Version of Record: https://www.sciencedirect.

com/science/article/pii/S0308814618320892
Manuscript_d516fdbdb6abb668e51f2a1e6b21202d

1 Dipeptidyl peptidase IV (DPP-IV) inhibitory properties of a camel whey

2 protein enriched hydrolysate preparation

4 Alice B. Nongonierma1,2, Cloé Cadamuro1, Aurélien Le Gouic1, Priti Mudgil3, Sajid Maqsood3*
5 & Richard J. FitzGerald1,2*
6

1
7 Department of Biological Sciences, University of Limerick, Limerick, Ireland.

2
8 Food for Health Ireland (FHI), University of Limerick, Limerick, Ireland.

3
9 Department of Food Science, College of Food and Agriculture, United Arab Emirates

10 University, Al Ain, 15551, United Arab Emirates

11

12

13

14

15

16 *Corresponding authors:

17 S. Maqsood: Email: sajid.m@uaeu.ac.ae; Tel: +971 37134519; Fax: +971 37675336.

18 R.J. FitzGerald: Email: dick.fitzgerald@ul.ie; Tel: +353 (0) 61 202598; Fax: + 353 (0) 61 331490.

19

© 2018 published by Elsevier. This manuscript is made available under the Elsevier user license
https://www.elsevier.com/open-access/userlicense/1.0/
20 Abstract

21 Camel milk proteins contain dipeptidyl peptidase IV (DPP-IV) inhibitory peptides. A camel

22 whey protein concentrate (WPC, 44.7 ± 3.4% (w/w) protein) was prepared and subsequently

23 hydrolysed with trypsin at different temperatures, enzyme to substrate (E:S) ratios and hydrolysis

24 times yielding fifteen hydrolysates (H1-H15). Their DPP-IV half maximal inhibitory

25 concentrations (IC50) ranged from 0.55 ± 0.05 to 1.52 ± 0.16 mg L-1 for H8 and H6, respectively.

26 E:S was the only factor having a significant effect on the DPP-IV IC50 value (p <0.05).

27 Relatively potent α-lactalbumin-derived DPP-IV inhibitory peptides (LAHKPL and

28 ILDKEGIDY) were detected in selected hydrolysates. Additionally, three potent β-CN-derived

29 peptides, VPV, YPI and VPF having DPP-IV IC50 values of 6.6 ± 0.5, 35.0 ± 2.0 and 55.1 ± 5.8

30 µM, respectively, were identified. After IPI, VPV is the second most potent DPP-IV inhibitory

31 peptide identified to date, which supports the role of camel milk as an antidiabetic agent.

32 Key words: dipeptidyl peptidase IV inhibition; camel milk; whey proteins; bioactive peptides;
33 trypsin; liquid chromatography tandem mass spectrometry (LC-MS/MS).

2
34 1 Introduction

35 Diabetes is a widespread disease worldwide which is increasing exponentially. Diabetes was

36 estimated to affect 422 million people in 2014 and to have caused the death of 1.5 million people

37 in 2012 (WHO, 2016). Different types of diabetes have been identified including type 1, type 2

38 and gestational diabetes. Type 2 diabetes is the most prevalent form of diabetes worldwide,

39 representing around 90% of cases (IDF, 2017). Type 2 diabetes is characterised by insufficient

40 insulin production and/or a peripheral resistance to the action of insulin. Different antidiabetic

41 drugs are commercially available to treat type 2 diabetes. These include gliptins, which are

42 synthetic inhibitors of dipeptidyl peptidase IV (DPP-IV). DPP-IV is an ubiquitous enzyme which

43 can degrade incretins including glucagon-like peptide-1 (GLP-1) and glucose-dependent

44 insulinotropic polypeptide (GIP). Degradation of these incretins causes a loss in their

45 insulinotropic activity. Therefore, the inhibition of DPP-IV may be used to increase insulin

46 secretion during the post-prandial phase, which can allow for a better glycaemic regulation.

47 Food proteins have been hydrolysed with a range of food-grade enzyme preparations to generate

48 peptides having the ability to inhibit DPP-IV in vitro (for reviews, see:Lacroix & Li-Chan, 2016;

49 Nongonierma & FitzGerald, 2017a). While various dietary proteins have been employed for the

50 generation of DPP-IV inhibitory hydrolysates, bovine milk proteins are the most studied for the

51 generation of DPP-IV inhibitory peptides. However, equine (Song, Wang, Du, Ji & Mao, 2017),

52 caprine (Tulipano, Cocchi & Caroli, 2012; Zhang, Chen, Zuo, Ma, Zhang & Chen, 2016) and

53 more recently camel milk proteins (Mudgil, Kamal, Yuen & Maqsood, 2018; Nongonierma,

54 Paolella, Mudgil, Maqsood & FitzGerald, 2017; Tagliazucchi, Martini, Shamsia, Helal & Conte,

55 2018) have been used as substrates for the generation of DPP-IV inhibitory peptides by

56 enzymatic hydrolysis.

3
57 The one-humped Arabian dromedary (Camelus dromedarius) is the main mammal used for the

58 production of milk in desert and semi-desert areas of the world. Annual volumes reaching 6

59 million tons were estimated for worldwide camel milk production (Faye, 2015). Camel milk does

60 not contain β-lactoglobulin (β-Lg), therefore in this regard, its protein composition is similar to

61 that of human milk (Hailu, Hansen, Seifu, Eshetu, Ipsen & Kappeler, 2016; Mati, Senoussi-

62 Ghezali, Ahmed Zennia, Almi-Sebbane, El-Hatmi & Girardet, 2016). Consumption of camel

63 milk has been linked with numerous benefits for human health conditions/disease such as

64 diabetes, autism, cancer, infections, colitis, heavy metal and alcohol-induced toxicity. More

65 particularly, the consumption of camel milk has been reported to decrease glycaemia in type 1

66 diabetic subjects (for review, see: Shori, 2015). Due to the presence of insulin and insulin-like

67 components, camel milk has been proposed as an adjunct to antidiabetic drugs for the

68 management of type 1 and type 2 diabetes (Agarwal et al., 2003; Ejtahed et al., 2015).

69 Enzymatic hydrolysis and fermentation processes have been applied to camel milk, which have

70 led to the release of peptides with a wide range of bioactivities including antioxidant,

71 antimicrobial, anti-inflammatory and antihypertensive properties (Hailu et al., 2016; Mati et al.,

72 2016). In silico analysis of camel milk proteins showed that they contain numerous known DPP-

73 IV inhibitory peptide motifs (Nongonierma et al., 2017). Notably, several potent DPP-IV

74 inhibitory Trp-containing dipeptides have been identified in α-lactalbumin (α-La). Whey from

75 camel milk is a particularly rich source of α-La, as it contains twice the amount of α-La than in

76 bovine milk, 2.3 vs. 1.1 g L-1 (Hailu et al., 2016). The amount of serum albumin is also

77 significantly higher in camel compared to bovine milk, i.e., 2.2 vs. 0.35 g L-1 (Hailu et al., 2016).

78 Currently, most DPP-IV inhibitory peptides identified within camel milk protein hydrolysates

79 appear to originate from caseins (CNs) (Nongonierma, Paolella, Mudgil, Maqsood & FitzGerald,

4
80 2018; Tagliazucchi et al., 2018). The contribution of the major camel whey proteins (α-La and

81 serum albumin) to DPP-IV inhibition does not appear to have been extensively studied to date. In

82 an earlier study conducted on camel milk proteins, most DPP-IV inhibitory peptides identified

83 originated from CNs. The difficulty in identifying whey protein-derived peptides during liquid

84 chromatography tandem mass spectrometry (LC-MS/MS) studies was likely to arise from the

85 lower abundance of whey proteins compared to CNs. It was therefore hypothesised that whey

86 protein derived peptides would be more easily detectable during LC-MS/MS analysis of

87 hydrolysates generated with a protein substrate enriched in camel whey proteins.

88 The aim of this study was to increase our understanding of the potential contribution of camel

89 whey peptides to inhibit DPP-IV. A camel milk protein preparation enriched in whey proteins

90 (WPC) was therefore generated. Optimisation of the generation of DPP-IV inhibitory peptides

91 originating from a camel WPC was carried out using a design of experiments (DOE) approach

92 combining three parameters, i.e., hydrolysis temperature, enzyme to substrate (E:S) ratio, and

93 time. These parameters were each studied at three different levels. The WPC hydrolysates were

94 characterised for their in vitro DPP-IV inhibitory properties and their physicochemical

95 characteristics (i.e., extent of hydrolysis, molecular mass distribution and peptide profile).

96 Simulated gastrointestinal digestion (SGID) was carried out on the intact and hydrolysed camel

97 milk proteins to study the stability of their DPP-IV inhibitory properties to hydrolysis by

98 gastrointestinal enzymes. Finally, peptide identification was carried out using LC-MS/MS in

99 selected samples followed by confirmatory studies with synthetic peptides.

5
100 2 Materials and methods

101 2.1 Reagents

102 High performance liquid chromatography (HPLC)-grade water and acetonitrile, hydrochloric

103 acid (HCl) and sodium hydroxide (NaOH) were from VWR (Dublin, Ireland). Sodium phosphate

104 monobasic, sodium phosphate dibasic, sodium dodecyl sulphate (SDS), trifluoroacetic acid

105 (TFA), Tris(hydroxymethyl)aminomethane (Tris), Gly-Pro p-nitroanilide (GP-pNA), leucine (L),

106 diprotin A (IPI), porcine DPP-IV (≥ 10 units mg-1 protein), MS-grade water, acetonitrile, formic

107 acid (FA) and standards for molecular mass distribution (i.e., bovine serum albumin (BSA), β-

108 Lg, α-La, aprotinin, bacitracin, LWMR and DE) were purchased from Sigma-Aldrich (Dublin,

109 Ireland). 2,4,6-Trinitrobenzenesulfonic acid (TNBS) from Pierce Biotechnology was obtained

110 from the Medical Supply Company (Dublin, Ireland). A micro bicinchoninic acid (BCA) protein

111 assay kit was obtained from Thermo Fisher Scientific (Rockford, IL, USA). Pepsin was provided

112 by Biocatalysts (Cefn Coed, UK, Wales), Corolase PPTM by AB enzymes (Darmstadt, Germany)

113 and trypsin (PTN 6.0STM) by Novozymes (Bagsvaerd, Denmark). Synthetic peptides (> 95%

114 purity, EPVK, FLNK, FYAPELL, IIF, LAHKPL, LLNEK, LQL, LQPK, VPF, VPV, YPI and

115 YPLR) were purchased from Genscript (Piscataway, NJ, USA).

116 2.2 Generation of the camel whey protein concentrate (WPC)

117 The raw milk of one-humped Arabian camels was procured from local camel farms in Al Ain,

118 United Arab Emirates (UAE). Milk was collected from 4 healthy camels (Camelus dromedarius,

119 local breed) and was pooled as a composite sample. Milk was then transported to the Laboratory

120 at the Food Science Department, UAE University in a chilled condition. Milk samples were then

121 stored at 4°C and processed within 2 h. The milk was skimmed via centrifugation at 4255 g for

122 10 min at 4°C (Allegra X-30R, Beckman Coulter Inc., Fullerton, CA, USA). The process of

6
123 skimming was repeated twice. The pH of the skimmed milk was adjusted to 4.6 with 6 N HCl

124 and milk samples were allowed to settle for 1 h. This was followed by centrifugation at 4700 g

125 for 10 min at 4°C which resulted in the separation of a casein pellet from a whey supernatant.

126 The whey protein preparation (WP) was frozen at -20°C and freeze-dried using a Telstar freeze-

127 drier (Telstar, Terrassa, Spain) set at -80°C and 0.1 bar. The freeze-dried camel whey powder

128 was stored at -20°C until further utilisation.

129 The freeze-dried WP was subsequently reconstituted in distilled water at a concentration of

130 20.0% (w/w). This solution was subjected to dialysis with distilled water using a SnakeSkinTM

131 dialysis tubing (Thermo Fisher Scientific) having a 3.5 kDa nominal molecular weight cut-off

132 (MWCO) for 33 h at 4°C. The aim of the dialysis was to reduce the content of acids, lactose and

133 salts in the WP in order to increase its protein content. The retentate of the dialysis was then

134 freeze-dried (Freezone 18L, Labconco, Kansas City, USA) yielding a camel whey protein-rich

135 concentrate (WPC).

136 The protein contents of WP and WPC were determined in triplicate (n=3) using the BCA method

137 as previously described (Nongonierma, Le Maux, Dubrulle, Barre & FitzGerald, 2015). The

138 absorbance of the samples was monitored at 562 nm with a plate reader (Biotek Synergy HT,

139 Winoosky, VT, USA) managed by Gen 5 software (Biotek Synergy HT). The BCA method was

140 calibrated using BSA standard solutions in the range of 25 to 2,000 mg mL-1. The protein content

141 and yield were determined as previously outlined in Nongonierma et al. (2015).

142 2.3 Enzymatic hydrolysis of the camel WPC and simulated gastrointestinal digestion of

143 WPC and WPC hydrolysates

144 Camel milk WPC hydrolysates were produced using PTN 6.0S as previously outlined by

145 Nongonierma et al. (2017). Briefly, camel WPC (0.5% (w protein/w)) was resuspended in

7
146 distilled water and incubated for 60 min in a water bath (Lauda E100, Lauda Brinkmann, Lauda-

147 Königshofen, Germany) set at reaction temperature. The pH of the WPC solution was adjusted to

148 7.0 using 2 M NaOH (VWR). Temperature (40, 50 and 60°C), enzyme to substrate ratio (E:S;

149 0.50, 1.25 and 2.00% (w/w)) and hydrolysis time (60, 150 and 240 min) were evaluated at three

150 different levels (-1, 0 and +1, corresponding to the centred and reduced (z-centred) values, Table

151 1). Fourteen hydrolysates (H1-H14) were generated once. A fifteenth hydrolysate corresponding

152 to the central point conditions (50°C, 1.25% E:S and 150 min) was generated as three

153 independent triplicates (H15A, H15B and H15C) to assess the reproducibility of the hydrolysis

154 reaction. A control sample (C40, C50 or C60) without addition of enzyme was prepared by

155 incubation for 240 min at each hydrolysis temperature. At the end of each reaction, the enzyme

156 was heat inactivated (90°C, 20 min) in a water bath.

157 For external validation of the model (Equation 1), four additional hydrolysates (H16-H19) were

158 generated using the conditions described in Table 1. The hydrolysis conditions which were

159 predicted to yield the lowest DPP-IV half maximal inhibitory concentration (IC50) value within

160 the experimental domain were determined (section 2.4.) and these were used to produce the

161 optimum hydrolysate in triplicate, yielding H20A, H20B and H20C.

162 Camel WPC and H20A were subjected to SGID (Walsh et al., 2004). Briefly, samples were

163 resuspended in distilled water to 2.0% (w protein equivalent/w) for 30 min at 37°C and the pH

164 was adjusted to 2.0 using 1 M HCl (VWR). Hydrolysis with pepsin (E:S 2.5% (w/w)) was

165 carried out under pH regulation (2.0) with HCl (pH stat Titrando 843, Tiamo 1.4 Metrohm,

166 Dublin, Ireland) for 90 min at 37°C. The pepsin hydrolysate was heat inactivated (90°C, 20 min)

167 and then brought to pH 7.5 using 1 M NaOH and hydrolysed with Corolase PP (E:S 1% (w/w))

168 for 150 min at 37°C, pH 7.5 using a pH stat (Metrohm), to yield WPC_SGID and H20A_SGID,

8
169 from camel WPC and H20A, respectively. The reaction was terminated by heat treatment (90°C,

170 20 min).

171 2.4 DPP-IV inhibition assay

172 The DPP-IV inhibition assay was carried out in triplicate as previously outlined bv Nongonierma

173 & FitzGerald (2013). Samples were dispersed in HPLC-grade water at concentrations ranging

174 from 2.8 × 10-2 to 2.8 mg mL-1 (final concentration expressed in w protein equivalent/w). IPI, the

175 positive control, was resuspended at final concentrations ranging from 36.6 × 10-2 to 36.6 µM.

176 Other synthetic peptides were resuspended at concentrations ranging from 1.3 to 2000.0 µM.

177 Essentially, 25 µL of sample was pipetted onto a 96-well microplate (Sarstedt, Dublin, Ireland)

178 containing the reaction substrate GP-pNA (final concentration 0.200 mM). The reaction was

179 initiated by the addition of DPP-IV (final concentration 0.0025 units mL-1). All reagents were

180 diluted in 100 mM Tris-HCl pH 8.0 buffer. The negative control consisted of 100 mM Tris-HCl

181 pH 8.0 buffer and GP-pNA. The microplate was incubated at 37°C for 60 min in a microplate

182 reader (Biotek Synergy HT) and the absorbance of the pNA released was monitored at 405 nm.

183 The DPP-IV IC50 values were determined by plotting the percentage inhibition as a function of

184 the test compound concentration. Each analysis was conducted in triplicate (n=3).

185 The DPP-IV IC50 values of the hydrolysates were used to develop a multilinear model (Equation

186 1) as previously described in Nongonierma et al. (2017). Modelling was carried out with Matlab

187 (version R2015b, MathWorks, Inc, Natick, MA, USA). Hydrolysis parameters yielding the

188 lowest DPP-IV IC50 values within the DOE boundaries were determined using Equation 1 as

189 described earlier (Nongonierma et al., 2017).

190 = + + + + + + + × + × + × + (Equation 1).

9
191 With Y, the DPP-IV IC50 value; β0 to β9: the coefficients of the MLR model (Equation 1); T

192 (temperature), ES (enzyme to substrate ratio) and t (time): the z-centred parameters of the DOE

193 and ε: the residual of the model.

194 2.5 Determination of the free amino group content of the camel WPC hydrolysates

195 The free amino group content (AN) of the hydrolysates (H1-H20) was determined in triplicate

196 (n=3) using the TNBS method of Adler-Nissen (1979) modified by Le Maux, Nongonierma,

197 Barre & FitzGerald (2016). Absorbance values at 420 nm (Cayot & Tainturier, 1997) were

198 measured with a microplate reader (Biotek Synergy HT). The AN was calculated using Equation

199 2:

200 AN = AN − AN (Equation 2)

201 Where AN1 and AN2 are the free amino group contents of the WPC before and after hydrolysis,

202 respectively.

203 2.6 Peptide profile by reverse-phase ultra-performance liquid chromatography (RP-

204 UPLC)

205 The peptide profile of the hydrolysates and controls was determined by RP-UPLC (Nongonierma

206 & FitzGerald, 2012). Briefly, samples were resuspended in mobile phase A (0.1% TFA in MS-

207 grade water) at 0.22% (w protein equivalent/w). Samples were filtered through 0.2 µm cellulose

208 acetate filters (VWR). The analysis was performed using an Acquity UPLC (Waters, Milford,

209 MA, USA) fitted with an Acquity UPLC BEH C18, 130 Å column (2.1 mm × 50 mm × 1.7 µm)

210 connected to an Acquity BEH C18 (1.7 µm) vanguard pre-column (both from Waters). The

211 separation of peptides was conducted at 30°C at a flow rate of 0.3 mL min-1 and an injection

212 volume of 10 µL. Mobile phase B consisted of 0.1% TFA, 80% MS-grade acetonitrile in MS-

10
213 grade water. A linear elution gradient as follows was used: 0-0.28 min: 100% A, 0.28-60 min:

214 100-40% A. Absorbance was monitored at 214 nm.

215 2.7 Molecular mass distribution by gel permeation high performance chromatography

216 (GP-HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-

217 PAGE)

218 Molecular mass distribution profiles of samples were obtained using GP-HLPC (Spellman,

219 O’Cuinn & FitzGerald, 2009). Isocratic mode of separation was used at 21°C with a TSK G2000

220 SW separating column (600 × 7.5 mm ID - Tosoh Bioscience, Tokyo, Japan) connected to a

221 TSKGEL SW guard column (75 × 7.5 mm ID - Tosoh Bioscience). The mobile phase consisted

222 of 0.1% (v/v) TFA and 30% HPLC-grade acetonitrile in HPLC water. The flow rate was set at

223 0.5 mL min-1 for 60 min. Samples (0.11% (w protein equivalent/v)) were resuspended in mobile

224 phase and filtered through 0.2 mm PTFE syringe filters (VWR)) before injection (20 µL). The

225 absorbance was monitored at 214 nm. Molecular mass standards were BSA, β-Lg, α-La,

226 aprotinin, bacitracin, LWMR and DE.

227 SDS-PAGE was performed using a Mini Protean II electrophoresis system (Bio-Rad, Hercules,

228 CA, USA) according to the procedure of Laemmli (1970). Briefly, samples were diluted in

229 distilled water at 0.5% (w/v) before further dilution in a 1:1 (v/v) ratio in 1% (w/v) SDS.

230 Samples were then resuspended in loading buffer (protein loading buffer blue 2X - National

231 Diagnostics, Atlanta, GA, USA) using a 1:1 (v/v) ratio. Samples (30 µL, i.e., 34 ng protein

232 equivalent) were added to each well of the gels (TruPage(R) precast polyacrylamide gels,

233 gradient 4-20%, Sigma-Aldrich). The samples were separated for 50 min using a voltage of 100

234 V. The molecular weight of the proteins and peptides was estimated by reference to the relative

235 migration of standard proteins (SigmaMarker®, 6,500-200,000 Da, Sigma-Aldrich). The SDS-

11
236 PAGE profiles were analysed with GelAnalyser version 2010a (http://www.gelanalyzer.com) to

237 determine the relative intensity of the bands within each lane.

238 2.8 Peptide identification by LC-MS/MS and in silico analysis of peptides identified

239 Peptide identification was performed for four samples, H8, H20A, SGID_H20A and SGID_WPC

240 using LC-MS/MS as previously outlined in Nongonierma et al. (2017). Briefly, an ultra-HPLC

241 (UHPLC) UltiMate 3000 (Dionex, Camberley, Surrey, UK) fitted with a security guard UHPLC

242 C18 PEPTIDE (2.1 mm, Phenomenex, Cheshire, UK) and an Aeris Peptide XB-C18 column

243 (150 × 2.1 mm, 1.7 µm, Phenomenex) was used for peptide separation at 25°C. The UHPLC was

244 coupled to a quadrupole time-of-flight mass spectrometer (Q-TOF, Impact HD™, Bruker

245 Daltonics GmbH, Bremen, Germany) using a 50-600 and 100-2500 m/z acquisition range. The

246 MS was fitted with an electrospray ionisation (ESI) source used in positive ion mode. Data

247 acquisition was performed with Hystar software (Bruker Daltonics). Samples were resuspended

248 in mobile phase A (0.1% FA in MS water) to a final concentration of 0.1 g L-1 and filtered with

249 regenerated cellulose filters (Minisart® RC15, Sartorius, Goettingen, Germany). A sample

250 volume of 5 (for ions ranging from 100-2500 m/z) and 10 µL (for ions ranging from 50-600 m/z)

251 was injected. Peptide identification was carried out with PEAKS Studio (version 7.5,

252 Bioinformatics Solutions Inc., Waterloo, Canada) software and its database search tools

253 including PEAKS DE NOVO sequencing, PEAKS BD (database search), PEAKS PTM (post

254 transitional modifications) and SPIDER (algorithm that matches sequence tags with errors to

255 database sequences for the purpose of protein and peptide identification). The database used was

256 UniProtKB/Swiss-Prot (http://www.uniprot.org), taxa Camelus dromedarius. The false discovery

257 rate (FDR), average local confidence (ALC) and MS/MS tolerance were set at 1, 60% and 0.4

258 Da, respectively. Sequences detected using PEAKS Studio software were manually checked to

12
259 verify that the peptide sequence was present in the camel milk proteins, and to analyse the

260 peptides fragmentation spectrum (as ion series continuity and major peaks identified as b or y

261 ions).

262 Peptides with known DPP-IV IC50 values were searched within the samples. In addition, other

263 sequences possessing DPP-IV inhibitory peptide features were determined as previously outlined

264 (Nongonierma et al., 2017). DPP-IV inhibitory peptide features consisted of hydrophobic amino

265 acids (W, L, I or F) at the N-terminus, P/A at position 2 and/or P at the C-terminus. The location

266 of peptides within mature camel milk protein sequences (αs1-, αs2-, β- and κ-CN as well as α-La)

267 was obtained from Uniprot on the ExPASy resource portal (http://www.expasy.org/).

268 2.9 Determination of the mode of inhibition of DPP-IV

269 The mode of DPP-IV inhibition was assessed using the Lineweaver and Burk double reciprocal

270 representation as previously described (Nongonierma et al., 2018). Briefly, DPP-IV inhibitory

271 peptides (IPI, EPVK, FLNK, FYAPELL, IIF, LAHKPL, LLNEK, LQL, LQPK, VPF, VPV, YPI

272 and YPLR) were evaluated at their IC50 value divided by 4, 8 and 16. The concentrations of GP-

273 pNA ranged from 0.1 to 0.6 mM (final concentration). The absorbance at 405 nm was recorded

274 for 30 min at 37°C using a microplate reader (Biotek Synergy HT). Each sample was evaluated 4

275 times.

276 2.10 Statistical analysis

277 A one-way analysis of variance (ANOVA) at a significance level of p < 0.05 was employed to

278 study differences between mean AN and DPP-IV IC50 values for the hydrolysates generated at

279 the central point conditions (H15A, H15B and H15C) and the optimum conditions (H20A, H20B

280 and H20C). ANOVA was followed by a post-hoc Student Newman Keuls test (p < 0.05)

281 conducted with SPSS (version 22, SPSS Inc., Chicago, IL, USA).

13
282 3 Results and discussion

283 3.1 Characteristics of the camel WPC

284 The protein content of the camel WP was 14.9 ± 1.1% (w/w). Following dialysis of WP, a WPC

285 was obtained which contained approximately three times more protein, i.e., 44.7 ± 3.4% (w/w)

286 protein. The extraction yield for protein during the dialysis was 63.9 ± 9.5%. The WPC was

287 subsequently used to produce various hydrolysates. In addition to being more concentrated in

288 proteins, the WPC was also enriched in α-La (Figure 1). Using relative band intensities of the

289 SDS-PAGE profile (Figure 1), it was estimated that the WPC contained an α-La:CN ratio of 0.92

290 ± 0.11:1.0. This ratio is higher than that present in camel milk which is of the order 0.10:1.0

291 according to the average concentration of individual proteins present in camel milk (Hailu et al.,

292 2016). This result demonstrated that a significant enrichment in α-La was achieved within the

293 WPC herein. The presence of CNs in the supernatants of camel milk which had been adjusted to

294 pH to 4.6 has previously been reported by Wangoh, Farah & Puhan (1998). This is consistent

295 with the results herein also showing that CNs were present in the WPC generated at pH 4.6 upon

296 centrifugation (Figure 1). The precipitation of CNs from camel milk during isoelectric focusing

297 or cheese-making has been described as challenging. This has been related to its low content of

298 κ-CN (~ 4 times less than in bovine milk) and relatively large CN micelle size (Berhe, Seifu,

299 Ipsen, Kurtu & Hansen, 2017; Wangoh et al., 1998), thus making it difficult for the whey

300 proteins to be completely separated from the CNs.

301 3.2 Physicochemical properties of the WPC hydrolysates

302 The AN of the 15 hydrolysates obtained from the experimental design varied between 0.35 ±

303 0.01 and 1.26 ± 0.02 mg NH3 g-1 for H1 and H8, respectively (Table 1). Significant differences

304 (p < 0.05) were seen for the AN of the central point hydrolysates H15A-H15C, which varied

14
305 between 0.73 ± 0.04 and 0.85 ± 0.07 mg NH3 g-1 (Table 1). These values were, however, of the

306 same order.

307 The molecular mass distribution determined by GP-HPLC is illustrated in Figure 2A. The WPC

308 contained significant proportions of material >10 kDa. This is consistent with the molecular mass

309 of the major proteins reported in camel milk (Mati et al., 2016). Following enzymatic hydrolysis,

310 the proportion of proteins (>10 kDa) reduced in all hydrolysates, yielding lower molecular mass

311 components. While all WPC hydrolysates still contained proteins (>10 kDa), they all mainly

312 contained components <5 kDa. Interestingly, camel milk hydrolysates manufactured using the

313 same hydrolysis conditions yielded samples containing 2-3 times less intact proteins than the

314 WPC hydrolysates generated herein (Nongonierma et al., 2017).

315 The results obtained using GP-HPLC are in agreement with those obtained by SDS-PAGE

316 (Figure 1). These results showed that intact proteins within WPC were digested during

317 hydrolysis, resulting in the appearance of bands with a lower molecular mass. However, bands

318 corresponding to CNs and whey proteins were still present within hydrolysates. This was

319 particularly noticeable for the band corresponding to α-La (14 kDa). The hydrolysis of camel

320 whey proteins and CNs by trypsin and chymotrypsin has been studied earlier. Salami et al.

321 (2008) reported that incubation with these two enzymes allowed a faster digestion of CNs than

322 whey proteins. This was explained by the differences in the structure of CNs and whey proteins.

323 CNs possess a more open structure than whey proteins. The globular structure of whey proteins

324 limits the accessibility of peptide bonds to enzymes, which explains their lower rate of

325 hydrolysis.

15
326 3.3 DPP-IV inhibitory properties of camel WPC hydrolysates

327 The camel WPC hydrolysates were assessed for their ability to inhibit DPP-IV in vitro. Their

328 IC50 values are summarised in Table 1. The DPP-IV IC50 values of the WPC hydrolysates

329 prepared within the experimental design varied between 0.55 ± 0.05 and 1.52 ± 0.16 mg L-1 for

330 H8 and H6, respectively. The hydrolysates manufactured using the central point conditions

331 (H15A-H15C) were reproducible in terms of their ability to inhibit DPP-IV and no significant

332 difference existed in their DPP-IV IC50 values (p > 0.05, Table 1).

333 Modelling of the experimental DPP-IV IC50 values as a function of temperature, E:S and time

334 was performed (Equation 1). The coefficient of correlation R2 of the model was 0.83 (Table 2).

335 The model was statistically significant (p = 0.0495), however, the lack of fit (p = 0.0244)

336 suggested that the model did not adequately fit the experimental data. The correlation between

337 the experimental and predicted DPP-IV IC50 values was low as presented in Supplementary Fig

338 S1. However, the model still allowed prediction, to a certain extent, of the DPP-IV IC50 values

339 on the basis of the hydrolysis parameters (temperature, E:S and time). Four additional

340 hydrolysates H16-H19 were manufactured and their DPP-IV IC50 values were used to externally

341 validate the model. Their IC50 values varied between 0.67 ± 0.11 and 1.00 ± 0.04 mg L-1 for H19

342 and H16, respectively (Table 1). The R2 linking experimental and predicted DPP-IV IC50 values

343 obtained when these four hydrolysates were taken into account was 0.80. This suggested that the

344 model allowed prediction of the DPP-IV IC50 value of hydrolysates manufactured within the

345 experimental domain of the DOE. The only coefficient of Equation 1 which was significant (p <

346 0.05) was that associated with the E:S as shown in Table 2.

347 The hydrolysis conditions predicted to yield the lowest DPP-IV IC50 value, i.e., 0.58 mg L-1 ,

348 were determined as being E:S, 1.775; temperature, 55°C and duration, 240 min. These hydrolysis

16
349 conditions were employed to generate H20A-H20C. Their DPP-IV IC50 values and their AN

350 contents were not significantly different (p > 0.05, Table 1). However, there was a significant

351 difference (p < 0.05), between the experimentally determined and predicted DPP-IV IC50 values,

352 i.e., 0.76 ± 0.04 vs. 0.58 mg L-1.

353 A limited number of studies have shown that camel milk protein hydrolysates inhibit DPP-IV in

354 vitro (Mudgil et al., 2018; Nongonierma et al., 2017; Tagliazucchi et al., 2018). Recently, potent

355 camel milk protein hydrolysates having DPP-IV IC50 values ranging from 0.09 ± 0.01 to 0.46 ±

356 0.04 mg L-1, for hydrolysates generated with AlcalaseTM and papain, respectively, have been

357 reported (Mudgil et al., 2018). In their study, Tagliazucchi et al. (2018) reported DPP-IV IC50

358 values of 17.2 ± 0.8 mg L-1 for the 3 kDa permeate of a whole camel milk SGID sample. The

359 DPP-IV IC50 values obtained with the camel WPC herein were of the same order of those

360 reported for camel milk protein hydrolysates obtained with the same enzyme preparation (PTN

361 6.0S), which ranged between 0.52 ± 0.06 and 1.26 ± 0.13 mg L-1 (Nongonierma et al., 2017).

362 The fact that hydrolysed WPC yielded relatively potent DPP-IV inhibitory hydrolysates indicates

363 a positive contribution of both α-La and CN-derived peptides to DPP-IV inhibition. In contrast

364 with the earlier study (Nongonierma et al., 2017), where all three parameters (temperature, E:S

365 and time) had a significant effect, E:S was the only parameter having a significant influence on

366 the DPP-IV inhibitory properties of camel WPC hydrolysates (Table 2). These results suggest

367 that enzyme concentration was the limiting parameter in the release of DPP-IV inhibitory

368 peptides from camel WPC. Hydrolysis temperature and time can affect the extent of protein

369 hydrolysis, however, they did not appear to play a role in the release of DPP-IV inhibitory

370 peptides herein. This may be due to a decreased accessibility of trypsin to the peptide bonds in

371 camel whey proteins and CNs. The camel WPC contains a higher ratio of α-La:CNs than camel

17
372 milk (i.e., 0.92:1.00 and 0.10:1.00, respectively). Therefore it is likely that α-La and CNs behave

373 like competitive substrates during hydrolysis with trypsin. Camel α-La is a globular protein

374 which contains 4 disulfide bridges (Mati et al., 2016) and is likely to be more complex to

375 hydrolyse than CNs. Vorob’ev, Rao & Kochetkov (2016) suggested that globular proteins need

376 to go through a demasking process prior to the enzyme being able to access peptide bonds.

377 Trypsin may therefore need to spend significant time to hydrolyse α-La. An increase in the E:S

378 would augment accessibility of both α-La and CN peptide bonds, resulting in a more efficient

379 peptide release. This may help explain why a positive effect of E:S on DPP-IV inhibition by the

380 hydrolysates herein was observed.

381 3.4 SGID of the WPC and H20

382 WPC and H20A were subjected to SGID, yielding, WPC_SGID and H20A_SGID, respectively.

383 The RP-UPLC profiles of WPC and H20C before and after SGID are illustrated in Figure 2B and

384 2C. Modifications in the peptide profile were seen following SGID for both samples. Peaks

385 corresponding to intact proteins were seen in the RP profile of WPC. After SGID of WPC, there

386 was an increase in the number of peptide peaks seen in the RP-UPLC profile Figure 2B. In

387 particular, peptide peaks eluted at lower retention times in WPC_SGID compared to WPC,

388 showing the generation of hydrophilic peptides. The RP-ULPC profile of H20A_SGID showed

389 more intense peptide peaks eluting at lower retention times in comparison with H20A (Figure

390 2C).

391 Unhydrolysed camel WPC was not able to inhibit DPP-IV at the highest concentration tested

392 (i.e., 2.8 mg mL-1). The DPP-IV IC50 value of WPC_SGID was 0.83 ± 0.10 mg L-1, showing that

393 SGID had a beneficial effect on increasing the DPP-IV inhibitory potency of WPC (Table 1).

394 The DPP-IV IC50 value of H20A appeared to be unaffected after SGID, i.e., 0.76 ± 0.03 vs. 0.99

18
395 ± 0.14 mg L-1 for H20A and H20A_SGID, respectively (Table 1). The DPP-IV IC50 value of

396 WPC_SGID was comparable to that obtained for camel milk SGID, i.e., 0.70 ± 0.13 mg L-1

397 (Nongonierma et al., 2017) and was lower than that reported for the 3 kDa permeate of whole

398 camel milk SGID 17.2 ± 0.8 mg L-1 (Tagliazucchi et al., 2018). These differences may come

399 from the origin (porcine vs rat) and level (Units) of DPP-IV activity used during the in vitro

400 inhibition assay.

401 3.5 Identification of DPP-IV inhibitory peptides within the camel WPC hydrolysates

402 Peptide identification in four samples (H8, H20A, SGID_H20A and SGID_WPC) was carried

403 out by LC-MS/MS. Seven known DPP-IV inhibitory sequences were found within the peptides

404 identified in all 4 samples. These peptides were present in H8, H20A, SGID_H20A and

405 SGID_WPC (Supplementary Table S1). The potency of these known DPP-IV inhibitory peptides

406 ranged from 44 to 3217 µM for LPVPQ and EK, respectively (Supplementary Table S1). Most

407 known DPP-IV inhibitory peptides within the hydrolysates originated from the CNs, with the

408 exception of EK, which is present within the primary sequence of camel α-La.

409 Novel sequences possessing the features of relatively potent DPP-IV inhibitory peptides were

410 predicted to be present using an in silico approach. Confirmatory studies were then conducted

411 with synthetic peptide sequences. Twelve sequences were synthesised and analysed for their

412 ability to inhibit DPP-IV in vitro. Ten of which (EPVK, FLNK, IIF, LLNEK, LQL, LQPK, VPF,

413 VPV, YPI and YPLR) originated from CNs and 2 from whey proteins (FYAPELL and

414 LAHKPL). FYAPELL was identified within H8 and H20A (Table 3). The amino acid sequence

415 of camel serum albumin has not been determined to date. However, FYAPELL is found within

416 serum albumin from other mammals, including human, bovine, sheep, goat, pig, Sumatran

417 orangutan, rhesus macaque, cat, dog, etc. Therefore, is likely that FYAPELL belongs to a

19
418 conserved sequence of serum albumin and may originate from camel serum albumin. LAHKPL,

419 an α-La-derived peptide (f105-110), was identified is all four samples.

420 For the 12 synthetic peptides, the % DPP-IV inhibition measured with 1000 µM peptide varied

421 between -7.4 ± 2.5 and 98.8 ± 0.1% for IIF and VPV, respectively (Table 3). The DPP-IV IC50

422 value of the most potent peptides was determined. An inhibition of < 50% was obtained for

423 FYAPELL and LQPK at the highest concentration tested in the dose-response experiment, i.e.,

424 2000 µM. The DPP-IV IC50 value of the other peptides evaluated (EPVK, FLNK, LAHKPL,

425 LLNEK, VPF, VPV, YPI and YPLR) varied between 6.6 ± 0.5 and 1367.2 ± 107.3 µM for VPV

426 and FLNK, respectively (Table 3). The majority of the potent DPP-IV inhibitory peptides

427 identified in the WPC hydrolysates originate from CNs. This suggests that CN are the major

428 source of peptides which contributes to the DPP-IV inhibitory properties of camel milk protein

429 hydrolysates. The relative contribution of tryptic peptides derived from camel whey proteins and

430 CNs to DPP-IV inhibition has not been mentioned in the literature, to date. Based on these

431 results, the generation of DPP-IV inhibitory camel milk protein hydrolysates with trypsin should

432 be more effective starting with protein substrates rich in CNs such as milk protein concentrate or

433 casein-based ingredients.

434 The mode of inhibition of the most potent DPP-IV inhibitory peptides (EPVK, LAHKPL, VPF,

435 VPV, YPI and YPLR), having DPP-IV IC50 values < 400 µM, was determined. Their

436 Lineweaver and Burke double reciprocal plots are shown in Figure 3. All six peptides studied

437 were competitive inhibitors of DPP-IV (Table 3).

438 As expected, most known and novel DPP-IV inhibitors identified within the camel WPC

439 hydrolysates (Supplementary Table S1 and Table 3) had sequences compatible with the cleavage

440 specificity of trypsin and chymotrypsin, which are the major activities present within PTN 6.0S

20
441 (Novozymes). Given the source of PTN 6.0S (porcine pancreas), it is likely that this preparation

442 also contain pancreatic elastase and other exopeptidase activities as shown previously with a

443 similar enzyme preparation (Mullally, O'Callaghan, FitzGerald, Donnelly & Dalton, 1994).

444 Trypsin preferentially cleaves peptide bonds at the C-terminal side of K/R, while chymotrypsin

445 and elastase have been reported to cleave peptide bonds post F/K/W/Y and A/I/L/V, respectively

446 (Bode, Meyer Jr & Powers, 1989; Godfrey, 1996).

447 A particularly potent DPP-IV inhibitory peptide, VPV, was identified for the first time in camel

448 WPC hydrolysates. IPI and VPL, also known as Diprotin A and B, respectively, are the most

449 potent DPP-IV inhibitory peptides reported to date (Nongonierma & FitzGerald, 2017a). VPV

450 had a DPP-IV IC50 value of 6.6 ± 0.5 µM. Its potency is ~ half that of IPI (3.59 ± 0.51 µM, Table

451 1) and twice that of VPL (15.8 µM (Umezawa, Aoyagi, Ogawa, Naganawa, Hamada &

452 Takeuchi, 1984)). VPV originates from camel β-CN (f213-215). VPV was reported during the

453 generation of a bovine CN hydrolysate as a potent DPP-IV inhibitor having an IC50 value of 3.19

454 µM (Yamada, Sakurai & Ochi, 2017). However, VPV does not appear in the sequence of the

455 major bovine milk proteins. In the study from Yamada et al., VPV may have originated from

456 minor milk proteins or a contaminant present within the bovine milk sample studied. To our

457 knowledge, VPV is the next most potent peptide to IPI, which has been identified within a food

458 (i.e. camel milk) protein hydrolysate. Interestingly, VPV can also be found within other

459 mammalian β-CNs, including those originating from donkey, chimpanzee, pig and human. Novel

460 peptides other than VPV, having a relatively high potency (DPP-IV IC50 value < 100 µM), were

461 identified within the camel WPC. These included VPF and YPI, both of which originated from

462 camel β-CN. LAHKPL and FYAPELL, which were novel whey protein-derived DPP-IV

463 inhibitory peptides, had DPP-IV IC50 values of 239.7 ± 24.7 and >2000.0 µM, respectively.

21
464 Overall, peptides originating from camel whey proteins, did not yield potent DPP-IV inhibition

465 (Supplementary Table S1 and Table 3). Similar results have been reported during the

466 identification of known DPP-IV inhibitory peptides in a camel milk SGID fraction, where most

467 peptides appeared to originate from CNs (Tagliazucchi et al., 2018). The most potent novel DPP-

468 IV inhibitory peptides identified within the camel WPC hydrolysates were VPV, VPF and YPI.

469 All three tripeptides possessed a P at position 2. This is in agreement with the features of DPP-IV

470 inhibitory tripeptides having DPP-IV IC50 values < 100 µM, which have been reported to always

471 possess a P at position 2 (Nongonierma & FitzGerald, 2017a).

472 The LC-MS/MS methodology used herein to identify the peptides within the camel milk protein

473 hydrolysates was based on a qualitative approach. Therefore, it does not allow quantification of

474 the peptides released during hydrolysis. Future work should include the development of an

475 analytical approach to specifically quantify those DPP-IV inhibitory peptides within selected

476 camel milk protein hydrolysates.

477 VPV and VPF were identified in H20A, SGID_H20A and SGID_WPC, which suggests that they

478 are relatively stable to hydrolysis by gastrointestinal enzymes. An earlier study has also

479 identified the known DPP-IV inhibitory β-CN-derived peptide, LPVPQ (f171-175), in the jejunal

480 contents of humans following the ingestion of bovine milk (Boutrou et al., 2013). The estimated

481 total concentration of the DPP-IV inhibitory peptide motifs present within camel milk was

482 calculated (Table 3 and supplementary Table S1). Potent DPP-IV inhibitory CN-derived peptides

483 are particularly rich within camel milk. For instance, VPF, VPV, YPI and VL motifs are present

484 within camel milk at levels > 18 times their DPP-IV IC50 value (Table 3 and supplementary

485 Table S1). In particular, VPV was estimated to be present in camel milk at levels 96 times higher

486 than its DPP-IV IC50 value. This analysis suggests that if these CN-derived peptides are

22
487 bioavailable in humans, they could be particularly interesting targets for release during the

488 generation of enzymatic hydrolysates of camel milk proteins.

489 The identification and quantification of peptides in the circulation of humans is critical to

490 determine their potential role in relation to the inhibition of key metabolic enzymes

491 (Nongonierma & FitzGerald, 2017b). To our knowledge, no study has identified DPP-IV

492 inhibitory peptides within human sera following the ingestion of food-protein derived

493 hydrolysates. However, LW which was identified in the camel WPC hydrolysates herein have

494 previously been found at ~ 1.5 nM in human sera following the ingestion of yoghurt with a

495 lactotripeptide preparation (Foltz, Meynen, Bianco, van Platerink, Koning & Kloek, 2007). A

496 number of studies have demonstrated a decrease in glycaemia following intake of camel milk in

497 humans (for review, see: Shori, 2015). However, camel milk protein hydrolysates do not appear

498 to have been evaluated in vivo for their antidiabetic properties. Therefore, evaluation of camel

499 WPC hydrolysates and peptides in vivo may provide some insights on their ability to regulate

500 insulin secretion and glycaemia.

501 4 Conclusions

502 The generation of a camel whey protein-rich preparation with a protein content of 44.7 ± 3.4%

503 (w/w) was described herein. Camel WPC hydrolysates possessing relatively potent in vitro DPP-

504 IV inhibitory properties were identified within this study. The only parameter of the DOE which

505 was significant was the E:S. This may be linked to a competition between CNs and α-La during

506 hydrolysis by trypsin and may also be related to the globular nature of α-La which makes it quite

507 resistant to hydrolysis as evidenced by the presence of residual intact α-La in the camel WPC

508 hydrolysates. Potent DPP-IV inhibitory peptides were identified within the hydrolysates.

509 Surprisingly, a limited number of the DPP-IV inhibitory peptides originated from whey proteins

23
510 with the most potent peptides originating from camel β-CN. In particular, five peptides, VPV,

511 VPF, LPVPQ, YPI and VL had DPP-IV IC50 values < 100 µM. VPV, which possessed a DPP-IV

512 IC50 of 6.6 ± 0.5 µM appears to be the second most potent DPP-IV inhibitory peptide identified

513 within a food protein hydrolysate to date. The stability of VPV to gastric and intestinal digestive

514 enzymes suggests that it may have potential as an antidiabetic agent for humans. The fact that

515 VPV is only present in the milk proteome from a few mammalian species, suggests that camel

516 milk may be a starting substrate of choice for the generation of potent DPP-IV inhibitory

517 peptides.

518 Acknowledgements

519 The authors wish to thank the United Arab Emirates University for funding this research through

520 University Program for Advanced Research (UPAR), fund code: 31F094. The work described

521 herein was partly supported by Enterprise Ireland under Grant Number TC2013-0001, and the

522 Science Foundation Ireland Research Infrastructure Fund. Cloé Cadamuro was funded by the

523 Erasmus programme. Aurélien Le Gouic was funded under the National Development Plan

524 through the Food Institutional Research Measure by the Department of Agriculture, Food and the

525 Marine, Ireland (grant number 14/F/873). The authors wish to thank Dr. Sara Paolella for her

526 assistance with the LC-MS/MS and for her suggestions for this manuscript. The authors wish to

527 acknowledge Anushree Dwivedi and Dr. Pat Kiely for their assistance with SDS-PAGE gel

528 scanning.

529 Conflicts of interests

530 The authors declare that they have no conflict of interest.

531

24
532 References

533 Adler-Nissen, J. (1979). Determination of the degree of hydrolysis of food protein hydrolysates
534 by trinitrobenzenesulfonic acid. Journal of Agricultural and Food Chemistry, 27, 1256-
535 1262.
536 Agarwal, R. P., Swami, S. C., Beniwal, R., Kochar, D. K., Sahani, M. S., Tuteja, F. C., &
537 Ghouri, S. K. (2003). Effect of camel milk on glycemic control, risk factors and diabetes
538 quality of life in type-1 diabetes: A randomized prospective controlled study. Journal of
539 Camel Practice and Research, 10, 45-50.
540 Berhe, T., Seifu, E., Ipsen, R., Kurtu, M. Y., & Hansen, E. B. (2017). Processing Challenges and
541 Opportunities of Camel Dairy Products. International journal of food science, 2017.
542 Bode, W., Meyer Jr, E., & Powers, J. C. (1989). Human leukocyte and porcine pancreatic
543 elastase: X-ray crystal structures, mechanism, substrate specificity, and mechanism-based
544 inhibitors. Biochemistry, 28, 1951-1963.
545 Boutrou, R., Gaudichon, C., Dupont, D., Jardin, J., Airinei, G., Marsset-Baglieri, A.,
546 Benamouzig, R., Tomé, D., & Leonil, J. (2013). Sequential release of milk protein–
547 derived bioactive peptides in the jejunum in healthy humans. American Journal of
548 Clinical Nutrition, 97, 1314-1323.
549 Cayot, P., & Tainturier, G. (1997). The quantification of protein amino groups by the
550 trinitrobenzenesulfonic acid method: A reexamination. Analytical Biochemistry, 249,
551 184-200.
552 Ejtahed, H. S., Naslaji, A. N., Mirmiran, P., Yeganeh, M. Z., Hedayati, M., Azizi, F., &
553 Movahedi, A. M. (2015). Effect of camel milk on blood sugar and lipid profile of patients
554 with type 2 diabetes: A pilot clinical trial. International Journal of Endocrinology and
555 Metabolism, 13.
556 Faye, B. (2015). Role, distribution and perspective of camel breeding in the third millennium
557 economies. Emirates Journal of Food and Agriculture, 27, 318.
558 Foltz, M., Meynen, E. E., Bianco, V., van Platerink, C., Koning, T. M. M. G., & Kloek, J.
559 (2007). Angiotensin converting enzyme inhibitory peptides from a lactotripeptide-
560 enriched milk beverage are absorbed intact into the circulation. Journal of Nutrition, 137,
561 953-958.
562 Godfrey, T. (1996). Protein modification. In T. Godfrey & S. West (Eds.), Industrial
563 Enzymology 2nd ed. (pp. 303-325). London: Macmillan Press.
564 Hailu, Y., Hansen, E. B., Seifu, E., Eshetu, M., Ipsen, R., & Kappeler, S. (2016). Functional and
565 technological properties of camel milk proteins: A review. Journal of Dairy Research,
566 83, 422-429.
567 IDF. (2017). IDF diabetes atlas, 8th edition. In).
568 Lacroix, I. M. E., & Li-Chan, E. C. Y. (2016). Food-derived dipeptidyl-peptidase IV inhibitors as
569 a potential approach for glycemic regulation – Current knowledge and future research
570 considerations. Trends in Food Science & Technology, 54, 1-16.
571 Laemmli, U. (1970). Most commonly used discontinuous buffer system for SDS electrophoresis.
572 Nature, 227, 680-686.
573 Le Maux, S., Nongonierma, A. B., Barre, C., & FitzGerald, R. J. (2016). Enzymatic generation
574 of whey protein hydrolysates under pH-controlled and non pH-controlled conditions:
575 Impact on physicochemical and bioactive properties. Food Chemistry, 199, 246-251.

25
576 Mati, A., Senoussi-Ghezali, C., Ahmed Zennia, S. S., Almi-Sebbane, D., El-Hatmi, H., &
577 Girardet, J.-M. (2016). Dromedary camel milk proteins, a source of peptides having
578 biological activities – A review. International Dairy Journal, 73, 25-37.
579 Mudgil, P., Kamal, H., Yuen, G. C., & Maqsood, S. (2018). Characterization and identification
580 of novel antidiabetic and anti-obesity peptides from camel milk protein hydrolysates.
581 Food Chemistry, 259, 46-54.
582 Mullally, M. M., O'Callaghan, D. M., FitzGerald, R. J., Donnelly, W., & Dalton, J. P. (1994).
583 Proteolytic and peptidolytic activities in commercial pancreatic protease preparations and
584 their relationship to some whey protein hydrolyzate characteristics. Journal of
585 Agricultural and Food Chemistry, 42, 2973-2981.
586 Nongonierma, A. B., & FitzGerald, R. J. (2012). Tryptophan-containing milk protein-derived
587 dipeptides inhibit xanthine oxidase. Peptides, 37, 263-272.
588 Nongonierma, A. B., & FitzGerald, R. J. (2013). Dipeptidyl peptidase IV inhibitory and
589 antioxidative properties of milk-derived dipeptides and hydrolysates. Peptides, 39, 157-
590 163.
591 Nongonierma, A. B., & FitzGerald, R. J. (2017a). Features of dipeptidyl peptidase IV (DPP-IV)
592 inhibitory peptides from dietary proteins. Journal of Food Biochemistry, In press, doi:
593 10.1111/jfbc.12451.
594 Nongonierma, A. B., & FitzGerald, R. J. (2017b). Strategies for the discovery and identification
595 of food protein-derived biologically active peptides. Trends in Food Science &
596 Technology, 69, 289-305.
597 Nongonierma, A. B., Le Maux, S., Dubrulle, C., Barre, C., & FitzGerald, R. J. (2015). Quinoa
598 (Chenopodium quinoa Willd.) protein hydrolysates with in vitro dipeptidyl peptidase IV
599 (DPP-IV) inhibitory and antioxidant properties. Journal of Cereal Science, 65, 112-118.
600 Nongonierma, A. B., Paolella, S., Mudgil, P., Maqsood, S., & FitzGerald, R. J. (2017).
601 Dipeptidyl peptidase IV (DPP-IV) inhibitory properties of camel milk protein
602 hydrolysates generated with trypsin. Journal of Functional Foods, 34, 49-58.
603 Nongonierma, A. B., Paolella, S., Mudgil, P., Maqsood, S., & FitzGerald, R. J. (2018).
604 Identification of novel dipeptidyl peptidase IV (DPP-IV) inhibitory peptides in camel
605 milk protein hydrolysates. Food Chemistry, 244, 340-348.
606 Salami, M., Yousefi, R., Ehsani, M. R., Dalgalarrondo, M., Chobert, J.-M., Haertlé, T., Razavi,
607 S. H., Saboury, A. A., Niasari-Naslaji, A., & Moosavi-Movahedi, A. A. (2008). Kinetic
608 characterization of hydrolysis of camel and bovine milk proteins by pancreatic enzymes.
609 International Dairy Journal, 18, 1097-1102.
610 Shori, A. B. (2015). Camel milk as a potential therapy for controlling diabetes and its
611 complications: A review of in vivo studies. Journal of food and drug analysis, 23, 609-
612 618.
613 Song, J. J., Wang, Q., Du, M., Ji, X. M., & Mao, X. Y. (2017). Identification of dipeptidyl
614 peptidase-IV inhibitory peptides from mare whey protein hydrolysates. Journal of Dairy
615 Science, 100, 6885-6894.
616 Spellman, D., O’Cuinn, G., & FitzGerald, R. J. (2009). Bitterness in Bacillus proteinase
617 hydrolysates of whey proteins. Food Chemistry, 114, 440-446.
618 Tagliazucchi, D., Martini, S., Shamsia, S., Helal, A., & Conte, A. (2018). Biological activities
619 and peptidomic profile of in vitro-digested cow, camel, goat and sheep milk.
620 International Dairy Journal, 81, 19-27.

26
621 Tulipano, G., Cocchi, D., & Caroli, A. M. (2012). Comparison of goat and sheep β-lactoglobulin
622 to bovine β-lactoglobulin as potential source of dipeptidyl peptidase IV (DPP-4)
623 inhibitors. International Dairy Journal, 24, 97-101.
624 Umezawa, H., Aoyagi, T., Ogawa, K., Naganawa, H., Hamada, M., & Takeuchi, T. (1984).
625 Diprotins A and B, inhibitors of dipeptidyl aminopeptidase IV, produced by bacteria.
626 Journal of Antibiotics, 37, 422-425.
627 Vorob’ev, M., Rao, N., & Kochetkov, K. (2016). Kinetic modeling of demasking and hydrolysis
628 of peptide bonds during proteolysis of β-lactoglobulin by trypsin. In Doklady
629 Biochemistry and Biophysics, vol. 471 (pp. 423-427): Springer.
630 Walsh, D. J., Bernard, H., Murray, B. A., MacDonald, J., Pentzien, A. K., Wright, G. A., Wal, J.
631 M., Struthers, A. D., Meisel, H., & FitzGerald, R. J. (2004). In vitro generation and
632 stability of the lactokinin β-lactoglobulin fragment (142–148). Journal of Dairy Science,
633 87, 3845-3857.
634 Wangoh, J., Farah, Z., & Puhan, Z. (1998). Iso-electric focusing of camel milk proteins.
635 International Dairy Journal, 8, 617-621.
636 WHO. (2016). Global report on diabetes. Available at
637 http://apps.who.int/iris/bitstream/10665/204871/1/9789241565257_eng.pdf. In).
638 Yamada, A., Sakurai, T., & Ochi, D. (2017). Dipeptidyl peptidase-IV inhibitor. Patent
639 application number US9617300B2. In USPTO (Ed.)). Japan: Morinaga Milk Industry Co
640 Ltd.
641 Zhang, Y., Chen, R., Zuo, F., Ma, H., Zhang, Y., & Chen, S. (2016). Comparison of dipeptidyl
642 peptidase IV-inhibitory activity of peptides from bovine and caprine milk casein by in
643 silico and in vitro analyses. International Dairy Journal, 53, 37-44.
644
645
646

27
647 Table 1. Extent of hydrolysis measured as the amino group content (AN) and dipeptidyl peptidase IV (DPP-IV) half maximal inhibitory

648 concentration (IC50) value of camel whey protein concentrate (WPC) hydrolysates (H1-H20) and the simulated gastrointestinal digestion (SGID) of

649 H20A (H20A_SGID) and of camel WPC (WPC_SGID).

1 2 2
Hydrolysate Variable levels AN DPP-IV IC50
Temperature (°C) E:S (%) Time (min) (mg NH3 g-1) (mg mL-1)
H1 40 (-1) 0.50 (-1) 60 (-1) 0.35 ± 0.01 1.37 ± 0.34
H2 40 (-1) 0.50 (-1) 240 (+1) 0.51 ± 0.01 1.22 ± 0.10
H3 40 (-1) 1.25 (0) 150 (0) 0.77 ± 0.03 1.09 ± 0.14
H4 40 (-1) 2.00 (+1) 60 (-1) 0.68 ± 0.01 1.06 ± 0.10
H5 40 (-1) 2.00 (+1) 240 (+1) 0.498 ± 0.02 0.85 ± 0.11
H6 60 (+1) 0.50 (-1) 60 (-1) 0.41 ± 0.02 1.52 ± 0.16
H7 60 (+1) 0.50 (-1) 240 (+1) 0.72 ± 0.03 1.15 ± 0.15
H8 60 (+1) 1.25 (0) 150 (0) 1.03 ± 0.02 0.55 ± 0.05
H9 60 (+1) 2.00 (+1) 60 (-1) 1.26 ± 0.02 0.57 ± 0.03
H10 60 (+1) 2.00 (+1) 240 (+1) 0.63± 0.01 0.62 ± 0.08
H11 50 (0) 1.25 (0) 60 (-1) 0.64 ± 0.01 0.58± 0.13
H12 50 (0) 0.50 (-1) 150 (0) 0.52 ± 0.01 0.80 ± 0.05
H13 50 (0) 1.25 (0) 240 (+1) 0.89 ± 0.01 0.60 ± 0.15
H14 50 (0) 2.00 (+1) 150 (0) 1.08 ± 0.02 0.73 ± 0.22
H15A 50 (0) 1.25 (0) 150 (0) 0.80 ± 0.04a ,b 0.76 ± 0.03a
H15B 50 (0) 1.25 (0) 150 (0) 0.85 ± 0.07b 0.73 ± 0.19a
H15C 50 (0) 1.25 (0) 150 (0) 0.73 ± 0.04a 0.80 ± 0.05a

28
 H16 50 (0) 0.50 (-1) 60 (-1) 0.34 ± 0.01 1.00 ± 0.04
H17 50 (0) 0.50 (-1) 240 (+1) 0.53 ± 0.01 0.93 ± 0.17
H18 50 (0) 2.00 (+1) 60 (-1) 0.89 ± 0.02 0.76 ± 0.07
H19 50 (0) 2.00 (+1) 240 (+1) 1.26 ± 0.03 0.67 ± 0.11
H20A 55 (0.45) 1.8 (0.70) 240 (1) 0.97 ± 0.03A 0.76 ± 0.03A
H20B 55 (0.45) 1.8 (0.70) 240 (1) 0.94 ± 0.02A 0.73 ± 0.19A
H20C 55 (0.45) 1.8 (0.70) 240 (1) 0.93 ± 0.01A 0.80 ± 0.05A
H20A_SGID - - - nd 0.99± 0.14
WPC_SGID - - - nd 0.83 ± 0.10
1
650 The z-centred values for each variable of the experimental design are provided into brackets.
2
651 Mean ± SD (n=3). Within the same column, values with different superscript letters are significantly different (p < 0.05). IC50: concentration inducing 50% DPP-IV inhibition,
652 expressed in mg protein equivalent per mL (mg mL-1). The IC50 value of the positive control Ile-Pro-Ile was of 3.59 ± 0.51 µM.
653 nd: not determined.
654

29
655 Table 2. Coefficients of the multilinear regression (MLR) model correlating the dipeptidyl peptidase IV (DPP-IV) half maximal inhibitory

656 concentration (IC50) of the camel milk protein hydrolysates to the parameters of the design of experiment (DOE) and their interactions.

Pa r a me t e r s Co e f f ic ie n t St a n d a r d t val ue p2
of t he e s t ima t e Er r o r
1
mo d e l
Complete Intercept 0.68 0.08 8.40 6.65E-05
ES -0.22 0.06 -3.74 7.22 E-03
T -0.12 0.06 -1.96 0.09
t -0.07 0.06 -1.12 0.30
ES² 0.16 0.12 1.39 0.21
T² 0.21 0.12 1.85 0.11
t² -0.02 0.12 -0.19 0.86
ES×T -0.10 0.07 -1.48 0.18
ES×t 0.05 0.07 0.69 0.51
T×t 4.50E-03 0.07 0.07 0.95
Root mean squared error = 0.188; R2 = 0.83; p-value model = 0.0495; p-value lack of fit = 0.0244
1
657 T: temperature; ES: enzyme to substrate ratio and t: time.
2
658 Parameters having a p < 0.05 are significantly different from 0.
659

660

30
661 Table 3. Dipeptidyl peptidase IV (DPP-IV) half maximal inhibitory concentration (IC50) and mode of inhibition of peptides identified within the

662 camel whey protein hydrolysates H8 and H20A and the simulated gastrointestinal digestion (SGID) of H20A (H20A_SGID) and of camel whey

663 protein concentrate (WPC_SGID). The estimated total concentration of the peptide motif in camel milk is also provided.

Pe pt id e Pr o t e in Pe pt id e s id e n t if ie d in 3: DPP-IV DPP-IV IC50 Es t ima t e d Mo d e o f

1 2 4
seq u en c e f r a g me n t in h ibit io (µM) t ot a l in h ibit io n 6
H8 H20A H20A_SGID WPC_SGID
n a t 1000 c o n c en t r a
µM (%) t io n in
c a me l
mil k (µM)5
EPVK αs1-CN (f147- nd nd 74.4 ± 2.9 330.1± 55.9 209.4 competitive
150)
FLNK αs2-CN (f141- nd 45.3 ± 4.0 1367.2 ± 108.2 ND
144) 107.3
FYAPELL in serum nd nd 34.5 ± 3.3 >2000.0 unknown ND
albumin from 6
mammalian
species
IIF α-La (f29-31) nd nd -7.4 ± 2.5 ND 159.4 ND
LAHKPL α-La (f105-110) 81.6 ± 2.1 239.7 ± 24.7 159.4 competitive
LLNEK κ-CN (f17-21) 37.0 ± 4.0 1360.7 ± 95.2 43.8 ND
LQL αs1-CN (f109- 3.5 ± 4.6 ND 209.4 ND
111)
LQPK β-CN (f89-92) 25.8 ± 3.5 >2000.0 632.8 ND
VPF β-CN (f86-88); 94.8 ± 0.7 55.1 ± 5.8 1265.7 competitive
β-CN (f118-120)
VPV β-CN (f213-215) 98.8 ± 0.1 6.6 ± 0.5 632.8 competitive
YPI β-CN (f69-71) nd nd 93.9 ± 0.1 35.0 ± 2.0 632.8 competitive
YPLR αs1-CN (f4-6) nd nd 74.6 ± 1.2 360.1 ± 28.1 209.4 competitive
1
664 Peptide sequences abbreviated with the one letter amino acid code.

31
 3
665 Peptide fragment in the mature protein sequence; α-La: α-Lactalbumin; CN: casein.
2
666 SGID samples were obtained by sequential digestion of H20A or camel whey with pepsin then Corolase PP hydrolysis as previously described in Walsh et al. (2004); nd: not
667 detected.
4
668 Mean ± SD (n=3). nd: not determined.
5
669 The estimated total concentration of peptide motif in camel milk was calculated using the concentration of individual milk proteins reported in Hailu et al. (2016).
6
670 Mode of DPP-IV inhibition determined using the Lineweaver and Burk double reciprocal plots. ND: not determined.
671

32
672 Figure captions

673

674 Figure 1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profile of the

675 camel whey protein, camel whey protein concentrate (WPC), WPC hydrolysates H1-H15 and

676 their associated negative controls (C40, C50 and C60). α-La: α-lactalbumin; CNs: caseins; LF:

677 lactoferrin.

678

679 Figure 2. (A) Molecular mass distribution determined by gel permeation high performance

680 liquid chromatography profile (GP-HPLC) of camel whey (WP), camel whey protein concentrate

681 (WPC), WPC hydrolysates H1-H120 and their associated negative controls (C40, C50 and C60)

682 and the simulated gastrointestinal digestion (SGID) of H20A (H20A_SGID) and of WPC

683 (WPC_SGID). Reverse-phase ultra-performance chromatography profile of (B) camel WPC

684 before and after SGID (WPC_SGID) and (C) hydrolysate H20A before and after SGID

685 (H20A_SGID).

686

687 Figure 3. Lineweaver and Burke double reciprocal plot representation for camel milk protein-

688 derived dipeptidyl peptidase IV (DPP-IV) inhibitory peptides (A) EPVK, (B) LAHKPL, (C)

689 VPF, (D) VPV, (E) YPI, and (F) YPLR.

690

33