Characterization and identification of novel antidiabetic and anti-obesity peptides from camel milk protein hydrolysates

Accepted Manuscript Characterization and identification of novel antidiabetic and anti-obesity peptides from camel milk protein hydrolysates Priti Mudgil, Hina Kamal, Gan Chee Yuen, Sajid Maqsood PII: DOI: Reference:

S0308-8146(18)30515-6 https://doi.org/10.1016/j.foodchem.2018.03.082 FOCH 22625

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

8 November 2017 18 March 2018 19 March 2018

Please cite this article as: Mudgil, P., Kamal, H., Chee Yuen, G., Maqsood, S., Characterization and identification of novel antidiabetic and anti-obesity peptides from camel milk protein hydrolysates, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.03.082

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Characterization and identification of novel antidiabetic and anti-obesity peptides from camel milk protein hydrolysates Priti Mudgil1, Hina Kamal1, Gan Chee Yuen2* and Sajid Maqsood1*$

1*$

Food Science department, College of Food and Agriculture, United Arab Emirates University, Al-Ain, 15551, United Arab Emirates. Corresponding authors: Email: [email protected] Tel: +971 37134519 Fax: +971 37675336

2

Analytical Biochemistry Research Centre (ABrC), Universiti Sains Malaysia, 11800 USM, Penang, Malaysia Email: [email protected]

Abstract In-vitro inhibitory properties of peptides released from camel milk proteins against dipeptidyl peptidase-IV (DPP-IV), porcine pancreatic α-amylase (PPA), and pancreatic lipase (PPL) was studied. Results revealed that upon hydrolysis by different enzymes, camel milk proteins displayed dramatic increase in inhibition of DPP-IV and PPL, only slight improvement in PPA inhibition was noticed. Peptide sequencing revealed a total of 20 and 3 peptides for A9 and B9 respectively, obtained the score of 0.8 or more on peptide ranker and were categorized as potentially active peptides eligible to be DPP-IV inhibitory peptide. KDLWDDFKGL in A9 and MPSKPPLL in B9 were identified as most potent PPA inhibitory peptide. For PPL inhibition only 7 and 2 peptides qualified as PPL inhibitory peptides from hydrolysates A9 and B9, respectively. The present study report for the first time PPA and PPL inhibitory and only second for DPP-IV inhibitory potential of protein hydrolysates from camel milk. Key words: dipeptidyl peptidase IV inhibition; porcine pancreatic α-amylase inhibition, pancreatic lipase inhibition, camel milk proteins; bioactive peptides; alcalase, papain, bromelain.

1. Introduction Milk proteins, apart from being an excellent source of nutrients and essential amino acids for humans, have other innnumerable functions like boosting immune system and protection from various diseases. Milk proteins within their primary structures encrypts various bioactive peptides possessing wide range of biological activities. Release of these latent bioactive peptides upon hydrolysis by gut or exogenous enzymes or microbial fermentation can impart a range of beneficial effects on the host. In quest of exploring more potent and novel bioactive peptides, a range of food proteins from various sources have been screened for their biological activities e.g. fish, soyabean, sunflower, milk etc (Li-Chan, 2015). Bioactive peptides from bovine milk proteins remained the most widely studied source (Nongonierma & FitzGerald, 2015). However, the bioactive potential of milk proteins from non-bovine sources predominantly camel milk are limited. One humped camel (Camelus dromedarius) milk has been utilized from ancient times for treatment of various ailments like autoimmune diseases and metabolic disorders. Scientifc literature regarding nutritional and health promoting benefits of whole camel milk have claimed for their anti-cancerous, anti-diabetic, anti-hypertensive and anti-infectious effects (Al Kanhal, 2010). The unique health benefits of camel milk in comparison to bovine milk might be linked to differences in their protein composition. Compositionally camel milk is very high in vitamin C, lactoferrin, lysozyme, lacto-peroxidase, minerals (calcium, magnesium, copper, iron, zinc, phosphorous, potassium and sodium) and immunoglobulins providing beneficial effect against pathogens and boosting immune responses (Mati, Senoussi-Ghezali, Zennia, Almi-Sebbane, ElHatmi, & Girardet, 2017). The above mentioned health promoting effect could also be due to the release of potential bioactive peptides from camel milk proteins during gastrointestinal digestion.

Therefore, this area demands further investigations in order to generate potent bioactive peptides from camel milk under optimised conditions and exploring them for their bioactive properties. In the recent years, camel milk derived peptides having potential health promoting effects are the focus of several investigations mostly related to in vitro anti-oxidant, anti-hypertensive and antimicrobial activities (AlShamsi, Mudgil, Hassan & Maqsood, 2018; Kumar, Chatli, Singh, Mehta, & Kumar, 2016a; Moslehishad et al., 2013). However, potential of camel milk derived bioactive peptides to demonstrate antidiabetic, antiobesity and anticancerous effects needs to be thoroughly investigated. The inhibition of some metabolic enzymes like dipeptidyl peptidase IV (DPP-IV), an enzyme involved in the cleavage of incretin hormones, amylase and pancreatic lipase (PL) metabolic enzymes involved in digestion of carbohydrates and fatty acids, respectively, have been an interesting target for the development of antidiabetic and antiobesity agents (Birari & Bhutani, 2007; Tundis, Loizzo, & Menichini, 2010). In our previous studies, trypsin derived camel milk protein hydrolysates has displayed potent DPP-IV inhibitory properties in vitro (Nongonierma, Paolella, Mudgil, Maqsood, & FitzGerald, 2017; 2018). However, generation of peptides with enzymes other than trypsin could yield more potent and novel DPP-IV inhibitory peptides upon hydrolysis. Moreover, to the best of our knowledge, camel milk proteins have never been studied for their ability to release amylase and pancreatic lipase inhibitory peptides. Therefore, the present study was undertaken to explore the potential of camel milk derived bioactive peptides generated using food grade proteolytic enzymes (alcalase, bromelain, and papain) towards inhibition of three key metabolic enzymes. A balanced design of experiment (DOE) was used to maximize the release of potential inhibitory peptides from camel milk proteins upon enzymatic hydrolysis. The hydrolysates generated were characterised from a

physicochemical perspective (degree of hydrolysis (DH) and peptide profile using RP-HPLC and SDS-PAGE and peptide identification was also carried out using Liquid Chromatography Mass Spectrometry-Linear Trap Quadropole (LTQ) Orbitrap (LCMS-LTQ Orbitrap) system. 2. Materials & Methods 2.1 Chemicals and reagents Alcalase (from Bacillus licheniformis), bromelain (from pineapple stem), papain (from papaya latex), porcine pancreatic α-amylase VI, lipase from porcine pancreas, dipeptidyl peptidase IV (DPP-IV), Gly-Pro-p-nitroanilide, p-Nitrophenyl Palmitate (PNPP), diprotin (Ile-Pro-Ile), acarbose, orlistat, o-pthaldehyde (OPA), trifluoroacetic acid (TFA), acetonitrile (HPLC grade), sodium tetraborate, β-mercaptoethanol, sodium dodecyl sulphate (SDS), trizma base were purchased from Sigma Aldrich (St. Louis, MO, USA). All reagents for electrophoresis were purchased from Bio-Rad (Richmond, CA, USA). 2.2 Design of experiment (DOE) for production of camel milk protein hydrolysates (CMPHs) Raw camel milk samples from different healthy camels (Camelius dromedaries) of same breed were procured from Al Ain Dairy Farm, Al Ain, Abu Dhabi, UAE. Camels were grown in a semi-intensive rearing system and fed with fed ad libitum on Rhodes grass (Chloris gayana) hay diet incorporated with date seed powder. The collected milk samples were immediately refrigerated and transferred to the laboratory at United Arab Emirates University. Camel milk samples were skimmed via centrifugation at 4500 rpm for 10 min at 4°C and pooled together to be used as a composite sample. The enzymatic hydrolysis of milk proteins was carried out with three different enzymes (i.e. alcalase, bromelain and papain). One portion of skimmed camel milk was kept as control while other portions were adjusted to pH 8 for alcalase, and pH 7 for

bromelain and papain, respectively, using a digital type pH meter (OHAUS, Starter 3100, USA). Protein hydrolysis was carried out at 50°C under constant stirring in a water bath at an enzyme/substrate (protein) ratio of 1:100 (w/w). The samples were removed after every 3 h interval up to 9 h, the enzymatic reactions were stopped by heating the samples in a water bath at 100 °C for 10 min. The samples were centrifuged (10,000xg, 15 min, 4 °C) and supernatant was collected and stored at -20 °C until further analysis. 2.3 Characterization of camel milk protein hydrolysate (CMPHs) 2.3.1 Degree of Hydrolysis (DH) Degree of hydrolysis (DH) was analyzed using the OPA method described by Nielsen et al. (2001) with a few modifications. The OPA reagent was freshly prepared by mixing 25 mL of sodium tetraborate buffer (100 mM; pH 9.3), 2.5 mL of sodium dodecyl sulphate (20%, w/w), 40 mg of OPA (dissolved in 1 mL of methanol), and 100 µL of β-mercaptoethanol. Final volume was raised to 50 mL with milli Q water. Small aliquots (100 µL) of the samples were added directly to cuvette containing 1 mL of OPA reagent, mixed gently for 5s. The absorbance was measured at 340 nm using a Nova-Spec-II Spectrophotometer (Pharmacia, England, UK) after two minutes of incubation in dark at room temperature. Degree of hydrolysis was determined by using following equation: DH (%) =h/ htot x 100 (Eq. 1). where, htot is the total number of peptide bonds per protein equivalent; and h is the number of hydrolyzed bonds, which was determined by using h = (SerineNH2 – β)/α. Where α, β and htot values were 1.039, 0.383 and 8.2 mEq/g protein, respectively (Nielsen et al., 2001). 2.3.2 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)

Camel milk and camel milk protein hydrolysates were characterized by SDS-PAGE following the method described by Laemmli (1976) with slight modification as described by Alshamsi, Mudgil, Hassan and Maqsood (2018) under reducing conditions on a 12.5% resolving gel and 4% stacking gel using the Mini Protean III apparatus (Bio-Rad, gel size 7 x 8 cm x 0.75 mm). 2.3.3 Characterization of CMPHs by Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) Camel milk protein (CMP) and CMPHs obtained after hydrolysis were analyzed by RP-HPLC by following the method described by Vincenzetti, Polidori, Mariani, Cammertoni, Fantuz, & Vita, (2008) with modifications. The separation was carried out on the reversed-phase C4 column (BIOshell™ A400 Protein C4, 3.4 μm HPLC Columns) (Sigma-Aldrich, USA). Trifluoroacetic acid (TFA)/methanol in the ratio of 1:1000 v/v (buffer A) was used for conditioning of column. Elution was achieved by carrying out step gradient with TFA/H2O/acetonitrile 1:100:900 v/v (buffer B) (Vincenzetti et al., 2008). The flow rate of the mobile phase (buffer A) was 1 mL/min, column was maintained at a temperature of 35ºC and 5 µL of sample was injected on to the injector port. The proteins and bioactive peptides eluted from RP-HPLC column were monitored at 220 nm using photodiode array detector (Dionex UltiMate 3000 RS Diode Array detector) included in the HPLC system (UltiMate 3000, Thermoscientific, Germering, Germany). Pure protein standards from bovine milk (SigmaAldrich, USA) were used to generated calibration curve for each single protein. 2.4 Bioactive properties of camel milk protein hydrolysates (CMPHs) 2.4.1 Inhibition of DPP-IV, porcine pancreatic α-amylase (PPA) and porcine pancreatic lipase (PPL)

Dipeptidyl peptidase (DPP-IV), porcine pancreatic α-amylase (PPA) and porcine pancreatic lipase (PPL) inhibitory activities were assessed according to the method as described by Nongonierma, Paolella, Mudgil, Maqsood, & FitzGerald, (2017); Chinedum, Sanni, Theressa, & Ebere, (2017) and Badmaev, Hatakeyama, Yamazaki, Noro, Mohamed, Ho, et al., (2015), respectively. Enzyme inhibition assays were performed for intact camel milk protein (CMP) and the hydrolysates (CMPHs) after in vitro digestion by different enzymes. Percentage of enzyme inhibition was calculated using Equation 2. The assays were performed in triplicate, and a curve of percentage inhibition against sample concentration (protein equivalent mg/mL) was plotted with the averaged values. IC50 (concentration of protein hydrolysates required to produce a 50% inhibition of the initial rate of reaction) of each sample was determined by interpolation from the curve. To eliminate the background absorbance produced from samples, appropriate controls without enzyme were included for each sample and enzyme inhibition was calculated as follows. (Eq. 2) where A: control, B: control blank, C: sample, and D: sample blank, are referring to the absorbance values of reaction vials containing live enzyme and buffer, dead enzyme and buffer, live enzyme and sample and dead enzyme and sample, respectively. Substrate was present in all reactions. The enzyme inhibitions of commercial inhibitors like Diprotin for DPP-IV, Acarbose for αamylase and Orlistat for pancreatic lipase were determined at different concentrations to calculate IC50 values.

2.5 Peptide identification and sequencing by Liquid Chromatography Mass SpectrometryLinear Trap Quadropole (LTQ) Orbitrap (LCMS-LTQ Orbitrap) system Peptide sequencing was conducted using mass spectrometry analysis for CMPHs (A9 and B9). Thermo LTQ/Orbitrap fusion mass spectrometer (Thermo Scientific, San Jose, CA, USA) was used for this purpose. The analysis conditions are as follows: Pre-column: Easy-Column C18 (20 × 0.1 mm i.d., 5 m; Thermo Scientific, San Jose, CA, USA); analytical column: Easy-Column C18 (100 × 0.75 mm i.d., 3 m; Thermo Scientific, San Jose, CA, USA); mobile phase: (A) deionized distilled water with 0.1% formic acid, and (B) acetonitrile with 0.1% formic acid; gradient: 0-3 min, 0-5% B; 3-73 min, 39%; 73-78 min, 80% B; and 78-81 min, 80% B; flow rate: 3 µL/min; injection volume: 1 µL; mass range: 400-1,600; resolving power: 120,000; injection time: 100 ms; spray voltage: 2.4 kV; ion transfer tube temperature: 275 °C; charge state: >2; mass tolerance: 10 ppm; fragmentation mode: collision induced dissociation; collision energy: 35 V. PEAKS studio version 6.0 was used for data analysis and de novo sequencing . 2.5.1 Identification of bioactive peptide using Bioinformatics analysis A total of 471 and 317 peptides (Supplementary data Table S1) in A9 and B9, respectively, which were obtained from the above section were subsequently screened and selected using Peptide Ranker

web

server

(Mooney,

Haslam,

Pollastri,

&

Shields,

2012)

accessed

at

http://bioware.ucd.ie/. Based on the score of above 0.80, peptides were selected as potential bioactive peptides. These peptides were then subjected to database search (i.e. BIOPEP, PeptideDB, SwePep and EROP-Moscow) for novelty check, followed by Peptide 2 ranking to predict the enzyme (i.e. DPP-IV or α-amylase or lipase) inhibitory potential. DPP-IV (PDB code: 4A5S), α-Amylase (PDB code: 1SMD) and lipase (ID number: 1ETH) from the RCSB Protein

Data Bank were used as enzyme models. The selection of the enzyme inhibitory peptide was performed according to the significance of binding (p-value < 0.05) and potential binding sites. 2.6 Statistical analysis The main hypothesis of this study was that camel milk proteins upon hydrolysis with different food grade enzymes will generate potential bioactive peptides capable of inhibiting enzymes responsible for diabetes (DPP-IV) and obesity (α-amylase and lipase). Camel milk protein hydrolysates were generated in three batches (triplicate) for each enzyme applied. All data were subjected to one-way analysis of variance (ANOVA) using SPSS 12.0 software (SPSS INC., Chicago, IL, USA,2002). All the assays were determined in triplicate. Significant treatment means were separated by Tukey’s New Multiple Range Test at significance level 0.05. 3. Results and Discussions 3.1 Characterization of CMPHs 3.1.1 Degree of hydrolysis The protein content of the skimmed camel milk was 3.23 ± 0.3% which is similar to those previously reported in the literature, with an average of 3.27 % proteins in camel milk (Al Kanhal, 2010). Extensive hydrolysis of milk proteins can adversely affect organoleptic, functional and bioactive properties of the resultant hydrolysates. Therefore, controlled hydrolysis is an important criterion to be adopted during production of hydrolysates (Kristinsson & Rasco, 2000). The skimmed camel milk was hydrolyzed with either of three enzymes (alcalase, bromelain or papain) for 3, 6 and 9 h at their optimal conditions. The E/S ratios as well as the hydrolysis times for all enzymes were kept similar. Therefore, the variation observed in the DH was only due to the ability of each enzyme to hydrolyze camel milk proteins differently. The DH

values of nine hydrolysates (A3-9; B3-9; P3-9) generated are presented in Fig. 1a. The DH achieved with all three enzymes showed a progressive increase with maximum DH achieved by papain at 9 h. (Fig. 1a). T

he DH of all 9 hydrolysates ranged from 8.84 (A3) to 81.01% (P9). After completion of

hydrolysis for 9 h, alcalase, bromelain and papain generated hydrolysates displayed 16.7, 35.0 and 81.0% of DH, respectively. Papain treated hydrolysates (P) showed highest DH among all hydrolysates at each time interval, which indicates its ability to hydrolyze the camel milk proteins more intensively than alcalase and bromelain. These differences in DH values among hydrolysates are primarily because of differences in enzyme reaction rate, enzyme specificity and substrate affinity (Dryakova, Pihlanto, Marnila, Curda, & Korhonen, 2010). Alcalase (a serine protease) preferentially cleaves at the carboxyl side of hydrophobic amino acid residues. Papain and bromelain both are cysteine proteases but have different specificity towards substrates. Papain hydrolyses peptide bonds contributed by an adjacent amino acid like lysine, arginine and phenyl-alanine while bromelain preferred amino acid next to lysine, arginine, phenylalanine and tyrosine. Furthermore, papain has overall higher proteolytic activity than bromelain which explains why papain generated hydrolysates had high DH in comparison to later (Jung, Yun, Lee, Kim, Ha, Yoo, et al., 2016). 3.1.2 Protein and peptide profile as determined by Reversed-Phase high performance liquid chromatography (RP-HPLC) Protein and peptide profile of intact camel milk proteins (CMP) and CMPHs (A9, B9 and P9) as displayed in HPLC chromatogram is shown in Fig. 1b. Camel milk contained intact protein peaks for α, β and κ- casein eluting around 5 to 6.10 min and a small peak for αlactalbumin eluting at 6.3 min. All the major proteins detected in intact camel milk were

hydrolyzed completely by different enzymes after 9 h of hydrolysis with exception of κ- casein for which some traces were still detected in all hydrolysates samples. In all the CMPHs, the major proteins were hydrolyzed to smaller peptides which eluted as small peaks towards lower retention time. The papain (P9) and alcalase (A9) generated hydrolysate possessed somehow similar peptide profile with exception that P9 displayed some additional peptides in the retention time between 0.9 to 1.5 min demonstrating wide range of hydrophobicity and molecular weight. Extensive degradation of proteins among papain (P9) hydrolysates was also reflected from the higher DH (Fig. 1a) as well as higher degradation of protein bands (Fig. 2) as displayed in SDSPAGE pattern than alcalase and bromelain generated hydrolysates. 3.1.3 SDS-PAGE of CMP and CMPHs The changes in protein pattern of intact camel milk proteins (CMP) and the CMPHs (A39;

B3-9; P3-9) as depicted with SDS-PAGE is presented in Fig. 1c. Protein bands corresponding to

lactoferrin, serum albumin, caseins (α, β- and κ) and α-lactalbumin were detected in the intact camel milk proteins. A light band of lactoferrin was retained in unhydrolysed camel milk which upon hydrolysis was completely degraded in all the hydrolysates except those generated with bromelain where some traces were still noticed after 3 and 6 h of hydrolysis. Serum albumin was found to be resistant towards enzymatic hydrolysis with exception of papain which hydrolyzed it to some extent. Casein proteins i.e. α-, β- and κ-casein in the camel milk appeared as clear separate band between 37-22 kDa. Hydrolysates generated by bromelain displayed slight degradation of casein proteins even after 9 h. While, for papain generated hydrolysates, casein protein bands were completely hydrolyzed and were not detected even after 3 h of hydrolysis (Fig. 1c). Moreover,

alcalase and bromelain showed limited degradation ability on -

lactalbumin which was even detected after 9 h of hydrolysis, indicating that -lactalbumin was

comparatively more resistant towards hydrolysis by alcalase and bromelain when compared to papain which hydrolyzed it noticeably just after 3 h. The structural differences between caseins and -lactalbumin could be a reason for the above results as peptidic cleavage sites of lactalbumin are buried deep inside the barrel structure in comparison to random and flexible structure of caseins. Moreover, differences in enzyme activity and specificity towards caseins and -lactalbumin might also cause these variations in term of hydrolysis or degradation of protein bands (Guo, Fox, Flynn, & Kindstedt, 1995). Similar findings are also reported by other researchers where caseins are more susceptible to enzymatic hydrolysis in comparison to lactalbumin and papain was reported to be more effective in degrading the camel milk proteins compared to alcalase (Salami, Yousefi, Ehsani, Dalgalarrondo, Chobert, Haertlé, et al., 2008). 3.2 Inhibitory effect of CMPHs towards DPP-IV, porcine pancreatic α-amylase (PPA) and porcine pancreatic lipase (PPL) 3.2.1 Inhibition of DPP-IV by CMPHs The DPP-IV IC50 values of the intact camel milk proteins (CMP), and camel milk protein hydrolysates (A3-9; B3-9 and P3-9) are provided in Table 1. CMP showed some inherent DPP-IV inhibitory activity with a DPP-IV IC50 of 3.51 mg/mL. Previous reports have demonstrated that raw camel milk has the ability to reduce blood glucose level in diabetic rats, compared to raw bovine milk and DPP IV inhibition by native milk proteins could be one of the reasons for the same (Kamal, Salama & El-Saied, 2007; Sboui, Djegham, Khorchani, Hammadi, Barhoumi & Belhadj, 2010). The DPP-IV IC50 value for commercial inhibitor diprotin was 0.001 mg/mL and was found to be similar to the values as reported previously by Nongonierma & FitzGerald (2013). Upon hydrolysis, a significant decrease in DPP-IV IC50 values for all hydrolysates was observed. The DPP-IV IC50 values of all hydrolysates vary between 0.09 (A9) to 0.46 (P9)

mg/mL (Table 1). In the present study, alcalase 9 h (A9), followed by alcalase 6h (A6) and papain 3h (P3) generated hydrolysates displayed highest DPP-IV inhibitory activity (lowest DPPIV IC50 values). Camel milk protein hydrolysates (CMPHs) generated in present study exhibit comparable or more potent DPP-IV IC50 values than those reported in the literature with milk proteins hydrolysates obtained from other species i.e. bovine, caprine and mare milk. For instance, peptic hydrolysates from bovine α-lactalbumin had comparatively similar DPP-IV IC50 values to hydrolysates of present study (Lacroix & Li-Chan, 2012). Similarly, hydrolysates obtained upon treatment of bovine whey proteins with papain, papain like enzymes and Corolase PP, had lower DPP-IV IC50 values than camel milk hydrolysates reported in the present study (Le Maux, Nongonierma, Barre, & FitzGerald, 2016; Nongonierma & FitzGerald, 2013). Moreover, higher DPP-IV IC50 values than CMPHs were observed for tryptic hydrolysates of caprine and bovine CN, respectively (Zhang, Chen, Zuo, Ma, Zhang, & Chen, 2016). Interestingly, in the present study CMPHs obtained with alcalase, bromelain and papain had more potent DPP-IV IC50 values as compared to trypsin generated CMPHs in our previous study (Nongonierma et al., 2017). These difference could be due to the difference in enzyme specificity, reactivity and substrate affinity (Dryakova, Pihlanto, Marnila, Curda, & Korhonen, 2010). 3.2.2 Porcine pancreatic α-amylase (PPA) inhibition by CMPHs Management of postprandial glucose (PPG) level is an important control point in early treatment of diabetes. As carbohydrates constitute the largest proportion (40-80%) of the human diet, therefore, selective and controlled release of glucose from dietary carbohydrates via inhibition of major carbohydrases (α-amylase and α-glucosidase) holds the key to specifically control PPG levels. As shown in Table 1 even the intact camel milk proteins (CMP) had very

potent PPA inhibitory activity suggesting that camel milk possess a strong and inherent PPA inhibitory activity. Upon enzymatic hydrolysis, slight but significant decrease in PPA-IC50 value from 0.03 to 0.02 mg/mL with the alcalase and bromelain generated hydrolysates was observed. The results indicated that new peptides generated during hydrolysis enhanced the inhibitory action towards PPA. Similar results were reported by (El, Karakaya, Simsek, Dupont, Menfaatli, & Eker, 2015) where simulated digestion of goat milk and kefir had a higher inhibitory effect on PPA compared to undigested samples. In the present study, papain generated hydrolysates (P3 and P6) were not effective in decreasing IC50 values for PPA inhibition while PPA-IC50 values of P9 increased to 0.07 mg/mL, which was even higher than CPM (P<0.05). It could be due to higher DH obtained for these hydrolysates that would have caused higher degradation of proteins to shorter peptides and some free amino acids which could have lead to loss of inhibitory activity. Similar results were reported in hydrolysis of sheep milk caseins for 3 h which displayed an increase in PPA inhibition while after 5 h PPA inhibition was found to be decreased and it was correlated with high DH (Jan, Kumar, & Jha, 2016). Moreover, hydrolysis of Pinto bean proteins with protamex for 1 h improved PPA inhibition while upon further hydrolysis for up to 1.5 h resulted in decline in PPA inhibition (Ngoh & Gan, 2017). However, the PPA-IC50 values obtained in present study are comparatively lower than (Siow & Gan, 2016), where three novel peptides derived from cumin seeds named CSP1, CSP2 and CSP3 had an PPA-IC50 of 0.02, 0.03 and 0.04 µg/mL respectively. To the best of our knowledge, direct studies which have evaluated the PPA inhibitory activities of camel milk protein hydrolysates are scarce. However, some studies conducted on the PPA inhibition of fermented camel milk has indicated that microbial fermentation leads to increased PPA inhibition in comparison to native and intact camel milk proteins and could be potentially

related to the release of different bioactive peptides (Ayyash, Al-Nuaimi, Al-Mahadin, & Liu, 2018). 3.2.3 Porcine pancreatic lipase (PPL) inhibition by CMPHs Pancreatic lipase is the most important enzyme responsible for digestion of dietary fat, so its inhibition can have beneficial effects in overweight and obese individuals (Birari & Bhutani, 2007; Podsedek, Majewska, Redzynia, Sosnowska, & Koziołkiewicz, 2014). The inhibition of PPL by camel milk protein hydrolysate generated by different enzymes at 3 different time intervals was tested (Table 1). PPL-IC50 for commercial inhibitor Orlistat was found to be 0.03 mg/mL and is in assortment with available literature (Adisakwattana, Moonrat, Srichairat, Chanasit, Tirapongporn, Chanathong, et al., 2010). IC50 value for CMP was 0.12 mg/mL and is higher in comparison to CMPHs indicating that newer peptides generated upon hydrolysis demonstrated more potent PPL inhibitory activity. For alcalase generated CMPHs, PPL-IC50 value decreased to 0.04 mg/mL after 3 h of hydrolysis, then remained constant until 6 h and finally a double fold decrease in PPL-IC50 value was observed after 9 h of hydrolysis. Similar results were obtained with Bromelain hydrolysates B3, B6, and B9. Just like PPA inhibition, hydrolysates obtained with papain treatment showed stronger inhibitory potential for P3 and P6 as suggested by low PPL-IC50 values but after 9 h of hydrolysis the IC50 increased to 0.07 mg/mL suggesting a potential degradation of lipase inhibitory peptides released and loss of PPL inhibitory activity. Very few studies have reported that the proteins could inhibit PPL (Gargouri, Julien, Sugihara, Verger, & Sarda, 1984). Moreover, literature regarding potential of protein hydrolysates possessing PPL inhibition is very limited. (Ngoh, Choi, & Gan, 2017) reported that peptides obtained upon pepsin hydrolysis of pinto bean proteins resulted in enhanced lipase inhibitory activity. As per our knowledge till now

no study has been carried out on the potential of milk proteins and protein hydrolysates on PPL inhibition and present report is one of the foundation stone suggesting the milk proteins upon hydrolysis can produce potent lipase inhibitory peptides. 3.3 Identification of peptides with DPP-IV, α-amylase and lipase inhibitory activities Based on the results of inhibitory potential of CMPHs towards DPP-IV, PPA and PPL (Table 1), hydrolysates A9 and B9 were selected for identification and sequencing of peptides responsible for these properties. The result showed that a total of 471 and 317 peptides were identified in A9 and B9 hydrolysates, respectively (Supplementary Data – Table 1). At this stage of screening, only peptide sequence with an average local confidence (ALC) value of at least 80% were selected because this value gives the high confidence that a particular amino acid is accurately presented in the sequence. Subsequently, the potential bioactive peptides were shortlisted based on Peptide Ranker score (>0.80). This software allowed us to predict the probability of every peptide for being bioactive using the impact of extracellular status and amino acid composition as the general peptide features (Mooney, Haslam, Pollastri, & Shields, 2012). Table 2 shows the list of peptides that obtained the score of more than 0.8 in A9 and B9 hydrolysates and were 20 and 3 in total number, respectively. However, the potential bioactivity which these selected peptides could exhibit still need to be explored. Therefore, Pepsite2 software was used to examine the targeted biological activities (i.e. DPP-IV, α-amylase and lipase inhibitory activities). Table 3, 4 and 5 show the p-value and the potential binding sites of the identified peptides with DPP-IV, α-amylase and lipase, respectively. All the peptides in Table 3 except ALWGAGGGGLGLSSGR and KDLWDDFKGL (p>0.05) were eligible to be potential DPP-IV inhibitory peptides. From the results obtained, it was expected that these peptides would bind strongly to the catalytic triad (i.e. Ser630, His740, and Asp708) and

substrate binding sites (i.e. Glu205, Glu206, and Tyr662) of DPP-IV enzymes (Metzler, Yanchunas, Weigelt, Kish, Klei, Xie, et al., 2008). Apart from that, Nabeno, Akahoshi, Kishida, Miyaguchi, Tanaka, Ishii, et al., (2013) had reported other important sites of DPP-IV (e.g. Val207, Ser209, Arg356, Phe357, Arg358, Glu403, Val404, Tyr547, Tyr585, Trp629, Ser630, Tyr631, and Tyr666) that are responsible for the inhibitory activity by the six well known inhibitors (i.e. vildagliptin, saxagliptin, alogliptin, linagliptin, sitagliptin, and teneligliptin). From the Table 3, it could be seen that these peptides have less interaction with the catalytic sites or substrate binding sites. However, there are a high number of inhibitor binding sites observed in these peptides. In general, DNLMPQFM (Glu206, Ser209, Phe357, Tyr547, Trp629, Ser630, Tyr631, and Tyr666) and WNWGWLLWQL (Glu205, Glu206, Phe357, Tyr547, Trp629, Tyr631 and Tyr666) in Sample A9 bind up to 7 important sites. It was also observed that peptides identified in the hydrolysates B9 seem to have a lower number of binding sites compared to those identified in A9. Table 4 shows that all identified peptides were eligible to be α-amylase inhibitor peptide except for ALWGAGGGGLGLSSGR (p>0.05) found in hydrolysate A9. The results showed that these peptides would be able to bind to the catalytic sites (i.e. Asp96, Arg195, Asp197, Glu233 and Asp300) or/and substrate binding sites (i.e. Trp58, Trp59, Tyr62, His101, Asp236, His299 and His305) of α-amylase (Ngoh, Choi, & Gan, 2017). Peptides that were present in hydrolysate A9 and B9 showed the ability to bind to 5 to 9 of these sites. In hydrolysate A9, KDLWDDFKGL was suggested to be the most potent sequence compared to other peptides because it can bind up to 9 sites (i.e. Trp58, Trp59, Tyr62, Arg195, Asp197, Glu233, His299, Asp300, and His305). Similarly, MPSKPPLL in hydrolysate B9 has the highest binding sites (i.e. Trp58, Trp59, Tyr62, Asp96, Arg195, Asp197, Glu233, His299, and Asp300). Therefore, it was proposed that these

identified peptides will be playing a leading role in inhibiting the activity of α-amylase by binding on the respective active segment of the enzyme. This will result in the hindrance of the catalytic site of α-amylase from approaching the substrates and/or the substrate to be captured by the substrate binding sites. For examples, Asp197, Glu233 and Asp300 were not be able to form a substrate binding cluster in the domain A, whereas Arg195 was not able to form the salt bridge interaction with the residues of Asp197 and Asp96, due to the binding of the peptides. Therefore, the substrate binding sites could not be formed eventually. Thus, the starch (substrate) will no longer be hydrolyzed into its monomers (glucose) by α-amylase, and this scenario would lead to a significant reduction in postprandial blood glucose (Tundis, Loizzo, & Menichini, 2010). Therefore, the peptides generated from camel milk could be promising ingredients which can prevent the development of diabetes. As for the lipase inhibitory peptides, a total of 7 peptides in hydrolysate A9 were identified as potent lipase inhibitors with significance level of p<0.05. (Table 5). FCLPLPLLK and KFQWGY possess the ability to bind onto the three significant binding sites (i.e. Ser153, Phe216 and His264) of lipase. It was reported that Ser153 and Phe 264 were the catalytic site of lipase, which are responsible for the triglyceride hydrolysis; whereas, Phe216 was reported to be one of the substrate binding sites (Hermoso, Pignol, Kerfelec, Crenon, Chapus, & FontecillaCamps, 1996). On the other hand, FMFFGPQ, MSKFLPLPLMFY, YWYPPK and YWYPPQ were able to bind to the same sites with an additional substrate binding site (Phe78), whereas LTMPQWW is reported to be bind to His152 site of lipase. Bromelain generated hydrolysate (B9) presented only peptide which qualified to possess the lipase inhibitory activity. TLMPQWW peptide sequence could successfully bound to Phe78, His152, Phe216 and His264, whereas MPSKPPLL could bound to Phe78, Ser153, Phe216 and His264. Previous study

suggested that the peptides possessing more of these binding sites, the higher the inhibitory activity will be for that peptide (Ngoh, Choi, & Gan, 2017). Overall, B9 derived peptides possessed lower number of binding sites for lipase compared to A9 generated peptides sequences. This results are reflected well in the Table 1 where hydrolysate A9 exhibited a higher lipase (PPL) inhibitory activity compared to hydrolysate B9. It could also be observed that the identified peptides generated from CMPHs (A9 & B9) consist of a high amount of hydrophobic amino acid residues, and therefore, they could easily bind to the active sites of lipase, being a lipophilic enzyme. For lipase to function effectively, flap of N terminal domain makes the active site accessible to substrate, form a functional oxyanion hole and generate interface binding site. However, these hydrophobic peptides in peptide sequence might prevent the N-terminal domain (catalytic site) flap from repositioning and therefore restrict the functionality of lipase enzyme (Hermoso, Pignol, Kerfelec, Crenon, Chapus, & Fontecilla-Camps, 1996). This scenario would be able to prevent the occurrence of obesity and hyper-lipidaemia because the lipase activity was suppressed and delayed the digestion/absorption of the triglyceride (Zhang et al., 2015). 4. Conclusions Camel milk has garnered the attention of scientific community because of their potential health benefits. In this study, it was demonstrated that camel milk proteins constitute an interesting source of bioactive peptides with potential of inhibiting key metabolic enzymes related to disorders like diabetes and obesity, therefore suggesting their further therapeutic potential. It was also observed that type of enzyme used for generating bioactive peptides and time of hydrolysis has profound effect on the bioactive properties of the peptides produced. Hydrolysis of camel milk proteins with alcalase and bromelain improved PPA inhibitory activity while papain

hydrolysis could not, thereby confirm the importance of controlled hydrolysis using specific enzymes. Highly potent DPP-IV inhibitory peptides were released upon treatment with all enzymes. Differences in hydrolytic conditions and enzyme specificity towards substrate may be responsible for the outcomes of this study. DNLMPQFM and WNWGWLLWQL peptides identified in A9 possessed the maximum DPP-IV binding sites, while as B9 derived peptides has lower number of binding sites for DPP-IV. KDLWDDFKGL and MPSKPPLL were predicted to be the most potent sequence compared to other peptides in A9 and B9, respectively binding up to 9 sites of α-amylase. Moreover, B9 derived peptides possessed lower number of binding sites for lipase compared to A9 generated peptides sequences. Future work demands investigating the potency of synthetic peptides possessing similar sequence obtained in the potential DPP-IV, PPA and PPL peptides from the present study and investigating their stability under simulated gastric and intestinal conditions. Acknowledgement Authors are thankful to United Arab Emirates University for the funding this research through a research grants (UPAR-31F094) awarded to PI, Sajid Maqsood.

Figure legends Figure 1. Degree of hydrolysis (a), Reverse phase-High performance liquid chromatography (RP-HPLC) profile of intact camel milk and hydrolysates A9, B9 and P9 (b) and Electrophoretic pattern (c) of intact camel milk proteins (CMP) and their hydrolysates (CMPHs) produced by alcalase, bromelain and papain after 3, 6 and 9 hrs of hydrolysis. Keynotes: CMP: Intact Camel milk proreins; P3, P6 and P9; A3, A6 and A9; and B3, B6 and B9: Papain, alcalase and bromelain generated CMPHs after 3, 6, and 9hrs of hydrolysis, respectively. α-Cs: α-caseins; κ-Cs: kappa-caseins; β-Ca: beta-caseins; α-La: α-lactalbumin.

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(a) α-Cs

Detector Response at 220 nm

250

κ-Cs

β-Cs α-La

200

CMP

150

A-9

100

B-9 50 P-9

(b)

0 0

2

4 6 Retention Time (mins)

8

(c) 1. Figure Table 1: Dipeptidyl peptidase (DPP-IV), procine pancreatic α-amylase (PPA) and porcine pancreatic lipase (PPL) inhibitory activity (IC50) of camel milk protein and hydrolysates obtained upon alcalase (A3-9), bromelain (B3-9) and papain (P3-9) hydrolysis. Enzyme inhibitory activity (IC50) (mg protein/mL) Samples DPP-IV PPA PPL e cd e CMP 3.51 ± 0.02 0.031 ± 0.001 0.123 ± 0.006 A3

0.18 ± 0.01

A6

0.12 ± 0.01

A9

0.09 ± 0.01

B3

0.32 ± 0.03

B6

0.17 ± 0.02

B9

0.18 ± 0.01

P3

0.16 ± 0.03

P6

0.18 ± 0.01

P9

0.46 ± 0.04

+ve control*

0.01 ± 0.03

bc ab a cd bc bc b bc de ab

0.027 ± 0.003 0.027 ± 0.001 0.027 ± 0.001 0.027 ± 0.008 0.027 ± 0.002 0.025 ± 0.002 0.030 ± 0.009 0.031 ± 0.002 0.074 ± 0.006 0.094 ± 0.004

ab ab ab ab ab a bcd cd e f

0.046 ± 0.005

c

0.042 ± 0.0009 0.029 ± 0.0008 0.034 ± 0.0007 0.037 ± 0.0008 0.029 ± 0.0003 0.025 ± 0.0005 0.026 ± 0.0001 0.079 ± 0.0002 0.036 ± 0.0002

c a b b a a a d b

* Diprotin for DPP-IV; Acarbose for PPA and Orlistat for PPL Values are means ± SD (n=3). Values in the same column with different superscript letters are significantly different (p < 0.05).

Table 2: Selected peptides derived from A9 and B9 from Peptide Ranker with a score of >0.8 A9 AEWLHDWKL ALWGAGGGGLGLSSGR AVVSPLKPCC CFLPLPLLK DNLMPQFM FCLPLPLLK FMFFGPQ GMAGGPPLL HCPVPDPVRGL KDLWDDFKGL KFQWGY LLPAPPLL LTMPQWW MMHDFLTLCM MSKFLPLPLMFY SQDWSFY WGLWDDMQGL WNWGWLLWQL YWYPPK YWYPPQ B9 TLMPQWW MPSKPPLL AVVSPLKPCC

Peptide Ranker Score 0.84 0.85 0.81 0.93 0.85 0.92 0.94 0.83 0.84 0.84 0.86 0.81 0.83 0.81 0.86 0.84 0.89 0.98 0.92 0.88 Peptide Ranker Score 0.86 0.83 0.81

Table 3: Potential binding sites between identified bioactive peptide and DPP-IV using Pepsite 2 Reactive residue in A9 p-value Bound residues of DPP-IV peptide Tyr48, Tyr547§, Trp627, Trp629§, Tyr AEWLHDWKL 0.03121 W3L4H5K8L9 Tyr666§, Tyr752 Tyr48, Val546, Trp627, Gly628, Ser6 ALWGAGGGGLGLSSGR 0.07364 L12S13S14R16 His740*, Ala743, Tyr752 Tyr547§, Val653, Trp627, Trp629§, Ty AVVSPLKPCC 0.006235 V2V3L6K7P8C9 Tyr666§, Ile752, Tyr752, Met755 Phe357§, Tyr547§, Tyr585§, Trp629§, CFLPLPLLK 0.002955 F2L3P4L5L7K9 Tyr666§, Tyr670, Tyr752 Glu206#, Ser209§, Phe357§, Pro550, T DNLMPQFM 0.003433 D1M4P5Q6M8 Trp629§, Ser630*, Tyr631§, Tyr666§, Tyr48, Phe357§, Tyr547§, Trp627, Trp FCLPLPLLK 0.004903 F1C2L5L7L8K9 Tyr631§, Tyr666§, Tyr670, Tyr752 Phe357§, Tyr547§, Pro550, Trp627, Tr FMFFGPQ 0.008375 F1M2P6Q7 Tyr666§, Tyr670, Tyr752 Phe357§, Tyr547§, Pro550, Trp629§, T GMAGGPPLL 0.01625 G1M2P6P7 Tyr670, His740*, Gly741, Tyr752 Tyr48, Phe357§, Tyr547§, Cys551, Trp HCPVPDPVRGL 0.002668 C2V4P5V8R9L11 Ser630*, Tyr631§, Val653, Tyr666§, G His748, Tyr752 Tyr48, Phe357§, Tyr547§, Trp627, Trp KDLWDDFKGL 0.05001 K1L3K8G9L10 Tyr631§, Tyr666§, Tyr670, His748, Ty Tyr547§, Trp627, Ser630*, Val653, T KFQWGY 0.004201 K1Q3W4G5Y6 Tyr752 Phe357§, Val546, Tyr547§, Trp627, Tr LLPAPPLL 0.01903 L1A4P5P6 Ser630*, Tyr666§, Tyr752 Tyr48, Trp627, Trp629§, Ser630*, Va LTMPQWW 0.001576 L1M3P4Q5W6 Ile703, His740*, Ile742, Tyr752, Met7 Tyr48, Phe357§, Val546, Tyr547§, Cy MMHDFLTLCM 0.03203 H3F5L8C9M10 Tyr585§, Trp627, Ser630*, Tyr631§, T Tyr752 Tyr48, Phe357§, Tyr547§, Tyr585§, Tr MSKFLPLPLMFY 0.001624 F4L5P6L9M10F11 Trp629§, Tyr666§, Tyr670, Gly741, T Ser209§, Phe357§, Tyr547§, Pro550, T SQDWSFY 0.02222 Q2D3W4F6Y7 Tyr631§, Tyr666§, Tyr670 Tyr48, Tyr547§, Trp627, Trp629§, Tyr WGLWDDMQGL 0.01692 W1G2L3W4Q8 Tyr666§, His740*, His748, Tyr752 Tyr48, Glu205#, Glu206#, Phe357§, Ty WNWGWLLWQL 0.002021 W1N2W3W8Q9L10 Trp627, Trp629§, Tyr631§, Val653, Ty Ile703, Ile742, His748, Ile751, Tyr752 Tyr48, Trp627, Trp629§, Gly741, His7 YWYPPK 0.001945 W2P4P5K6 Tyr752 Phe357§, Tyr547§, Pro550, Tyr631§, T YWYPPQ 0.001992 Y3P4P5Q6

31

Tyr670 B9

p-value

Bound AA within peptide

Bound residues on DPP-IV

Tep48, Val546, Trp627, Gly628, Trp6 Ser630*, His748, Tyr752 Tyr48, Phe357§, Tyr547§, Trp627, Trp MPSKPPLL 0.005656 M1P2K4L7L8 Tyr631§, Tyr666§, His748, Tyr752 Tyr547§, Trp627, Trp629§, Tyr631§, V AVVSPLKPCC 0.006235 V2V3L6K7P8C9 Tyr666§, Ile703, Ile742, Ile751, Tyr7 Met755 Note: *catalytic sites of DPP-IV; #substrate binding site of DPP-IV; sites of DPP-IV that bound by other inhibitors§ TLMPQWW

0.001513

L2M3P4Q5W6

32

Table 4: Potential binding sites between identified bioactive peptide and pancreatic αamylase (PPA) using Pepsite 2 Reactive residue in Sample A9 p-value Bound residues of PPA peptide His15, Phe17, Glu18, Gln41, Ser43, Pro44, AEWLHDWKL 0.01009 A1W3L4H5D6W7 Trp58#, Trp59#, Tyr62#, Asp96*, Arg195*, Asn298, His299#, Asp300*, Arg337, Tyr342 Phe17, Glu18, Trp58#, Trp59#, Tyr62#, His299#, ALWGAGGGGLGLSSGR 0.07058 L10G11L12S13S14R16 Asp300*, His305#, Tyr342 His15, Phe17, Gln41, Val42, Ser43, Pro44, AVVSPLKPCC 0.000873 V3P5K7P8C9C10 Trp58#, Trp59#, Tyr62#, Asp96*, His299#, Asp300* Phe17, Glu18, Trp58#, Trp59#, Tyr62#, His299#, CFLPLPLLK 0.001283 C1F2P4L5P6L7 Asp300*, Tyr342 His15, Phe17, Glu18, Gln41, Val42, Ser43, DNLMPQFM 0.001751 D1L3M4P5Q6F7 Pro44, Trp58#, Trp59#, Tyr62#, Asp96*, His299#, Asp300*, Tyr342 Phe17, Glu18, Trp58#, Trp59#, Tyr62#, His299#, FCLPLPLLK 0.001381 C2L3P4L5P6L7 Asp300*, Tyr342 His15, Gln41, Val42, Ser43, Pro44, Trp58#, FMFFGPQ 0.003609 F1M2F4P6Q7 Trp59#, Tyr62#, Asp96*, His299#, Asp300* His15, Gln41, Val42, Ser43, Pro44, Trp58#, GMAGGPPLL 0.0006525 M2A3G4P6P7L8 Trp59#, Asp96*, Arg195*, Asp197*, Glu233*, His299#, Asp300* Phe17, Gln41, Val42, Ser43, Pro44, Trp58#, HCPVPDPVRGL 0.0007958 H1P3P5D6P7V8 Trp59#, Tyr62#, Asp96*, His305#, Lys352, Asp353, Val354, Asp356, Trp357 Phe17, Glu18, Trp58#, Trp59#, Tyr62#, Arg195*, KDLWDDFKGL 0.03514 W4D5F7K8G9L10 Asp197*, Glu233*, His299#, Asp300*, His305#, Tyr342, Lys352, Asp356 Trp58#, Trp59#, Tyr62#, Arg195*, Asp197*, KFQWGY 0.01317 K1Q3W4G5Y6 Glu233*, His299#, Asp300*, His305#, Lys352, Asp356 Phe17, Glu18, Trp58#, Trp59#, Tyr62#, His299#, LLPAPPLL 0.000304 L2P3P5P6L7L8 Asp300*, Tyr342 Phe17, Glu18, Trp58#, Trp59#, Tyr62#, His299#, LTMPQWW 0.0004868 L1M3P4Q5W6W7 His305#, Tyr342, Lys352, Asp356 His15, Gln41, Ser43, Pro44, Trp58#, Trp59#, MMHDFLTLCM 0.00775 M2H3D4L6C9M10 Tyr62#, Asp96*, Arg195*, Asn298, His299#, Asp300* His15, Phe17, Glu18, Gln41, Val42, Ser43, MSKFLPLPLMFY 0.001794 F4P6L7P8L9M10 Pro44, Trp58#, Trp59#, Tyr62#, Asp96*, His299#, Asp300*, Tyr342 His15, Gln41, Val42, Ser43, Pro44, Trp58#, SQDWSFY 0.006842 S1Q2D3W4F6Y7 Trp59#, Tyr62#, Asp96*, Arg195*, Asn298, His299#, His305#, Arg337, Lys352, Asp356

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WGLWDDMQGL

0.03314

W1L3W4D5D6M7

WNWGWLLWQL

0.01938

W3W5L7W8Q9L10

YWYPPK

0.0005587

W2Y3P4P5K6

YWYPPQ

0.0002076

W2Y3P4P5Q6

Sample B9

p-value

Reactive residue in peptide

His15, Phe17, Glu18, Gln41, Val42, Trp58#, Trp59#, Tyr62#, Asp96*, Tyr231, Asn298, His299#, Asp300*, Arg337, Tyr342 His15, Gln41, Trp58#, Trp59#, Tyr62#, Arg195*, Asn298, His299#, Asp300*, His305#, Arg337, Lys352, Asp356, Phe17, Trp58#, Trp59#, Tyr62#, Asp300*, Arg303, His305#, Asp356 Phe17, Trp58#, Trp59#, Tyr62#, His299#, Asp300* Bound residues of PPA

Phe17, Glu18, Trp58#, Trp59#, Tyr62#, His299#, TLMPQWW 0.0004868 L2M3P4Q5W6W7 Asp300*, His305#, Tyr342, Lys352, Asp356 His15, Gln41, Ser43, Pro44, Trp58#, Trp59#, MPSKPPLL 0.0005927 M1K4P5P6L7L8 Tyr62#, Asp96*, Arg195*, Asp197*, Glu233*, His299#, Asp300* His15, Phe17, Gln41, Val42, Ser43, Pro44, AVVSPLKPCC 0.000873 V3P5K7P8C9C10 Trp58#, Trp59#, Tyr62#, Asp96*, His299# Note: *catalytic sites of α-amylase; #substrate binding site of α-amylase

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Table 5: Potential binding sites between identified camel milk bioactive peptide and procince pancreatic lipase (PPL) using Pepsite 2 server and their p-values Peptide sequence p-value Reactive residue in Bound amino acid residues of PPL (Sample A9) peptide AEWLHDWKL 0.243 W3L4H5W7K8 Phe78#, Tyr115, His152#, Ser153*, Leu154, Gly155, Ala179, Phe216#, His264* ALWGAGGGGLGLSSGR 0.3576 A1L2W3G9 Lys81, Glu84, Tyr115, Ser153*, Phe216#, Trp253 AVVSPLKPCC 0.1105 A1V2V3C9C10 Gly77, Phe78#, Lys81, Tyr115, Leu154, Glu180, Pro181, Phe216#, Trp253, His264* CFLPLPLLK 0.05954 L5P6L7K9 Gly77, Phe78#, Lys81, Tyr115, Ser153*, Leu154, Phe216#, Trp253, His264* DNLMPQFM 0.0804 M4P5F7M8 Gly77, Phe78#, Tyr115, His152#, Ser153*, Leu154, Gly155, Pro181, Phe216#, His264* FCLPLPLLK 0.04992 F1C2L3P4K9 Lys81, Glu84, Tyr115, Ser153*, Pro181, Phe216#, Trp253, His264* FMFFGPQ 0.01719 F1M2F3F4G5 Gly77, Phe78#, Tyr115, Ser153*, Leu154, Pro181, Phe216#, His264* GMAGGPPLL 0.2516 P6P7L8 Phe78#, Tyr115, Ser153*, Leu154, Ala179, Phe216#, His264* HCPVPDPVRGL 0.07047 H1C2R9G10 Phe78#, Lys81, Glu84, Tyr115, Ser153*, Phe216#, Trp253 KDLWDDFKGL 0.1108 K1F7K8G9L10 Gly77, Phe78#, Lys81, Glu84, Tyr115, His152#, Leu154, Gly155, Ala179, Pro181, Phe216#, Trp253, His264* KFQWGY 0.04202 K1F2W4G5Y6 Gly77, Ile79, Tyr115, Ser153*, Leu154, Pro181, Phe216#, His264* LLPAPPLL 0.08178 A4P5P6L7 Gly77, Phe78#, Tyr115, Ser153*, Leu154, Ala179, Pro181, Phe216#, His264* LTMPQWW 0.01416 T2M3P4Q5W6 Gly77, Tyr115, His152#, Ser153*, Leu154, Gly155, Ala179, Pro181, Phe216#, Ala261, His264* MMHDFLTLCM 0.05348 M1M2H3L8C9M10 Gly77, Phe78#, Lys81, Glu84, Tyr115, Ser153*, Leu154, Phe216#, Thr256, His264* MSKFLPLPLMFY 0.01198 K3F4L5P6L7Y12 Gly77, Phe78#, Tyr115, Ser153*, Leu154, Pro181, Phe216#, Trp253, His264* SQDWSFY 0.3656 W4S5F6Y7 Gly77, Ile79, Tyr115, Ser153*, Leu154, Ala179, Pro181, Phe216#, His264* WGLWDDMQGL 0.1707 W1G2L3W4Q8 Gly77, Phe78#, Lys81, Tyr115, Leu154, Pro181, Phe216#, Trp253, His264* WNWGWLLWQL 0.1837 W1N2W3W5Q9 Gly77, Phe78#, Lys81, Tyr115, Leu154, Phe216#, Trp253, His264* YWYPPK 0.006533 W2Y3P4P5K6 Phe78#, Tyr115, Ser153*, Pro181, Phe216#, His264* YWYPPQ 0.02162 Y3P4P5Q6 Phe78#, Tyr115, Ser153*, Pro181, Phe216#, Ile210, His264*

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Peptide sequence (Sample B9) TLMPQWW

p-value

Reactive residue in peptide L2M3P4W6W7

Bound residues of PPL

Gly77, Phe78#, Tyr115, His152#, Leu154, Phe216#, Ala261, His264* MPSKPPLL 0.03829 S3K4P5P6L7 Gly77, Phe78#, Tyr115, Ser153*, Leu154, Ala179, Phe216#, His264* AVVSPLKPCC 0.1105 A1V2V3C9C10 Gly77, Phe78#, Lys81, Tyr115, Leu154, Glu180, Pro181, Phe216#, Trp253, His264* Note: *catalytic sites of lipase; #substrate binding site of lipase 0.04701

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Highlights 

Camel milk protein hydrolysates (CMPHs) effectively inhibited DPP-IV and lipase



20 and 3 novel peptides were identified from A9 and B9 derived CMPHs, respectively



7 and 2 peptides from A9 and B9 qualified to be PPL inhibitors, respectively



KDLWDDFKGL in A9 and MPSKPPLL in B9 were the potent α-amylase inhibitory peptides

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