Thermal denaturation kinetics of whey proteins in reverse osmosis and nanofiltration sweet whey concentrates

Accepted Manuscript Thermal denaturation kinetics of whey proteins in reverse osmosis and nanofiltration sweet whey concentrates Melanie Marx, Ulrich Kulozik PII:

S0958-6946(18)30123-7

DOI:

10.1016/j.idairyj.2018.04.009

Reference:

INDA 4311

To appear in:

International Dairy Journal

Received Date: 14 February 2018 Revised Date:

19 April 2018

Accepted Date: 23 April 2018

Please cite this article as: Marx, M., Kulozik, U., Thermal denaturation kinetics of whey proteins in reverse osmosis and nanofiltration sweet whey concentrates, International Dairy Journal (2018), doi: 10.1016/j.idairyj.2018.04.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

ACCEPTED MANUSCRIPT

Thermal denaturation kinetics of whey proteins in reverse osmosis and nanofiltration sweet whey concentrates Melanie Marx*,a,b and Ulrich Kulozika, b a

Technical University of Munich, Chair for Food and Bioprocess Engineering, Freising,

ZIEL Institute for Food & Health, Technical University of Munich, Weihenstephaner Berg

1, Freising, Germany

*Corresponding author: Melanie Marx [email protected]

Tel.:

+49 8161-71 3855

M AN U

E-Mail:

SC

b

RI PT

Germany

Abstract:

Shelf-stable whey concentrates produced by membrane filtration represent an energy efficient alternative to whey powder. However, to obtain products with sufficient microbiological shelf-life, preservation of concentrates is necessary. The idea of this study was, therefore, to

TE D

investigate the influence of ionic composition and dry matter (DM) of whey concentrates on thermal stability of the major whey proteins. Taking the thermal impact of heating up into account, denaturation kinetics of β-lactoglobulin and α-lactalbumin were determined in reverse osmosis (RO) and nanofiltration (NF) whey concentrates with DM contents of 12-30%

EP

at heating temperatures of 90 and 125 °C. Denaturation caused by heating up was strongly increased with increasing DM of both types of concentrates. During holding at 90 °C, whey proteins showed a higher thermal stability in RO concentrates as compared to NF concen-

AC C

trates. However, denaturation rates at 125 °C of whey proteins were lower in NF than in RO concentrates.

ACCEPTED MANUSCRIPT 1

1. Introduction Whey proteins are important additives for various food applications because of their techno-

3

functional properties and their high content of essential amino acids. Evaporation and subse-

4

quent spray drying are still the methods of choice to obtain whey proteins in a stable form.

5

However, spray drying is the most energy intensive process step in dairy industry. The solu-

6

bility of α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) is, furthermore, strongly affected at

7

high outlet gas temperatures (100-120°C) during spray drying (Anandharamakrishnan, Rielly,

8

& Stapley, 2008).

9

Using energy-efficient membrane filtration performed at cold temperatures (10 °C), the whey

10

proteins are kept in the native state in contrast to evaporation performed at much higher tem-

11

peratures (60-71 °C) (Jelen; Plock, 1994). The composition of the concentrate is determined

12

by the retention behavior of the used membrane and, thus, can be adapted to its further appli-

13

cation. By means of reverse osmosis (RO), all solutes of the whey are retained by the mem-

14

brane. Therefore, RO whey concentrates and concentrates produced by evaporation show al-

15

most the same composition. Using nanofiltration (NF), however, concentration and partial

16

demineralisation of whey can be combined in one process. A reduction in ash content of 40-

17

60% is obtainable for a concentration factor of 4-5 during NF of whey (Gernigon, Schuck,

18

Jeantet, & Burling, 2011).

19

By skipping the energy-intensive spray-drying step after concentration, huge amounts of en-

20

ergy could additionally be saved. In contrast to whey powder, the water activity of whey con-

21

centrate, however, is too high to prevent microbial growth. Therefore, thermal treatment of

22

concentrates is necessary to guarantee sufficient shelf-life.

23

To keep whey protein denaturation during heat treatment of whey concentrates as low as pos-

24

sible, it is necessary to determine the denaturation rate of the major whey proteins in the re-

25

spective heating medium. The thermal denaturation of α-La and β-Lg is a complex process

26

described in detail by De Wit (2009), Tolkach & Kulozik (2007), Havea, Singh, & Creamer

27

(2000), Permyakov & Berliner (2000) and Schokker, Singh, & Creamer (2000).

28

In previous studies, the thermal denaturation behavior of whey proteins in whey, whey con-

29

centrated by evaporation, and whey protein solutions has been investigated (Dickow, Kauf-

30

mann, Wiking, & Hammershøj, 2012; Tolkach & Kulozik, 2007; Plock, Spiegel, & Kessler,

31

1997; Dannenberg, 1986). However, based on these studies, it is not possible to predict

32

thermal denaturation behavior of whey proteins in whey concentrated by RO or NF up to 30%

33

DM. On the one hand, whey concentrated by membrane filtration contains a higher concentra-

34

tion of native whey proteins than whey concentrated by evaporation which influences thermal

AC C

EP

TE D

M AN U

SC

RI PT

2

1

ACCEPTED MANUSCRIPT stability. On the other hand, nature and concentration of solutes in the heating medium greatly

36

influence the thermal stability of whey proteins.

37

Lactose accounts for about 75% of the dry matter of whey and whey concentrates and is

38

known to stabilize protein structures (Shimizu & Smith, 2004). However, the stabilizing ef-

39

fect of lactose on α-La is strongly dependent on the type of the heating medium (Anema, Lee,

40

& Klostermeyer, 2006; Plock, Spiegel, & Kessler, 1998). Another factor affecting whey

41

protein denaturation is the protein concentration itself; the higher the concentration of whey

42

proteins, the higher the reaction rate for the denaturation (Wolz & Kulozik, 2015; Verheul,

43

Roefs, & Kruif, 1998). By concentrating whey using RO or NF, the concentration of whey

44

proteins in whey increases from 0.7% to 4% in whey concentrates with 30% DM. Further-

45

more, the salt concentration of whey concentrates represents an important factor on whey pro-

46

tein denaturation. The effect influencing the thermal stability of whey proteins is also deter-

47

mined by the nature of the ionic components. Using NF, the concentration of monovalent ions

48

in the whey concentrates decreases strongly, whereas losses of multivalent ions and lactose

49

are negligible. Calcium is generally known to accelerate the heat-induced aggregation of β-Lg

50

(Petit, Herbig, Moreau, & Delaplace, 2011; Simons et al., 2002; Visser, 1992), but high con-

51

centrations of calcium can also result in a deceleration of protein unfolding (Visser, 1992).

52

Sodium chloride increases the denaturation temperature of β-Lg with increasing concentration

53

(Nicorescu et al., 2008). Different ions in variable concentrations influence the shielding ef-

54

fect of electric charges of proteins in general which vary as a function of pH. Antagonistic

55

interactions between the different salts and lactose are expected to affect the protein stability

56

in whey concentrates. The lower ionic strength of NF concentrates compared to RO concen-

57

trates might have a positive effect on the thermal stability of whey proteins. The hypothesis,

58

therefore, is that the thermal stability of whey proteins in whey, RO and NF whey concen-

59

trates varies because of the differences in their low molecular solute composition.

60

In addition to the composition of the heating medium, the reaction rate of the denaturation

61

reaction is strongly influenced by the applied process conditions. The treatment temperature

62

has a strong effect on the denaturation reaction of the whey proteins. With increasing tem-

63

perature, the reaction rate increases, whereby the rate is determined by the unfolding reaction

64

at lower temperatures (≤ 90 °C) and by the aggregation reaction at higher temperatures

65

(˃ 90 °C) (Tolkach & Kulozik, 2007; Anema et al., 2006; Schokker, Singh, & Creamer, 2000;

66

Plock et al., 1997).

AC C

EP

TE D

M AN U

SC

RI PT

35

2

ACCEPTED MANUSCRIPT Thus, depending on the heating temperature and rate as well as the composition of the medi-

68

um, the heating-up phase to the desired temperature can already lead to thermal denaturation

69

of whey proteins.

70

In previous studies, in which also the reaction kinetics of whey protein denaturation were in-

71

vestigated by means of the same heating system as in this study, the thermal impact of the

72

heating-up phase was not considered. This means that the heat-holding phase starts when the

73

heating-up phase of the samples is started (Leeb, Haller, & Kulozik, 2018; Spiegel & Huss,

74

2002). However, the heating media in these studies were protein solutions (protein concentra-

75

tion 5-10%) without salts or with only low ionic strength. At these protein concentrations,

76

proportions of denatured proteins after the heating-up phase are negligible. Due to the high

77

concentration of solutes and the high ionic strength of whey concentrates, thermal denatura-

78

tion of whey proteins is, however, expected to be enhanced and might occur to a considerable

79

extent during the phase of heating-up to high temperatures (> 100 °C).

80

Therefore, we decided to follow the recommendation of Whitaker (2003) and take the heat-

81

ing-up phase into account by immediate cooling of the samples after the heating-up phase.

82

The remaining concentration of native protein after heating up was used as reference value for

83

the calculation of the reaction kinetics.

84

The aim of this study was, therefore, to investigate the influence of the ionic composition and

85

the DM content of whey concentrates on thermal denaturation kinetics of whey proteins, con-

86

sidering the thermal impact of the heating-up phase.

SC

M AN U

TE D

87

RI PT

67

2. Materials & Methods

2.1 Preparation of RO and NF whey concentrates with defined dry matter contents

89

Sweet whey from hard cheese production was obtained from the dairy MEGGLE Wasserburg

90

GmbH & Co. KG (Wasserburg, Germany). Table 1 shows the composition of the sweet whey.

91

Sweet whey was concentrated by means of RO or NF up to a DM content of 30% on a pilot

92

scale at a temperature of 10 °C and transmembrane pressure of 40 bar (RO) or 20-30 bar

93

(NF). Permeates of RO and NF were collected and used for redilution of the retentates to de-

94

fined DM contents of 12-30%. The salt retention of the used RO membranes was > 99.2%

95

based on standard testing conditions (FILMTEC™ SW30-2540, FilmTec Corporation, Edina,

96

MN, USA, and Koch TFC®-SW 2.5”, Koch Membrane Systems, Aachen, Germany). Table 1

97

summarizes the composition of the RO whey concentrates.

98

The salt rejection of the used NF membrane of 98% was slightly lower as compared to the RO

99

membrane (GE DK Series Thin Film Membrane, General Electric Company, Fairfield, CT,

100

AC C

EP

88

USA). The composition of the NF whey concentrates is depicted in Table 2. 3

ACCEPTED MANUSCRIPT After concentration, the pH-value of the whey concentrates was adjusted to 6.5 by dropwise

102

addition of 1 M or 0.1 M NaOH or HCl with vigorous stirring.

103

2.2 Analytical Methods

104

Lactose concentration of feed, permeate and retentate from RO and NF whey concentrates

105

was analyzed by high-performance liquid chromatography using an Aminex HPX-87H

106

(300 x 7.8 mm) column (Bio-Rad Laboratories GmbH, München, Germany) and an Agilent

107

1100 chromatograph (Santa Clara, CA, USA). Using a nitrogene analyser vario MAX cube,

108

the total protein content was measured by determination of the total nitrogen concentration

109

with conversion to protein content (Elementar Analysensysteme GmbH, Langenselbold,

110

Germany) according to the method of Dumas. For determining the concentration of calcium,

111

sodium and potassium flame photometry was performed by means of a flame photometer

112

ELEX 6361 (Eppendorf AG, Hamburg, Germany). The total dry matter content was measured

113

by differential weighing using a smart turbo micro wave dryer (CEM GmbH, Kamp-Lintfort,

114

Germany). To determine and adjust the pH-value of the solutions, a pH meter (Xylem Analyt-

115

ics Germany Sales GmbH & Co. KG, Weilheim, Germany) was used.

116

2.3 Thermal treatment of whey and whey concentrates

117

Four mL of whey or whey concentrates were filled in stainless steel tubes for thermal treat-

118

ment. Heat treatment was performed by means of a water bath (WB 22, Memmert

119

GmbH + Co. KG, Schwabach, Germany) at heating temperatures < 100 °C. After the thermal

120

treatment, the samples were put into ice water for fast cooling. A pressure vessel heated by

121

saturated steam was used for treatment temperatures > 100 °C. Using cold tap water, the sam-

122

ples were cooled. In the study of Behringer (1989), the pressure vessel is explained in detail.

123

By use of a data logger connected to a temperature sensor in a reference tube, the temperature

124

profile of the thermal treatment was recorded. Marx & Kulozik (2018) presented a typical

125

temperature profile of heat treatment in the pressure vessel at a temperature of 125 °C. When

126

the desired treatment temperature, e.g., 125 °C, was reached, the heat holding phase started.

127

To subtract the effect of the heating phase, the heat treatments were also performed without

128

holding phase. In this case, the samples were immediately cooled down after reaching the

129

desired treatment temperature. This treatment was used as zero point whose concentration is

130

defined as c0 as proposed by Whitaker (2003). All heat treatment experiments were conducted

131

at least in triplicate.

AC C

EP

TE D

M AN U

SC

RI PT

101

4

ACCEPTED MANUSCRIPT 2.4 Analysis of the concentration of native whey proteins

133

The concentrations of native α-La and β-Lg were determined by means of reversed phase-

134

high performance liquid chromatography (RP-HPLC) as described by Toro-Sierra, Tolkach,

135

& Kulozik (2013). To evaluate the effect of the heating-up phase on the nativity of the whey

136

proteins, the concentrations of native protein were measured in the untreated sample cuntreated

137

and after the heating-up phase cheat up.

138

For the calculation of reaction kinetics of thermal denaturation of whey proteins, the protein

139

concentration after defined heat-holding times ct was additionally measured. As already men-

140

tioned, the concentration of native whey proteins after heating up cheat up was defined as initial

141

concentration c0 for calculation of reaction kinetics in Section 2.5.

142

The contents of native α-La and β-Lg were determined after the denatured proteins were pre-

143

cipitated at pH 4.6 and separated using a syringe filter with a cut-off of 0.45 µm, while the

144

native whey proteins remained soluble and permeated through the filter.

145

The degree of denaturation (DD) after heating up was calculated by equation (1):

= 1−

M AN U

SC

RI PT

132

× 100%

(1)

where Cuntreated is the initial protein concentration and Ct is the concentration of native β-Lg

147

after a heating time, t.

148

2.5 Evaluation of reaction kinetics

149

The changes in the native protein concentration caused by thermal treatment can be mathe-

150

matically described. The reaction rate of the denaturation reaction is determined by the reac-

151

tion order, n, the native protein concentration, c, and the temperature-dependent rate constant,

152

kT/n, and can be described by equation (2):

153

−

154

For a reaction order of n ≠ 1.0 the integration of equation (2) results in equation (3):

=

=

155



.

AC C

/

EP

TE D

146

/

× ! × "# − 1$ ×

(2)

%

"

$

+ 1

(3)

156

and for n = 1.0, equation (4) is obtained by integrating equation (2):

157

ln

158

The reaction order of the denaturation reaction of α-La and β-Lg was adjusted to a value re-

159

sulting in a fit of the data points by straight lines according to the linear equations (2) or (3).

160

The rate constants, kT/n, of the thermal denaturation of α-La and β-Lg at the different tempera-

161

tures were calculated from the slopes of the regression lines.

= −

/

× !

(4)

5

ACCEPTED MANUSCRIPT 162

The temperature dependence of the concentration independent reaction constant kT/n can be

163

described by the Arrhenius equation:

164

=

/

%/

× '

()* +×,

(5)

where k0/n is the pre-exponential factor, Ea is activation energy, R is the universal gas constant

166

(8.314 J mol-1 K-1) and T is absolute temperature.

167

2.6 Statistical analysis

168

All experiments were at least performed in triplicate. Linear regression of the protein denatur-

169

ation data was performed using OriginPro 2015 (OriginLab Corporation, Northampton, MA,

170

USA) for Windows. All data points in figures show mean values ± standard deviation calcu-

171

lated by use of the Student’s t-distribution. The temperature dependent rate constants kT/n

172

(Tables 4 and 5) are given with a 95% confidence interval calculated from the regression

173

lines.

SC

3. Results and Discussion

M AN U

174

RI PT

165

In the present study, the denaturation kinetics of the major whey proteins α-La and β-Lg in

176

RO and NF whey concentrates were investigated. Since environmental conditions are known

177

to affect the thermal stability of whey proteins, the impact of the DM content (12-30% DM)

178

and the mineral composition of whey concentrates on heat stability was studied during heat

179

treatment at 90 °C and 125 °C. There is a change in temperature dependence of the denatura-

180

tion reaction at temperatures close to 95 °C observed in milk and cheese whey (Hillier & Lys-

181

ter, 1979). Thus, the influence of both temperature ranges was studied. Variations of the min-

182

eral composition were achieved by concentration of whey by means of NF. By concentrating

183

whey, the water content is reduced not only, but also the pH-value. During the concentration

184

of whey by RO from 6 to 30% DM, the pH decreases from 6.5 to about 5.9. The effect of the

185

NF on the pH is similar, but a little less pronounced. Thermal resistance of the whey proteins

186

is strongly affected by the pH of the heating medium (O'Kennedy & Mounsey, 2009; Leeb et

187

al., 2018). To avoid interactive effects, we adjusted the pH of all concentrates to the initial pH

188

of the whey (pH 6.5) prior to the heat treatment.

189

The integral method was used to identify the reaction order of the denaturation reaction of α-

190

La and β-Lg in whey and the differently composed whey concentrates. When the results are

191

plotted according to equation (3) or (4) with reaction order n as a variable, n is done such that

192

the data fulfill the requirement of a linear equation yielding a straight line in the data plot. For

193

the range of investigated denaturation temperatures, a reaction order for β-Lg of n=1.5 was

AC C

EP

TE D

175

6

ACCEPTED MANUSCRIPT found to be suitable for the approximation of the measured data by equation (3), which is in

195

agreement with the results of previous studies carried out in concentrated whey, skim milk

196

and whey protein solutions at neutral pH (Leeb et al., 2018; Wolz & Kulozik, 2015; Tolkach

197

& Kulozik, 2007; Anema et al., 2006; Plock et al., 1998). The denaturation reaction of α-La

198

was, however, best described by a reaction of the order of n=1.0. This is in accordance with

199

the results of Anema & McKenna (1996) and Dannenberg & Kessler (1988), who investigated

200

the reaction kinetics of whey proteins in milk. In addition, the thermal impact of the heating-

201

up phase was assessed by immediate cooling after the heat-holding temperature was reached.

202

The remaining concentration of native whey protein was used as reference value for determi-

203

nation of reaction kinetics.

204

3.1 Thermal denaturation of whey proteins during heating-up phase

205

Thermal denaturation of whey proteins starts already during the heating-up phase to the

206

desired heat-holding temperature. Therefore, we determined the degrees of denaturation (DD)

207

of α-La and β-Lg after heating up to 90 and 125 °C (Fig. 1). As shown in Fig. 1 A, the DD of

208

β-Lg after heating up to 90 °C is dependent on the DM content and the ionic composition of

209

the concentrates. In RO concentrates, an increase of the DM from 6 to 18% results in an in-

210

crease of the DD from 40 to 50%, whereas further increase of the DM up to 30% leads to in-

211

creased thermal stability of β-Lg, which might be due to a protective effect of the non-protein

212

soluble components, which Anema et al. (2006) also observed in milk.

213

However, by increasing the DM from 12 to 30% in NF whey concentrates, the DD of β-Lg is

214

increasing by 15% in total. Thus, no protective effect of the increased DM content can be ob-

215

served in NF concentrates, which might be explained by the reduced concentration of NaCl in

216

NF concentrates (Suárez, Lobo, Alvarez, Riera, & Álvarez, 2009; Suárez, Lobo, Álvarez,

217

Riera, & Álvarez, 2006).

218

By heating up to 125 °C (Fig. 1 C), the DD of β-Lg rises from 75% to 95% with increasing

219

the DM from 6 to 30% independent of the used membrane method. We explain this accelera-

220

tion of the thermal denaturation of β-Lg at 125 °C by the increased initial protein content of

221

concentrates with higher DM contents on the one hand and the reduced stabilizing effect of

222

lactose at temperatures > 110 °C (Anema et al., 2006).

223

Table 3 shows the residual native protein concentration of β-Lg after the heating-up phase to

224

125 °C without heat holding in whey, RO and NF whey concentrates. The remaining native

225

protein concentration of RO concentrates is decreasing with increasing DM content from 12

226

to 30%, although the RO whey concentrate with 30% DM content contains the highest initial

227

protein concentration before heating (Tables 1 and 2). In NF concentrates, the native protein 7

AC C

EP

TE D

M AN U

SC

RI PT

194

ACCEPTED MANUSCRIPT concentration is increasing with increasing DM content from 12 to 24%. Compared to the NF

229

concentrates with 12-24% DM, only the NF concentrate with 30% DM shows a significantly

230

reduced remaining protein concentration, which is similar to that of whey. The accelerated

231

denaturation reaction of β-Lg in NF concentrate with 30% DM might be explained by the

232

higher initial protein content and the reduced concentration of monovalent ions as also de-

233

scribed for a heating temperature of 90 °C. Since the initial protein concentration of whey is

234

lower than the protein concentration of the concentrates, β-Lg shows the lowest DD at 125 °C

235

after the heating-up phase in whey.

236

As can be seen from Figs. 1 B and D, α-La showed a higher thermal stability than β-Lg at

237

both heating temperatures. By heating up to 90 °C, only 10% of α-La are denatured in whey

238

as well as RO and NF whey concentrates with 12% DM. With increasing DM from 18 to

239

30%, the DD increases by about 10% for RO and NF whey concentrates. The thermal stability

240

of α-La is slightly higher in RO than in NF concentrates which show the highest DD of 22%

241

in NF concentrate with 30% DM. During the heating-up phase to 125 °C, the influence of the

242

DM as well as of the composition of the concentrate on the thermal stability of α-La is in-

243

creased as compared to 90 °C. The lowest DD of 24% was measured in whey. In RO and NF

244

whey concentrates, the DD of α-La was significantly increased for higher DM contents and

245

reached a maximum value of 64% in RO concentrate with 30% DM. In NF concentrates, the

246

DD showed a linear dependence on the DM content, which was slightly less pronounced than

247

in RO concentrates. The remaining concentration of native α-La after heating up to 90 or

248

125 °C is shown in Table 3. In contrast to β-Lg, the remaining concentration of native α-La

249

increased with increasing DM contents in all RO and NF concentrates analyzed.

250

Based on the remaining protein concentrations after heating up, reaction kinetics for the dena-

251

turation of whey proteins during heat-holding at 90 or 125 °C were calculated. Since the main

252

objective of the study was to compare RO and NF concentrates with different DM contents,

253

we also determined the reaction kinetics for the concentrates showing already high DD after

254

heating up to 125 °C.

255

3.2 Reaction kinetics of β-Lg in whey and RO whey concentrates

256

Figs. 2 A and B show the denaturation of β-Lg in sweet whey as a control, i.e., prior to the

257

concentration, and RO whey concentrates (12-30% DM) at a heating temperature of 90 and

258

125 °C plotted as a reaction of the order of n=1.5 in dependence of the heat-holding time,

259

which starts when the desired heating temperature is reached in the respective sample. At a

260

heating temperature of 90 °C, β-Lg showed a similar denaturation behavior in whey and RO

261

concentrates with 12, 18 and 30% DM and in whey, whereas an increase of the DM from 18 8

AC C

EP

TE D

M AN U

SC

RI PT

228

ACCEPTED MANUSCRIPT to 24% leads to increased denaturation. As several studies have shown, the denaturation reac-

263

tion of β-Lg is greatly dependent on the environmental conditions of the heating medium,

264

especially in the unfolding limited temperature area (≤ 90 °C) (Dissanayake, Ramchandran,

265

Donkor, & Vasiljevic, 2013; Erabit, Flick, & Alvarez, 2013). When β-Lg is heated in pure

266

protein solutions, an increase of the protein concentration results in an acceleration of the de-

267

naturation reaction (Anema et al., 2006; Wolz & Kulozik, 2015). However, in complex solu-

268

tions, such as whey or whey concentrates, the non-protein soluble components, which account

269

for 85-90% of the total DM content (Tables 1, 2 and 3), also influence the denaturation behav-

270

ior of β-Lg to a great extent. Lactose has a protective effect on the whey proteins that can

271

overlay the negative effect of increasing native protein concentration with increasing dry mat-

272

ter (Plock et al., 1998; Anema et al., 2006). Depending on their nature and concentration, salts

273

can either promote or decelerate whey protein denaturation. However, by using RO for con-

274

centration of whey, the ratio between protein content and the content of non-protein soluble

275

components remains constant during concentration. It is assumed that this protective effect of

276

the non-protein dry matter, especially the lactose, is the reason for the similar denaturation

277

rates of β-Lg in RO whey concentrates (12, 18, 30% DM) and whey at 90 °C. However, β-Lg

278

shows a decreased thermal stability in RO whey concentrate with 24% DM.

279

By increasing temperature from 90 to 125 °C, the denaturation reaction of β-Lg is accelerated

280

according to the Arrhenius equation (equation (5)) which becomes apparent by the steeper

281

slopes of the regression lines (Fig. 2 B). Furthermore, the impact of the DM content on the

282

denaturation reaction of β-Lg changes by the temperature increase. As can be seen from Fig. 2

283

B, higher DM contents seem to lead to retarded denaturation of β-Lg at 125 °C, except for

284

whey. Knowing that the amount of native β-Lg at the beginning of the heat holding time (0 s)

285

(Table 3) decreases with increasing DM content of the RO concentrates due to strong dena-

286

turation during heating up (Fig. 1 C), it becomes clear that the native protein concentration is

287

the main influencing factor for the different slopes of the regression lines in Fig. 2 B. Due to

288

its low initial protein concentration, the protein concentration of whey was very similar to that

289

of RO concentrates with 24 and 30% DM after heating up. The number of collisions per time

290

unit is responsible for the extent of denaturation in the aggregation limited temperature area.

291

3.3 Reaction kinetics of β-Lg in NF whey concentrates

292

Figs. 3 A and B show the denaturation of β-Lg in NF whey concentrates (12-30% DM) at a

293

heating temperature of 90 and 125 °C plotted as a reaction of the order of n=1.5. Changes in

294

the DM content of NF concentrates have a stronger impact on the denaturation of β-Lg as

295

compared to RO concentrates at a heating temperature of 90 °C. By increasing the dry matter 9

AC C

EP

TE D

M AN U

SC

RI PT

262

ACCEPTED MANUSCRIPT content of NF concentrate from 12 or 18 to 24% DM, the thermal stability of β-Lg decreases

297

significantly (P < 0.05). However, when the DM content was further increased, we detected

298

the lowest level of β-Lg denaturation in NF concentrates with 30% DM at 90 °C (Fig. 3 A).

299

Since the denaturation of β-Lg is greatly influenced by the composition of the heating medi-

300

um at temperatures ≤ 90 °C as described for RO concentrates, we suppose that the complex

301

and changing composition of protein, lactose and salt concentration in the NF whey concen-

302

trates is responsible for the lower heat stability of β-Lg. Compared with RO whey concen-

303

trates with the same DM contents, NF whey concentrates are characterised by a reduced con-

304

centration of monovalent ions and, consequently, show slightly higher lactose and protein

305

concentrations (Tables 2 and 3). Depending on the DM content, the concentration of the mon-

306

ovalent anion chloride is reduced by 10-46% (data not shown) and the monovalent cations

307

sodium and potassium are reduced by 33-48%. In contrast, the reduction in multivalent cati-

308

ons, e.g., calcium, as a result of concentration by NF is negligible. Consequently, the ratio of

309

protein to lactose to the different salts is changing during concentration by means of NF. The

310

findings in literature show that the influence of the ionic strength on the denaturation behavior

311

of β-Lg is complex, because it is strongly dependent on the nature and the concentration of

312

the present ions (O'Kennedy & Mounsey, 2009; Simons et al., 2002; Rham & Chanton, 1984;

313

Petit et al., 2011). O'Kennedy & Mounsey (2009) observed that the level of β-Lg denaturation

314

and aggregation in a 1% (w/w) β-Lg solution was promoted by increasing concentrations of

315

calcium ions, while an increase of the ionic strength by use of NaCl overlaid this effect. How-

316

ever, Plock (1994) found a stabilizing effect of calcium at concentrations between 0.3 mg mL-

317

1

318

again to accelerated denaturation. We suppose that the complex interaction of ionic strength,

319

lactose and protein concentration in NF whey concentrates results in a differing thermal sta-

320

bility of β-Lg in the concentrates with increasing DM from 18 to 30%.

321

At a treatment temperature of 125 °C the DM also significantly affects thermal stability of β-

322

Lg (Fig. 3 B). β-Lg shows the highest thermal stability in NF whey concentrate with a DM

323

content of 30%. Minimal thermal stability of β-Lg was observed for NF whey concentrates

324

with 18 and 24% DM content. As shown in Fig. 1 C, a high proportion of β-Lg in NF whey

325

concentrates undergoes thermal denaturation during the heating-up phase to 125 °C. Except

326

for 24% DM (P < 0.05), RO and NF whey concentrates show the same DD after the heating-

327

up phase (Fig. 1 C). Thus, the remaining native concentration of β-Lg in RO and NF whey

328

concentrates after heating up only differs significantly in concentrates with 24% DM (Table

329

3). NF concentrates with 18% and 24% DM show the highest concentrations of native β-Lg at

TE D

M AN U

SC

RI PT

296

AC C

EP

and 2-3 mg mL-1 in evaporated whey. Further increases in the calcium concentration led

10

ACCEPTED MANUSCRIPT the beginning of the heat-holding phase followed by 12% and 30% DM content. As already

331

described for RO concentrates, it is assumed that the concentration of native β-Lg after the

332

heating-up phase determines the denaturation rate during the heat-holding phase at 125 °C.

333

The effect of the protein concentration seems to be more important than the effect of the com-

334

position of the heating medium at 125 °C, which is in accordance with the results of Plock

335

(1994) and Dissanayake et al. (2013).

336

3.4 Temperature-dependent rate constants of the denaturation reaction of β-Lg in whey, RO

337

and NF whey concentrates at 90 °C and 125 °C

338

The temperature-dependent reaction rate constants kT/1.5 for the thermal denaturation of β-Lg

339

were calculated from the regression lines shown in Figs. 2 and 3. To illustrate the effect of the

340

DM content and of the composition of whey concentrates on the thermal denaturation of β-

341

Lg, the temperature-dependent reaction rate constants for β-Lg in whey, RO and NF whey

342

concentrates (12-30 % DM) are summarized in Table 4.

343

At 90 °C the lowest reaction rate constants were found for NF whey concentrate with 30%

344

DM (11.2 x 10-3 s-1), whey (11.7 x 10-3 s-1) and RO whey concentrate with 12% DM

345

(12.6 x 10-3 s-1), which were not statistically different (P ≥ 0.05). In NF whey concentrate with

346

24% DM, we found the highest reaction rate constant with 30.4 x 10-3 s-1. In both types of

347

concentrates, the highest rate constants were found for a DM of 24% at a heating temperature

348

of 90 °C. Comparing concentrates with the same DM content, it becomes clear that thermal

349

denaturation of β-Lg is accelerated in NF concentrates for 12, 18 and 24% DM. Leeb et al.

350

(2018) determined a temperature dependent rate constant of 31.4 x 10-3 s-1 for a 5% β-Lg so-

351

lution at pH 6.8 and a heating temperature of 90 °C, which is in good agreement with our re-

352

sults for NF whey concentrate with 24% DM, which had a total protein content of 3.16%.

353

At 125 °C, the lowest reaction rate constants of 33.7 x 10-3 s-1 were again found for NF whey

354

concentrate with 30% DM. We determined the highest reaction rate constants in RO whey

355

concentrates with 12 (98.9 x 10-3 s-1) and 18% DM (85.6 x 10-3 s-1), which were not statistical-

356

ly different (P ≥ 0.05). In contrast to the results obtained at a treatment temperature of 90 °C,

357

the rate constants in RO concentrates tend to be higher than in NF whey concentrates for the

358

denaturation of β-Lg at 125 °C. In our opinion, this difference in the thermal stability of β-Lg

359

could be caused by the reduced salt concentration of NF concentrates as compared to RO con-

360

centrates. Our findings are of the same order of magnitude as the results of Tolkach

361

& Kulozik (2007), who determined a rate constant of 45 x 10-3 s-1 for a 5% β-Lg solution at

362

neutral pH and at a temperature of 120 °C.

AC C

EP

TE D

M AN U

SC

RI PT

330

363 11

ACCEPTED MANUSCRIPT 3.5 Reaction kinetics of α-La in whey and RO whey concentrates

365

Fig. 4 shows the time dependent denaturation behavior of α-La in whey and RO whey concen-

366

trates (12-30% DM) at 90 °C and 125 °C as a reaction of the order of n=1. The denaturation

367

rates of α-La are very similar for the different DM at 90 °C. Only in RO whey concentrate

368

with 12% DM and whey, α-La showed a slightly increased thermal stability.

369

The denaturation rate of α-La was significantly accelerated at 125 °C compared to 90 °C. In

370

contrast to β-Lg, α-La showed a higher thermal stability and, therefore, much lower degrees

371

of denaturation after the heating-up phase. Thermal stability of α-La during heat-holding at

372

125°C was only marginally influenced by the DM of the whey concentrates. Solely for con-

373

centrate with 30% DM, the denaturation of α-La was slightly increased compared to concen-

374

trate with 12, 18 or 24% DM and whey, although the initial native protein concentration at the

375

beginning of the heat-holding time continuously increased with increasing DM (Table 3).

376

Anema & McKenna (1996) also reported that the rate constants for the denaturation of α-La

377

in reconstituted whole milk were essentially independent of the protein concentration for tem-

378

peratures > 85 °C.

379

3.6 Reaction kinetics of α-La in NF whey concentrates

380

The denaturation of α-La in NF whey concentrates (12-30% DM) at 90 °C (A) and 125 °C (B)

381

is depicted in Fig. 5. At 90 °C, we found the highest thermal stability of α-La in NF whey

382

concentrates with 30% DM, followed by NF whey concentrate with 12% DM. The fastest

383

denaturation reaction characterized by the steepest slope was determined for NF whey con-

384

centrate with 24% DM. An increase of the temperature to 125 °C results in significantly

385

steeper slopes of the regression lines. From Fig. 5 B it is evident that the denaturation reaction

386

is dependent on the DM content of the NF whey concentrates. α-La shows the same denatura-

387

tion rate for whey concentrates with 12 and 30% DM. However, the denaturation rate was

388

increased for NF concentrates with 18 and 24% DM.

389

As presented in Figs. 4 A and B, the denaturation of β-Lg showed a similar dependence on the

390

DM content of NF whey concentrates as α-La at 90 °C and 125 °C. It is known from literature

391

that the denaturation of α-La is strongly influenced by the presence of β-Lg and the availabil-

392

ity of free and reactive thiol groups. Increasing concentrations of unfolded β-Lg molecules

393

facilitate thiol disulfide interchange reactions between α-La and β-Lg and result in increased

394

levels of irreversible denaturation and aggregation of α-La. Furthermore, the degree of revers-

395

ibility of the denaturation reaction of α-La depends on the ionic environment of the solution

396

(McGuffey, Otter, van Zanten, & Foegeding, 2007). Thus, the differences in α-La denatura-

AC C

EP

TE D

M AN U

SC

RI PT

364

12

ACCEPTED MANUSCRIPT tion rates in NF whey concentrates are probably a consequence of the denaturation of β-Lg,

398

which is greatly influenced by the varying solute concentrations at the different DM contents.

399

3.7 Temperature-dependent rate constants of the denaturation reaction of α-La in whey, RO

400

and NF whey concentrates at 90 °C and 125 °C

401

The temperature dependent denaturation rate constants kT/1.0 for the thermal denaturation of α-

402

La were calculated from the regression lines in Figs. 4 and 5. The reaction rate constants for

403

the thermal denaturation of α-La in whey, RO and NF whey concentrates (12-30 % DM) are

404

presented in Table 5. The lowest reaction rate constants for the denaturation of α-La were

405

found for whey with 3.1 x 10-3 s-1 and for RO whey concentrate with 12% DM with 3.2 x 10 -

406

3 -1

407

(1988) determined for the denaturation of α-La in milk at 90 °C.

408

In RO whey concentrates with 18, 24 and 30% DM, as well as in NF whey concentrates with

409

12 and 30% DM, the denaturation rates were not significantly different and ranged between

410

4.3 x 10-3 and 4.9 x 10-3 s-1. NF whey concentrate with 24% DM showed the highest denatura-

411

tion rate of 8.4 x 10-3 s-1 for α-La at 90 °C.

412

At a heating temperature of 125 °C, we found the lowest reaction rate constant again for

413

whey, with 13.2 x 10-3 s-1 which is in good agreement with the rate constant, which Dannen-

414

berg & Kessler (1988) determined for α-La in milk at 120 °C. In RO and NF whey concen-

415

trates, the reaction rate constants for the denaturation of α-La were significantly higher than in

416

whey. The rate constants in RO whey concentrates, that range between 41.0 x 10-3 and

417

53.8 x 10-3 s-1, increased compared to the rate constants in NF whey concentrates, which

418

ranged between 20.3 x 10-3 and 32.6 x 10-3 s-1.

TE D

M AN U

SC

at 90 °C. These findings are in agreement with the results of Dannenberg & Kessler

EP

419

s

RI PT

397

4. Conclusions

The findings of this study provide new insights in the denaturation behavior of the major

421

whey proteins α-La and β-Lg in RO and NF whey concentrates with DM contents of 12-30%.

422

It could be shown that the method used for membrane concentration as well as the DM con-

423

tent have a significant influence on the whey protein denaturation which is dependent on the

424

heating temperature. A positive effect of demineralisation by NF on whey protein stability

425

could be determined for heat treatment at 125 °C. The influence of increasing DM contents on

426

the whey protein denaturation was particularly pronounced during the heating-up phase.

427

Based on the obtained results, it is possible to control the denaturation behavior of α-La and

428

β-Lg in complex whey based solutions.

AC C

420

13

ACCEPTED MANUSCRIPT However, especially β-Lg already shows high DD after heating up to the desired temperature.

430

For industrial applications, where very high proportions of native whey proteins are desired, it

431

therefore appears to be necessary to develop a gentler preservation process for fluid whey

432

concentrate. To produce tailor-made whey concentrates showing the desired shelf-life as well

433

as native whey protein, a two-step preservation process consisting of cold microfiltration and

434

gentle heat treatment could be more suitable in terms of native whey protein. This combined

435

preservation process and its suitability for whey concentrates will therefore be investigated in

436

further studies.

437

Acknowledgement

438

The authors gratefully thank Theodora Brodbeck, Corinna Karl, Franziska Kurz, Melanie

439

Groß, Julia Hintermayer, Anna Müller and Isabella Tallarita for experimental support. This

440

research project was supported by funds of the Federal Ministry of Food and Agriculture

441

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

442

Federal Office for Agriculture and Food (BLE) under the innovation support program (grant

443

number 313.06.01-28-1-74.005-11).

AC C

EP

TE D

444

M AN U

SC

RI PT

429

14

ACCEPTED MANUSCRIPT 445

References

446

Anandharamakrishnan, C., Rielly, C. D., & Stapley, A.G.F. (2008). Loss of solubility of α-

447

lactalbumin and β-lactoglobulin during the spray drying of whey proteins. LWT - Food

448

Science and Technology, 41, 270–277. doi:10.1016/j.lwt.2007.03.004 Anema, S. G., Lee, S. K., & Klostermeyer, H. (2006). Effect of protein, nonprotein-soluble

450

components, and lactose concentrations on the irreversible thermal denaturation of beta-

451

lactoglobulin and alpha-lactalbumin in skim milk. Journal of Agricultural and Food Chem-

452

istry, 54, 7339–7348. doi:10.1021/jf061508+

453

RI PT

449

Anema, S. G., & McKenna, A. B. (1996). Reaction Kinetics of Thermal Denaturation of

Whey Proteins in Heated Reconstituted Whole Milk. Journal of Agricultural and Food

455

Chemistry, 44, 422–428. doi:10.1021/jf950217q

457 458 459 460

Behringer, R. (1989). Über das Absterbeverhalten von Bacillus-Sporen in Milch und

M AN U

456

SC

454

Milchkonzentraten. (PhD thesis). Technical University of Munich, Freising. Dannenberg, F. (1986). Zur Reaktionskinetik der Molkenproteindenaturierung und deren technologischer Bedeutung (Dissertation). Technical University of Munich, Freising. Dannenberg, F., & Kessler, H. G. (1988). Reaction Kinetics of the Denaturation of Whey Proteins in Milk. Journal of Food Science, 53, 258–263. doi:10.1111/j.1365-

462

2621.1988.tb10223.x

463

TE D

461

Dickow, J. A., Kaufmann, N., Wiking, L., & Hammershøj, M. (2012). Protein denaturation and functional properties of Lenient Steam Injection heat treated whey protein concentrate.

465

Innovative Food Science & Emerging Technologies, 13, 178–183.

466

doi:10.1016/j.ifset.2011.11.005

EP

464

Dissanayake, M., Ramchandran, L., Donkor, O. N., & Vasiljevic, T. (2013). Denaturation of

468

whey proteins as a function of heat, pH and protein concentration. International Dairy

469

Journal, 31, 93–99. doi:10.1016/j.idairyj.2013.02.002

AC C

467

470

Erabit, N., Flick, D., & Alvarez, G. (2013). Effect of calcium chloride and moderate shear on

471

β-lactoglobulin aggregation in processing-like conditions. Journal of Food Engineering,

472

115, 63–72. doi:10.1016/j.jfoodeng.2012.09.020

473

Gernigon, G., Schuck, P., Jeantet, R., & Burling, H. (Eds.). (2011). Encyclopedia of Dairy

474

Sciences (Second Edition): Whey Processing | Demineralization. (2nd ed.): Elsevier.

475 476

Hillier, R. M., & Lyster, R. L. J. (1979). Whey protein denaturation in heated milk and cheese whey. Journal of Dairy Research, 46, 95. doi:10.1017/S0022029900016897 15

ACCEPTED MANUSCRIPT 477 478 479

Jelen, P. Encyclopaedia of dairy sciences: Whey processing - utilization and products., pp. 731–737. Leeb, E., Haller, N., & Kulozik, U. (2018). Effect of pH on the reaction mechanism of ther-

480

mal denaturation and aggregation of bovine β-lactoglobulin. International Dairy Journal,

481

78, 103–111. doi:10.1016/j.idairyj.2017.09.006

483 484

Marx, M., & Kulozik, U. (2018). Spore inactivation in differently composed whey concen-

RI PT

482

trates. International Dairy Journal, 76, 1–9. doi:10.1016/j.idairyj.2017.08.007

McGuffey, M. K., Otter, D. E., van Zanten, J. H., & Allen Foegeding, E. (2007). Solubility and aggregation of commercial α-lactalbumin at neutral pH. International Dairy Journal,

486

17, 1168–1178. doi:10.1016/j.idairyj.2007.04.003

487

SC

485

Nicorescu, I., Loisel, C., Vial, C., Riaublanc, A., Djelveh, G., Cuvelier, G., & Legrand, J. (2008). Combined effect of dynamic heat treatment and ionic strength on denaturation and

489

aggregation of whey proteins – Part I. Food Research International, 41, 707–713.

490

doi:10.1016/j.foodres.2008.05.003

M AN U

488

O'Kennedy, B. T., & Mounsey, J. S. (2009). The dominating effect of ionic strength on the

492

heat-induced denaturation and aggregation of β-lactoglobulin in simulated milk ultrafil-

493

trate. International Dairy Journal, 19, 123–128. doi:10.1016/j.idairyj.2008.09.004

494

Petit, J., Herbig, A.-L., Moreau, A., & Delaplace, G. (2011). Influence of calcium on β-

TE D

491

495

lactoglobulin denaturation kinetics: Implications in unfolding and aggregation mecha-

496

nisms. Journal of Dairy Science, 94, 5794–5810. doi:10.3168/jds.2011-4470 Plock, J. (1994). Zum Einfluss von Lactose auf die Denaturierung von Molkenproteinen in

EP

497

Molkenkonzentrat und auf die Ausbildung thermisch induzierter Gelstrukturen.

499

Fortschritt-Berichte VDI Reihe 14, Landtechnik, Lebensmitteltechnik: Vol. 69. Düsseldorf:

500

VDI-Verlag GmbH.

501

AC C

498

Plock, J., Spiegel, T., & Kessler, H. G. (1997). Reaction kinetics of the thermal denaturation

502

of whey proteins in sweet whey concentrated by evaporation. Milchwissenschaft, 85, 678–

503

681.

504

Plock, J., Spiegel, T., & Kessler, H. G. (1998). Influence of the dry matter on the denaturation

505

kinetics of whey proteins in concentrated sweet whey. Milchwissenschaft, 53, 327–331.

506

Rham, O. de, & Chanton, S. (1984). Role of Ionic Environment in Insolubilization of Whey

507

Protein During Heat Treatment of Whey Products. Journal of Dairy Science, 67, 939–949.

508

doi:10.3168/jds.S0022-0302(84)81392-2 16

ACCEPTED MANUSCRIPT 509

Schokker, E. P., Singh, H., & Creamer, L. K. (2000). Heat-induced aggregation of β-

510

lactoglobulin A and B with α-lactalbumin. International Dairy Journal, 10, 843–853.

511

doi:10.1016/S0958-6946(01)00022-X

512

Shimizu, S., & Smith, D. J. (2004). Preferential hydration and the exclusion of cosolvents from protein surfaces. The Journal of Chemical Physics, 121, 1148–1154.

514

doi:10.1063/1.1759615

RI PT

513

Simons, J.-W. F. A., Kosters, H. A., Visschers, W. R., de Jongh, H. H. J., Simons, J.-W. F.A.,

516

Kosters, H. A., … (2002). Role of calcium as trigger in thermal β-lactoglobulin aggrega-

517

tion. Archives of Biochemistry and Biophysics, 406, 143–152. doi:10.1016/S0003-

518

9861(02)00429-0

SC

515

Spiegel, T., & Huss, M. (2002). Whey protein aggregation under shear conditions - effects of

520

pH-value and removal of calcium. International Journal of Food Science & Technology,

521

37, 559–568. doi:10.1046/j.1365-2621.2002.00612.x

522

M AN U

519

Suárez, E., Lobo, A., Alvarez, S., Riera, F. A., & Álvarez, R. (2009). Demineralization of

523

whey and milk ultrafiltration permeate by means of nanofiltration. Desalination, 241, 272–

524

280. doi:10.1016/j.desal.2007.11.087

Suárez, E., Lobo, A., Álvarez, S., Riera, F. A., & Álvarez, R. (2006). Partial demineralization

526

of whey and milk ultrafiltration permeate by nanofiltration at pilot-plant scale. Desalina-

527

tion, 198, 274–281. doi:10.1016/j.desal.2005.12.028

TE D

525

Tolkach, A., & Kulozik, U. (2007). Reaction kinetic pathway of reversible and irreversible

529

thermal denaturation of β-lactoglobulin. Le Lait, 87, 301–315. doi:10.1051/lait:2007012

530

EP

528

Toro-Sierra, J., Tolkach, A., & Kulozik, U. (2013). Fractionation of α-Lactalbumin and βLactoglobulin from Whey Protein Isolate Using Selective Thermal Aggregation, an Opti-

532

mized Membrane Separation Procedure and Resolubilization Techniques at Pilot Plant

533

Scale. Food and Bioprocess Technology, 6, 1032–1043. doi:10.1007/s11947-011-0732-2

534

Verheul, M., Roefs, S.P.F.M., & Kruif, K. G. de. (1998). Kinetics of heat-induced aggrega-

AC C

531

535

tion of β-lactoglobulin. Journal of Agricultural and Food Chemistry, 46, 896–903.

536

Visser, H. (Ed.). (1992). Protein interactions: Developed from the Symposium on Protein

537

Interactions, held at the 201st annual meeting of the American Chemical Society, Atlanta,

538

GA, USA, April 15 - 17, 1991. Weinheim: VCH.

539 540

Whitaker, J. R. (Ed.). (2003). Food Science and Technology: Vol. 122. Handbook of food enzymology: Inactivation of enzymes. New York, NY: Dekker. 17

ACCEPTED MANUSCRIPT 541

Wolz, M., & Kulozik, U. (2015). Thermal denaturation kinetics of whey proteins at high pro-

542

tein concentrations. International Dairy Journal, 49, 95–101.

543

doi:10.1016/j.idairyj.2015.05.008

AC C

EP

TE D

M AN U

SC

RI PT

544

18

ACCEPTED MANUSCRIPT Table 1.

546

Composition of whey and reverse osmosis whey concentrates with dry matter contents of 12,

547

18, 24 and 30%. Data represent mean values ± standard deviations of at least three repli-

548

cates. a

550 551 552

549 Item Whey RO 12 RO 18 RO 24 RO 30 DM, % 5.72 ± 0.12a 12.17 ± 0.24b 18.11 ± 0.18c 24.08 ± 0.13d 30.09 ± 0.31e Total protein, % 0.73 ± 0.08a 1.50 ± 0.09b 2.20 ± 0.14c 2.96 ± 0.12d 3.75 ± 0.11e thereof: α-La, g L-1 0.78 ± 0.04a 1.55 ± 0.14b 2.28 ± 0.15c 3.22 ± 0.26d 4.06 ± 0.27e β-Lg, g L-1 3.27 ± 0.30a 6.53 ± 0.59b 9.33 ± 0.39c 13.04 ± 0.49d 16.46 ± 0.95e -1 Lactose, g L 39.45 ± 2.52a 81.91 ± 5.86b 120.89± 2.86c 162.43 ± 3.24d 220.27± 4.94e Na+, g L-1 0.45 ± 0.08a 0.91 ± 0.17a,b 1.32 ± 0.16b,c 1.60 ± 0.03c 2.07 ± 0.18c K+, g L-1 1.52 ± 0.07a 2.86 ± 0.02b 4.14 ± 0.32c 5.82 ± 0.08d 6.86 ± 0.47e Ca2+, g L-1 0.40 ± 0.01a 0.78 ± 0.02b 1.15 ± 0.05c 1.57 ± 0.02d 1.94 ± 0.04e a Abbreviations are: RO, reverse osmosis whey concentrate at 12%, 18%, 24% and 30% dry matter content; DM, dry matter; Na+, sodium; K+, potassium; Ca2+, calcium. Measured values with the same superscript in one row are not statistically different (P ≥ 0.05).

M AN U

SC

RI PT

545

AC C

EP

TE D

553

19

ACCEPTED MANUSCRIPT 554

Table 2.

555

Composition of nanofiltration whey concentrates with dry matter contents of 12, 18, 24 and

556

30%. Data represent mean values ± standard deviations of at least three replicates. a

1.56 ± 0.09a 6.67 ± 0.37a 84.28 ± 4.84a 0.61 ± 0.05a 2.19 ± 0.02a 0.77 ± 0.03a

3.32 ± 0.23c 14.47 ± 0.79c 184.85 ± 2.46c 0.85 ± 0.07a 2.86 ± 0.078b 1.46 ± 0.04c

2.35 ± 0.08b 9.91 ± 0.31b 131.57 ± 11.12b 0.78 ± 0.07a 2.48 ± 0.14a,b 1.08 ± 0.04b

a

NF 30 30.08 ± 0.65d 3.93 ± 0.39c

RI PT

NF 24 24.41 ± 0.14c 3.16 ± 0.21b,c

4.17 ± 0.31d 18.51 ± 2.07d 230.09 ± 5.58d 1.09 ± 0.13b 3.62 ± 0.13c 1.86 ± 0.08d

Abbreviations are: NF, nanofiltration whey concentrate at 12%, 18%, 24% and 30% dry matter content; DM, dry matter; Na+, sodium; K+, potassium; Ca2+, calcium. Measured values with the same superscript in one row are not statistically different (P ≥ 0.05).

M AN U

557 558 559

NF 12 NF 18 a 12.14 ± 0.23 18.32 ± 0.58b 1.45 ± 0.20a 2.41 ± 0.19a,b

SC

Item DM, % Total protein, % thereof: α-La, g L-1 β-Lg, g L-1 Lactose, g L-1 Na+, g L-1 K+, g L-1 Ca2+, g L-1

AC C

EP

TE D

560

20

ACCEPTED MANUSCRIPT Table 3.

562

Concentration of remaining native β-Lg and α-La in whey, RO and NF whey concentrates

563

(12-30% DM) after heating up to 125 °C. Data represent mean values ± standard deviations of

564

at least three replicates. a

565 566

Concentration of native α-La (g L-1) 0.60 ± 0.03 1.09 ± 0.01 0.96 ± 0.09 1.15 ± 0.30 1.30 ± 0.15 1.30 ± 0.18 1.69 ± 0.05 1.44 ± 0.38 1.92 ± 0.06

a

SC

Whey RO 12 NF 12 RO 18 NF 18 RO 24 NF 24 RO 30 NF 30

Concentration of native β-Lg (g L-1) 0.82 ± 0.02 1.14 ± 0.20 1.17 ± 0.11 1.12 ± 0.40 1.32 ± 0.25 0.79 ± 0.05 1.50 ± 0.06 0.77 ± 0.26 0.89 ± 0.04

M AN U

Item

RI PT

561

Abbreviations are: RO, reverse osmosis whey concentrate at 12%, 18%, 24% and 30% dry matter content; NF, nanofiltration whey concentrate at 12%, 18%, 24% and 30% dry matter content.

AC C

EP

TE D

567

21

ACCEPTED MANUSCRIPT 568

Table 4.

569

Temperature dependent reaction rate constants kT/1.5 for the denaturation of β-Lg in whey, RO

570

and NF whey concentrates. Data represent mean values of three replicates with a 95% confi-

571

dence interval. a

a

Abbreviations are: RO, reverse osmosis whey concentrate at 12%, 18%, 24% and 30% dry matter content; NF, nanofiltration whey concentrate at 12%, 18%, 24% and 30% dry matter content. Measured values with the same superscript in one row are not statistically different (P ≥ 0.05).

M AN U

572 573 574

RI PT

Whey RO 12 NF 12 RO 18 NF 18 RO 24 NF 24 RO 30 NF 30

Temperature dependent reaction rate constants kT/1.5 (10-3 s-1) in whey, RO and NF whey concentrates 90 °C 125 °C 11.7 ± 0.6 a, d 68.2 ± 1.8 a a, d 12.6 ± 2.1 98.9 ± 9.7 b 18.0 ± 0.8 b 45.7 ± 2.7 e a 13.2 ± 1.1 85.6 ± 6.4 b, c 18.3 ± 0.4 b 65.9 ± 5.5 a, d b 19.9 ± 1.2 79.0 ± 9.8 a, c 30.4 ± 1.0 c 65.5 ± 3.5 a, d a 12.7 ± 1.7 60.1 ± 5.0 d 11.2 ± 0.7 d 33.7 ± 4.4 f

SC

Item

AC C

EP

TE D

575

22

ACCEPTED MANUSCRIPT 576

Table 5.

577

Temperature dependent reaction rate constants kT/1.0 for the denaturation of α-La in whey, RO

578

and NF whey concentrates. Data represent mean values of three replicates with a 95% confi-

579

dence interval. a

a

Abbreviations are: RO, reverse osmosis whey concentrate at 12%, 18%, 24% and 30% dry matter content; NF, nanofiltration whey concentrate at 12%, 18%, 24% and 30% dry matter content. Measured values with the same superscript in one row are not statistically different (P ≥ 0.05).

M AN U

580 581 582

RI PT

Whey RO 12 NF 12 RO 18 NF 18 RO 24 NF 24 RO 30 NF 30

Temperature dependent reaction rate constants kT/1.0 (10-3 s-1) in whey, RO and NF whey concentrates 90 °C 125 °C 3.1 ± 0.3 a 13.2 ± 3.0 a a 3.2 ± 0.3 43.1 ± 8.5 b, c, e 4.9 ± 0.4 b, c 20.3 ± 1.6 d b 4.4 ± 0.3 42.1 ± 7.0 b, c, e 5.8 ± 0.5 c 32.0 ± 3.6 e b 4.3 ± 0.7 41.0 ± 3.4 b 8.4 ± 0.8 d 32.6 ± 4.5 e b 4.3 ± 0.5 53.8 ± 7.9 c 4.4 ± 0.5 b 20.7 ± 6.1 a, d

SC

Item

AC C

EP

TE D

583

23

ACCEPTED MANUSCRIPT Figure legends.

585

Fig. 1. Degree of denaturation (DD) of β-Lg (A, C) and α-La (B, D) in whey (●), RO (■) and

586

NF (◆) concentrates after heating-up phase to (A, B) 90 °C or (C, D) 125 °C of the thermal

587

treatment as affected by the DM content. Data points represent means values ± standard devi-

588

ations of three replicates.

589

Fig. 2. Denaturation of β-Lg in whey (○) and RO whey concentrates with 12% (□), 18% (△),

590

24% (▽) and 30% (◇) DM as a reaction of the order of 1.5 at (A) 90 °C and (B) 125 °C. Co-

591

efficients of determination are for 90 °C: ○, R2 = 0.983; □, R2 = 0.953; △, R2 = 0.926;

592

0.981; ◇, R2 = 0.963. Coefficients of determination are for 125 °C: ○, R2 = 0.979; □, R2 =

593

0.922; △, R2 = 0.929;

594

three replicates with standard deviation.

595

Fig. 3. Denaturation of β-Lg in NF whey concentrates with 12% (□), 18% (△), 24% (▽) and

596

30% (◇) DM as a reaction of the order of 1.5 at (A) 90 °C and (B) 125 °C. Coefficients of

597

determination are for 90 °C: □, R2 = 0.977; △, R2 = 0.995; ▽, R2 = 0.989; ◇, R2 = 0.963. Co-

598

efficients of determination are for 125 °C: □, R2 = 0.980; △, R2 = 0.926;

599

R2 = 0.897. Data points represent mean values of at least three replicates with standard devia-

600

tion.

601

Fig. 4. Denaturation of α-La in whey (○) and RO whey concentrates with 12% (□), 18% (△),

602

24% (▽) and 30% (◇) DM as a reaction of the order of n=1.0 at (A) 90 °C and (B) 125 °C.

603

Coefficients of determination are for 90 °C: ○, R2 = 0.962; □, R2 = 0.949; △, R2 = 0.984;

604

R2 = 0.943; ◇, R2 = 0.968. Coefficients of determination are for 125 °C: ○, R2 = 0.912; □,

605

R2 = 0.933; △, R2 = 0.957;

606

of at least three replicates with standard deviation.

607

Fig. 5. Denaturation of α-La in NF whey concentrates with 12% (□), 18% (△), 24% (▽) and

608

30% (◇) DM as a reaction of the order of n=1.0 at (A) 90 °C and (B) 125 °C. Coefficients of

609

determination are for 90 °C: □, R2 = 0.979; △, R2 = 0.980; ▽, R2 = 0.978; ◇, R2 = 0.973. Co-

610

efficients of determination are for 125 °C: □, R2 = 0.990; △, R2 = 0.977;

611

R2 = 0.906. Data points represent mean values of at least three replicates with standard devia-

612

tion.

RI PT

584

SC

0.984; ◇, R2 = 0.922. Data points represent mean values of at least

▽,

R2 = 0.981; ◇,

AC C

EP

TE D

M AN U

▽,

▽,

▽,

▽,

R2 = 0.960; ◇, R2 = 0.960. Data points represent mean values

▽,

R2 = 0.969; ◇,

24

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT