Antioxidant and cytoprotective effect of peptides produced by hydrolysis of whey protein concentrate with trypsin

Journal Pre-proofs Antioxidant and cytoprotective effect of peptides produced by hydrolysis of whey protein concentrate with trypsin María Belén Ballatore, Marina del Rosario Bettiol, Noelia L. Vanden Braber, Carla Aylen Aminahuel, Yanina Estefanía Rossi, Gabriela Petroselli, Rosa ErraBalsells, Lilia René Cavaglieri, Mariana Angélica Montenegro PII: DOI: Reference:

S0308-8146(20)30334-4 https://doi.org/10.1016/j.foodchem.2020.126472 FOCH 126472

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

28 July 2019 18 February 2020 22 February 2020

Please cite this article as: Belén Ballatore, M., del Rosario Bettiol, M., Vanden Braber, N.L., Aylen Aminahuel, C., Estefanía Rossi, Y., Petroselli, G., Erra-Balsells, R., René Cavaglieri, L., Angélica Montenegro, M., Antioxidant and cytoprotective effect of peptides produced by hydrolysis of whey protein concentrate with trypsin, Food Chemistry (2020), doi: https://doi.org/10.1016/j.foodchem.2020.126472

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Antioxidant and cytoprotective effect of peptides produced by hydrolysis of whey protein concentrate with trypsin

María Belén Ballatore1,5†, Marina del Rosario Bettiol2,5†, Noelia L. Vanden Braber2,5, Carla Aylen Aminahuel2,5, Yanina Estefanía Rossi2,5, Gabriela Petroselli3,4, Rosa Erra-Balsells3,4, Lilia René Cavaglieri1,5, Mariana Angélica Montenegro2,5* †

They contributed equally in the development of this investigation.

1Departamento

de Microbiología e Inmunología, Universidad Nacional de Río Cuarto, Río Cuarto,

Córdoba, Argentina. 2Centro

de Investigaciones y Transferencia de Villa María (CITVM-CONICET), Universidad Nacional

de Villa María, Villa María, Córdoba, Argentina. 3Universidad

de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Química

Orgánica, Pabellón II, 3er P., Ciudad Universitaria, 1428, Buenos Aires, Argentina. 4CONICET,

Universidad de Buenos Aires, Centro de Investigación en Hidratos de Carbono

(CIHIDECAR), Facultad de Ciencias Exactas y Naturales Pabellón II, 3er P., Ciudad Universitaria, 1428, Buenos Aires, Argentina. 5Member

of Consejo Nacional de Investigaciones Científicas y Tecnológicas (CIC-CONICET),

Argentina.

* Corresponding author. Tel.: +54 353 4539106. E-mail address: [email protected] (M. A. Montenegro).

1

Abstract Whey protein is one of the most relevant co-products manufactured by the dairy industry and it is a powerful environmental pollutant. Therefore, the enzymatic hydrolysis of whey protein concentrate (WPC 35) to produce antioxidant peptides is an innovative approach which can provide added value to whey. The WPC 35 hydrolysis with trypsin was carried out for 4.31 h at 41.1 °C with an enzyme/substrate ratio of 0.017. Under such hydrolysis conditions, the peptides produced have the highest radical scavenging activity and cytoprotector effect. The WPC hydrolysate and a permeate  3 kDa were characterized by SDS-page, RP-HPLC and MALDI-TOF-MS. Furthermore, O2• and HO• scavenging activity and the cytoprotective effect against a stress agent in epithelial cells of the rat ileum (IEC-18) were determined. In this study, strong antioxidant and cytoprotective peptides were obtained from a lowcost dairy industry product, which could improve consumers’ health when used as functional ingredients.

Keywords: whey protein, hydrolysis, trypsin, peptides, antioxidants, cytoprotection.

2

1. Introduction Whey is a co-product of casein and cheese-making in the dairy industry and it is composed of proteins with high nutritional value (Smithers, 2008). The principal protein components of whey are βlactoglobulin (-Lg), α-lactalbumin (-La), bovine serum albumin (BSA), lactoferrin, and immunoglobulin (Ig) (Mollea, Marmo, & Bosco, 2013). Besides, whey is rich in lactose as well as minerals and vitamins, which are present in smaller quantities. However, whey is a strong environmental pollutant because it contains a high quantity of organic matter (Brandelli, Daroit, & Corrêa, 2015). Thus, whey is no longer considered as waste and, instead, it has become a prized raw material (Bacenetti, Bava, Schievano, & Zucali, 2018). However, the industry has not yet developed functional products based on its own co-products and derivative products such as WPC35; therefore, its usage is innovative from an economic, environmental and nutraceutical point of view. It is known that molecular oxygen O2(3Ʃ-g) is vital for aerobic life processes. However, the inhaled O2(3Ʃ-g) is converted into reactive oxygen species (ROS), as a by-product of normal oxygen metabolism, because they are necessary for developing certain important functions inside the human body. The most popular ROS are hydroxyl radical (HO•), superoxide anion radical (O2•) and hydrogen peroxide (H2O2) (Valko et al., 2007). Consequently, cellular systems are exposed to permanent oxidative stress produced by ROS. Fortunately, the organism has the ability of rapidly eliminating free radicals through the action of antioxidant enzymes such as catalase, superoxide dismutase (SOD) and glutathione peroxidase. However, when there is an imbalance between the ROS generated and the capacity of the biological system to eliminate them quickly, oxidative stress occurs. This stress has been associated with a variety of chronic diseases in humans including cancer, Alzheimer, Parkinson, cardiovascular diseases, among others (McGarry, Biniecka, Veale, & Fearon, 2018; Scicchitano, Pelosi, Sica, & Musarò, 2018). Therefore, the excess of ROS must be neutralized as soon as they are formed to avoid such collateral cellular damage. In that sense, bioactive peptides have demonstrated to be powerful antioxidant 3

compounds with the ability to protect the human body from ROS effects. Furthermore, they can improve body functions through different mechanisms of action (Kitts, & Weiler, 2003). Previous studies reveal that the antioxidant capacity of peptides is related with amino acid composition and sequence in which they are present (Nielsen, Beverly, Qu, & Dallas, 2017). Bioactive peptides can be produced through a number of different routes, such as protein hydrolysis, microbial fermentation and digestive processes. The most extensively studied route was in vitro enzymatic hydrolysis employing pepsin, chymotrypsin and especially trypsin (Toldrá, Reig, Aristoy, & Mora, 2018). In recent years, bioactive peptides have been widely studied due to their potential as a functional ingredient capable of producing beneficial effects on the health of their consumers (Li-Chan, Hunag, Jao, Ho, Hsu, 2015). At the same time, production of hydrolysates can be an interesting approach to add value to whey protein and, at the same time, protect the environment from their pollutant effects. It is important to take into account that the cost of whey powder manufacturing vary widely depending on the production procedures (Dullius, Goettert, & de Souza, 2018). In that respect, whey protein concentrate with 35% of protein (WPC 35) is the most economical product derived from cheese industries: approximately 3 times cheaper than WPC80 and 7 times cheaper than whey protein isolate (WPI). Hence, the main goal of this work is to produce strong antioxidant peptides through the enzymatic hydrolysis of WPC 35, one of the cheapest products derived from the dairy industry. After optimizing WPC 35 enzymatic hydrolysis conditions, the protein content and the degree of hydrolysis (DH) were determined. Subsequently, the hydrolysate was ultrafiltered through a 3 kDa membrane to separate peptides of lower molecular weight. The peptides were characterized by reversed-phase high-performance liquid chromatography (RPHPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS). Finally, the ability of peptides to scavenge HO• and O2• was evaluated, as well as the cytoprotective effect against a stress agent in epithelial cells of the rat ileum (IEC-18). Nowadays, whey is vastly used as an ingredient 4

in several food formulations such as fortified drinks, whey protein shakes and products with a high content of conjugated linoleic acid. Therefore, the whey protein hydrolysate rich in antioxidant peptides is an added-value product that could be very attractive for industries. Furthermore, the new product could be a functional ingredient with the ability to prevent a large number of diseases associated with ROS. As a result, this product could not only be interesting for industries, but also for consumers.

2. Materials and methods 2.1. Materials Molfino Hermanos S.A. (Buenos Aires, Argentina) provided WPC from bovine milk with 35% w/w (WPC 35). 2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), bovine trypsin (activity 10,000 units/mg), angiotensin I, angiotensin II, apomyoglobin, bradykinin, bromophenol blue, BSA, SOD (activity 3,277 units/mg), α-ciano-4-hydroxycinnamic acid (CHCA), 2,5dihydroxybenzoic acid (DHBA), di-natetraborate decahydrate, 2-deoxy-D-ribose (DoR), dithiothreitol 99% (DTT), E-3,5-dimethoxy-4-hydroxycinnamic acid (E-sinapinic acid, SA), insulin, β-lactoglobulin (β-Lg),

α-lactalbumin

(α-La),

norharmane

(nHo),

o-phthaldialdehyde

97%

(OPA),

1,2,3-

trihydroxybenzene (pyrogallol), sodium dodecyl-sulfate (SDS), trichloroacetic acid (TCA), trifluoroacetic acid (TFA), 6-hydroxy-2,5,7,8-tetramethylchroman-2-acid (trolox), and menadione (MEN) were obtained from Sigma-Aldrich (MO, USA). Copper sulfate pentahydrate, sodium-potassium tartrate, ferric chloride, dibasic sodium phosphate, monobasic sodium phosphate, sodium hydroxide, sodium carbonate, hydrochloric acid, glycerol, D-mannitol and ascorbic acid, all analytical grade, were obtained from Cicarelli (Santa Fe, Argentina). The Folin-Ciocalteu reactive, thiobarbituric acid (TBA), and ethylenediaminetetraacetic (EDTA) were provided by Merck (Darmstadt, Germany). Potassium persulphate, 2-mercaptoethanol, acrylamide, tricine and trimethamine (TRIS), by Bio-Rad (California, USA). Acetic acid, acetonitrile, hydrogen peroxide and methanol, by Anedra (Argentina). Dulbecco's 5

Modified Eagle Medium (DMEM), phosphate buffered saline (PBS), trypsin–EDTA (0.05%) and GlutaMAX™-I Supplement were purchased from Gibco (Invitrogen, Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Natocor (Argentina). IEC18 cells (epithelial cells of the rat ileum) were obtained from the American Type Culture Collection, USA.

2.2. Preparation of heat pre-treated WPC The necessary amount of WPC 35 was dissolved in phosphate buffer (pH 8.0) in order to obtain a solution with a final protein concentration of 5.71 % (w/v). The chemical composition of WPC 35 is presented in Table S1 of supplementary material. In a thermostat bath, 50 mL of the WPC solution (final pH 7.6) was heated at 90 °C for 10 min. Subsequently, the solution was cooled in a water bath at 25 °C.

2.3. Optimization of enzymatic hydrolysis of pre-treated WPC 2.3.1. Experimental design and response surface analysis The response surface analysis (RSM) was used to optimize the enzymatic hydrolysis of pretreated WPC by trypsin. The central composite design (CCD) with three factors was selected to identify relationships between the response variables and the process parameters (Myers, Montgomery & Anderson-Cook, 2016). The independent variables were enzyme to substrate ratio (E/S) (0.003 – 0.030), temperature (Tº) (37 – 50 °C) and time (t) (0.5 – 4.5 h) according to the hydrolysis conditions defined by the first experimental design (Table 1). The pH for each experiment was 7.6. Immediately after hydrolysis, the enzyme was inactivated by heating solutions at 100 °C for 10 min. The response variable was antioxidant activity expressed as free radical scavenging activity by the ABTS assay (section 2.5.). The statistical analysis was performed with Design Expert version 10.0.3 free trial (Minneapolis, USA). The quadratic response surface analysis was based on multiple linear regressions taking into account the main, the quadratic and the interaction effects, according to Eq. (1). 6

5

5

5

𝑌 = 𝑏0 + ∑𝑖 = 1𝑏𝑖𝑋𝑖 + ∑𝑖 = 1𝑏𝑖𝑖𝑥𝑋2𝑖 + ∑𝑖 < 𝑗 = 2𝑏𝑖𝑗𝑋𝑖𝑋𝑖𝑗 + 𝑒

(1)

where Y is the response, b0 is the constant term, bi represents the coefficients of the linear parameters, Xi represents the factors, bii represents the coefficients of the quadratic parameter, and bij represents the coefficients of the interaction parameters. The significance of the b-coefficients calculated by regression analysis was tested with the Student t-test. To obtain the regression models, the experimental results were assessed by analysis of variance (ANOVA). The fitness of the polynomial models was estimated through both the determination coefficient (R2) and the lack-of-fit (LOF) test. To avoid bias errors, all experiments were performed in triplicate and randomly. The replicas of the central point were done to allow estimation of pure error as square sums.

2.3.2. Hydrolysis of WPC with trypsin Hydrolysis of pre-treated WPC samples was carried out with an E/S, T° and t according to the hydrolysis conditions defined by the first experimental design (Table 1). The pre-treated WPC was diluted to a concentration of 4% w/v which, expressed in terms of protein content, was constant throughout the experiment; then, trypsin was added form a stock solution produced in phosphate buffer (pH 8). Different volumes of enzyme stock solution were added to reaction tubes in order to reach the E/S required by the software. The system was stirred in a MaxQ Shaker (Thermo Scientific, Massachusetts, USA) throughout the hydrolysis process (180 rpm). Immediately after hydrolysis, the enzyme was inactivated by heating the solution at 100 °C for 10 min. Then, the reaction tube was cooled down to 25 °C. The protein hydrolyzed was subjected to ultracentrifugation (ST 16R, Thermo Fisher Scientific, Walthman, Massachusetts, USA) at 10,000 rpm for 30 min at 4 °C. Afterwards, the 7

supernatant with the peptides was collected and stored at 4 °C for further analyses and treatments. The DH was determined by the OPA method following the protocol presented by Nielsen, Petersen & Dambmann (2001), and were expressed as percentage of DH. Protein contents of WPC and hydrolysates were estimated according to the method of Lowry, Rosebrough, Farr & Randall (1951) using BSA as standard.

2.3.3. ABTS decolorizing assay as a response for RSM The antioxidant activity was evaluated as free radical scavenging activity by using the cation radical of ABTS (ABTS•+) decolorizing assay following the method described by Re et al. (1999). The reaction mixture contained 1 mL ABTS•+ dissolved in deionized water, and 15 µL of hydrolysate solution diluted (1/4) with buffer phosphate (pH 8). The final absorbance in each sample was read at 734 nm on a Specord S600 UV–Vis diode array spectrophotometer (Analytik Jena, Germany). The ABTS•+ scavenging activity percentage (SA (%)) was determined by Eq. (2).

ABTS• + SA (%) =

(

A(0) ― A(x) A(0)

)x 100

(2)

where A(0) is the absorbance of ABTS•+ before the addition of the hydrolysate solution (A ~ 0.7), and A(x) is the absorbance after the addition of the hydrolysate solution after 16 min of incubation in the dark.

2.3.4. Hydrolysis of WPC under one optimal reaction condition Pre-treated WPC was dissolved in phosphate buffer (pH 8) at a protein concentration of 4% (w/v) and the enzymatic hydrolysis was carried out in a solution at a final pH of 7.6. Then, WPC was incubated at 41.1 °C under agitation at 180 rpm with an E/S of 0.017 for 4.3 h. This is one of the optimal conditions defined by the experimental design. When the trypsin enzymatic reaction time was over, the solution was 8

heated at 100 °C for 10 min. Afterwards, suspensions were cooled down to room temperature in a water bath. The protein hydrolysate WPH was ultracentrifuged (10,000 rpm, 30 min, 4 °C) and the supernatant was collected and saved at 4 °C for further purification. The centrifuged hydrolysate (WPH) was separated by ultrafiltration (UF) membranes (Vivaflow 200 Sartorius AG, Göttingen, Germany, cutoff of 3 kDa). Permeate was collected, and a fraction with molecular weight equal to or less than 3 kDa (F  3 kDa) was obtained. Subsequently, WPH and F  3 kDa were lyophilized using a RIFICOR model L-IE300-CRT lyophilizer system (Buenos Aires, Argentina). The chemical composition of WPH and F  3 kDa after the lyophilization process is presented in Table S1 of the supplementary material.

2.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done on a MiniPROTEAN® Tetra System (Bio-Rad, California, United Sates) using 15% polyacrylamide gel slabs (ExcelGel SDS Homogeneous) at pH 8.8, with 10% (w/v) SDS. The samples were prepared as follows: 20 µL of peptides dissolved in deionized water (1 mg/mL in function of protein content) together with 5 µL of 5x Sample Buffer (0.5 mL of glycerol saturated with bromophenol blue, 250 µL Tris-HCl 1 M (pH 6.8), 25 µL deionized water, 150 µL 2-mercaptoethanol and 0.01 g SDS) were mixed inside Eppendorf vials. The vials were subsequently heated at 80 °C for 10 min and then cooled down to room temperature. The gels were run with a constant current (20 mA) and at room temperature for 2.5 h. After the running, the protein bands were stained with Coomassie Blue R-250 (Phastgel Blue R., Pharmacia). Distaining was performed in an aqueous solution with 10% (v/v) of methanol and 10% (v/v) of acetic acid. The high molecular weight calibration kit used as standard was Precision Plus Protein™ All Blue Standards (BioRad, California, USA).

2.5. Reversed-phase high-performance liquid chromatography analysis 9

WPC, WPH and F  3 kDa were analyzed by RP-HPLC. All samples were filtered through a 0.45 μm polyvinylidene fluoride (PVDF) membrane and stored at -20 °C prior to RP-HPLC analysis. The samples were injected into a Thermo Scientific Ultimate 3000 (Waltham, Massachusetts, USA) equipped with degasser, pump, auto-sampler, and UV detector (set at 214 nm). Protein and peptide profiles were obtained using a PhenoSphere-NEXT 5 μm C18 120 Å LC column of 4.6 mm i.d. × 250 mm (Phenomenex Inc., California, United Sates) with a pre-column of the same characteristics. Solvent A was TFA dissolved in Mili-Q water to a final concentration of 0.1% v/v. On the other hand, solvent B was a mix of three solvents in different proportions: acetonitrile (80% v/v), Mili-Q water (20% v/v) and TFA (0.1% v/v). Solvents A and B were used for elution at a flow rate of 0.5 mL min−1 at 25 °C. A linear gradient of solvent B was used from 10% to 80% in 40 min. The serum proteins, BSA, -Lg and -La, were identified by comparison with standards. The degradation of -Lg and -La was evaluated through consumption in areas of peaks analyzed with the software PeakFit Version 4.12 (SeaSolve Software Inc., 1999-2003).

2.6. MALDI-TOF-MS analysis Ultraviolet matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) was performed on the Bruker Ultraflex Daltonics TOF/TOF mass spectrometer. Mass spectra were acquired in linear and reflectron positive modes and with the LIFT device in the MS/MS mode. External mass calibration was carried out using commercial peptides and proteins (bradykinin (1-7) (757.3997), angiotensin II (1046.5423), angiotensin I (1296.6853), insulin (5730.6087), apomyoglobin (16.9529) and albumin (66.430)) in positive ion mode. On the other hand, norharmane (nHo), gentisic acid (2.5dihydroxybenzoic acid, DHBA), α-ciano-4-hydroxycinnamic acid (CHCA), and E-3.5-dimethoxy-4hydroxycinnamic acid (E-sinapinic acid, SA) were used as MALDI matrices. The WPC was dissolved in phosphate buffer (pH 8) before the heat treatments. Two samples of heat-treated WPC were analyzed 10

in order to evaluate the effect of thermal treatment on peptide formation: WPCT1 including the heat pretreatment (90 °C for 10 min) and WPCT2 including the heat pre-treatment and thermal inactivation of the enzyme (90 °C for 10 min and 100 °C for 10 min). Additionally, F  3 kDa was analyzed to evaluate the effect of trypsinolysis on peptide formation. All samples were lyophilized for the MALDI-MS analysis. The WPCT1, WPCT2 and F  3 kDa were suspended in TFA 0.1% or acetonitrile: TFA 0.05% 50:50 (v/v). The sample solutions were spotted on an MTP 384 polished steel target plate from Bruker Daltonics (Germany). For MALDI-MS, matrix solutions were prepared as nHo (1 mg/mL), DHBA (1 mg/mL) in water, E-SA (1 mg/mL) and CHCA (1 mg/mL) in acetonitrile: TFA 0.05% 50:50 (v/v). For MALDI-MS experiments, the sandwich method was used according to Nonami, Fukui & Erra‐Balsells (1997); 0.5 L of matrix solution, analyte solution and matrix solution (x2) were successively loaded after drying each layer at normal atmosphere and room temperature. The matrix to analyte ratio was 3:1 (v/v), and the matrix and analyte solution loading sequence was: i) matrix, ii) analyte, iii) matrix, iv) matrix. Desorption/ionization was carried out by using the frequency-tripled Nd:YAG laser (355-nm). The experiments were performed using firstly the full-range setting for laser firing position in order to select the optimal position for data collection, and secondly fixing the laser firing position in the sample sweet spots. The laser power was adjusted to obtain a high signal-to-noise ratio (S/N) while ensuring minimal fragmentation of the parent ions, and each mass spectrum was generated by averaging 100 laser pulses per spot. The spectra were obtained and analyzed with FlexControl and FlexAnalysis softwares, respectively.

2.7. Biological activities 2.7.1. Antioxidant activity The antioxidant activity was determined with the ABTS•+ decolorization assay (section 2.5). The WPC, WPH and F  3 kDa were evaluated in triplicate and phosphate buffer (pH 7.4) was used as blank. 11

The final antioxidant activity (TEAC) was expressed in trolox equivalent (µmol TE/mg protein sample) according to Eq. (3). Protein contents of WPC, WPH and F  3 kDa were estimated according to the method of Lowry, Rosebrough, Farr & Randall (1951).

Slope for each sample (mg ―1L)

(3)

TEAC = Slope for TE standard (μmol ―1L)

Here, the slope for each sample corresponds to the linear regression fit of ABTS•+ SA (%), Eq. (2) versus concentration of the protein solution expressed as mg/L. On the other hand, the slope for TE standard corresponds to the regression fit of SA (%) in function of trolox concentration expressed as µmol/L. Additionally, the 50% scavenging of ABTS•+ (EC50) was obtained graphically from the linear regression fit of ABTS•+ SA (%) (Eq. 2) versus concentration of the protein solution, and it was expressed as protein concentration (mg/L).

2.7.2. Hydroxyl radical scavenging activity The HO• was generated via Fenton reaction at pH 7.4. The scavenging effect of WPC, WPH and F  3 kDa was determined via the 2-deoxyribose degradation assay as previously described by Vanden Braber et al. (2018); the colored adduct was measured at 532 nm, and the HO• SA (%) was calculated according to Eq. (4):

HO•SA (%) =

(

)x100%

A532nm,control ― A532nm,sample A532nm,control

(4)

where A532nm,control is the absorbance at 532 nm of the mixture without the sample, and A532nm,sample is the absorbance at 532 nm of the mixture with the sample. The final HO• radical SA was expressed in mannitol equivalent activity (MEA) (µmol mannitol/mg protein sample) according to Eq. (5): 12

Slope for each sample (mg ―1L)

HO• MEA = Slope for MEA standard (mmol ―1L)

(5)

where the slope for each sample corresponds to the slope of the linear regression fit of HO• SA (%) versus concentration of the protein sample expressed as mg/L. On the other hand, the slope for MEA standard corresponds to the linear regression fit of HO• SA (%) versus mM concentration of mannitol expressed as mmol/L. Furthermore, the 50% scavenging of HO• (EC50) was determined graphically and expressed as protein concentration (mg/L), previously determined by the method of Lowry, Rosebrough, Farr, & Randall (1951).

2.7.3. Superoxide radical anion scavenging activity The O2•scavenging activity was carried out using the pyrogallol autoxidation method which was originally designed by Marklund & Marklund (1974) specifically for superoxide dismutase (SOD). The determination was done following the methodology previously described by Li (2012). The value of absorbance for each sample was measured against the Tris-HCl buffer (pH 8.2) at a wavelength of 325 nm. The O2• SA (%) was calculated according to Eq. (6):

O•2 ― SA (%) =

(

)x100%

ΔA325nm,control ― ΔA325nm,sample ΔA325nm,control

(6)

where ΔA325nm,control correlates with the increase in absorbance at 325 nm of the mixture reaction with Tris-HCl buffer. On the other hand, ΔA325nm,sample corresponds to the increase in absorbance at 325 nm of the mixture reaction with protein samples. 13

The final O2• scavenging activity was expressed in superoxide dismutase equivalent activity (SODEA) (nmol SOD/mg protein sample) by Eq. (7). Additionally, the results were expressed as the protein concentration (mg/L) EC50 leading to 50% scavenging of O2•. Protein contents of WPC, WPH and F  3 kDa were estimated according to the method of Lowry, Rosebrough, Farr & Randall (1951).

Slope for each sample (mg ―1L)

O•2 ― SODEA = Slope for SOD standard (μg ―1L)

(7)

Here, the slope for each sample corresponds to the relationship between the slopes of the linear regression fit of O2• SA (%) versus concentration of the protein sample expressed as mg/L. On the other hand, the slope for SOD standard corresponds to the linear regression fit of O2• SA (%) versus concentration of the SOD solution expressed in µg/L.

2.7.4. Cytoprotection assay in IEC-18 cells With the aim of studying the WPH and F  3kDa cyprotection against a stressor agent, IEC-18 cells (5×104/mL) were seeded in 96-well plates and then incubated in a growing medium for 24 h. The cells were exposed to stressor MEN (Marchionatti et al., 2009) at a concentration of 25 μM (showing 40−60% inhibition) to examine the bioprotective effects of peptides. To assess the cytoprotective effects of WPC, WPH and F  3 kDa against MEN-induced oxidative stress, the cells were pre-treated with 0.1, 0.25, 0.50 and 1 mg protein/mL of samples for 24 h and then exposed to MEN (25 µM) for 24 h. After each assay, cell viability was measured using the MTT assay as follows: after incubation, the supernatant was discarded and replaced with 0.5 mg/mL of MTT solution in DMEM without FBS. The plates were incubated at 37 °C for 4 h in darkness. The MTT solution was discarded and the blue crystals were solubilized with DMSO. The absorbance was read at 570 nm on the microplate spectrophotometer Multiskan GO (ThermoFisher Scientific). It was directly proportional to the production of formazan and 14

it represents the viable cells. The cytotoxic effect was calculated as the percentage of cell viability values with respect to the control cells. Data represent the mean ± standard deviation (SD) of three wells per treatment and are representative of the three experiments. On the other hand, cell morphology was analyzed by phase contrast using a Nikon Eclipse TI-S inverted microscope equipped with a digital camera.

3. Results 3.1. Optimization of hydrolysis conditions of WPC Before hydrolysis, the WPC 35 was pre-treated at 90 °C for 10 min. The pre-heating was carried out to cause a slight denaturation and a conformational change of whey globular proteins and thus facilitate its hydrolysis by trypsin (Adjonu, Doran, Torley, & Agboola, 2013). The effect produced by heat on the structure of β-Lg and α-La was previously studied (Patrick, Geoffrey, & Jameson, 2014). Briefly, β-Lg dimer dissociates at ∼ 70 °C and the constituent molecules begin to unfold. On the other hand, α-La undergoes a thermal unfolding at a temperature lower than that for β-Lg. Nowadays, the thermal treatment prior to enzymatic hydrolysis is a well-established and widely used procedure (Peng, Xiong, & Kong, 2009; Adjonu et al., 2013). The enzyme chosen to carry out the hydrolysis of WPC was trypsin because it presents some advantages in comparison with other proteolytic enzymes. It was demonstrated that trypsin is highly active, has an elevated cleavage specificity, and is very stable under different experimental conditions (Olsen, Ong, & Mann, 2004). However, there are few reports on peptide release by trypsin away from their optimum performance conditions (pH 7.8 and 37 °C). In order to evaluate the antioxidant activity of hydrolysates, the ABTS•+ decolorization assay was carried out. The ABTS•+ SA (%) (Eq. 2) of hydrolysates varied from 37% to 56% (Table 1). It is important to note that the hydrolysate solutions were diluted (1/4) with buffer phosphate (pH 8) prior to 15

ABTS•+ SA (%) determination. All the samples treated with trypsin had significantly higher ABTS•+ SA (%) values than the un-hydrolyzing WPC (p < 0.05). This result indicates that antioxidant peptides were released after trypsin hydrolysis of WPC 35. The difference between non-heat pre-treated WPC (ABTS•+ SA 11.75%) and heat pre-treated WPC (ABTS•+ SA 15.45%) was significant (p > 0.05). This result shows that denaturation of whey globular proteins has occurred by the heat effect. The effects of the three different parameters (E/S, T° and t) were analyzed by the multiple regression technique. The application of RSM produced the following regression Eq. (8) of empirical relationship between the ABTS•+ SA (%) values and the variables:

𝐸

𝐸

ABTS• + SA (%) = 51.90 + 1.61 𝑥 𝑆 ―1.87 𝑥 𝑇° + 1.62 𝑥 𝑡 ― 1.82 𝑥 𝑆 𝑥 𝑇° ― 2.68 𝑥 𝑇° 𝑥 𝑡 ― 0,91 𝐸 2

𝐸

𝑆

𝑆

( ) 𝑥 𝑇° + 2.12 𝑥

(𝑇°)2 +5.05 𝑥

𝑥 (𝑇°)2

(8)

where E/S is the enzyme to substrate ratio, T° is temperature, and t is time. The Model F-value of 22.32 and values of “Probe > F” less than 0.05 implied that the model is significant. The estimated model for WPC hydrolysis with trypsin was found acceptable to describe the data of ABTS•+ SA (%). Under these conditions, 94.20% of the variation in the response variables can be explained by this model (R2 = 0.9420) (Table S1 of the supplementary material). The “LOF F-value” of 1.14 implies that the LOF is not significantly related to the pure error. Non-significant LOF is good. The “Pred R-Squared” of 0.8990 is in reasonable agreement with the “Adj R-Squared” of 0.8998 (Figure 1a). This model is adequate to describe the data of ABTS•+ SA (%) of the hydrolysates of WPC 35. The regression coefficients listed in Eq. (8) were analyzed in order to evaluate the effects of T°, t and E/S on ABTS•+ SA (%). The significance of each coefficient was determined by p-values which are listed in Table S2 of the supplementary material. The relationship between the three

16

parameters studied (T°, t and E/S) are illustrated in the response surface plot shown in Figure 1b and in the two response contour plot shown in Figure 1c-d. Figure 1b shows that ABTS•+ SA (%) became greater with increasing E/S and T° at first, and then decreased with increasing E/S and T° within a given range. High values of E/S and T° achieve high values of ABTS•+ SA (%). This result could be explained taking into account that the enzymatic hydrolysis of proteins is an endothermic reaction and, therefore, a rise in temperature increases the reaction rate. However, this positive influence by temperature is reversed at higher temperatures because proteins begin to denature. Figure 1b shows that high values of E/S and T° increase the value of ABTS•+ SA (%). In order to explain this result, it is important to consider the conformational changes produced in protein structure at high temperatures. In a preview report, Cheison, Schmitt, Leeb, Letzel, & Kulozik (2010) showed that the hydrolysis of β-Lg with trypsin carried out at high temperatures (50 °C) produce nonspecific peptides. Therefore, at a high temperature, a nonspecific trypsin hydrolysis occurred. Figure 1c shows three optimum zones highlighted in red, where SA (%) is a maximum. Hence, discarding the hydrolysis conditions in which conformational changes of the whey proteins could occur due to high hydrolysis temperatures (E/S < 0.005 and T° > 44 °C; E/S > 0.025 and T° > 44 °C), the optimal conditions for trypsin hydrolysis of WPC 35 were selected. Thereby, a temperatures of 41.1 °C and an E/S between 0.0170.025 were the hydrolysis conditions chosen to perform the enzymatic hydrolysis of WPC 35. On the other hand, Figure 1d showed that SA (%) was higher with increasing hydrolysis time with an E/S between of 0.0170.030. After 3.4 h at 37.1 °C, the highest ABTS•+ SA (%) was achieved. The ANOVA analysis showed that the p-value of the model was <0.0001, indicating that it was significant at the 99% confidence level. In order to verify the optimized results, the validity and suitability of the model described above was tested. Thus, three optimal enzymatic reaction conditions were chosen and the confirmation 17

experiments were conducted in triplicate (Table S3 of the supplementary material). The experimental values were in agreement with the predicted values of the model within a 95% confidence interval, indicating that the model was powerful and suitable for estimation of experimental values.

3.2. Degree of hydrolysis After the enzymatic hydrolysis of WPC 35 following the hydrolysis conditions defined by the first experimental design (Table 1), the number of amino groups (NH2) released by breaking the peptide bonds was quantified as percentage of hydrolysis degree DH (%). The difference of DH (%) between the hydrolyzed (WPH) and unhydrolyzed (WPC) samples provides the number of amino groups produced during the trypsin reaction. Trypsin’s preferred cleavage sites in β-Lg are peptide bonds at the C-terminus of lysine and arginine residues, and it was reported that the maximum DH (%) expected for this reaction is 10.56% (Cheison et al., 2011). After trypsin hydrolysis of WPC 35, the maximum DH (%) reached was 18 ± 1%, and the lowest was 8 ± 1%. Therefore, the DH (%) values determined (Table S4 of the supplementary material) are due to the degradation of β-Lg, as well as other proteins in WPC, such as α-La, BSA, lactoferrin and Ig. It was found that DH (%) increased when E/S and hydrolysis time was higher. However, when the hydrolysis reaction was carried out with a constant E/S (0.0167) and t (2.5 h), but varying the temperature from 37 °C to 50 °C, the DH (%) decreased from 13.71 to 11.67, respectively. These results could be explained taking into account that 50 °C is the highest possible temperature under which trypsin could remain stable (Cheison et al., 2011). On the other hand, it was observed that pre-treated WPC (90 °C, 10 min) produces a minimum increase in DH (%) (1.52% higher) compared to WPC (Table S5 of the supplementary material). This result is in accordance with the increase in the value of ABTS•+ SA (%), which could be attributed to protein structural changes during the thermal pre-treatment of WPC. Moreover, it was found that there is no thermal hydrolysis of

18

trypsin when the reaction mixture is heated in a boiling water bath for 10 min in order to finish the enzyme hydrolysis. These results are shown in Table S5 of the supplementary material. The DH (%) was determined for the three optimized hydrolysis conditions and the results are shown in Table S6 of the supplementary material. The highest DH (%) was found when the hydrolysis temperature was the optimum one for trypsin reaction.

3.3. RP-HPLC and SDS-PAGE analysis RP-HPLC was used to study the enzymatic hydrolysis of WPC. Therefore, the peptide formation and the degradation of β-Lg, α-La and BSA were evaluated (Figure 2a-b). Figure 2a shows the typical WPC's HPLC profile identifying β-Lg, α-La and BSA peaks. These results are consistent with recently published data (Corrochano, Sariçay, Arranz, Kelly, Buckin & Giblin, 2018). The chromatogram of WPC 35 shows that the β-Lg peak has the highest intensity, which is in accordance with the fact that β-Lg is the major whey protein in milk (Antal et al., 2001). On the other hand, Figure 2b shows that heated pretreatment and the subsequent trypsin hydrolysis of WPC at optimal conditions cause the degradation of 99% and 96% of β-Lg and of α-La, respectively. At the same time, peptides with higher polarity than WPC proteins (retention times between 10 and 25 min) were formed. After 4.3 h of hydrolysis, twelve major peaks corresponding to peptides were observed at the RP-HPLC chromatogram. A comparison of Figures 2b and 2c shows that four peaks, named P3, P9, P11 and P12, are not present in the fraction collected by the UF membrane passage of 3 kDa. This result suggests that these peaks correspond to peptides with a molecular mass greater than 3 kDa. The SDS-PAGE analysis confirmed the degradation of β-Lg and α-La, as well as the cleavage of other higher molecular weight proteins from WPC, such as Ig and BSA (Figure 2d). The protein profile for WPC is in agreement with previous studies performed with whey (Önay-Uçar et al., 2014). TricineSDS-PAGE is commonly used to separate proteins in the mass range 1-100 kDa. However, in this work, 19

the SDS-PAGE without tricine was carried out to study the degradation of the principal protein components of whey. Peptide formation was studied by RP-HPLC and MALDI-TOF-MS analysis. The results obtained with RP-HPLC and SDS-PAGE are in accordance with DH (%) achieved for the trypsin hydrolysis of whey. Ferreira et al. (2007) carried out the enzymatic hydrolysis of WPC 80 from bovine milk using trypsin. The reaction was performed at a constant temperature (37 °C) and pH (8.0), and it was stopped after five different times of incubation (15, 30, 60, 120 and 180 min). The RPHPLC results demonstrated that the hydrolysis of α-La was almost complete after 15 min. In contrast, degradation of β-Lg was about 58% after 180 min of incubation.

3.4. MALDI-TOF-MS analysis The MALDI mass spectrometry was used to characterize the representative fractions obtained from bovine milk whey and to attempt to characterize their peptide content. As detailed in Table 2, quite characteristic fingerprint is shown by each sample analyzed: fraction F  3 kDa characteristic fingerprint in the m/z 2000 and 2400 region; WPCT1 characteristic fingerprint in the m/z 700 and 840 region, and no signals in the 2000 and 2400 region; WPCT2, characteristic fingerprint in the m/z 1000 and 1750 region. The obtained spectra for each fraction are displayed in Figures S1, S2 and S3 of the supplementary material. Among the complex pattern of signals shown by WPC heat treatments, the signals corresponding to low molecular polypeptides were observed, i.e. ALC (carbamidomethyl) SEK and ELKDLK, both formed by degradation of α-La (Brandelli et al., 2015; Wada & Lönnerdal, 2015). The carbamidomethyl-modified cysteine residues in the polypeptide ALC (carbamidomethyl) SEK can be considered a product of the treatment of the samples with MALDI-MS matrices and alkylating agents (CHCA and sinapic acid) (Gundry et al., 2009); for this reason, the sequence of interest would be LACSEK. On the other hand, the signal assigned to ALPMHIR (calculated m/z =837.48, experimental data m/z 837.65; formed by trypsin digestion of β-Lg) (Brandelli et al., 2015; Mao, Krischke, Hengst & 20

Kulozik, 2018) was detected in WPC and also in F  3 kDa (experimental m/z = 837.86). The complex pattern of signals shown by WPCT1 and WPCT2 could be associated with glycolipids and/or lipo-peptide compounds. All the spectra obtained from the three fractions described above were compared with the corresponding control samples. The antioxidant activity of the peptides depends, among other factors, on the type of amino acids that constitute them (Rajapakse, Mendis, Jung, Je, & Kim, 2005). It was also reported that amino acids such as cysteine (C), methionine (M), lysine (K), histidine (H), tyrosine (Y), and tryptophan (W) are responsible for the antioxidant properties of whey peptides (Sarmadi & Ismail, 2010). The amino acid sequences presented in Table 2 show that the produced peptides are composed of amino acids with antioxidant properties (C, M, K and H).

3.5. Biological activities of hydrolysates 3.5.1. Antioxidant activity Antioxidant activities of WPC, WPH and F  3 kDa were measured by the ABTS•+ decolorization assay. When the ABTS•+radical is formed, the solution turns blue and then the deactivation reaction could be followed spectroscopically at 734 nm. Figures S4b and S4c of the supplementary material show the change in color of the ABTS•+ solution due to the radical scavenging activity of F  3 kDa. For all the samples evaluated, the antioxidant activity values were expressed as TEAC, Eq. (3). The linear regression fit of ABTS•+ SA (%) are shown in Figure S4a of the supplementary material. On the other hand, the EC50 values of each sample are listed in Table 3. The WPH and F  3 kDa have significantly higher antioxidant activity values (about 4 times) than the WPC (p < 0.05) (Table 3). This result indicates that after trypsin hydrolysis of WPC, the antioxidant peptides are released. Furthermore, WPH is ~ 1.3 times better ABTS•+ scavenging than F  3 kDa. This result could be explained taking into account that, in addition to peptides, WPC contains a number of 21

other proteins with antioxidant activity such as lactoferrin and Ig, among others (Smithers, 2008). Thus, the antioxidant activity observed in WPH results from the combination of the antioxidant activity of the inherent WPC constituents and the peptides released during trypsin hydrolysis. Adjonu et al. (2013) have studied the trypsin hydrolysis of whey protein isolate (WPI) at a constant pH (7.8) and E/S (0.4) after 24 h of hydrolysis. They found that the hydrolyzed WPI has an antioxidant activity of 0.31 μmol TE mg−1 protein, and the trypsin unhydrolysate has a value of 0.07 μmol TE mg−1 protein. These results are similar to those obtained with WPC 35 (Table 3), a substrate with a lower percentage of pure protein and a greater number of additional compounds (lactose, carbohydrates and fat, among others) (Table S6 of the supplementary material).

3.5.2. Scavenging activity of HO• radical The HO• radical is the most active ROS generated in biological systems and foods. The DoR assay was used to determine the ability of peptides to scavenge HO• produced by Fenton reaction. The change of color in the Fenton-DoR reaction medium due to the presence of WPC, WPH and F  3 kDa is shown in Figures S5b and S5c of the supplementary material. The scavenging activity of HO• by WPC, WPH and F  3 kDa was expressed as HO• MEA, Eq. (5). The linear regression fit of HO• SA (%) versus concentration of protein is shown in Figure S5a of the supplementary material. Mannitol was used as a positive control because it is a good HO• scavenger (Maisch, Bosl, Szeimies, Lehn & Abels, 2005). On the other hand, the EC50 was determined and their values are presented in Table 3. From the data shown in Table 3, it can be concluded that F  3 kDa is ~ 1.9 times better at HO• scavenging than WPH. Moreover, although WPC showed some antihydroxyl radical activity, WPH is twice a better antioxidant than WPC. One more time, the antioxidant assays revealed that the enzyme hydrolysis of WPC releases peptides responsible for the antioxidant activity of the resulting material. 22

Peng et al. (2009) used electron spin resonance (ESR) in order to determine the HO• scavenging by peptide fractions obtained after the enzymatic hydrolysis of WPI. They argue that the antioxidant activity of WPI hydrolysates was related to their radical quenching capability because peptides could react with free radicals to convert them into stable products.

3.5.3. Scavenging activity of O2• radical Numerous biological reactions generate O2•−, which is a precursor of highly ROS, such as HO• (Valko et al., 2007). For that reason, studying the capability of hydrolysates to scavenge O2•− is very important. Thus, O2•−was generated from autoxidation of pyrogallol to quinone, which has a characteristic absorbance maximum at 325 nm. The reaction was followed spectroscopically and the scavenging activity of O2• for each sample was expressed as O2• SODEA, Eq. (7). The SOD was used as a standard molecule for the pyrogallol assay (Valko et al., 2007). All the O2• SODEA and EC50 results are summarized in Table 3. The O2• SA (%) could not be determined for WPC and WPH due to experimental limitations. Therefore, we decided to evaluate the O2• scavenging activity of WPH after separation into fractions of different molecular weight by UF. Hence, the effect of the molecular weight of peptides on the antioxidant activity was analyzed. Three protein fractions were produced: F1  3 kDa, F2 = 3 - 5 kDa, and F3 = 5 - 10 kDa. Numbers were assigned to each fraction to identify them. The EC50 values (mg protein /mL) obtained for F1, F2 and F3 were 1.460.03, 1.620.03 and 1.440.02, respectively. The hydrolyzed fractions present similar scavenging activities and they are concentration-dependent (Figure S6a of the supplementary material). Peng et al. (2009) evaluated the O2• scavenging activity of the fractions obtained after the enzymatic hydrolysis of WPI. They found SA (%) between 47.2% and 14.3% for a protein concentration of 0.5 mg/mL. The authors conclude that the antioxidant activity is higher for the fraction of peptides 23

with molecular weight between 0.1 kDa and 2.8 kDa. Önay-Uçar et al. (2014) obtained similar results some years later. They carried out the trypsin hydrolysis of WPC and found that the protein fraction, with peptides of low molecular weight, presented the highest superoxide radical scavenging activity.

3.5.4. Cytoprotector effect on IEC-18 cells The cytoprotective effect of WPC, WPH and F  3 kDa against the oxidative stress generated by MEN was tested (Figure 3a). The percentage of viable cells in IEC-18 induced by 25 μM MEN was 52%. However, pre-treated cells with 0.1–1.0 mg protein/mL WPC, WPH and F  3 kDa significantly restored cell viability up to 82%, 98% and 88%, respectively. This indicates a high cytoprotective effect being higher for WPH and F  3 kDa, although without significant differences with WPC and control. Dose-dependent effects were not observed. Morphological changes of cells after 24 h of incubation were observed with an inverted microscope (Figure 3b-f). The IEC-18 cells treated with MEN showed a reduction in cell numbers and a loss of cell-to-cell contact (Figure 3c). These alterations in MEN-induced IEC-18 cells were attenuated by pre-treatment with 0.1 mg/mL of WPC, WPH and F  3 kDa (Figure 3d-f). The morphological analysis confirms the results obtained by the MTT assay. Therefore, peptides released after WPC 35 enzymatic hydrolysis have a strong antioxidant activity both in solution and on IEC-18 cells. O’Keeffe & FitzGerald (2014) performed the enzymatic hydrolysis of WPC 80 with alcalase and neutrase. The hydrolysate fractions (F  5 kDa) showed greater antioxidant capacity and, moreover, increased cellular glutathione and catalase activity when they are incubated with human umbilical vein endothelial cells (HUVECs). Subsequently, Corrochano et al. (2018) demonstrated that peptides released after simulated gastrointestinal digestion of bovine whey proteins, protected Caco-2 and HT-29 cells from the formation of free radicals.

24

4. Conclusion Industrially produced WPC 35 was hydrolyzed with trypsin in order to obtain peptides with high bioactivity. It was found that optimal parameters to obtain peptides with powerful antioxidant activity were 4.31 h, 41.1 °C and an E/S ratio of 0.017. This condition was selected taking into in account cost manufacture in dairy industries. The optimization was done with RSM and the originality of this study lies in the broad reaction conditions assayed for hydrolysis of WPC with trypsin. From the hydrolysis of WPC 35 with trypsin, two products were obtained: WPH and F  3 kDa. The peptides with a molecular weight lower than 3 kDa have shown a high antioxidant activity, being more effective in the HO• radical scavenging, which is of great importance considering that this radical has a high reactivity with biological systems. Additionally, both WPH and F  3 kDa showed a high cytoprotection against oxidative stress generated by MEN (48% inhibition in cell viability) on IEC-18 cells, raising the viability above 88% in pre-treatments. The protection produced by WPH and F  3 kDa against in vitro-induced oxidative stress (buffer solutions and cell culture) with different methodologies reveals that hydrolysates could be used as powerful antioxidant compounds. To the best of our knowledge, this is the first report in which WPC with 35% w/w of protein is used as a raw material for producing antioxidant peptides. Perhaps, this discovery may provide dairy industries new economic opportunities because they can add value to a cheap co-product such as WPC 35. Since the hydrolysis of WPC 35 with trypsin produced peptides with a high antioxidant activity, a next important step consists in carrying out in vitro studies of simulated gastrointestinal digestion to know if the antioxidant peptide properties could change after being consumed. Taking into account the results obtained by the digestion model, the design of a vehiculization system may be necessary. Thus,

25

the peptides generated in this work could imply an important technological development with direct transfer to the food industry in the near future.

26

Acknowledgements We thank to Ministerio de Ciencia y Tecnología (PIODO, 0058/18) of Argentina, Universidad Nacional de Río Cuarto (UNRC-SECYT PPI-2018 331/16), Universidad de Buenos Aires (UBACyT 20020130100166BA and X 0055BA), Consejo Nacional de Investigación Científica y Tecnológica (CONICET PIP 0072CO, PIO 20320150100052CO) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT, PICT 2033/15 and 0130/16) of Argentina for their financial support. We kindly acknowledge Molfino Hermanos S.A. for providing the commercial WPC. We gratefully thank Dr. Mariana Bonaterra from Universidad Nacional de Villa María (UNVM- CONICET) for her technical assistance with the RP-HPLC analysis. We also appreciate the technical assistance with the SDS-PAGE studies of Dr. María Laura Breser and Dr. Telma Eleonora Scarpeci, both of them from the National Universidad Nacional de Villa María (UNVM- CONICET). Y.R., L.R.C., G.P, R.E.B and M.M.A are research members of CONICET. M.B.B., M.R.B., N.L.V.V, C.A.A., thanks CONICET for the research fellowship. The Ultraflex II (Bruker) TOF/TOF mass spectrometer was supported by a grant from ANPCYT, PME 125.

27

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Figure Captions Figure 1. (a) Predicted results in rational accordance with the actual results. (b) Response surface 3-D plot for ABTS•+ scavenging activity percentage activity of WPHs on temperature versus E/S. (c) Response contour plot for ABTS•+ scavenging activity percentage activity of WPHs on temperature versus E/S. (d) Response contour plot for ABTS•+ scavenging activity percentage activity of WPHs on time versus E/S. Figure 2. (a) RP-HPLC chromatogram of WPC 35 before the enzymatic reaction. (b) Peptide profiles of WPH. (c) Peptide profiles of F 3 kDa. (d) SDS-PAGE analysis of WPC 35 at a concentration of 1.5 mg/mL (1), WPC 35 1 mg/mL (2), β-Lg (3), BSA (4), and WPH (5).

Figure 3. (a) Cytoprotection assays in IEC-18 cells pre-treated with WPC, WPH and F  3 kDa at 0.1– 1.0 mg/mL for 24 h before exposure to 25 µM MEN for 24 h. The values are presented as the mean ± SD (n = 3) (values with the same superscript letters are not significantly different from each other at p < 0.05). Morphological analysis of IEC-18 cells pre-treated with WPC, WPH and F < 3 kDa (0.1 mg/mL) for 24 h before treatment with 25 μM MEN for 24 h. Representative images of (b) non-stressed cells, (c) MEN-induced, cells pre-treated with (d) WPC, (e) WPH, (f) F  3 kDa, before MEN-induced.

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Table 1. Experimental design for the optimization of inhibition activity in CCD showing the ABTS•+ SA (%) of WPHs from WPC prepared under different hydrolysis conditions. Assay

Independent variable

Response variable

E/S

T° (°C)

time (h)

ABTS•+ SA (%)

1

0.0030

43.50

2.50

49.85

2

0.0167

37.00

2.50

53.76

3

0.0167

43.50

0.50

49.47

4

0.0248

39.64

1.31

48.30

5

0.0167

43.50

4.50

55.39

6

0.0167

43.50

2.50

52.84

7

0.0167

43.50

2.50

49.76

8

0.0248

47.36

3.69

54.04

9

0.0167

43.50

2.50

53.43

10

0.0248

39.64

3.69

56.56

11

0.0167

50.00

2.50

47.48

12

0.0085

39.64

1.31

37.09

13

0.0167

43.50

2.50

53.36

14

0.0167

43.50

2.50

52.45

15

0.0085

47.36

3.69

50.13

16

0.0303

43.50

2.50

55.25

17

0.0248

47.36

1.31

56.26

18

0.0085

39.64

3.69

45.59

19

0.0167

43.50

2.50

51.91

20

0.0085

47.36

1.31

52.56

35

Table 2. MALDI-TOF-MS fingerprints of three fractions obtained from bovine milk and peptides identified F ≤ 3 kDa, WPCT1 (after treatment 90°C 10 min), and WPCT2 (after treatment 90 °C 10 min and 100 °C 10 min). The corresponding spectra (Figures S4, S5 and S6) are included in the supplementary material. n.d.: not detected Signals observed in each fraction (m/z) F WPCT1 WPCT2 ( 3 kDa) n.d. n.d. 658.79 671.06 n.d. 671.10 n.d. n.d. 677.10 n.d. 689.06 689.09 n.d. 706.98 707.00 717.44 n.d. 717.04 726.76 726.35 727.11 n.d. n.d. 734.96 n.d. 745.02 745.08 n.d. 782.90 782.97 n.d. 788.68 788.00 n.d. 796.89 796.99 n.d. 809.97 809.65 n.d. 824.90 n.d. 837.86 837.65 n.d. n.d. n.d. 1026.49 n.d. n.d. 1041.42 n.d. n.d. 1055.47 n.d. n.d. 1068.33 n.d. n.d. 1082.31 n.d. n.d. 1371.71 n.d. n.d. 1388.99 n.d. n.d. 1402.13 n.d. n.d. 1416.40 n.d. n.d. 1428.31 n.d. n.d. 1541.24 n.d. n.d. 1566.93 n.d. n.d. 1749.56 2033.11 n.d. n.d. 2050.38 n.d. n.d. 2116.05 n.d. n.d. 2318.22 n.d. n.d. 2339.75 n.d. n.d. 2362.61 n.d. n.d.

Calculated mass

Protein fragment

Amino acid sequence

707.34

-La

ALC(carbamidomethyl)SEK

745.44

-La

ELKDLK

837.48

-Lg

ALPMHIR

36

Table 3. ABTS•+, HO• and O2• scavenging activity of WPC, WPH and F  3 kDa. Sample

ABTS•+ TEAC 1*

WPC

EC50 4*

0.078±0.006a 228.3±0.9a

WPH

0.40±0.02b

46.9±0.4b

F  3 kDa

0.30±0.01c

59.7±0.5c

HO• MEA 2*

O2• SODEA 3*

EC504*

0.046±0.006a 0.29±0.02a

n.d.

n.d.

0.090±0.008b 0.17±0.01b

n.d.

n.d.

0.17±0.02c

EC50 4*

0.09±0.01c

n.d.: not detected. 1 (µmol TE/mg protein sample) 2 (mmol mannitol/mg protein sample) 3 (µg SOD/mg protein sample) 4 (mg protein sample/mL) *All data were expressed as means of triplicate measurements ± standard deviation (SD). a,b,c Different letters indicate significant differences from each other at p < 0.05.

37

0.205±0.009 1.46±0.03

Figure 1. Ballatore et al.

38

Figure 2. Ballatore et al.

39

Figure 3. Ballatore et al.

40

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

41

Highlights   

Hydrolysis conditions by trypsin regulate the antioxidant activity of WPC hydrolysates. Peptides with molecular weight less than 3 kDa shown high antioxidant activities. WPC hydrolysis increases cytoprotection against oxidative stress on IEC-18 cells.

42