached. Surface dilatational moduli were in most, but not all, instances related to FS, implying

20

that, while the formation of a visco-elastic protein hydrolysate film is certainly important, this is

21

not the only phenomenon which determines FS. In contrast to what might be expected, surface

22

charge was not a major factor contributing to FS, except when close to the point-of-zero-charge.

23

Surface hydrophobicity and intrinsic fluorescence measurements suggested that changes in

24

protein conformation take place when the pH is varied, which can in turn influence foaming.

25

Finally, hydrolyzed gluten proteins formed relatively large particles, suggesting that protein

26

hydrolysate aggregation probably influences its foaming properties.

27

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Keywords: Gluten hydrolysates, pH, foam, air-water interface, protein conformation, interfacial

29

properties


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1. INTRODUCTION

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A foam is a dispersion of a gaseous phase in a liquid. Its creation requires energy input is. Foams

32

destabilize rapidly as they are thermodynamically unstable 1. Both foaming capacity (FC) and

33

foam stability (FS) are important foam properties which can be influenced by the presence of

34

low molecular weight surfactants or proteins 2. Low molecular weight surfactants and proteins

35

tend to diffuse to and adsorb at an air-water (A-W) interface. Once adsorbed, they stabilize the

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interface in different ways1-3:

37

-

tension,

38 39

-

-

proteins can sterically hinder gas bubbles from approaching each other and merging, and

42 43

proteins and ionic surfactants promote electrostatic repulsion between the gas bubbles which they coat,

40 41

proteins and low molecular weight surfactants at the interface lower the surface

-

proteins likely undergo changes in conformation upon adsorption at the A-W interface.

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They expose their more hydrophobic regions towards the air phase and form a

45

viscoelastic film around the gas cells through protein – protein interactions which

46

stabilize the A-W interface. While such interactions can be hydrogen bonds or of the van

47

der Waals type, especially electrostatic and hydrophobic interactions are considered to

48

be important in this context 1, 2, 4.

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Not all (plant) proteins are evenly suited as foaming agents. For example, wheat (Triticum

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aestivum L.) gluten protein, a co-product of the industrial starch isolation, has very low solubility

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in aqueous media and very low ability to form and stabilize foams 5-7. Enzymatic hydrolysis not 3 ACS Paragon Plus Environment


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only strongly increases the solubility of gluten but also improves its foaming properties 8. Such

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enzymatic hydrolysis induces three major structural changes in proteins: a decrease in average

54

molecular mass, a better accessibility of hydrophobic regions and a higher level of ionizable

55

groups 9. These structural changes affect intermolecular interactions between the peptides in a

56

hydrolysate and consequently their foaming and interfacial behavior.

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Also, the structure of peptides as well as inter-peptide interactions at an A-W interface are

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strongly influenced by environmental conditions such as pH. The effect of pH on plant protein

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hydrolysate foaming properties has been addressed by some researchers. The FC of pea protein

60

isolates hydrolyzed with papain is lower at pH 8.0 than at pH 3.0, 5.0 and 7.0

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observation was made for peanut protein isolates hydrolyzed with Alcalase, which not only have

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good FC but also high FS at pH 3.0

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with subtilisin) increases with pH (up to pH 8.0), after which it decreases again (at pH 11.0) 12.

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For gluten protein chymotrypsin hydrolysates, Popineau et al.

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properties (FC and FS) at pH 4.0 than at pH 6.5, while Drago & González 14 observed an opposite

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trend for such hydrolysates when obtained using a fungal protease. They reported better

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foaming properties at pH 6.5 and pH 9.0 than at pH 4.0. Another study noted no differences in

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foaming capacity at pH 5.0, 7.0 and 8.0 of papain gluten hydrolysates 15. The impact of pH on

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foaming of wheat gluten hydrolysates is thus complex and depends on the peptidase used and

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the hydrolysis conditions. The latter determine which peptides are solubilized, the properties of

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which (including their charge, conformation and aggregation propensity) depend on conditions

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such as pH and temperature. For non-hydrolyzed proteins extracted from e.g. hemp seed

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lentil

17

and soy

18

11

10

. A similar

. In contrast, FS of barley protein hydrolysates (produced

13

reported better foaming

16

,

, pH-induced structural changes and their effect on foaming have been


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described. For plant protein hydrolysates, similar studies relating pH to structural changes are

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rare. Some authors, including some of the ones mentioned above 10-12, 14 have speculated on the

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molecular phenomena taking place at the A-W interface. However, it has often only been

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assumed but not experimentally shown [for example by measuring zeta potential (ZP)], that the

78

surface charge of proteins and peptides plays a key role in pH-dependent foaming

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example, Thewissen et al.

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from which they concluded this had to be related to the surface charge of the peptides. In

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addition to the above, pH-induced conformational changes in hydrolyzed plant protein which

82

affect A-W interfacial properties remain to be investigated.

83

This background shows that in order to better understand the structure – function relationship

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of wheat gluten hydrolysates, a comprehensive study is necessary. Here, foaming, A-W

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interfacial (decrease of surface tension and surface dilatational moduli) and structural (surface

86

charge, protein conformation and aggregation) properties of wheat gluten hydrolysates were

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evaluated at various pH values. The work reported has increased our understanding of how

88

hydrolyzed proteins at A-W interfaces impact FS.

19

10, 14

. For

observed pH and salt dependent foaming of gliadin hydrolysates

89 90 91

2. MATERIALS AND METHODS 2.1 Materials

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Commercial wheat gluten was kindly provided by Tereos Syral (Aalst, Belgium). It contained

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82.4% protein (N x 5.7) on dry matter basis when determined using an adaptation of AOAC

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Official Method 990.03

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Waltham, MA, USA). Trypsin (EC 3.4.21.4) from porcine pancreas (reference from the distributor

20

to an EA1108 Elemental Analyzer (Carlo Erba/Thermo Scientific,



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T0303) and pepsin (EC 3.4.23.1) from porcine gastric mucosa (reference from the distributor

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P6887) were purchased from Sigma-Aldrich (Bornem, Belgium), as were all other chemicals,

98

solvents and reagents.

99 100

2.2 Enzymatic hydrolysis

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A 6.0% (wprotein/v) wheat gluten aqueous dispersion was incubated with trypsin or pepsin at pH-

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stat conditions in a Titrino 718 device (Metrohm, Herisau, Switzerland). For each enzyme, gluten

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was hydrolyzed to degrees of hydrolysis (DH) 2 and 6. The DH reflects the percentage of initially

104

present peptide bonds which have been hydrolysed (see below). For tryptic hydrolysis, pH-stat

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conditions were 50 °C, pH 8.0 and an enzyme to substrate ratio of 1:480 (DH 2) or 1:20 (DH 6)

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on protein mass basis was used. For peptic hydrolysis, the reactions were carried out at 37 °C,

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pH 3.5 and an enzyme to substrate ratio of 1:1200 (DH 2) or 1:300 (DH 6) on protein mass basis

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was used. When the desired DH was reached, the pH was adjusted to 6.0 and proteolysis was

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stopped by heating the protein suspension for 15 min at 95 °C. It should be noted that the

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heating procedure may cause limited peptide aggregation. This will be further discussed in

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section 3.6. The mixtures were then centrifuged (10 min, 12,000 g) at room temperature, and

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the supernatants filtered and then freeze-dried. All further analyses, including those of protein

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contents (carried out as outlined in Section 2.1), were conducted on the freeze-dried

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supernatants of DH 2 or DH 6 tryptic (further referred to as T2 and T6, respectively) and peptic

115

(further referred to as P2 and P6, respectively) hydrolysates.

116 117

2.3 Determination of degree of hydrolysis


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DH is the percentage of peptide bonds hydrolyzed (h) relative to the total number of peptide

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bonds (htot) per unit weight present in wheat gluten protein. It was calculated from the quantity

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of NaOH (trypsin) or HCl (pepsin) solution used to keep the pH constant during hydrolysis:

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% =

. .

=

. .

(1)

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With X the consumption (mL) of NaOH or HCl solution needed to keep the pH during hydrolysis

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constant and Mx the molarity of the acid or base (respectively 0.50 and 0.20 M). The term α is a

124

measure for the degree of dissociation of the α-NH3+ (neutral or alkaline conditions) or α-COOH

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group (acidic conditions). Under the given conditions, for tryptic hydrolysis α is 0.89 21, whereas

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for peptic hydrolysis it is 0.29

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[milli-equivalents (meqv)/g protein] and htot is the theoretical number of peptide bonds per unit

128

weight present in gluten protein. Nielsen, Petersen and Dambmann 23 calculated the latter to be

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8.3 meqv/g protein.

22

. Mp is the mass of protein used, h are hydrolysis equivalents

130 131

2.4 Measurement of protein solubility

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First, solutions of T2, T6, P2 and P6 [0.075% (wprotein/v)] in 0.050 M sodium phosphate buffer (pH

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6.8) containing 2.0% of sodium dodecyl sulfate (SDS) were made. Under such conditions, the

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samples were completely soluble and are further referred to as reference samples. Then, T2, T6,

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P2 and P6 [0.15% (wprotein/v)] were suspended in deionized water and the pH was adjusted to pH

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3.0, 5.0 and 7.0 by adding small amounts of 1.0 M NaOH or 1.0 M HCl. The samples were

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centrifuged (10 min, 10,000 g) to remove any insoluble material. An aliquot of the obtained

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supernatants (400 µL) was diluted with an extra 400 µL 0.100 M sodium phosphate buffer (pH

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6.8) containing 4.0% SDS. An aliquot (40 µL) of these diluted samples was loaded on a BioSep 7 ACS Paragon Plus Environment


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SEC-S3000 column (Phenomenex, Torrance, CA, USA) and analyzed using a Shimadzu (Kyoto,

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Japan) LC-2010 integrated HPLC system with peptide elution monitoring at 214 nm. Samples

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eluted at a flow rate of 0.50 mL/min using 0.050 M sodium phosphate buffer (pH 6.8) containing

143

2.0% SDS at 30 °C. Peptide solubility was determined by calculating the area in the

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chromatographic profiles and expressing it relative to the area in the profile of a reference

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sample (100% soluble).

146 147

2.5 Analysis of foaming properties

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Foaming properties were determined with a standardized whipping test based on Caessens et

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al. 24. Solutions of T2, T6, P2 and P6 [0.050% (wprotein/v)] were prepared and the pH was adjusted

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to pH 3.0, 5.0 and 7.0 as above. An aliquot (50.0 mL) of these solutions was placed in a

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graduated glass cylinder (internal diameter 60.0 mm) in a water bath at 20 °C. After

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temperature equilibration for 15 min, it was whipped for 70 s using a rotating propeller (outer

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diameter 45.0 mm, thickness 0.4 mm) at 2,000 rpm. After whipping, the propeller was

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immediately removed and the glass cylinder sealed with Parafilm M (Bemis, Neenah, WI, USA)

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to avoid foam disruption by air circulation. The FC was the foam volume exactly 2 min after the

156

start of whipping. The foam volume was then also measured at 4, 10, 15, 30, 45 and 60 min

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after the start of whipping. The decrease of foam volume over time was an indication for the FS

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of a given sample. Based on the foam height and the cylinder internal diameter, foam volume

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was calculated and expressed in mL.

160 161

2.6 (Oscillating) pendant drop measurements


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Solutions [0.050% (wprotein/v)] of T2, T6, P2 and P6 in deionized water, adjusted to pH 3.0, 5.0

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and 7.0 as above, were introduced in a Theta optical tensiometer (Biolin Scientific Attension,

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Stockholm, Sweden) to create a pendant drop with a fixed volume of 8 µL. For every drop, the

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decrease in surface tension was measured over a 10 min time interval to assess protein

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adsorption and rearrangement at the A-W interface. In doing so, images were taken (1 frame

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per 7 seconds). A sinusoidal oscillation (50 cycles) was then performed at a frequency of 1 Hz

168

with an amplitude set at 1.00 in the OneAttension software (Biolin Scientific Attension), which

169

corresponded to a volume of ± 1 µL. During oscillation, images were recorded (7 frames per

170

second). The surface dilatational elastic modulus E’ of the interfacial film could be calculated

171

from the drop shape analysis data during oscillation. Measurements were conducted at room

172

temperature (22 ± 2 °C). After each measurement, the device was thoroughly cleaned and the

173

surface tension of pure water was checked to be 72.0 ± 0.5 mN/m, before initiating the next

174

measurement.

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2.7 Measurements of zeta potential

176 177

Solutions of T2, P2, T6 and P6 [0.15% (wprotein/v)] in deionized water, adjusted to pH 3.0, 4.0, 5.0,

178

6.0 and 7.0 as above, were transferred to a disposable capillary zeta cell (Malvern Instruments,

179

Malvern, United Kingdom) to determine the ZP in a Zetasizer Nano ZS (Malvern) based on laser

180

Doppler micro-electrophoresis. Measurements were conducted at room temperature (22 ± 2

181

°C).

182 183

2.8 Determination of surface hydrophobicity



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The surface hydrophobicity of all samples was determined with 1-anilino-8-naphtalene sulfonic

185

acid (ANS) as fluorescent probe. Samples were dissolved in deionized water and the pH adjusted

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to pH 3.0, 4.0, 5.0, 6.0 and 7.0 as above. They were then diluted with deionized water (adjusted

187

to the same pH) to protein concentrations of 0.18, 0.36, 0.54, 0.72 and 0.90 mg/mL. Of each

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diluted sample, 200.0 µL was transferred to a 96-well plate in duplicate, and 10.0 µL ANS

189

solution (8.0 mM in deionized water) was added. The fluorescence emission intensity of the

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protein samples was recorded at 480 nm with a Synergy Multi-Mode Microplate Reader (BioTek,

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Winooski, VT, USA) after excitation at 390 nm. The relative fluorescence intensity was calculated

192

as the intensity of the fluorescence of the protein-ANS mixture minus that of the control sample

193

(ANS with water) fluorescence, which was then divided by the fluorescence intensity of the

194

control sample. The slope of the plot of relative fluorescence intensity as a function of protein

195

concentration for each sample represents the surface hydrophobicity. Measurements were

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conducted at room temperature (22 ± 2 °C).

197 198

2.9 Analysis of intrinsic tryptophan fluorescence

199

Changes in the local environment of tryptophan residues in a protein chain can be assessed

200

from the fluorescence spectrum of the tryptophan indole ring. The intrinsic tryptophan

201

fluorescence of T2, T6, P2 and P6 solutions [0.010% (wprotein/v) in deionized water] adjusted to

202

pH 3.0, 5.0 and 7.0 as above was evaluated with a Fluoromax 4 fluorospectrometer (Horiba

203

Jobin Yvon, Edison, NJ, USA). The excitation wavelength was 295 nm with a 2 nm slit width,

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while the emission spectrum was recorded from 300 to 450 nm using a 3 nm slit width.

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Measurements were conducted at room temperature (22 ± 2 °C). 10 ACS Paragon Plus Environment

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2.10 Dynamic light scattering

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Solutions of T2, P2, T6 and P6 [0.15% (wprotein/v) in deionized water] adjusted to pH 3.0, 5.0 and

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7.0 as above were placed in disposable cuvettes to measure dynamic light scattering in a

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Zetasizer Nano ZS (Malvern). The particle size distribution (PSD) was calculated from this

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scattering pattern with the Stokes-Einstein relationship using the Malvern Zetasizer software.

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Measurements were conducted at room temperature (22 ± 2 °C).

213 214

2.11 Statistical analysis

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Protein solubility of the samples at different pH values was determined in duplicate. All foam

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related experiments, ZP, dynamic light scattering, surface hydrophobicity, pendant drop and

217

intrinsic tryptophan fluorescence measurements were carried out at least in fourfold. All data

218

were analyzed using statistical software JMP Pro 11 (SAS Institute, Cary, NC, USA), with a

219

Student t test at a significance level α = 0.05.

220 221 222

3. RESULTS AND DISCUSSION 3.1 Protein solubility

223

The solubility of the peptides ranged from 94 to 100% (Table 1). No impact of pH on solubility

224

was noted for any of the samples (Table 1, capital letters) except for P2 which had a marginally

225

lower solubility at pH 5.0 and pH 7.0 than at pH 3.0. In addition, at pH 5.0 there were no

226

significant (P < 0.05) differences in solubility between the four different samples. At pH 7.0, P6 11 ACS Paragon Plus Environment


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had slightly lower protein solubility than the other samples while at pH 3.0, it was slightly less

228

soluble than P2. Thus, none of the differences observed in foaming or structural properties

229

described in the following paragraphs can be attributed to differences in peptide solubility. The

230

reason that only minor differences were observed is that all insoluble material had already been

231

removed directly after hydrolysis, while in most other studies on the impact of pH on plant

232

protein hydrolysates (see introduction) this was not the case 10, 12-15. We believe that the merit

233

of our approach is that it allows drawing conclusions about the relationship between gluten

234

hydrolysate structural properties and their foaming, thereby surpassing mere differences in

235

levels of solubilized protein.

236 237

3.2 Foaming properties

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Foaming properties of the four hydrolysates were impacted differently by pH (Figure 1). At a

239

given pH value, the FC did not differ much between different samples (Figure 1). The FC of all

240

samples was significantly (P < 0.05) higher at pH 7.0 than at pH 3.0 or pH 5.0, except for T2,

241

which had higher FC at pH 7.0 than at pH 3.0 but not than that at pH 5.0. Overall, these

242

differences were minor. In terms of FS, all samples sustained at least some foam after 60 min at

243

pH 7.0. However, DH 2 hydrolysates clearly had better FS than DH 6 hydrolysates. At pH 5.0,

244

foams from all hydrolysates destabilized very quickly as illustrated by the steep, fast decrease in

245

foam volume. At pH 3.0, tryptic and peptic hydrolysates clearly behaved differently. While both

246

foams of peptic hydrolysates very rapidly collapsed at pH 3.0, those of tryptic hydrolysates did

247

not. The foam of T2 at pH 3.0 was the most stable of all foam samples produced. Although the

248

foam volume of T6 at pH 3.0 after 60 min was zero, it did not rapidly lose stability the way


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foams of P2 and P6 did. Thus, a pH decrease from 7.0 to 5.0 led to a steep decrease in FS for all

250

hydrolysates. A further pH drop from 5.0 to 3.0 improved FS of tryptic but not peptic

251

hydrolysates. These differences in FS, not only for different samples at a given pH, but also for a

252

given sample at different pH values, might be attributed to the nature of protein films formed at

253

the interface at different pH values. Electrostatic and hydrophobic interactions, because of

254

differences in charge of ionizable groups and peptide conformation at varying pH, probably play

255

a role in this context. All these hypotheses will be explored in the following paragraphs.

256 257

3.2 Air-water interfacial properties

258

The foaming of protein hydrolysates depends on their adsorption at the A-W interface and the

259

nature of the film they there form. Differences in pH values may alter the surface-activity and

260

the ability of gluten hydrolysates to form a strong viscoelastic protein film. Figure 2 shows the

261

decrease of surface tension over time after formation of a pendant drop. The rate and extent of

262

this decrease are indicative for the rates of diffusion and adsorption of the hydrolysate

263

constituents at the interface, as well as for their re-arrangement at the A-W interface, thereby

264

orienting their more hydrophobic regions towards the air phase. For both T2 and T6, the

265

decrease in surface tension was similar at pH 3.0, 5.0 and 7.0 (Figure 2), so the rate and ability

266

of their peptides to diffuse to and continuously adsorb at the A-W interface remained

267

unchanged. Proteins continue to adsorb and re-arrange at the interface until an equilibrium is

268

reached. Data was collected up to 10 min after drop formation. At this time, the surface tension

269

approached a plateau, and it was possible to estimate a value representative for the equilibrium

270

surface tension (Figure 2). To this end, surface tension versus 1/√t (t = Wme) plots (not shown 13 ACS Paragon Plus Environment


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here) were made using the data in Figure 2. From a certain time onwards (and thus at low 1/√t

272

values) linear relationships were observed, which were used to make extrapolations to 1/√t = 0

273

(which accords to t  ∞). The laZer yielded surface tension values, indicaWve for equilibrium

274

surface tensions (Table 2). Even though there were some significant differences in these

275

estimated equilibrium surface tension values of either T2 or T6 at different pH values (Table 2,

276

capital letters), these differences were minor. In contrast, peptic hydrolysates were much less

277

able to decrease surface tension at pH 3.0 than at pH 5.0 and 7.0. Also, estimated equilibrium

278

surface tension values for P2 and P6 were much higher at pH 3.0 than at pH 5.0 or pH 7.0 (Table

279

2, capital letters), suggesting that the hydrolysates had a lower affinity for the A-W interface or

280

that they were not able to arrange themselves very efficiently at the A-W interface at this lower

281

pH value. These observations are in line with the fact that both tryptic hydrolysates led to a

282

decent FS at pH 3.0, while peptic hydrolysates did not. After subsequent sinusoidal volume

283

oscillations of the hanging drops, surface dilatational moduli could be determined (Figure 2). E’,

284

which is indicative for the elasticity of the film at the interface, is often related to foam stability,

285

in the sense that elevated film elasticities in many instances are responsible for high stability in

286

protein foams 3, 25. T2 and to a lesser extent T6 had very high E’ at pH 3.0. As expected from the

287

low FS of peptic hydrolysates at pH 3.0, P2 and P6 had very low E’ at pH 3.0. With increasing pH,

288

E’ of both tryptic hydrolysates gradually decreased, while the opposite was observed for both

289

peptic hydrolysates. These had the highest E’ values at pH 7.0. As already stated above, foams

290

of both DH 2 hydrolysates at pH 7.0 were more stable than those of both DH 6 hydrolysates.

291

This is in accordance with the fact that T2 and P2 had significantly (P < 0.05) higher E’ than T6

292

and P6 at pH 7.0. However, not all observations regarding FS were fully in line with E’ values. For


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example, all samples had relatively high E’ at pH 5.0, even though their foams rapidly lost

294

stability at this pH. This suggests that the elasticity obtained through oscillating drop

295

measurements of the protein film in some but not all, and especially not at pH 5.0, cases can

296

explain differences in FS.

297 298

3.4 Surface charge

299

As pH evidently impacts the charge of the ionizable groups in the hydrolysates and thereby may

300

alter their ability to interact at the A-W interface and to stabilize the foam, it may well be at the

301

basis of differences in foaming. At pH 7.0, all samples had comparable ZPs (P > 0.05), with values

302

ranging from -2 mV to -4 mV, except for T6, which had a much lower ZP (-16 mV) (Figure 3). This

303

observation cannot directly be related to differences in foaming properties. Indeed, while P6

304

and T6 had similar low FS at pH 7.0, the former had a much higher ZP than the latter. In

305

addition, these two DH 6 hydrolysates had comparable surface tension (Table 2) and surface

306

dilatational moduli (Figure 2) at pH 7.0. As expected, for all hydrolysates, the zeta potential

307

increased with decreasing pH, as the ionizable groups were gradually protonated. The point-of-

308

zero-charge (PZC), i.e. the pH at which a sample has a ZP of zero, of T2, P2 and P6 was about 5.0,

309

while that recorded for T6 was about 4.0. At this pH, electrostatic repulsion between peptide

310

chains is minimal, so aggregation - with or without loss of solubility - is most probable. These

311

observations are largely in line with the very low FS at pH 5.0 of all samples. However, as

312

discussed in section 3.1, there was no decrease in protein solubility at pH 5.0. It is indeed

313

possible that aggregation occurs at the PZC but apparently only to such extent that it does not

314

impact peptide solubility. Furthermore, in spite of the low FS readings at pH 5.0, all hydrolysates 15 ACS Paragon Plus Environment


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still had relatively high E’ values. Thus, the lack of an overall net charge did not prevent the

316

hydrolysates from interacting and forming rather strong protein films. At pH 3.0, while there

317

were some statistically significant (P < 0.05) differences in ZP between some of the samples,

318

these should not be over-interpreted as all ZP values were rather close to zero and differences

319

were only minor. In contrast, there were large differences in FS as well as E’ values between

320

tryptic and peptic hydrolysates at pH 3.0. Thus, the assumption that the pH dependence of

321

foaming is dictated by the charge of ionizable groups in the hydrolysates does not hold true in

322

all cases. Only at the PZC, where FS is very low for all samples, surface charge seems to be

323

directly related to differences in foaming. We hypothesize that the low FS observed at pH 5.0

324

(and thus close to the PZC) is likely related to a lack of electrostatic repulsion between different

325

gas bubbles, which makes their coalescence more likely, rather than to a weakening of the

326

protein films around the gas bubbles. This would mean that at pH 3.0 and 7.0, repulsion

327

(because of the overall positive or negative charge of the protein films) occurs to such extent

328

that it prevents gas bubbles from easily merging. At these pH values, the strength and integrity

329

of the protein films (see section 3.3) probably determine the stability of gluten hydrolysate

330

foams.

331

3.5 Surface hydrophobicity and intrinsic tryptophan fluorescence

332

As pH changes, not only the charge of the peptides varies, but also their conformation may be

333

affected. For various native plant proteins, pH-induced conformational changes have been

334

studied

335

case, irrespective of pH, peptic hydrolysates had a higher surface hydrophobicity than tryptic

336

hydrolysates, and DH 2 hydrolysates had a higher surface hydrophobicity than their DH 6

16-18

, but this has not yet been reported for plant protein hydrolysates. In the present


Page 17 of 37


337

counterparts (Figure 4). These results are in accordance with RP-HPLC evidence from our team

338

26

339

surface hydrophobicity was observed at pH 3.0 for all samples. Compared to the surface

340

hydrophobicity at pH 4.0, an 8-fold increase of surface hydrophobicity was observed for T2 at

341

pH 3.0, followed by P6 (5.5-fold increase), P2 (5-fold increase) and finally T6 (2-fold increase).

342

These surface hydrophobicity readings (Figure 4) at pH 3.0 seem to suggest that hydrophobic

343

regions are more accessible by ANS at this pH value, possibly indicative for strong differences in

344

conformation. With regard to the surface hydrophobicity readings, it should be mentioned that

345

due to the negative charge on the sulfonic acid group of ANS, electrostatic interactions between

346

proteins and ANS may influence surface hydrophobicity measurements

347

transition from pH 4.0 to pH 3.0, there are barely any functional groups that become positively

348

charged, there is an increase in net positive charge of the peptide mixture due to the

349

protonation of negatively charged carboxylic acid groups. However, while the carboxylic groups

350

of the side chains of glutamic acid and aspartic acid have pKa values of around 4.0, the

351

carboxylic groups originating from the backbone of the protein chain have a pKa of around 2.0

352

and for a significant part will not occur in their protonated form at pH 3.0 29. Additionally, if this

353

were the sole factor involved, it would have been expected that all hydrolysates would have had

354

a similar response, as they had comparable ZPs at pH 3.0. Clearly, this is not the case. Thus,

355

there are some arguments which suggest that the peptides in the hydrolysates might in fact

356

undergo pH-induced conformational changes. These could then contribute to the pH-

357

dependence of their foaming, especially since the surface hydrophobicity increased drastically

358

at pH 3.0, which is the pH where tryptic and peptic hydrolysates had distinctly different foam

that at pH 6.4, P2 and P6 have higher overall hydrophobicity than T2 and T6. The highest

27, 28

. While, upon the



Page 18 of 37

359

stabilities. However, as similar ZP values (and therefore net surface charges) don’t always

360

correspond to equal levels of ionizable groups, such possible interference by electrostatic

361

interactions between ANS and the peptides at pH 3.0 could not be completely ruled out.

362

Therefore, changes in protein conformation were also studied by analysis of tryptophan

363

fluorescence. Changes in the local environment of this amino acid affect both the wavelength at

364

which the emission intensity is highest (i.e. the maximum emission wavelength) as well as the

365

fluorescence emission spectrum intensity. Shifts in maximum emission wavelength usually

366

indicate changes in polarity of the environment of the amino acid, while changes in intensity are

367

often related to a higher mobility and accessibility of tryptophan for quenchers. Thus, both

368

phenomena report on changes in protein conformation 30. Figure 5 shows the impact of pH on

369

the emission spectra (300-450 nm) of all four hydrolysates. There were notable shifts in the

370

maximum emission wavelengths neither for different samples at a given pH nor within one

371

sample at different pH values. Values of maximum emission wavelengths all ranged between

372

355 and 359 nm, which is a value typical for tryptophan residues which are completely exposed

373

to a polar environment. At the wavelength of maximum emission and at any given pH value, P2

374

had the highest fluorescence intensity, followed by T2, T6 and P6, respectively (all differences

375

were significant, P < 0.05). Additionally, for any given sample, the fluorescence emission

376

intensities at pH 7.0 were significantly (P < 0.05) higher than at pH 5.0, which in turn were

377

significantly higher than the values recorded at pH 3.0. All this confirmed that it is likely that

378

changing the pH induces changes in the conformation of the proteins. Furthermore, as all

379

samples had a similar change in fluorescence emission intensity, this and the surface


Page 19 of 37


380

hydrophobicity values at different pH values did not explain why tryptic hydrolysates had

381

relatively high FS, while peptic hydrolysates had very low FS at pH 3.0.

382 383

3.6 Particle size distribution

384

In the previous paragraphs, it was suggested that the peptides may occur as aggregates. Even

385

when it would not affect the overall peptide solubility, such aggregation may heavily influence

386

the A-W interfacial properties of gluten hydrolysates. PSDs were measured with dynamic light

387

scattering. There was quite some variation between different measurements of the same

388

sample at a given pH. This may be due to the fact that aggregates present in such hydrolysates

389

are loosely assembled, and re-organize rather easily upon changes in the environment. The

390

PSDs shown in Figure 6 are averages of at least 12 repeated measurements, and are indicative

391

of the aggregation of different samples under these conditions. In all cases, samples contained

392

particles ranging from 10 up to 4,000 nm in size (Figure 6). Thus, enzymatic hydrolysis probably

393

yielded peptides which have a tendency to form (soluble) aggregates. It should be mentioned

394

that larger particles scatter more light meaning that their relative abundance based on the PSDs

395

in Figure 6 should be interpreted with care. PSDs of P2 and P6 were not or only slightly affected

396

by varying the pH. It seemed that, although intrinsic fluorescence and, even more so, surface

397

hydrophobicity measurements indicated that conformational changes occurred in P2 and P6,

398

these changes did not impact their aggregation, as measured by dynamic light scattering. In

399

contrast, the impact of pH on the PSDs of tryptic hydrolysates was much more pronounced. T2

400

had polydisperse and rather similar PSDs at pH 3.0 and 7.0. While at pH 7.0 T2 and T6 both had

401

polydisperse PSDs, the latter had a higher average particle size. At pH 3.0, a monodisperse peak 19 ACS Paragon Plus Environment


Page 20 of 37

402

of particles at around 530 nm was observed for T6. At pH 5.0, all samples had rather

403

monodisperse PSDs. One could hypothesize that the aggregates at pH 5.0 would be larger than

404

those at other pH values because of the proximity to the PZC, where no electrostatic repulsion

405

occurs, thereby leading to formation of an impaired protein film at the A-W interface. However,

406

this was not always the case. For example, T6 solutions contained even larger (> 1,000 nm)

407

particles at pH 3.0 than at pH 5.0, but led to better FS at pH 3.0 than at pH 5.0. It is difficult to

408

assess how the presence of these aggregates affects the A-W interfacial and foaming properties

409

of gluten hydrolysates. Aggregation may slow down the diffusion to and adsorption at the

410

interface but may also affect the ability of the hydrolysate constituents to re-arrange

411

themselves at the A-W interface. Rullier et al.

412

efficient at forming foams in a non-aggregated state than when present in a heat-induced

413

aggregated form. In contrast, the presence of aggregated proteins led to higher thin film and

414

foam stability. Such effects have also been described for food proteins such as those of pea 33,

415

soy 34, egg white 35 and whey 35, 36. Thus, the presence of aggregated as well as non-aggregated

416

peptides in gluten hydrolysates may contribute to their overall foaming characteristics. From

417

the results in Figure 6, no direct relationship between the aggregation state of the hydrolysates

418

at different pH values and their A-W interfacial behavior (see section 3.3) could be established.

419

However, given the relatively large variation of the particle size distributions (as described

420

above), it is likely that the aggregates observed here are not very compact and that they re-

421

arrange or dissociate in the whipping procedure to produce foams. A more systematic study on

422

the role of controlled aggregation of protein hydrolysates on the way they stabilize A-W

423

interfaces is desirable.

31, 32

showed that β-lactoglobulins are more


Page 21 of 37


424

In conclusion, depending on the enzyme used for their production and the degree of hydrolysis

425

reached, different gluten hydrolysates were affected in a very different way by changes in pH.

426

At pH 7.0, DH 2 hydrolysates had better FS than DH 6 hydrolysates, while at pH 3.0, tryptic

427

hydrolysates had better FS than peptic hydrolysates. At pH 5.0, which was close to the PZC of

428

the hydrolysates, they all had relatively high E’ values, but very low FS. We believe this is due to

429

a lack of electrostatic repulsion between gas bubbles, making their coalescence more likely,

430

irrespective of the strength of the protein films. At pH 3.0 and 7.0 the hydrolysates had an

431

overall positive or negative charge so there was some repulsive effect between gas bubbles. At

432

these pH values, protein film elasticity values were in line with their FS. Thus, surface charge

433

was a major factor which directly contributed to FS in the proximity of the PZC. At other pH

434

values, FS depended on the strength and integrity of the protein films at the A-W interface, to

435

which hydrophobic interactions probably contributed significantly. While pH-induced changes in

436

protein conformation for native (plant) proteins have been studied in various instances, prior to

437

the present work this was not the case for protein hydrolysates. Surface hydrophobicity and

438

intrinsic fluorescence measurements performed here did in fact suggest that changes in protein

439

conformation took place in gluten hydrolysates when varying the pH. Such changes seemed

440

most pronounced when changing the pH to 3.0, which is the pH value where striking differences

441

in FS between peptic and tryptic hydrolysates were observed. However, at this pH, there was a

442

clear distinction neither in surface hydrophobicity nor in or fluorescence intensity between

443

tryptic and peptic hydrolysates. It is of course possible that different conformational re-

444

arrangements lead to a similar response in fluorescence intensity, but with a completely

445

different effect on interfacial stabilization. At the same time, the techniques used here only



Page 22 of 37

446

allow evaluating conformational changes of the hydrolysates in solution, while peptide behavior

447

at the A-W interface might still be different. Furthermore, it is important to consider protein

448

hydrolysate aggregation, as it became clear that hydrolyzed gluten formed relatively large but

449

probably loose aggregates which may influence foaming. Such aggregates can to a certain

450

extent be considered ‘particles’. Upon adsorption at an A-W interface, inorganic particles

451

provide excellent stability against disproportionation and coalescence by forming a strong

452

mechanical barrier 37. However, research on biodegradable, food-grade, protein-based particles

453

has only recently emerged and much is still to be learned

454

assess the role of peptide aggregation on the A-W interfacial and foaming behavior of protein

455

hydrolysates.

456

Finally, we conclude that all measurements performed here were necessary to better

457

understand how structural properties of plant protein hydrolysates change with pH, and how

458

this is related to their foaming behavior. Still, not all observations could be explained,

459

illustrating the complexity of studying the behavior of proteins at the A-W interface.

38-40

. More research is needed to

460 461 462 463

ABBREVIATIONS USED

464

FC, foaming cacapity; FS, foam stability; A-W, air-water; ZP, zeta potential; DH, degree of

465

hydrolysis; h, number of hydrolyzed peptide bonds; htot, total number of peptide bonds present;

466

Mp, mass of protein; Mx, molarity of acid or base; meqv, milli-equivalents; SDS, sodium dodecyl


Page 23 of 37


467

sulphate; ANS, 1-anilino-8-naphthalene sulphonic acid; PSD, particle size distribution; t, time;

468

PZC, point-of-zero-charge.

469 470

ACKNOWLEDGEMENTS

471

I. Rombouts thanks the Research Foundation – Flanders (FWO, Brussels, Belgium) for financial

472

support. K. Brijs acknowledges the Industrial Research Fund (KU Leuven, Leuven, Belgium) for a

473

position as Industrial Research Manager. J.A. Delcour is W.K. Kellogg Chair in Cereal Science and

474

Nutrition at KU Leuven. This work is part of the Methusalem program “Food for the Future” at

475

KU Leuven.



Page 24 of 37

476

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479 480 481

2. R66.

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3. Rodríguez Patino, J. M.; Carrera Sánchez, C.; Rodríguez Niño, M. R. Implications of interfacial characteristics of food foaming agents in foam formulations. Adv Colloid Interface Sci. 2008, 140, 95-113.

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4. Hunter, T. N.; Pugh, R. J.; Franks, G. V.; Jameson, G. J. The role of particles in stabilising foams and emulsions. Adv Colloid Interface Sci. 2008, 137, 57-81.

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5. Van Der Borght, A.; Goesaert, H.; Veraverbeke, W. S.; Delcour, J. A. Fractionation of wheat and wheat flour into starch and gluten: overview of the main processes and the factors involved. J Cereal Sci. 2005, 41, 221-237.

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6. Day, L.; Augustin, M. A.; Batey, I. L.; Wrigley, C. W. Wheat gluten uses and industry needs. Trends Food Sci Technol. 2006, 17, 82-90.

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7. Delcour, J. A.; Joye, I. J.; Pareyt, B.; Wilderjans, E.; Brijs, K.; Lagrain, B. Wheat Gluten Functionality as a Quality Determinant in Cereal-Based Food Products. Annu Rev Food Sci T. 2012, 3, 469-492.

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8. Adler-Nissen, J. Enzymatic Hydrolysis of Proteins for Increased Solubility. J Agric Food Chem. 1976, 24, 1090-1093.

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9. Wouters, A. G. B.; Rombouts, I.; Fierens, E.; Brijs, K.; Delcour, J. A. Relevance of the Functional Properties of Enzymatic Plant Protein Hydrolysates in Food Systems. Compr Rev Food Sci Food Saf. 2016, 15, 786-800.

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10. Barac, M.; Cabrilo, S.; Stanojevic, S.; Pesic, M.; Pavlicevic, M.; Zlatkovic, B.; Jankovic, M. Functional properties of protein hydrolysates from pea (Pisum sativum, L) seeds. Int J Food Sci Technol. 2012, 47, 1457-1467.

Damodaran, S. Protein stabilization of emulsions and foams. J Food Sci. 2005, 70, R54-

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11. Jamdar, S. N.; Rajalakshmi, V.; Pednekar, M. D.; Juan, F.; Yardi, V.; Sharma, A. Influence of degree of hydrolysis on functional properties, antioxidant activity and ACE inhibitory activity of peanut protein hydrolysate. Food Chem. 2010, 121, 178-184.

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12. Yalcin, E.; Celik, S.; Ibanoglu, E. Foaming properties of barley protein isolates and hydrolysates. Eur Food Res Technol. 2008, 226, 967-974.

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13. Popineau, Y.; Huchet, B.; Larre, C.; Berot, S. Foaming and emulsifying properties of fractions of gluten peptides obtained by limited enzymatic hydrolysis and ultrafiltration. J Cereal Sci. 2002, 35, 327-335.

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14. Drago, S. R.; González, R. J. Foaming properties of enzymatically hydrolysed wheat gluten. Innov Food Sci Emerg Technol. 2000, 1, 269-273.

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15. Wang, J. S.; Zhao, M. M.; Bao, Y.; Hong, T.; Rosella, C. M. Preparation and characterization of modified wheat gluten by enzymatic hydrolysis-ultrafiltration. J Food Biochem. 2008, 32, 316-334.

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16. Malomo, S. A.; He, R.; Aluko, R. E. Structural and Functional Properties of Hemp Seed Protein Products. J Food Sci. 2014, 79, C1512-C1521.

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17. Jarpa-Parra, M.; Bamdad, F.; Tian, Z.; Zeng, H. B.; Temelli, F.; Chen, L. Impact of pH on molecular structure and surface properties of lentil legumin-like protein and its application as foam stabilizer. Colloids Surf B Biointerfaces. 2015, 132, 45-53.

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18. Ruíz-Henestrosa, V. P.; Sánchez, C. C.; Escobar, M. d. M. Y.; Jiménez, J. J. P.; Rodríguez, F. M.; Patino, J. M. R. Interfacial and foaming characteristics of soy globulins as a function of pH and ionic strength. Colloids Surf Physicochem Eng Aspects. 2007, 309, 202-215.

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19. Thewissen, B. G.; Celus, I.; Brijs, K.; Delcour, J. A. Foaming properties of tryptic gliadin hydrolysate peptide fractions. Food Chem. 2011, 128, 606-612.

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20. AOAC. Official Methods of Analysis. Method 990.03. In Association of Official Analytical Chemists: Washington, DC, USA, 1995.

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21. Adler-Nissen, J. Enzymic hydrolysis of food proteins. Elsevier Applied Science Publishers: New York, USA, 1985; p 427.

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22. Diermayr, P.; Dehne, L. Controlled Enzymatic Hydrolysis of Proteins at Low Ph Values .1. Experiments with Bovine Serum-Albumin. Z Lebensm Unters Forsch. 1990, 190, 516-520.

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23. Nielsen, P. M.; Petersen, D.; Dambmann, C. Improved method for determining food protein degree of hydrolysis. J Food Sci. 2001, 66, 642-646.

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24. Caessens, P. W. J. R.; Gruppen, H.; Visser, S.; van Aken, G. A.; Voragen, A. G. J. Plasmin hydrolysis of beta-casein: Foaming and emulsifying properties of the fractionated hydrolysate. J Agric Food Chem. 1997, 45, 2935-2941.

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25. Murray, B. S. Rheological properties of protein films. Curr Opin Colloid Interface Sci. 2011, 16, 27-35.

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26. Wouters, A. G. B.; Rombouts, I.; Legein, M.; Fierens, E.; Brijs, K.; Blecker, C.; Delcour, J. A. Air–water interfacial properties of enzymatic wheat gluten hydrolyzates determine their foaming behavior. Food Hydrocolloid. 2016, 55, 155-162.

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27. Gasymov, O. K.; Glasgow, B. J. ANS Fluorescence: Potential to Augment the Identification of the External Binding Sites of Proteins. Biochim. Biophys. Acta. 2007, 1774, 403-411.

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28. Alizadeh-Pasdar, N.; Li-Chan, E. C. Y. Comparison of Protein Surface Hydrophobicity Measured at Various pH Values Using Three Different Fluorescent Probes. J Agric Food Chem. 2000, 48, 328-334.

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29. Belitz, H.-D.; Grosch, W.; Schieberle, P. Food Chemistry. fourth ed.; Springer-Verlag: Berlin, Germany, 2009; p 1070 p.

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30. Ghisaidoobe, A.; Chung, S. Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques. Int J Mol Sci. 2014, 15, 22518–22538.

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31. Rullier, B.; Axelos, M. A. V.; Langevin, D.; Novales, B. beta-Lactoglobulin aggregates in foam films: Correlation between foam films and foaming properties. J Colloid Interface Sci. 2009, 336, 750-755.

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32. Rullier, B.; Novales, B.; Axelos, M. A. V. Effect of protein aggregates on foaming properties of β-lactoglobulin. Colloids Surf Physicochem Eng Aspects. 2008, 330, 96-102.

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33. Liang, H.-N.; Tang, C.-h. Pea protein exhibits a novel Pickering stabilization for oil-inwater emulsions at pH 3.0. LWT - Food Sci Technol. 2014, 58, 463-469.

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34. He, Z. Y.; Li, W. W.; Guo, F. X.; Li, W. Y.; Zeng, M. M.; Chen, J. Foaming Characteristics of Commercial Soy Protein Isolate as Influenced by Heat-Induced Aggregation. Int J Food Prop. 2015, 18, 1817-1828.

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35. Nicorescu, I.; Vial, C.; Talansier, E.; Lechevalier, V.; Loisel, C.; Della Valle, D.; Riaublanc, A.; Djelveh, G.; Legrand, J. Comparative effect of thermal treatment on the physicochemical properties of whey and egg white protein foams. Food Hydrocolloid. 2011, 25, 797-808.

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36. Nicorescu, I.; Riaublanc, A.; Loisel, C.; Vial, C.; Djelveh, G.; Cuvelier, G.; Legrand, J. Impact of protein self-assemblages on foam properties. Food Res Int. 2009, 42, 1434-1445.

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37. Binks, B. P. Particles as surfactants—similarities and differences. Curr Opin Colloid Interface Sci. 2002, 7, 21-41.

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38. Xiao, J.; Li, Y.; Huang, Q. Recent advances on food-grade particles stabilized Pickering emulsions: Fabrication, characterization and research trends. Trends Food Sci Technol. 2016, 55, 48-60.

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39. Lam, S.; Velikov, K. P.; Velev, O. D. Pickering stabilization of foams and emulsions with particles of biological origin. Curr Opin Colloid Interface Sci. 2014, 19, 490-500.

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40. Tavernier, I.; Wijaya, W.; Van der Meeren, P.; Dewettinck, K.; Patel, A. R. Food-grade particles for emulsion stabilization. Trends Food Sci Technol. 2016, 50, 159-174.

614 615 616



Page 28 of 37

617

FIGURE CAPTIONS

618

Figure 1: Impact of pH on the foaming properties of 0.050% (wprotein/v) solutions of T2, T6, P2

619

and P6 hydrolysates. The codes T and P refer to gluten trypsin and pepsin hydrolysates, the

620

codes 2 and 6 refer to degrees of hydrolysis of 2 and 6.

621 622

Figure 2: Impact of pH on the decrease of surface tension at the air-water (A-W) interface after

623

forming a hanging drop with 0.050% (wprotein/v) solutions of T2, T6, P2 and P6 hydrolysates.

624

After 10 min, the drops were sinusoidally oscillated to determine surface dilatational moduli

625

(E’). Codes T2, T6, P2 and P6 as in Figure 1.

626 627

Figure 3: Impact of pH on the zeta potential (ZP) of 0.15% (wprotein/v) solutions of T2, T6, P2 and

628

P6 hydrolysates. Codes T2, T6, P2 and P6 as in Figure 1.

629 630

Figure 4: Impact of pH on the surface hydrophobicity of solutions of T2, T6, P2 and P6

631

hydrolysates. Codes T2, T6, P2 and P6 as in Figure 1.

632 633

Figure 5: Impact of pH on the intrinsic tryptophan fluorescence emission spectra of solutions of

634

T2, T6, P2 and P6 hydrolysates after excitation at 295 nm. Codes T2, T6, P2 and P6 as in Figure 1.

635 636

Figure 6: Impact of pH on the particle size distribution (PSD), measured with dynamic light

637

scattering) of 0.15% (wprotein/v) solutions of T2, T6, P2 and P6 hydrolysates. Codes T2, T6, P2 and

638

P6

as

in

Figure

1. 28

ACS Paragon Plus Environment

Page 29 of 37


TABLE 1

Table 1: Impact of pH on the protein solubility of T2, T6, P2 and P6 hydrolysates. Capital letters indicate significant (p < 0.05) differences between different pH values for a given sample. Lowercase letters indicate significant (p < 0.05) differences between samples at a given pH value. The codes T and P refer to gluten trypsin and pepsin hydrolysates, the codes 2 and 6 refer to degrees of hydrolysis of 2 and 6.

pH 3.0

pH 5.0

pH 7.0

T2

99 ± 3 A,ab

95 ± 2 A,a

99 ± 0 A,a

T6

97 ± 1 A,ab

94 ± 2 A,a

98 ± 1 A,a

P2

101 ± 1 A,a

97 ± 1 B,a

98 ± 0 B,a

P6

95 ± 1 A,b

96 ± 1 A,a

94 ± 1 A,b



Page 30 of 37

TABLE 2 Table 2: Estimation of equilibrium surface tension of 0.050% (wprotein/v) solutions of T2, T6, P2 and P6, at pH 3.0, 5.0 and 7.0, at t ∞ based on surface tension vs. 1/√t plots using the same data as displayed in Figure 2. Capital letters indicate significant (p < 0.05) differences for a given sample at different pH values. Lowercase letters indicate significant (p < 0.05) differences between samples at a given pH value. Codes T2, T6, P2 and P6 as in Table 1.

pH 3.0

pH 5

pH 7

T2

48.6 ± 0.5 B,d

49.0 ± 1.0 B,c

51.1 ± 0.6 A,ab

T6

50.8 ± 0.4 A,c

50.0 ± 0.7 B,b

50.4 ± 0.5 AB,b

P2

54.4 ± 1.3 A,b

49.7 ± 0.3 B,bc

49.5 ± 0.5 B,c

P6

58.7 ± 0.7 A,a

52.1 ± 0.6 B,a

51.8 ± 0.9 B,a


Page 31 of 37


FIGURE 1



Page 32 of 37

FIGURE 2


Page 33 of 37


FIGURE 3



Page 34 of 37

FIGURE 4


Page 35 of 37


FIGURE 5



Page 36 of 37

FIGURE 6


Page 37 of 37


² TABLE OF CONTENT GRAPHIC


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