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Modifications induced by controlled storage conditions on whey protein concentrates:

effects on whey protein lactosylation and solubility

Alessandra Gasparini, Martine P. Van Gool, Miranda Bultsma, Sara Cutroneo,

Stefano Sforza, Tullia Tedeschi

PII: S0958-6946(20)30135-7
DOI: https://doi.org/10.1016/j.idairyj.2020.104765
Reference: INDA 104765

To appear in: International Dairy Journal

Received Date: 28 November 2019

Revised Date: 9 May 2020
Accepted Date: 9 May 2020

Please cite this article as: Gasparini, A., Van Gool, M.P., Bultsma, M., Cutroneo, S., Sforza, S.,
Tedeschi, T., Modifications induced by controlled storage conditions on whey protein concentrates:
effects on whey protein lactosylation and solubility, International Dairy Journal, https://doi.org/10.1016/
j.idairyj.2020.104765.

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 1 Modifications induced by controlled storage conditions on whey protein concentrates: effects

2 on whey protein lactosylation and solubility

7 Alessandra Gasparinia, Martine P. Van Goolb, Miranda Bultsmab, Sara Cutroneoa, Stefano Sforzaa,

8 Tullia Tedeschia*

10

11

12

a
13 Department of Food and Drug, University of Parma, Parco Area delle Scienze 27/A, I-43124,

14 Parma, Italy.
b
15 FrieslandCampina, Amersfoort, The Netherlands

16

17

18

19

20 * Corresponding author. Tel.: +39 0521 905406

21 E-mail address: tullia.tedeschi@unipr.it (T. Tedeshi)

22

23

24
25 ________________________________________________________________________________

26 ABSTRACT

27

28 Food processing and storage conditions play an important role in product stability and quality.

29 Chemical and structural modifications may be induced on food proteins. In this context, a study on

30 the effect of storage conditions on milk whey proteins was performed on whey protein

31 concentrates (WPC-35). Samples were stored up to 2 wk and up to 60 °C. Kjeldahl and SDS-PAGE

32 analysis confirmed that the protein content was not affected. With UPLC-MS analysis, lactosylated

33 forms of the proteins were identified and quantified. It was observed that the level of protein

34 glycation increases due to both temperature and duration of storage. All samples were also

35 characterised for amino acid composition and nutritional value determination. Results showed

36 that the nutritional value of the products was not altered, in a consistent way, even in the extreme

37 conditions applied (14 d 60 °C).

38 ________________________________________________________________________________

39
40 1. Introduction

41

42 In the food production chain, storage conditions play an important role in product stability

43 and quality. Trade requires that food must be stored in a suitable way to maintain its quality and

44 shelf life until the final commercialisation. Whey protein concentrates and isolates are usually

45 treated to be stored as a dry powder (i.e., through freeze-drying or spray-drying techniques). In

46 the dry state as compared with liquid products, they are less prone to physicochemical reactions,

47 they are easier to handle and have lower costs of storage and transportation (Norwood,

48 Croguennec, Le Floch-Fouéré, Schuck, & Jeantet, 2018).

49 During storage, crucial parameters for product stability are temperature, relative humidity,

50 water activity, light and oxygen (Moschopoulou, Moatsou, Syrokou, Paramithiotis, & Drosinos,

51 2019). For instance, it is known that storage temperature affects protein structure, inducing

52 protein unfolding and denaturation. Aggregation phenomena could occur, both reversible and

53 irreversible, due to intermolecular covalent (i.e., disulphide bonding) and non-covalent

54 interaction, affecting protein solubility (Nishanthi, Chandrapala, & Vasiljevic, 2017). Moreover, the

55 presence of the same protein in a denatured, native and aggregated state influences the possible

56 reaction between lactose and proteins triggered by the processing. Elevated storage temperature

57 can induce protein lactosylation, i.e., the conjugation between lactose and the proteins’ lysine

58 residues, initiating the Maillard reaction. With respect to the native protein, a denatured protein is

59 supposed to have more residues available for lactosylation, while aggregation reduces them

60 (O’Mahony, Drapala, Mulcahy, & Mulvihill, 2019). Protein lactosylation is an important factor

61 relating to nutritional quality. The nutritional value of the product is affected by lactosylation, that

62 reduces the amount of bioavailable lysine (Pellegrino, Masotti, Cattaneo, Hogenboom, & de Noni,

63 2013) and also decreases protein digestibility (Deng, Wierenga, Schols, Sforza, & Gruppen, 2017).
64 In this context, it is important to determine the right storage conditions to guarantee products’

65 shelf life and quality.

66 To study the correlations of possible molecular modifications induced by processing, it was

67 noted that market products do not report the detailed specifications concerning the way in which

68 they were produced (particularly concerning thermal conditions) and the way in which they have

69 been stored.

70 This paper presents the data from a study performed on a set of bovine whey protein

71 concentrates (WPC-35), prepared and provided by FrieslandCampina (Amersfoort, The

72 Netherlands). These samples were subjected to a controlled time of storage (up to two weeks) and

73 controlled temperatures (up to 60 °C). The overall purpose was to investigate the possible effect

74 of the applied storage conditions on protein content and protein structure. Higher storage

75 temperatures, up to 60 °C, hardly used in a real context, were selected to verify molecular

76 modifications in extreme conditions. The study was developed assessing the effect of storage

77 conditions on: (i) protein content, by nitrogen analysis and SDS-PAGE, (ii) soluble protein fraction,

78 (iii) degree of lactosylation of proteins and (iv) total amino acid profile. This set of analyses allowed

79 to investigate the molecular modifications induced by the storage conditions, and how the

80 nutritional value of the final product can be affected by them.

81

82 2. Materials and methods

83

84 2.1. Reagents

85

86 Sodium sulphate, boric acid, dithiothreitol (DTT), α-chymotrypsin from bovine pancreas, DL-

87 norleucine, L-cysteic acid, hydroxyproline, iodacetamide, hydrobromic acid, 5-methyl tryptophan,

 88 hydrochloric acid, ammonium hydrogen carbonate (NH4HCO3), β-lactoglobulin (98% purity) and α-

89 lactalbumin (92% purity) standards were purchased from Sigma–Aldrich (St. Louis, MO, USA).

90 Kjeldahl defoamer was purchased from Merck (Darmstadt, Germany). The XT sample buffer, XT

91 reducing agent 20×, protein standards, CriterionTM XT 12% bis-Tris precast gel, XT MES running

92 buffer and Coomassie Brilliant Blue were purchased from BIO-RAD (Hercules, CA, USA). Quant-iT™

93 Protein assay kit was purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA).

94 Doubly deionised water was obtained using a MilliQ system (Millipore, Bedford, MA, USA). Formic

95 acid and Amino Acid Standard H were purchased from Thermo Fisher Scientific. SepPack Plus C18

96 cartridges, AccQ Fluor borate buffer and AccQ Fluor reagent were purchased from Waters

97 (Milford, MA, USA). Sodium hydroxide, HPLC grade acetonitrile, copper(II)oxide powder, sulfuric

98 acid 96%, hydrochloric acid 0.1N and methanol were purchased from VWR International (Milan,

99 Italy). Acetic acid and hydrogen peroxide were purchased from Carlo Erba (Milan, Italy).

100

101 2.2. Sample selection

102

103 In this study samples of whey protein concentrate (WPC-35), obtained from cheese whey,

104 were used provided by FrieslandCampina (Amersfoort, The Netherlands). Product composition is:

105 lactose 50.5%, protein 35.0%, minerals 6.5%, fat 2.5%, moisture 3.5%, organic milk salts 2.0%. It

106 was freeze-dried to reduce the chance of chemical modifications. This sample was used as the

107 starting point for all model heat treatments. Samples were stored for different days at different

108 controlled temperatures, trying to mimic situations arising during storage and transportation,

109 after the production and before commercialisation. A high temperature of 60 °C was included as

110 an extreme storage situation to be used as reference for an exaggerated condition. In addition, a

111 spray-dried sample of whey protein concentrate (WPC-35) was also provided. This sample was
112 compared with the freeze-dried sample stored for 3 d at 20 °C (used as a control sample) to

113 investigate the effect of this drying technique on protein content. Indeed, during spray drying

114 samples are exposed at high temperatures that might affect protein structure. As an example, it

115 has been demonstrated that the temperature of the spray dryer has a catalytic effect on protein

116 lactosylation (Guyomarc’h, Warin, Muir, & Leaver, 2000).

117

118 2.3. Sample preparation

119

120 Samples were collected from FrieslandCampina (Amersfoort, The Netherlands), which

121 provided whey protein concentrates that were obtained after bovine cheese production followed

122 by ultrafiltration and freeze-drying. From the same batch of freeze-dried WPC-35, samples were

123 collected and immediately stored for different durations (3, 8, 10, 14 d) at different temperatures

124 (20, 30, 40, 50, 60 °C). Samples were kept in closed containers under nitrogen atmosphere to

125 prevent changes in the relative humidity and water activity. In addition, also another sample of

126 whey protein concentrate (WPC-35) was provided, a sample collected from the same batch of

127 liquid whey and which was spray-dried instead of freeze-dried. All the samples were flushed with

128 nitrogen to minimise oxidation reactions during further storage. To preserve the samples during

129 the whole study they were stored at –20 °C in nitrogen atmosphere. The freeze-drying process

130 requires 3 days to be completed, thus the sample stored for 3 d at 20 °C was used as control in the

131 analyses.

132

133 2.4. Total nitrogen determination

134
135 For the total nitrogen determination, and consequently the determination of the protein

136 content, the Kjeldahl instrument was used following the standard protocol, according to the

137 European Regulation EC 152/20096. In short, approximately 300 mg of sample was weighed and

138 poured in digestion tubes among with a catalyst (sodium sulphate), a defoamer, a small spoon of

139 copper(II)oxide, and sulphuric acid (98%). Tubes were heated for 30 min at 420 °C, then cooled to

140 room temperature. Samples were distilled with a solution of 35% sodium hydroxide. A solution of

141 boric acid is used for dissolving the generated ammonia gas into ammonium ions again. After

142 adding 0.1 N HCl until the change of colour, the nitrogen content was determined. For these

143 samples a conversion factor 6.41 was used to determine the protein content, representative for

144 whey proteins as reported in literature (Maubois & Lorient, 2016).

145

146 2.5. Determination of the water content

147

148 The water content was determined by loss on drying. Samples were heated at 104 °C for

149 24h to determine the weight loss due to moisture loss. All the analyses were performed in

150 duplicate.

151

152 2.6. Protein characterisation by SDS-PAGE analysis

153

154 SDS-PAGE analysis was performed to characterise proteins in the samples. The amount of

155 sample needed was determined with the Quant-iT™ Protein Assay (Invitrogen, Thermo Scientific,

156 Waltham, MA, USA). Samples (approximately 40 μg of protein) was mixed with the XT sample

157 buffer and the XT reducing agent. The marker was prepared mixing the protein standard, the XT

158 sample buffer and the XT reducing agent. After 5 min at 95 °C and 5 min at –20 °C, samples were
159 loaded on a CriterionTM XT bis-Tris precast gel. Using an XT MES running buffer, gels were run for

160 almost 60 min at constant voltage (150 V). After the run, gels were stained with a Coomassie blue

161 solution (50% water MilliQ, 40% methanol, 10% Coomassie Brilliant Blue) for 2 h to visualise

162 protein bands. Gels were de-stained with a de-staining solution (50% MilliQ water, 40% methanol,

163 10% acetic acid) for 20 min, repeating the operation 3–4 times. Gels were then scanned using a

164 GS-800 calibrated imaging densitometer (BIO-RAD).

165

166 2.7. Whey protein quantification with UPLC-MS analysis

167

168 Quantification of whey proteins in the samples was performed with UPLC-MS analysis. For

169 each sample, 2 mg was dissolved in 1 mL of Milli-Q water. The solutions were centrifuged before

170 the analysis. For protein quantification a calibration curve was prepared, using standards of β-

171 lactoglobulin and α-lactalbumin. For the calibration curve, solutions were prepared in duplicate

172 with concentrations ranging from 0.125 mg mL-1 to 4 mg mL-1 for β-lactoglobulin, and from 0.0625

173 mg mL-1 to 2 mg mL-1 for α-lactalbumin. All the samples were prepared and analysed in duplicate.

174 UPLC-ESI-MS analysis was performed as described in literature (Buhler et al., 2019) with

175 the only difference that the injection volume was 4 μL and samples were injected directly. The

176 software used for data processing was MassLynxTM V4.0 (Waters).

177 After the identification, the total amount of whey protein in the samples, the amount of

178 unmodified proteins and the amount of lactosylated forms could be determined. Since proteins in

179 the unmodified and modified forms co-elute, extract-ion chromatograms (XICs) of the proteins in

180 the different forms were used for the quantification. XICs were obtained by extracting from the

181 total ion current chromatograms (TICs) the characteristics MS ions of the single proteins (see

182 Supplementary material). For the extraction of the XICs, for each protein form the most abundant
183 eight ions of the multicharged pattern were used. The areas of the chromatographic peaks in the

184 XICs were integrated with the MassLynx™ software. The quantification was performed using an

185 external calibration curve (and setting the value of the intercept as zero). The total amount of a

186 protein was determined by the sum of the areas obtained from the XICs of the same protein in the

187 unmodified and modified forms.

188 The two isoforms of β-lactoglobulins were quantified together assuming an identical

189 response factor, both for the quantification of the unmodified and lactosylated forms (Norwood et

190 al., 2016). Protein forms for which the signal to noise ratio was found equal or lower to 10 were

191 not considered in the quantification. For spectra deconvolution the MaxEnt tool (MassLynx™,

192 Waters) was used. Quantification data are expressed based on dry matter content. Analysis of

193 variance (ANOVA) was performed at a significance level of α = 0.05. Significant differences among

194 the mean values were calculated using Tukey's Honestly Significant Difference test (p ≤ 0.05). All

195 experimental data were statistically analysed using SPSS software (IBM company, Armonk, NY,

196 USA).

197

198 2.8. Total amino acid profile determination

199

200 For the determination of the total amino acid profile, sample preparation was performed

201 as reported in literature (Caligiani et al., 2018) with little modifications. Indeed, since 100 mg

202 sample was weighed volumes of the other reagents were adjusted: 1.2 mL 6 M HCl; 1.5 mL 5mM

203 nor-leucine; 1 mL performic acid and 0.15 mL hydrobromic acid. Samples were filtered and diluted

204 with deionised water to a final volume of 50 mL. The calibration curve required was prepared

205 starting from a standard solution obtained mixing in a 1:1 ratio the amino acid standard mixture

206 (2.5 mM) with a mixture of other amino acids (norleucine hydroxyproline, cysteic acid, methionine
207 sulfone in 0.1 N HCl) 2.5 mM, to reach the final concentration of 1.25 mM. The calibration curve

208 was obtained preparing solutions with different concentration from 0.078 mM to 1.25 mM in

209 duplicate.

210 Derivatisation with the AccQ Fluor reagent kit (Waters) was performed according to

211 manufacturer instructions and using 10 μL of each hydrolysed sample or standard solution. The

212 derivatised solutions obtained were diluted with 100 μL deionised water before injecting in the

213 UPLC system. UPLC-ESI-MS analysis was performed using an ACQUITY UPLC® separation system

214 with an Acquity UPLC© Protein BEH C18 (300 Å, 1.7 μm, 2.1 mm × 150 mm, Waters) column with

215 an ACQUITY UPLC® Peptide CSH™ C18 VanGuard™ Pre-column (130 Å, 1.7 μm, 2.1 mm × 5 mm,

216 Waters). Mobile phase was composed of water + 0.1% formic acid (eluent A) and acetonitrile +

217 0.1% formic acid (eluent B). Gradient elution was performed according to the following steps:

218 isocratic 100% A for 7 min, from 100% A to 75.6% A by linear gradient in 21 min plus washing step

219 at 100% B (4 min) and reconditioning (13 min at 100% A). Flow rate was set at 0.20 mL min-1,

220 injection volume 4 μL, column temperature 35 °C and sample temperature 18 °C. Detection was

221 performed as described in section 2.7.

222 Total tryptophan amount was determined following a protocol reported in literature

223 (Delgado-Andrade, Rufián-Henares, Jiménez-Pérez, & Morales, 2006) with little modification: 100

224 mg of sample was weighed and hydrolysis was performed for 4 h. UPLC/ESI-MS analysis was

225 performed using the same column and eluents used for the determination of the other amino

226 acids. Gradient elution was performed according to the following steps: isocratic 100% A for 1.8

227 min, from 100% A to 50% A by linear gradient in 11.4 min and 0.8 min at 50% A plus washing step

228 at 100% B (2.1 min) and reconditioning (13.4 min at 100% A). Flow rate was set at 0.25 mL min-1,

229 injection volume 4 μL, column temperature 35 °C and sample temperature 23 °C. Detection was
230 performed as described in section 2.7 with SIR acquisition mode at 188.0 and 205.0 for trp; 202.1

231 and 219.1 for 5-methyl-tryptophan m/z, scan duration 1 s.

232 The software used for data processing was MassLynxTM V4.0 (Waters). All the samples

233 were prepared and analysed in duplicate. Analysis of variance (ANOVA) was performed test at a

234 significance level of α = 0.05. Significant differences among the mean values were calculated using

235 Tukey's Honestly Significant Difference test (p ≤ 0.05). All experimental data were statistically

236 analysed using Matlab software.

237 The amount of amino acids determined was used to calculate the chemical score of the

238 product. For the calculation only the nine essential amino acids were considered. For each of these

239 amino acids the percentage on the total amount of amino acids was determined and divided for

240 the value of the corresponding essential amino acid amount calculated for the milk reference

241 protein (Institute of Medicine, 2005). The chemical score is expressed as the value of the limiting

242 amino acids, the essential amino acid present in the lowest amount.

243

244 3. Results and discussion

245

246 3.1. Protein content and composition determination

247

248 Initial analyses were performed to verify the effect of the storage conditions applied on

249 gross protein content. From the determination of the moisture content, a mean value of 2.91 ±

250 0.43% was determined for the freeze-dried samples, while a value of 4.73 ± 0.46% was determined

251 for the spray-dried sample. Kjeldahl analysis allowed determining the total nitrogen content from

252 which it was possible to calculate the percentage of protein. Data, calculated on the dry matter,

253 indicated that the protein content was not affected by the storage conditions applied, indeed a
254 mean value of 35.2 ± 0.20% was determined for the freeze-dried samples and a value of 35.1 ±

255 0.29% for the spray-dried sample.

256 SDS-PAGE analyses were also conducted to obtain more information on the protein

257 distribution in the samples (Supplementary material Fig. S1).

258 All the samples showed the presence of the two main whey proteins, α-lactalbumin at

259 around 14 kDa and β-lactoglobulin at around 18 kDa. Sample did not show differences in protein

260 distribution; some bands seem more diffused or lower in the intensity than the others (in

261 particular for samples stored at 50 and 60 °C), but confirmation with LC-MS is required.

262

263 3.2. Whey protein quantification and degree of lactosylation

264

265 UPLC-MS analysis was used to determine the amount of soluble whey proteins and identify

266 their lactosylated forms, following a method previously set up in our group (Buhler et al., 2019). As

267 an example of the UPLC chromatograms obtained, Fig. 1 shows the sample stored for 3 d at 20 °C,

268 8 d at 40 °C and 14 d at 60 °C.

269 It is possible to identify the α-lactalbumin peak at 11.6 min and two peaks corresponding to

270 β-lactoglobulin between 16 and 20 min. The two signals are relative to the two main isoforms of β-

271 lactoglobulin, isoform A at 18.5 min and isoform B at 17.1 min. The identification of the two

272 isoforms was confirmed with the MS data. These two isoforms differ for only two amino acids

273 (position 64: D (isoform A)↔G (isoform B)) and 118 (V (isoform A)↔A (isoform B)). Although the

274 two isoforms can be well separated by UPLC analysis, it was not possible to separate the

275 lactosylated forms of these proteins from the non-lactosylated ones. Comparing the

276 chromatographic profiles, it is possible to observe a little decrease in the peak intensity related to

277 the increase in the days and temperature of storage. Beside in the profile of the sample stored for
278 14 d at 60 °C, peaks have a little shift in the retention time. This might be due to the presence of a

279 higher amount of lactosylated forms of the proteins. There was also broadening of the whey

280 protein peaks that were presumed to be due to increasing degrees of protein lactosylation. The

281 lactosylated forms were nonetheless identified in the MS spectra corresponding to each

282 chromatographic peak. The MS spectra of β-lactoglobulin A and α-lactalbumin, as shown in Fig. 2,

283 are representative examples of the chromatograms of the samples stored at 20 °C for 3 d, 40 °C

284 for 8 d and 60 °C for 14 d, respectively.

285 The identification of the proteins in their different forms (lactosylated and non

286 lactosylated) was possible according to the MS ions, reported in Supplementary material Table S1.

287 In the MS spectra it was possible to identify the proteins in the unmodified forms, with one

288 lactose bound (+324 Da), with two lactose bound (+648) and with three lactose bound (+972). Di-

289 lactosylated and tri-lactosylated forms were clearly identified only in β-lactoglobulin. In particular

290 the tri-lactosylated form of this protein was identified in samples stored for longer days at higher

291 temperatures. From the MS spectra, the MS ions were used to extract from the chromatogram

292 (XIC method) the peak relative to the single modified/unmodified protein. Since the protein in the

293 native and modified form co-elute in the chromatogram, extracting the MS ions allowed

294 determination of the peak area for the native, monolactosylated and di-lactosylated forms. In this

295 way we could detect the various lactosylated forms at the best of the sensitivity allowed by the

296 instrument. In some samples, it was possible to identify also the protein with 4 (+1296 Da) lactose

297 bound for β-lactoglobulin and with 2 (+648) lactose bound for α-lactalbumin. The identification

298 was confirmed after spectra deconvolution (MaxEnt, MassLynx™ software). However,

299 quantification of these modified forms was not possible since their very low abundance. Mass

300 peak deconvolution might yield reconstructed MS spectra, but the deconvolution software suffers

301 from biases when deconvoluting multicharged pattern of compounds having very different
302 concentrations, as it is the case here (the non lactosylated and the monolactosylated forms are

303 much more abundant than highly lactosylated forms).

304 In Figs. 3 and 4 the amount of proteins (non lactosylated and lactosylated) is reported. The

305 samples show both a decrease in total protein amount and an increase in lactosylation in a

306 time/temperature dependent way. Figs. 3 and 4 show a decrease in the total amount of whey

307 proteins due to the high temperatures applied. Analysis of variance (ANOVA) was performed at a

308 significance level of α = 0.05, as described in Material and Methods section.

309 The same trend was observed grouping data due to the time of storage (data not shown).

310 This decrease may be partially due to the presence of whey proteins with more lactose bound that

311 were not identified due to instrumental limitations. Alternatively, the decrease may also be the

312 effect of protein denaturation and formation of aggregates that decrease the soluble proteins

313 content. Indeed, the effect of temperature on aggregation/denaturation phenomena was

314 previously demonstrated by several authors. It is well known that temperature plays a crucial role

315 during storage; increased temperatures can induce aggregation/denaturation phenomena and

316 catalyse the Maillard reaction favouring the binding between lactose and lysine residues in

317 proteins (Norwood et al., 2016; van Lieshout, Lambers, Bragt, & Hettinga, 2019). In the storage of

318 whey protein isolates (WPI), it was observed that powders (aW = 0.23) are stable up to 15 months

319 at 20 °C, while storing at 40 °C induced a decrease in the native content after 3 months and an

320 increase in the amount of denatured/aggregate proteins (Norwood et al., 2016). Storage of four

321 different types of whey (acid, native, salty and sweet whey protein concentrates powders, with

322 controlled relative humidity of 22 or 33%) showed a dependence of the aggregation with the

323 temperature applied, with the highest effect at 45 °C (Nishanthi et al., 2017). Furthermore, storage

324 of skim milk powders at +37 °C showed a higher extent of lactosylation in two weeks with respect
325 to powders stored at room temperature, while an advanced state of the Maillard reaction was

326 observed when storing samples at +52 °C for 3.5 weeks (Guyomarc’h et al., 2000).

327 The results shown here indicated that the larger effects were obtained for samples stored

328 at 50 °C and 60 °C, whereas for the samples up to 40 °C the amount of soluble whey proteins and

329 lactosylation remained relatively stable. These findings are in agreement with the data reporting

330 that the amount of native protein decreased by 50% after 7 d of storage at 80 °C (Morr & Ha,

331 1993), suggesting that with higher storage temperatures the effects on protein content appears in

332 shorter storage times.

333 Samples stored for 3 and 8 d had limited modifications in their content and limited

334 lactosylation was observed, even at high temperatures. The same was observed for samples

335 stored at 10 and 14 d until 40 °C. In these latter samples, higher temperatures might have

336 promoted large aggregation and precipitation phenomena.

337 As far as lactosylation was concerned, unmodified protein content decreased with

338 increasing temperature, while the lactosylated protein content increased, as expected due to the

339 catalytic action of elevated temperature on the Maillard reaction. In our samples, β-lactoglobulin

340 seemed to be more susceptible to lactosylation than α-lactalbumin, with the lactosylated form

341 overcrossing the amount of the unmodified forms when stored at high temperatures even for

342 short times.

343 The present findings demonstrate that the effect of storage is triggered by the increasing

344 temperature: when samples are stored at higher temperatures (50–60 °C) it is possible to see a

345 more pronounced increase in the lactosylated protein content with increasing duration of storage.

346 In addition, samples stored until 14 d at 20 °C and 30 °C do not show relevant differences in the

347 native protein content, indicating that the temperature of storage, more than the time, is

348 responsible for the increased lactosylation.

349 By integration of the XICs peaks areas of the modified protein forms the degree of

350 lactosylation was also determined as percentages (data not shown). For β-lactoglobulin the di-

351 lactosylated form showed the same increasing trend with higher temperatures of the mono-

352 lactosylated one. Looking singularly to the two isoforms of this whey protein, A and B, they

353 showed the same trend with no differences among them, meaning that there is no difference in

354 the lactosylation degree between the two isoforms (data not shown).

355 In the present study, a very small difference in the total amount of protein was observed

356 comparing products processed with freeze-drying or spray-drying (Fig. 5), indicating that the

357 different techniques applied do not influence protein aggregation/denaturation.

358 Fig. 5 shows the comparison between the two different drying processes. Results shows

359 that lactosylation degree was little affected, after 3 d at 20 °C the degree of lactosylation was very

360 small in both instances, meaning that the spray-drying technique applied does not induce more

361 lactosylation compared with the freeze-drying technique. The effect of drying processes on

362 proteins are less clear in literature, since the product in the liquid state usually undergoes other

363 treatments, beside simple drying, that may induce changes in proteins. It has been previously

364 reported that dry heating (at 100 °C for 24 h, with a water activity of 0.23) of α-lactalbumin and β-

365 lactoglobulin powders induces nearly 30% of protein aggregation (Gulzar, Bouhallab, Jardin,

366 Briard-Bion, & Croguennec, 2013).

367 Present findings here demonstrate that when samples are stored at higher temperatures it

368 is possible to see a higher decrease in native protein with the days of storage. This behaviour is

369 mainly caused by the accelerating effect of temperatures on the lactosylation mentioned above.

370 These results could be useful for the evaluation of the shelf life markers or a marker of harshness

371 of product treatment.

372
373 3.3. Total amino acid profile determination

374

375 To know the nutritional value of the product and to investigate the influence of the

376 selected storage conditions on it and an eventual shelf life marker, the total amino acid

377 composition was determined. The obtained results are presented in Fig. 6.

378 Data are reported on 100 g dry matter and are grouped for d of storage. As usually

379 observed in foods rich in milk proteins, the most abundant amino acids are glutamic acid, aspartic

380 acid, leucine, threonine, valine, proline and lysine. However, one-way ANOVA statistical analysis

381 showed that significant differences (p ≤ 0.05), small as they were, were present for some amino

382 acids that are marked with an asterisk in the figures. These differences mostly affected sensitive

383 amino acids: serine, tyrosine and tryptophan were the first amino acids, even from the 3rd day of

384 storage, to show some decrease. These amino acids are known to be susceptible for degradation

385 during food processing, for dehydration and oxidative reactions (Sforza, Tedeschi, & Wierenga,

386 2016).

387 By increasing the days of storage, more amino acids seemed to be affected. In particular

388 lysine shows significant variations starting from the 10th day of storage. This finding can be related

389 to protein lactosylation, which decreases the amount of detectable lysine. Lysine residues are

390 likely blocked in the Amadori compound and their biological availability is reduced (Pellegrino et

391 al., 2013).

392 Moreover, a little decrease in the content of Phe, Tyr and His was also observed with the

393 increase in days and temperature of storage. Evidence for the decrease in content of these amino

394 acids due to storage conditions were previously not reported in literature. Oxidation phenomena

395 occurring at high temperatures might degrade these amino acids reducing their amounts (Sforza

396 et al., 2016).

397 The values obtained for amino acid quantifications were used to calculate the true protein

398 content for each sample (Supplementary material Table S2), obtaining results that are somewhat

399 lower (average 25%) compared with the data obtained with the Kjeldahl analysis performed. Part

400 of this lower value can be explained by the possible degradation of amino acids during acid

401 hydrolysis. In addition, a non-protein nitrogen (NPN) in the samples is being annotated as nitrogen

402 from protein. It has been reported that the NPN content of cows’ milk is about 5% of the total

403 nitrogen (Maubois & Lorient, 2016).

404 Total amino acid content was also determined for the spray-dried sample and, based on

405 ANOVA statistical analysis, small significant variation was only observed for few amino acids (Phe,

406 Ser, Tyr). This confirms the assumption made before that the spray-drying process produces

407 similar effects as the freeze-drying process (Guyomarc’h et al., 2000). Data are shown in

408 Supplementary Material Fig. S2.

409

410 3.4. Determination of the chemical score for the evaluation of the nutritional quality

411

412 The amino acid content was also evaluated in terms of quality, considering the relative

413 amount of the nine essential amino acids (phenylalanine, valine, threonine, tryptophan,

414 methionine, leucine, isoleucine, lysine and histidine) on the total amino acids (Loveday, 2019).

415 The amount of essential amino acids did not seem to be affected by the storage and

416 temperature applied (data not shown), but to get more insights in the nutritional value, the

417 chemical scores for essential amino acids were calculated in each sample (Supplementary material

418 Table S3) according to Mitchell and Block, (1946) and Boye, Wijesinha-Bettoni, and Burlingame

419 (2012), using the milk reference protein (Institute of Medicine, 2005). The chemical score is a

420 measure of protein quality based on chemical analysis of its amino acid composition, and it is a
421 parameter commonly used for the evaluation of the nutritional quality of proteins. It is obtained

422 by comparing the amount of each essential amino acid in a sample with the amino acids in a

423 reference protein that is known to be well balanced in the amino acid content related to human

424 requirements (Pellegrino et al., 2013).

425 In all the samples Phe and Tyr are the limiting amino acids, with a chemical score that

426 ranges mainly between 61 and 91. The only exception is represented by the sample stored for 14 d

427 at 30 °C where they are the second limiting amino acids and the first one is his, with a calculated

428 value of 87. Results are in agreement with data reported in literature for whey protein

429 concentrates (Forsum, 1974; Sindayikengera & Xia, 2006) which indicates Phe+Tyr being the

430 limiting amino acids of whey proteins, with scores ranging between 70 and 90%. No clear

431 correlation emerged between the conditions of storage (time or temperature) with the nutritional

432 value. Thus, even if the modifications induced by storage temperature and time affected the

433 amino acid content, the nutritional value of the product was still adequate and was not

434 significantly affected by them.

435

436 4. Conclusions

437

438 The present study, performed on a selected WPC sample subjected to a controlled time

439 and temperature of storage, allowed the observation that the selected storage conditions have an

440 effect on protein composition, inducing protein denaturation/aggregation and favouring lysine

441 lactosylation. From the LC-MS quantification data, it can be concluded that elevated temperature

442 affect proteins, as expected, in particular for temperatures above 30 °C, probably inducing protein

443 denaturation. In all the samples lactose was bound to whey proteins. For both α-lactalbumin and

444 β-lactoglobulin the same effect was observed: the lactosylation degree increases with the applied
445 temperature and with the number of days. That is true also for the di-lactosylated forms that were

446 identified for both β-lactoglobulin isoform A and isoform B, with the same trend. Thus, the results

447 obtained suggest that storing samples for a short period of time at moderate temperatures avoids

448 the formation of the lactosylated forms and denatured proteins.

449 However, the impact of the storage conditions on the amino acid composition, and thus on

450 the nutritional value, is low. Losses were found only for few amino acids and to a very minor

451 extent. Lysine residues were mostly affected, confirming that this amino acid bound lactose in the

452 sample and reduces its availability, but the amount of available lysine remained more than

453 adequate. The nutritional value determined by the limited score of the Phe+Tyr group in whey

454 proteins, was not significantly affected by the storage conditions. Concerning the drying

455 technique, minimal differences among the spray-dried sample and the freeze-dried one were

456 observed. These results suggest that the different drying technique applied have a little influence

457 on the quality of the product. Results here presented can be useful for the assessment of milk

458 protein damages and milk nutritional value after storage. Finally, molecular characterisation of

459 glycated products and amino acids determination, can also be useful as shelf life markers and as a

460 marker for the harshness of product treatment.

461

462 References

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524
 1 Figure legends

3 Fig. 1. UPLC-MS profile obtained for the analysed samples: sample stored for 3 d at 20 degrees

4 (starting sample, a), sample stored for 8 d at 40 °C (mid-point sample, b), sample stored for 14 d at

5 60 °C (end-point sample, c). Intensity is shown on the y axis, time on the x axis.

7 Fig. 2. MS spectra of chromatographic peak at 11.6 min (α-lactalbumin) and 18.5 min (β-

8 lactoglobulin A) of the samples shown in Fig. 1. A, 3 d 20 °C (β-lactoglobulin A), B, 8 d 40 °C (β-

9 lactoglobulin A), C, 14 d 60°C (β-lactoglobulin A); D, sample 3d 20 °C (α-lactalbumin); E, 8 d 40 °C

10 (α-lactalbumin); F, 14 d 60 °C (α-lactalbumin). Relative intensity is shown on the y axis, mass–to-

11 charge ratio (m/z) on the x axis. A single asterisk highlights the mono-lactosylated identified forms,

12 two asterisks the forms with two lactose bound, three asterisks when three lactose were found

13 bound to the protein.

14

15 Fig. 3. Amount of β-lactoglobulin in the samples, total amount (black bars), unmodified form (grey

16 bars) and glycated forms (light-grey bars) based on initial concentration. Amounts are expressed

17 on dry matter; different letters in the same group are significantly different (p ≤ 0.05).

18

19 Fig. 4. Amount of α-lactalbumin in the samples, total amount (black bars), unmodified form (grey

20 bars) and glycated forms (light-grey bars) based on initial concentration. Amounts are expressed

21 on dry matter; different letters in the same group are significantly different (p ≤ 0.05).

22

23 Fig. 5. Amount of β-lactoglobulin and α-lactalbumin in spray-dried sample compared to sample 1,

24 total amount (black bars), unmodified form (grey bars) and glycated forms (light-grey bars) based
25 on initial concentration. Amounts are expressed on dry matter; different letters in the same group

26 are significantly different (p ≤ 0.05).

27

28 Fig. 6. Total amino acid profile obtained for sample sets stored for 3, 8, 10 and 14 d. Asterisks

29 indicate amino acids that are significantly different (p ≤ 0.05).

30

31
100

β-LG
18.50

17.13

α-LA
11.55 c ba

0
6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.0019.00 20.00 21.00
Amount (g g-1 dry matter)

Amount (g g-1 dry matter)

Amount (g 100 g-1 sample) Amount (g 100 g-1 sample)

Amount (g 100 g-1 sample) Amount (g 100 g-1 sample)

A B C

D E F