Impact of microfluidization on the microstructure and functional properties of pea hull fibre

Journal Pre-proof Impact of microfluidization on the microstructure and functional properties of pea hull fibre R. Morales-Medina, D. Dong, S. Schalow, S. Drusch PII:

S0268-005X(19)32065-X

DOI:

https://doi.org/10.1016/j.foodhyd.2020.105660

Reference:

FOOHYD 105660

To appear in:

Food Hydrocolloids

Received Date: 25 September 2019 Revised Date:

10 January 2020

Accepted Date: 10 January 2020

Please cite this article as: Morales-Medina, R., Dong, D., Schalow, S., Drusch, S., Impact of microfluidization on the microstructure and functional properties of pea hull fibre, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/j.foodhyd.2020.105660. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

AUTHOR CONTRIBUTION STATEMENT Dr Morales-Medina conceived the workplan and wrote the manuscript with support from Dr Schalow and Prof. Drusch. Mr Dong carried out the experiments. Prof Drusch supervised the project and conceived the original idea.

GRAPHICAL ABSTRACT: VISCOELASTICITY

D90 = 80 µm

D90 = 120 µm

CELULLOSE

WATER RETENTION CAPACITY

PECTIN & HEMICELLULOSE

D90 = 60 µm

1

IMPACT OF MICROFLUIDIZATION ON THE MICROSTRUCTURE AND

2

FUNCTIONAL PROPERTIES OF PEA HULL FIBRE

3

Morales-Medina, R*a., Dong, D.a, Schalow S.a, Drusch Sa.

4 5

a

6

ABSTRACT

7

The goal of the present work was to evaluate the impact of the particle size (D90) of

8

microfluidized pea hull fibre suspensions on the physical microstructure, composition

9

and functional properties (i.e. water retention capacity and rheological behaviour). To

10

that end, fibre suspensions with D90 of 120, 100, 80 and 60 µm were produced. Among

11

them, the suspensions of 100 and 80 µm were produced employing two different

12

processing conditions to evaluate the impact of processing on the microstructure and

13

functionality. Suspensions with smaller particles (D90 ≤ 80 µm) presented a thermal

14

stable, pseudoplastic, thixotropic and viscoelastic behaviour varying their elastic

15

modulus in the linear viscoelastic region from 19 to 89 Pa for suspensions with D90 of

16

80 and 60 µm. By decreasing the D90, it was observed a continuous defibrillation of

17

aggregates of cellulosic macrofibrils, into macrofibrils and microfibrils. Consequently,

18

the number of particles and their interactions by electrostatic and friction forces

19

increased. Alcohol insoluble substances, released from the hemicellulosic and pectic

20

networks, were only detected for the suspensions with D90 = 60 µm. In this case,

21

particles presented inter-fibrillar voids that may increase their flexibility and

22

functionality. For a given particle size distribution, the processing conditions had

23

negligible effect on the functionality. In summary, functionality of microfluidized fibre

24

suspensions may be tuned by controlling the release of alcohol insoluble substances and

25

the defibrillation of the cellulosic network, process conditions then can follow energetic

26

or economic criteria.

27

Keywords: dietary fibre, pea hull, viscoelastic properties, microfluidization, particle

28

size distribution, water binding capacity

Technische Universität Berlin, Department of Food Technology and Food Material Science, Königin-

Luise Str. 22, 14195 Berlin.

*

Correspondence: [email protected], Tel: +49-30-214-71425

1

29

1. INTRODUCTION

30

In the last decade, the worldwide production of dried peas has increased from 10.3 to

31

16.2 million tonnes (FAO, 2017). During the industrial processing of peas, cotyledon

32

and hulls are separated; cotyledons are employed to extract starch and protein, while

33

hulls are by-products (Tiwari et al., 2010). Ground pea hulls are bright, tasteless and

34

have a high content of dietary fibre (Guillon, & Champ, 2002). This makes them an

35

interesting raw material for food application, since there is an increasing interest to

36

incorporate dietary fibre into food matrixes (European Food Safety Authority, 2010).

37

However, due to poor techno-functional properties of pea hulls (i.e. poor capacity to

38

bind water), their incorporation into the food matrix has a negative impact on the food

39

consistency, texture, and sensorial characteristics (Guillon, & Champ, 2002).

40

The low technological functionality of pea hulls is related to the compact structure of

41

the cell wall polysaccharides (i.e. cellulose, hemicellulose and pectin) (Nechyporchuk,

42

& Belgacem, 2016). Cellulose interact with hemicellulose by crosslinking and

43

electrostatic interactions (Fry, 2001) and both polysaccharide networks are embedded in

44

a pectic matrix (Carpita, & Gibeaut, 1993). As a consequence, the vegetable cell wall

45

has a strong and highly cohesive structure. Cellulose is organized in a multi-scale

46

structure presenting a well-ordered architecture of fibrillar elements (i.e. elementary

47

fibrils,

48

Nechyporchuk, & Belgacem, 2016). Defibrillation of the cellulose structure into the

49

fibrillar elements is conducted to considerably improve the functionality by decreasing

50

the particle size, increasing the specific area of the particles and the number of exposed

51

hydrophilic groups (Liu et al., 2016).

52

Mechanical treatments can effectively reduce the particle size; however, not all of them

53

lead to a proper defibrillation without damaging the cellulosic fibres. For instance,

54

intense size reduction by dry milling can break micropores and microcapillaries,

55

damaging the fibre structure and resulting in a loss of functionality (Jacobs et al., 2015).

56

Contrary, size reduction by high pressure homogenization in aqueous media has been

57

successfully employed to improve the water binding properties of cellulosic suspensions

58

by defibrillating the cellulose (Agoda-Tandjawa et al., 2010; Rezayati Charani et al.,

59

2013; Shogren et al., 2011). Microfluidization is a high pressure homogenization

60

technique in which a pressurized suspension is forced to pass through a microchannel

microfibrils

and

macrofibrils)

(Chinga-Carrasco,

2011;

Fry,

2001;

2

61

(diameter < 400 µm) resulting in the disruption of particles due to the high shear-stress,

62

cavitation, expansion and turbulence (Martínez-Monteagudo et al., 2017; Siqueira et al.,

63

2010). To control the intensity of the treatment, the pressure, number of runs,

64

concentration and diameter of the microchannels can be modified. It is common to

65

employ several runs and pressure until 200 MPa, which implies a high energetic cost

66

and high energy dissipation as heat (Martínez-Monteagudo et al., 2017).

67

Most of the studies focused on the microfluidization of purified cellulosic suspensions

68

to produce stable viscoelastic gels whose strength increased with increasing intensity of

69

microfluidization (Pääkkö et al., 2007; Rezayati Charani et al., 2013). Regarding pectin

70

and hemicellulose, both polysaccharide networks can be broken by microfluidization

71

(Jun Chen et al., 2012; Tu et al., 2014); more specifically microfluidization of high-

72

methoxyl pectic suspensions decreased the particle size, average molecular weight and

73

apparent viscosity (Jun Chen et al., 2012). Few studies focused on the microfluidization

74

of suspensions of complex matrixes, as for instance wheat bran suspensions whose

75

swelling capacity increased with decreasing D90 (Wang et al., 2012). Hence, the state of

76

art of size reduction by microfluidization is mostly related to the defibrillation of the

77

cellulose macrostructure rather than to the interplay of the breakage of the three

78

polysaccharides networks present in the cell wall. Consequently, little is known about

79

the impact of the release of pectic and hemicellulosic-like substances on the

80

defibrillation of the cellulosic macrostructure and on the functionality of the fibres

81

suspensions. Additionally, most of studies describe the interplay between fixed

82

processing conditions (i.e. pressure, number of runs) and functionality, composition

83

and/or microstructure. However, there is a lack of a systematic study focused on the

84

impact of the processing conditions on the particle size, results that may lead to an

85

energetic optimization of the process by decreasing the energetic loss by dissipation.

86

Also, there is no systematic studies where the particle size is employed as a key

87

parameter that can define the microstructure and functionality of microfluidized

88

suspensions.

89

In this context, the goal of this work is to produce pea hull fibre suspensions with

90

specific particle size (D90) to evaluate the impact on the fibre microstructure,

91

composition and functional properties (i.e. water retention capacity and rheological

92

behaviour). Additionally, by comparing microfluidized suspensions with same particle

3

93

size distribution produced under different processing conditions, we also evaluate the

94

feasible impact of the mechanical energy on the functionality and microstructure.

95

We hypothesise that the key parameter that defines the functionality of microfluidized

96

suspensions is the particle size, whereas the processing conditions will have a negligible

97

effect on the microstructure. To produce a given particle size, an increase of the

98

mechanical energy employed during microfluidization may just increase the energy

99

dissipation. Additionally, we hypothesise that a decrease of particle size will be linked

100 101

to: (i)

102 103

a higher release of soluble fibre due to the breakage of the pectin and hemicellulose networks;

(ii)

an improvement of the water binding capacity due to a high cellulosic

104

defibrillation which increases the surface area and the exposition of

105

hydrophilic groups

106

(iii)

the formation of stronger fibre networks with viscoelastic properties due to a

107

higher soluble fibre content and stronger interactions among particles due to

108

electrostatic and frictional forces.

109

To test these hypotheses, a design of experiments was conducted to correlate the

110

processing conditions (i.e. pressure and number of runs) to the particle size (D90). Then,

111

fibre suspensions with D90 of 120, 100, 80 and 60 µm were produced. Among them, the

112

suspensions of 100 and 80 µm were produced employing two different processing

113

conditions. Finally, for each microfluidized fibre-based suspension, we determined the

114

fibre fraction composition, the water retention capacity and characterize the rheological

115

behaviour by conducting amplitude, frequency and temperature sweeps and shear stress

116

and thixotropic tests.

117

2. MATERIALS AND METHODS

118

2.1 Materials

119

The commercial product Empet E5 B10 (batch: 310907) was kindly donated by

120

Emsland-Stärke GmbH (Emlichheim, Germany) in dried and milled state. This product

121

is mainly composed of yellow pea hulls obtained after physical dehulling of grains.

122

According to the product specifications, the pea hulls contained 86% of total dietary

123

fibre and contained 60 % of cellulose, 7 % of hemicellulose and 10 % of pectin. 4

124

Additionally, pea hulls contained protein, starch, and ashes in a much lower percentage

125

(0.3; 3 and 3 % respectively). Hemicellulose from pea hulls has been described to be

126

mainly composed of xyloglucan (Scheller, & Ulvskov, 2010) and pectin presents a low

127

content of rhamnose (Ralet et al., 1993).

128

2.2 Fractionation of pea hulls

129

The pea hulls were homogenized in an ultra-centrifugal miller ZM 1, (Retsch

130

Technology GmbH, Haan, Germany) equipped with a 12 tooth SS rotor (Retsch

131

Technology GmbH, Haan, Germany) and with exchangeable steel ring sieves of sizes

132

500 and 250 µm (Retsch Technology GmbH, Haan, Germany). The sample was ground

133

in three passes: first one employing a ring sieve of 500 µm and, then, conducting two

134

passes with the ring sieve of 250 µm. Finally, 50 g of pea hulls were fractionated

135

employing a vibratory mechanical siever (VIBRO, Retsch Technology GmbH, Haan,

136

Germany) equipped with sieves (mesh sizes of 50 µm and 140 µm) for 20 min and 80 %

137

of amplitude. The recovered mass collected between the mesh sizes of 50 µm and

138

140 µm was employed as feed for the next steps. This range was chosen considering

139

that: (i) particles must be small enough to prevent the clogging of the inner channels of

140

the microfluidizer (d = 200 µm) (ii) intense grinding in dry state favours fibre shredding

141

(Nechyporchuk et al., 2014) damaging the structure of the particles (Iwamoto et al.,

142

2007).

143

2.3 Dynamic high pressure microfluidization

144

The homogenized pea hull fibre was dispersed in distilled water (1 wt%) and pre-shared

145

in an ULTRA-TURRAX T25 digital (IKA-Werke GmbH & Co. KG, Germany) for

146

2 min (level 4). Subsequently, it was stirred (300 rpm) at room temperature and

147

overnight. Then, high pressure microfluidization experiments were conducted

148

employing a LM20 Microfluidizer, (Microfluidics Co., MA, USA) equipped with two

149

exchangeable Z-type interaction chambers with inner channel diameters of 200 and

150

100 µm (H30z 200µ Ceramic S#17403 and H10z 100µ Diamond S#17406,

151

Microfluidics Co, MA, USA). To avoid the clogging of the narrower interaction

152

chambers, as pre-treatment, fibre suspensions were microfluidized employing the

153

200 µm interaction chamber for 2 runs at 2000 bar. After that, both interaction

154

chambers were connected in series, from bigger to smaller inner channel diameter. The

5

155

microfluidization was conducted following the conditions of the design of experiments

156

and keeping the temperature of the feed below 35 ºC.

157 158

2.4 Experimental Design: prediction of D90 and D50 as a function of pressure and number of runs.

159

To produce pea hull suspensions with specific particle size distribution, a mathematical

160

model was calculated to predict the percentiles of the particle size distribution (i.e. D90,

161

D50) as a function of the operational variables of the microfluidization (i.e. pressure and

162

number of runs). To this end, the operational variables were set at three levels (pressure

163

500; 1250 and 2000 bar; and number of runs 2; 5 and 8) following a two-factor central

164

composite design, with five repetitions of the central point; resulting in 13 experiments.

165

For each suspension, the D90 and D50 were measured as described in section 2.5.1. The

166

results were employed to perform an analysis of variance (ANOVA) and to estimate a

167

polynomic equation (Eq 1) that correlates the inverse of percentile (i.e. Di) to the

168

number of runs (N) and pressure (P):

169

1/Di (N, P) = α0 + α1·N + α11·N2 + α2·P + α22·P2+ α12·N·P

170

The coefficients αij were calculated by multiple regression using the software Design

171

Expert 8.0 (Stat-Ease Inc., Minneapolis, USA).

172

2.4.1

Eq.1

Particle size distribution

173

The particle size distribution (PSD) of the micro-fluidized fibre suspensions was

174

measured by using laser light diffraction using a particle size analyser LA-950, (Horiba,

175

Retsch Technology GmbH, Haan, Germany). The volumetric PSD was employed to

176

calculate the 10th, 50th and 90th percentiles (D10, D50, D90) according to the Fraunhofer

177

optical model using the instrument's software. These analyses were done per triplicate.

178 179

2.4.2

Fibre suspensions composition: insoluble, alcohol-insoluble and soluble fractions.

180

The composition of the dry matter of the fibre suspensions was characterized by

181

determining: (i) the insoluble fraction (IF), which represents the water insoluble

182

compounds, (ii) the alcohol-insoluble fraction (AIF) which represents the compounds

183

soluble in water but insoluble in ethanol and (iii) the soluble fraction (SF) which

184

includes the compounds soluble in water and in ethanol. These fractions were quantified

185

by adapting the official method 993.19 (AOAC Official Method 993.19, 1995). 6

186

Briefly, 25 g of fibre suspension were centrifuged (3000 g, 20 min) in a Sigma 6K10

187

centrifuge (Sigma Laborzentrifugen, GmbH, Osterode, Germany) and the pellets and

188

supernatant were carefully separated. The insoluble mass was re-dispersed in 40 mL of

189

distilled water and filtered in a FibreBag S (10-0142, Gerhardt GmbH &Co KG,

190

Königswinter, Germany). The insoluble mass was, then, flushed twice with 10 mL of

191

ethanol 95 v% and with 10 mL of acetone. The FibreBag containing the insoluble mass

192

was dried at 105 ºC overnight and weighted to determine the insoluble mass. The

193

supernatant was mixed with 100 mL of ethanol 95 v% at 60 ºC. After 1 h at room

194

temperature, the precipitate (i.e. AIS) was recovered by filtration through a FibreBag S

195

and cleaned twice with 10 mL of ethanol 78 v %, 10 mL of ethanol 95 v % and 10 mL

196

of acetone. Then, the Fibrebag containing the precipitated was processed as explained

197

for the insoluble mass. The soluble mass was calculated employing Eq. 3. MDM = MIF + MAIF + MSF

(Eq. 3)

198

Where MDM is the total dry matter content, MIF is the mass of the insoluble fraction and

199

MAIF the mass of the alcohol insoluble fraction. The percentage of dry matter was

200

determined by weighing 1 g of suspension and drying at 105 °C overnight. Hence, the

201

exact dried matter content of the 25 g can be calculated and, consequently, the soluble

202

fraction (Eq. 3).

203

The insoluble, soluble and alcohol-insoluble fractions were presented as percentage of

204

the total dried matter. All determinations were conducted per duplicate. In this work, IF

205

can be considered as an approximate determination of the insoluble dietary fibre and the

206

AIF as an approximation of the soluble dietary fibre.

207

2.4.3

Microscopy analysis

208

The morphology of the six pea fibre suspensions was visualized by using light and

209

scanning electronic microscopy (SEM).

210

Light microscopy was performed using an Optihop Eclipse E400 microscope (Nikon,

211

Chiyoda, Japan) equipped with a Nikon DS-Fi2 digital sight DS-U3 camera.

212

Microscopy was conducted in liquid state after storage for 24 h at 6 ºC. Micrographs

213

were taken with magnification objectives 4, 10 and 20x to have an overview of the

214

microfluidized samples.

7

215

The microstructure of the six suspensions was analysed by SEM at the Center for

216

Electron Miscroscopy (ZELMI, Technische Universität Berlin, Berlin, Germany) with a

217

S-2700 scanning electron microscope (Hitachi, Tokyo, Japan), with magnification of

218

50, 300 and 1000 x employing an accelerating voltage of 5.0 kV. Prior to microscopy,

219

15 mL of sample was transferred into 60 mL plastic beakers and frozen by immersion

220

into liquid nitrogen. Frozen samples were stored at -20 ºC until being freeze dried in a

221

lyophilizer Beta1-8LSCplus (Christ alpha 2e4, Osterode, Germany) and stored at room

222

temperature. Finally, prior to microscopic analysis, samples were carefully broken and

223

sputter coater with a gold-palladium alloy (SCD 030, Balzers, Wiesbaden-Nordenstadt,

224

Germany).

225

2.5 Functional properties

226

2.5.1

Water retention capacity

227

Water retention capacity (WRC) was determined by following the method described by

228

(Robertson et al., 2000), with some modifications. An aliquot of 25 g of fresh fibre

229

suspension was centrifuged at 3000 g for 20 min in a centrifuge Sigma 6K10 (Sigma

230

Laborzentrifugen, GmbH, Osterode, Germany). Then, the supernatant of each tube was

231

carefully decanted and the excess of liquid was drained by turning the tubes upside

232

down on a fine-meshed paper for 10 min. Sample fresh weight (mhydrated) was recorded

233

immediately after draining and also after drying overnight at 105 ºC (mdried). The WRC

234

was calculated as the mass of water which is retained by the pellet (g water/g dried

235

mass) (Eq.4). All analyses were conducted per duplicate. WRC

236

m g = g

2.5.2

m

− m

(Eq. 4)

Rheological measurements

237

Rheological analyses were conducted with a rheometer MCR 502 (Anton Paar GmbH,

238

Ostfildern, Germany) equipped with a measuring cup (C-PTD200, diameter 28.9 mm)

239

and a coaxial measuring cylinder (CC27/P6 36734, diameter 26.7 mm). Amplitude

240

sweeps, frequency sweeps and thixotropic analysis were done per duplicate at 20 ºC

241

within 24 h after microfluidization.

242

Dynamic shear rheological analyses were conducted to analyze the viscoelastic behavior

243

of the fibre-based suspensions by monitoring the storage and loss moduli (G’ and G’’).

244

Initially, an amplitude test from 0.1 to 100 % deformation at an angular frequency of 8

245

1 Hz was performed to identify the linear-viscoelastic regime (LVR). The deformation

246

that defines the end of the LVR was calculated as the one in which the complex

247

modulus (G*) deviates from the initial value in a 5 % (Mezger, 2014) and was

248

employed for characterization. Frequency sweep tests were conducted, within the LVR,

249

at a constant deformation of 0.1 % and varying the frequency from 0.01 to 100 Hz.

250

To evaluate the temperature-dependent viscoelastic behaviour, the loss factor (i.e.

251

tan(δ) = G’’/G’), was monitored while conducting a shearing oscillatory test

252

(γ = 0.001 %; f = 1 Hz) varying the temperature. The temperature was linearly increased

253

(1ºC/min) from 20 to 80 ºC, kept constant for 10 min and, finally, decreased (1ºC/min)

254

to 20 ºC. To minimize the water evaporation, the surface of the sample was covered

255

with silicon oil and a metallic lid.

256

Flow curve were measured strain-controlled from 1 to 200 s-1 (forwards) and then 200 to

257

1 s-1 (backwards) recording a point each 2 s. The backwards curves were then fitted to

258

the Herschel-Bulkley model (Eq. 5): =

+ ∙

(Eq. 5)

259

Where τ represents shear stress [Pa], τ0 the yield stress [Pa], k the consistency index

260

[Pa·s],

261

Thixotropic tests were conducted by monitoring tan(δ) while applying oscillation at

262

γ = 0.1 % and f = 1 Hz for 100 s, followed by shearing at a rate of 3000 s-1 for 10 s and

263

finally, 10 min of oscillation at γ = 0.1 % and f = 1 Hz.

the shear rate [s-1] and n [-] the flow index.

264

3. RESULTS AND DISCUSSION

265

3.1 Production of microfluidized pea fibre suspensions with specific D90

266

To obtain a mathematical correlation between D90, D50 and the processing variables

267

(pressure and number of runs) a full factorial design was conducted (Table 1,

268

supplementary data). Main parameters of the analysis of variance (ANOVA) are shown

269

in Table 2 (supplementary data); in both cases, a non-significant (p< 0.05) lack of fit

270

was obtained. Hence, the predicted models fitted adequately to the experimental data

271

with high determination coefficient (R2 > 0.9). Both, D90 and D50 were related to the

272

operational variables by a polynomial equation (Eq, 2 and Eq 3) as follows: 1/D90 = 3.5·10-3 + 1.4·10-3·N+5.0·10-6·P+9.9·10-8·P·N - 7.2·10-5·N2 - 1.5·10-9·P2

Eq.2

9

1/D50 = -8.6·10-3 + 6.3·10-3·N+3.0·10-5·P-1.6·10-8·P·N - 3.7·10-4·N2-9.10.0·10-9·P2

Eq.3

273

The Eq.2 and Eq.3 were employed to predict the range of D90 and D50 that can be

274

produced by microfluidization and to select the particle size distributions of the selected

275

suspensions for further characterization. In Fig.1, predicted data of D90 and D50 as a

276

function of pressure and number of runs are shown. The curves of predicted D90 (Fig

277

1A) showed a double asymptotic behavior with increasing pressure and number of runs.

278

Contrary, the curves of predicted D50 showed a minor increased for pressures ranging

279

between 1500 and 2000 bar (Fig 1B). Bigger particles might be disrupted into particles

280

which are bigger than the initial D50, increasing the value of D50, despite the overall

281

particle size distribution decreased.

282

Regardless the pressure and number of runs employed, the D90 and D50 varied following

283

a common trend; for instance, suspensions with D90 varying from 90 to 100 µm

284

presented D50 between 35 and 43 µm. Hence, by fixing the D90, the D50 is also indirectly

285

fixed, that implies that the particle size distribution of the fibre-based suspensions could

286

be characterized by merely employing D90 values.

287

In Fig 1, it can also be seen that several combinations of pressure and number of runs

288

can lead to suspensions with similar D90, for instance, a fibre suspension with a D90 of

289

100 µm can be produced by employing 1090 bar and 2 runs or 680 bar and 3 runs. This

290

fact might be related to the percentage of energy which is thermally dissipated

291

(Karagiannidis et al., 2017). The loss of energy by dissipation might be influenced by

292

the processing conditions and it might increase with increasing the mechanical energy

293

employed during microfluidization.

294

Based on these results, six pea hull based-suspensions with D90 120, 100, 80 and 60 µm

295

were produced for further characterization. Suspensions with D90 of 100 and 80 µm

296

were produced with (i) the highest pressure (HP) and (ii) the lowest pressure (LP)

297

predicted by the equation. As a result, four suspensions were obtained: S-100HP, S-

298

100LP, S-80HP and S-80LP. Suspensions with D90 of 120 and 60 µm (S-120 and S-60)

299

could only be produced under one condition. In the Table 1, the number of runs and

300

pressure required to produce the six suspensions with their predicted and measured D90

301

are listed. All produced suspensions presented a deviation lower than 5% when

302

compared to the predicted value calculated employing Eq.2 (Fig 1).

10

303

3.2 Microstructural and chemical characterization (mass fraction)

304

3.2.1

Particle size distribution (PSD) and light microscopy

305

The volumetric PSD of the six pea hull-based suspensions are shown in Fig. 2. All

306

suspensions had a monomodal distribution and those with D90 ≥ 80 µm presented a wide

307

shoulder between 10 and 30 µm. With decreasing D90, the volumetric fraction of

308

particles with an equivalent diameter between 10 and 40 µm increased. Contrarily, the

309

percentage of particles with sizes between 50 and 200 µm decreased. Furthermore, all

310

suspensions had a similar D10 (10.93 ± 1.46 µm), which might indicate that the

311

mechanical energy required for decreasing size until this scale is much higher than the

312

one employed in this work. When comparing the PSD of the suspensions with the same

313

D90 but produced with different operational variables (i.e. S-80HP, S-80LP, S-100HP

314

and S-100LP), the curves almost overlapped proving that similar PSD can be produced

315

under several processing conditions.

316

The degree of cell wall disruption and cellulose defibrillation can be qualitatively

317

followed by light microscopy in the liquid state (Fig. 3A to 3D). Some pieces of the cell

318

wall were detected in S-120 and S-100 when a magnification of 4 was employed

319

(Supplementary data, Fig S.1.) while for the rest of the tested suspensions the cell tissue

320

was completely disrupted. All suspensions with D90 ≥ 80 µm contained particles in

321

process of defibrillation (in Fig. 3A to C). Cellulose has a complex macromolecular

322

structure which is organized in several levels: cellulose molecule, elementary fibrils,

323

microfibrils and macrofibrils (Nishiyama et al., 2002). During microfluidization the

324

aggregates of macrofibrils are defibrillated into macrofibrils (width >1µm) and,

325

subsequently, into microfibrils that can have a length over 100 µm (Fry, 2001),

326

depending on the source. Hence, the decrease of the percentage of particles with

327

equivalent diameter between 50 and 200 µm might be mainly the result of: (i) disruption

328

of remaining cell wall fractions and (ii) defibrillation of aggregates of macrofibrils,

329

macrofibrils and release of microfibrils. In suspension S-60, where the microfluidization

330

was conducted with the highest mechanical energy, there was a considerable increase of

331

particles with sizes between 10 and 50 µm (Fig. 2) which is related to the partial

332

defibrillation of microfibrils.

11

333

3.2.2

Chemical characterization: mass fractions (IF, SF, AIF) and SEM

334

In the Fig.4, the mass fractions (i.e. insoluble, alcohol insoluble and soluble fractions)

335

of the six pea-fibre based suspensions are shown. All suspensions with D90 ≥ 80 µm had

336

a similar content of insoluble and soluble mass (average values of suspensions:

337

87.2 ± 0.9 % and 12.8 ± 0.9 %, respectively) with non-detectable alcohol insoluble

338

fraction. Contrarily, in the S-60 suspension AIF was detected while the amount of IF

339

and SF was lower than in the suspensions with D90 ≥ 80 µm. The insoluble mass might

340

be mainly composed of insoluble dietary fibre, negligible amount of ashes and proteins.

341

On the other hand, the alcohol insoluble fraction is composed of substances that are

342

soluble in water and insoluble in alcohol. Finally, the soluble fraction refers to

343

compounds which are soluble in both alcohol and water. Regarding, insoluble dietary

344

fibres, it has been described that they are mainly composed of celluloses and some type

345

of hemicelluloses (Elleuch et al., 2011). Alcohol insoluble fraction, that might be an

346

approximation of the soluble dietary fibre, has been described to be composed of pectin-

347

like substances, and sections of hemicellulose and cellulose with a degree of

348

polymerization higher than 12, so they precipitate in alcohol and not in water (Ohkuma

349

et al., 2000). Cell wall polysaccharides are firmly and tightly bounded, and,

350

consequently, they are water insoluble (Fry, 2001). However, after processing their

351

solubility can be modified due to an intense size reduction that increases the porosity

352

and capillary attraction (Huang et al., 2010). Regarding suspensions with D90 ≥ 80 µm,

353

produced under relatively mild processing conditions (Table 1), the PSD indicated the

354

defibrillation of macrofibrils into microfibrils, both insoluble substances. However, in

355

the case of S-60, the conditions of microfluidization were more intense (8 runs,

356

2000 bar, Table 1). The energy, then, was high enough to (i) partially defibrillate

357

microfibrils (releasing insoluble fibrils), (ii) break the interactions of hemicellulose-

358

cellulose, hemicellulose-pectin and pectin-cellulose and (iii) break the glycosidic bonds 12

359

within the polysaccharides (i.e. cellulose, hemicellulose and pectin). The decomposition

360

of hemicellulose and pectin into smaller molecules might result in an increase of AIF, as

361

shown in Fig.4. The release of soluble fibre during microfluidization has already been

362

reported when employing soybean or purified apple pectin (Jun Chen et al., 2012; Tu et

363

al., 2014) and was also explained by the breakage of glycosidic bonds by intense

364

mechanical stress.

365

The impact of composition on the structure of the particles can be also observed in the

366

SEM pictures. Samples of suspensions with D90 ≥ 80 µm presented a flake-like lamella

367

network (Fig. 5A-C). In the case of S-60 (Fig. 5D), the network presented a high

368

amount of voids that could be the result of the release of AIF. Hence, by releasing

369

alcohol insoluble substances, these networks are disrupted. This type of structural voids

370

has been previously observed for microfluidized apple pectin and they were related to

371

the release of pectin and breakage of the pectin network (Jun Chen et al., 2012).

372

As a summary, the impact of size reduction on the extent of cellulosic defibrillation, on

373

the feasible disruption of the hemicellulosic and pectic networks (release of AIF) and

374

the microstructure is shown in Fig 6. For suspensions with D90 ≥ 80 µm, the mechanical

375

energy was enough to break electrostatic interactions among macrofibrils and their

376

aggregates, resulting in the gradual defibrillation of macrofibrils. In that case, the

377

insoluble mass was constant, showing that it was not degraded enough to release

378

alcohol insoluble substances. However, for the suspension S-60, the macrofibrils were

379

almost totally defibrillated, the insoluble mass decreased and alcohol insoluble

380

substances were detected. Hence, the mechanical energy was high enough to break

381

glycosidic bonds. Presumable, the pectic and hemicellulosic networks were disrupted,

382

which resulted in the presence of inter-fibrillar voids within the particles.

383

3.3 Functional properties

384

3.3.1

Water retention capacity (WRC)

385

Water retention capacity refers to the mass of water that can be retained by the insoluble

386

mass of the fibre-based suspensions. As general trend, the WRC increased with

387

decreasing particle size (Supplementary Data, Fig. S.2). For suspensions between 120

388

and 80 µm, there was a clear linear trend (R2 = 0.95) between the WRC and the D90. In 13

389

that case, if D90 decreased by 20 µm, the WRC increased by 2 g of water/ g insoluble

390

mass. However, the WRC of S-60 was 33.9 g of water/g of insoluble mass, almost the

391

double as the one obtained for suspensions with D90=80 µm (i.e. 18.4 g of water/ g of

392

insoluble mass). This is mainly linked to the almost complete defibrillation of

393

macrofibrils, that leads to a high number of particles (consequently, high surface area)

394

(Fig 3D), and to the release of AIF (Fig 4). An increase of WRC with decreasing

395

particle size was also described for microfluidized and dried samples of wheat bran

396

(Wang, Sun, Zhou, & Chen, 2012). Additionally, microfluidized suspensions of

397

insoluble fibre of peach and oat with an average size of ~ 110 and ~ 75 µm reached

398

values of WRC of ~ 8 and ~ 6 g of water/ g of fibre, which significantly improved the

399

functionality of the untreated sample (Jialun Chen et al., 2013). The main differences

400

might be related to the source of fibre which defines the cell wall structure. The WRC

401

of those suspensions produced with the same D90 but modifying the processing

402

conditions (i.e. S-100HP and S-100LP, S-80HP and S-80LP) only showed low variation

403

with an average value of 16.7 ± 0.2 and 18.4 ± 0.4 g water/ g insoluble mass for

404

suspensions S-100 and S-80, respectively.

405

Water associated to fibres can be trapped within interstices and capillaries (free or bulk

406

water), tightly bound to fibre surface by hydrogen bonds (adsorbed water), and it can be

407

part of the chemical or crystalline structure (bonded water) (Taipale et al., 2010).

408

Additionally, the pore volume of fibres exerts a crucial role in the WRC, showing a

409

direct and linear correlation (Jaturapiree et al., 2008). Suspensions with a D90 > 80 µm

410

showed some cell wall residues and defibrillation of macrofibrils. Then, it is feasible

411

that water might be retained in the macromolecular structure as large voids, lumen

412

(intra-fibre areas) or inter-fibre pores (Déléris, & Wallecan, 2017). In the case of S-60,

413

the higher content of microfibrils with inter-fibrillar voids might increase porosity.

414

Furthermore, the inter-fibrillar voids may also favour the flexibility of the

415

polysaccharide network and they can give water the possibility to penetrate into the

416

fibrils (Redgwell et al., 1997).

417

Additionally, the crystallinity index of the cellulose (ratio between crystalline and

418

amorphous regions) has an impact on the WRC of the fibre suspensions (Déléris, &

419

Wallecan, 2017), since water molecules are not able to enter into the crystalline region

420

of the cellulose (Déléris, & Wallecan, 2017). Microfluidization can decrease the

421

crystallinity index (Iwamoto et al., 2007; Taheri, & Samyn, 2016). The degree of 14

422

reduction depends on the size of the diameters on the interaction chambers, the number

423

of runs and pressure. To disrupt the crystalline region, interactions chambers with a

424

small diameter (i.e. ≤ 200 µm) are needed to produce mechanical shear forces and

425

friction forces. Since we employed two interaction chambers in series (inner diameters:

426

200 and 100 µm; section 2.3), it is expected that the crystalline cellulose has been

427

disrupted. An increase of number of runs and pressure, required to decrease the D90 of

428

the suspensions, can decrease the amount of amorphous cellulose by orientation of

429

smectic regions under shear (Taheri, & Samyn, 2016). Hence, suspensions with smaller

430

D90 shall have lower crystallinity favouring the water binding capacity.

431

3.3.2

Rheological characterization and stability

432

Among the six suspensions prepared by microfluidization, those with D90 > 80 µm

433

showed phase separation. Hence, the rheological characterization was conducted only

434

for samples S-80HP, S-80LP and S-60.

435

Amplitude, frequency and temperature sweep

436

The amplitude sweep curves of storage (G’) and loss (G’’) moduli are shown in Fig 7A.

437

Both moduli were around one order of magnitude higher for S-60 when compared to S-

438

80 until the crossover (G’= G’’). Macrofibrils and microfibrils are able to

439

electrostatically interact, resulting in a physical cross-link that leads to the formation of

440

a highly entangled elastic network (Agoda-Tandjawa et al., 2010; Iotti et al., 2011;

441

Pääkkö et al., 2007).

442

In Fig 8A, it can be seen that, once the G’ begins to decrease, G’’ slightly increases

443

until reaching the flow point from where both moduli decreases. This behavior has

444

already been shown for microcrystalline cellulose suspensions (Zhao et al., 2011) and it

445

is the result of the relative motion between particles, flexible end-pieces of chains or

446

agglomerates which are not fixed in the network (Mezger, 2014).

447

The deformation, G’ and G’’ at which the linear-viscoelastic regime (LVR) ends (i.e.

448

end of the elastic reversible deformation region (Mezger, 2014)) are listed in Table 2. At

449

the end of the LVR, all samples showed viscoelastic behavior being G’ ~4.5-fold bigger

450

for S-60 when compared to both of S-80. For S-60, the higher density of smaller

451

particles eases the electrostatic interactions between them. Also, the presence of alcohol

452

insoluble substances, specifically the pectic-like ones containing residual uronic acids,

453

might prevent the particle aggregation (Agoda-Tandjawa et al., 2010). 15

454

In Fig 7B, G’ and G’’ are depicted as a function of the frequency while the suspensions

455

are subjected to low strain deformation. The storage modulus slightly increases with the

456

frequency in the whole range studied. The loss modulus is independent to the frequency

457

at low frequency regions (f < 0.1 Hz) and begins to increase considerably for f > 1 Hz.

458

Then, the increase of the frequency results in a loss of elasticity of the network until

459

breaking down (Rezayati Charani et al., 2013). Similar behavior has been previously

460

described in the literature for microfibrillated cellulosic samples (Shogren et al., 2011).

461

When comparing the loss factor for amplitude and frequency test, the three curves

462

overlapped (Supplementary data, Fig S.3). The loss factor values lower than 1 refer to

463

elastic behavior; on the contrary, values higher than 1 describe viscous behavior. This

464

suggests that, in all cases, the structure of the network is similar whereas the strength of

465

the electrostatic interactions between particles and particle and water are stronger with

466

decreasing D90.

467

The impact of the temperature on the particle network was evaluated by monitoring the

468

loss factor while temperature was swept from 20 to 80 ºC (f = 1 Hz, γ= 0.001 %). For

469

the three suspensions, tan(δ) was inversely proportional to the temperature and

470

presented similar values (Fig 8C). Hence, an increase of temperature resulted in a

471

reversible improvement of the elasticity of the network, as the decrease of tan(δ) shows.

472

The reduction of the loss factor can be related to a decrease of viscosity due to: (i) the

473

deswelling of fibres with increasing temperature and (ii) an increase of mobility of the

474

fibrils of the network (Iotti et al., 2011). This result agrees with the behavior described

475

to microfibrillated cellulose suspensions produced from bleached sulfite softwood

476

cellulose pulp or sugar-beet cellulose, where a slightly stronger network was observed

477

increasing temperature (Lowys et al., 2001; Pääkkö et al., 2007).

478

Flow Curve and thixotropy

479

As shown in Fig 8A, all suspensions presented a shear thinning behavior; i.e. the

480

viscosity decreases with increasing shear rate in the whole range studied. Additionally,

481

a hysteresis loop was observed, proving the thixotropic behavior of the pea fibre

482

suspensions. Thixotropy and shear thinning behavior has been already described for

483

suspensions of microfibrillated cellulose (Agoda-Tandjawa et al., 2012; Pääkkö et al.,

484

2007; Saarikoski et al., 2012). Regarding the impact of the particle size, S-60 presented

485

higher viscosity than S-80 in the whole range studied (Fig. 8A). 16

486

A high degree of defibrillation, as shown for sample S-60, results in a network structure

487

which has a high resistance to flow and, consequently, a high viscosity (Henriksson et

488

al., 2007; Iotti et al., 2011; Pääkkö et al., 2007). Additionally, the shear thinning

489

behavior can be related to the presence of aggregates which under shear conditions are

490

disrupted (Saarikoski et al., 2012). Under shearing, the particles orientate in parallel to

491

the shear force and, with increasing shear rate, the network is broken; resulting in a

492

decrease of viscosity. By decreasing the shear rate, the network is re-built, and the

493

viscosity increases. Effectively, this behavior has been described for microfibrillated

494

cellulose suspensions (Karppinen et al., 2012), where at intermediate share rates (~10 s-

495

1

496

flocculate state.

497

In the case of S-80 suspensions, the curves showed a constant slope (Ln(η) / Ln(γ))

498

while for S-60, the forwards line (where shear rate is increasing) presented a minor

499

sigmoidal behavior, reaching a constant viscosity region between 20 and 30 s-1. Some

500

authors relates this Newtonian plateau to the formation of a shear induced structure

501

(Iotti et al., 2011) while other explains it by wall-slip and shear banding phenomena

502

during the rheological analysis (Barnes, 1995; Nechyporchuk et al., 2014; Saarikoski et

503

al., 2012). In this study, further rheological configurations have to be tested to clarify

504

the cause of this behaviour.

505

To characterize the flow behaviour of the suspensions, the flow curve was fitted to the

506

Herschel-Bulkley equation (Eq 5) and three parameters i.e. the yield stress (τ0),

507

consistency index (k) and flow index (n) were calculated with a high correlation

508

coefficient (r2 > 0.99) (Table 2). All suspensions presented a yield stress with the same

509

order of magnitude. Hence, the network structure is able to elastically deform, until

510

reaching a strain large enough where plastic deformation begins and the suspension

511

begins to flow (Saarikoski et al., 2012). The consistency index (k) and the flow index

512

(n) describe the viscosity characteristics, the consistency index increased with

513

decreasing particle size while the fluid index decreased (Table 1). The consistency

514

index is proportional to the particles and molecular interactions, (Zhang et al., 2018),

515

while the flow index defines the type of fluid, being n = 1 for Newtonian fluids and

516

n < 1, for shear thinning behaviours.

), large flocs appeared. By increasing the share rate, the suspension broke resulting a

17

517

Thixotropy refers to the ability of a structural regeneration after the total breakage of the

518

structure (Mezger, 2014), a specific thixotropic test was conducted to monitor the loss

519

factor after the breakage of the structure by intense shearing rate. In the Fig. 8B, it can

520

be seen that all suspensions presented a highly elastic behavior (i.e. tan(δ) << 1) that

521

was rapidly recovered after the breakage, at 115 s. Hence, the network built by the pea

522

hulls particles was reversible broken and was able to re-build once the mechanical stress

523

disappeared. After the intense shearing, S-80 suspensions presented a loss factor bigger

524

than one, which indicates a viscous behavior. Therefore, the structure was totally broken

525

and viscoelasticity lost. Contrary the S-60 suspension was not totally disrupted since the

526

loss factor was lower than 1; value that indicates that the elastic behavior is

527

predominant, proving the higher strength of the network of particles with smaller sizes.

528

4. CONCLUSIONS

529

Particle size distribution (D90) of pea hull based-suspensions is a key parameter that

530

determines the composition (soluble and insoluble dietary fibre), extent of defibrillation

531

of the cellulosic network, disruption of pectin and hemicellulose and functionality (i.e.

532

water binding properties and rheological properties). Several processing conditions (i.e.

533

pressure and number of runs) can produce suspensions with similar D90, composition

534

and functionality. Consequently, for further industrial applications, the production of

535

microfluidized suspensions with fixed D90 could be optimized following energetic or

536

economic criteria. The water retention capacity increased with decreasing D90.

537

Additionally, suspensions with D90 ≤ 80 µm showed a thermal stable, pseudoplastic,

538

thixotropic and viscoelastic behaviour, that was improved with decreasing particle size.

539

The better functionality of S-60 is related to the release of soluble dietary fibre and to

540

inter-fibrillar voids observed in the particles that might increase their flexibility

541

improving the functionality. Hence, for food applications, microfluidization of

542

suspensions of complex vegetal matrixes may lead to both nutritional and functional

543

advantages. However, for a global understanding of the pea based suspensions a more

544

detailed characterisation of the mechanical properties shall be conducted taking into

545

account the role of the degree of the cellulose crystallinity.

546

To optimize the functionality of microfluidized pea hulls suspensions, future studies

547

should focus on reaching a higher extent of defibrillation (i.e. more intense size

548

reduction) and on increasing the release of soluble fibre. To that end, the combination of

549

enzymatic and/or thermal pre-treatments with microfluidization can be used to lose the 18

550

cell

wall

polysaccharide

networks

and

partially

hydrolyse

them

prior

to

551

microfluidization. A better understanding of the interplay among the disruption of the

552

three polysaccharide networks during microfluidization is needed to efficiently

553

functionalize fibre from high cellulosic cell wall sources. Finally, further

554

characterisation of the mechanical properties of the gels and the influence

555

ACKNOWLEDGEMENTS

556

The project is supported by funds of the Federal Ministry of Food and Agriculture

557

(BMEL) based on a decision of the Parliament of the Federal Republic of Germany via

558

the Federal Office for Agriculture and Food (BLE) under the Protein Crop Strategy

559

under the project 2815EPS010.

560

FIGURES AND TABLES CAPTION

561

Table 1. Processing conditions of the microfluidization (pressure and number of runs)

562

and D90 of the fibre-based suspensions produced. Rheologial parameters that define the

563

end of the linear viscoelastic region (obtained from amplitude test) and value of the

564

coefficients predicted for Herschel-Bulkley model.

565

Table 2. Value of the coefficients predicted for Herschel-Bulkley model and rheologial

566

parameters that define the end of the linear viscoelastic region (obtained from amplitude

567

test).

568

Figure 1. Predicted and measured (A) D90 and (B) D50 of 1 wt% fibre-based

569

suspensions varying the pressure and the number of runs obtained after

570

microfluidization based on the analysis of two-factor central composite design.

571

Figure 2. Particle size distribution (volumetric fraction) of the produced fibre-based

572

suspensions

573

Figure 3. Light microscopy images at 20-fold of the 1 wt% fibre-based suspensions:

574

(A) S-120, (B) S-100HP, (C) S-80HP and (D) S-60. Arrows indicate macrofibrils in

575

process of defibrillation. The scale bar represents a length of 100 µm.

576

Figure 4. Mass composition in terms of insoluble, alcohol insoluble and soluble

577

fraction of the produced fibre-based suspensions

19

578

Figure 5. SEM images at 1000-fold of the 1 wt% fibre-based suspensions: (A) S-120,

579

(B) S-100HP, (C) S-80HP and (D) S-60. The scale bar represents a length of 20 µm.

580

Figure 6. Schematic diagram showing the impact of the particle size on the degree of

581

defibrillation of the cellulosic structure and the state of the hemicellulosic and pectin

582

networks.

583

Figure 7. Viscoelastic behaviours of the 1 wt % fibre-based suspensions S-80HP, S-

584

80LP and S-60. (A) Amplitude sweep, (B) frequency sweep and (C) thermal stability.

585

Legend of figure B is the same as in figure A.

586

Figure 8. (A) Flow curves with increasing and decreasing shear rate and (B) thixotropy

587

test of the 1wt% fibre based suspensions S-80HP, S-80LP and S-60.

588

ELECTRONIC SUPPLEMENTARY DATA:

589

Table S1. Experimental design and measured values for the particle size distribution

590

(D90, D50 and D10).

591

Table S2. ANOVA of the effects that the number of runs and pressure in the inverse of

592

the D50 and D90. A refers to the number of runs and B to the pressure. (*p-values < 0.05

593

indicate significant effects).

594

Fig S1. Light microscopy images at 4-fold of the 1 wt% fibre-based suspensions: (A) S-

595

120, (B) S-100HP, (C) S-80HP and (D) S-60. Arrows indicate pieces of the cell

596

structure. The scale bar represents a length of 500 µm.

597

Fig S.2. Water retention capacity (g of water / g of insoluble matter) of the 1 wt% fibre-

598

based suspensions: S-120, S-100HP, S-80HP and S-60.

599

Fig S.3. Loss factor (tan (δ)) of the 1 wt % fibre-based suspensions S-80HP, S-80LP

600

and S-60. (A) Amplitude sweep, (B) frequency sweep. Legend of figure A is the same

601

as in figure B.

602

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Table 1. Sample

N

P [bar]

D90 [µm]

S-120 S-100 HP S-100 LP S-80 HP S-80 LP S-60

2 2 3 4 7 8

520 1090 680 1270 560 2000

119.6 95.5 98.4 81.5 82.5 60.2

Table 2. Sample S-80 HP S-80 LP S-60

Herschel-Bulkley Parameters k [Pa·s] n τ0 [Pa] 1,35 0,11 0,65 0,79 0,10 0,88 1,82 0,88 0,43

Yield point (LVRend) G' [Pa] G'' [Pa] 23,40 2,80 17,20 2,12 91,10 10,50

γ [%] 0,04 0,16 0,63

Figure 1. 1

Figure 2.

2

Figure 3.

3

Figure 4.

4

Figure 5.

5

Figure 6

6

Figure 7

7

Figure 8

8

HIGHLIGHTS •

A particle size (D90) between 60 and 120 µm was achieved by microfluidization

•

Viscoelastic and thermal stable suspensions were obtained for D90 ≤ 80 µm.

•

At a D90 of 60 µm release of soluble fibre improved structure formation.

•

Functionality of pea fibre may be modulated by tuning particle size.

IMPACT

OF

MICROFLUIDIZATION

ON

THE

FUNCTIONAL PROPERTIES OF PEA HULL FIBRE Morales-Medina, R., Dong, D., Schalow S., Drusch S.

Compliance with ethical standards: The authors declare no conflict of interest.

MICROSTRUCTURE

AND