Effect of digestive enzymes on the bioactive properties of goat milk protein hydrolysates

1 EFFECT OF DIGESTIVE ENZYMES ON THE BIOACTIVE 1 PROPERTIES OF GOAT MILK PROTEIN HYDROLYSATES 2 F. Javier Espejo-Carpio*, Pedro J. García-Moreno, Raúl Pérez-Gálvez, Rocío Morales-Medina, 3 Antonio Guadix, Emilia M. Guadix. 4 Department of Chemical Engineering, University of Granada, 18071 Granada, Spain 5 *Corresponding author: Tel.: +34 958 241329; Fax: +34 958 248992; Email: [email protected] 6 7 ABSTRACT 8 The aim of this research was to study the influence of the gastrointestinal digestion on the 9 bioactivity of goat milk protein hydrolysates prepared with subtilisin, trypsin and a combination 10 of these two enzymes. All hydrolysates had excellent ACE inhibitory, antioxidant and bile acid11 binding capacity. Peptide profiles and bioactivities were mainly altered during the intestinal 12 digestion, whereas the effect of the gastric digestion was negligible. The influence of the 13 intestinal digestion varied depending on the hydrolysate and the bioactivity studied. In the case 14 of ACE inhibitory activity, it exclusively decreased when peptides were produced with trypsin. 15 Contrarily, antioxidant activity and bile acid-binding capacity improved after the gastrointestinal 16 digestion, regardless the enzymatic treatment conducted. Hydrolysis employing mixtures of 17 subtilisin and trypsin is considered a good approach to produce peptides that maintain, or even 18 enhance, their bioactivity after digestion. 19 20 2 1. INTRODUCTION 21 Food proteins, and specially milk proteins, have been described as an important source of 22 bioactive peptides (Korhonen & Pihlanto, 2006). Apart from their nutritional value, these 23 compounds present a physiological activity which might have beneficial effects on health. To be 24 considered as bioactive, the compounds should generate a measurable biological effect and 25 have the potential of generate health benefits without presenting toxicity, allergenicity or 26 mutagenicity (Möller, Scholz-Ahrens, Roos, & Schrezenmeir, 2008). Bioactive peptides are 27 encrypted in the sequence of the proteins and should be released to exert their activity. 28 Enzymatic hydrolysis is the main procedure for liberating active peptides. Enzymes such as 29 subtilisin and trypsin generate peptides with hydrophobic or positively-charged amino acids at 30 the C-terminus, respectively. These features have been shown to be favourable for exerting 31 bioactivities (Hernández-Ledesma, Del Mar Contreras, & Recio, 2011; Power, Jakeman, & 32 FitzGerald, 2013). Regarding the substrates, most of the studies dealing with the production of 33 bioactive peptides from milk proteins employ cow milk as a raw material (Nagpal et al., 2011). 34 However, more recently, other types of milk, such as goat milk have also shown its potential as 35 a source of bioactive peptides (Ahmed, El-Bassiony, Elmalt, & Ibrahim, 2015; Espejo-Carpio, 36 De Gobba, Guadix, Guadix, & Otte, 2013). Compared to cow milk, goat milk has a slightly 37 higher casein fraction, which is mainly composed by β-casein (50% of total caseins) while, in 38 bovine casein fraction, the α-caseins represent almost 50% of the total (Bernaka, 2011). 39 Moreover, goat milk proteins have better digestibility and hypoallergenic properties (Park, 40 2009). The bioactivities observed in milk protein derived peptides cover a wide range, including 41 antihypertensive, antioxidant and cholesterol lowering (Korhonen & Pihlanto, 2006; Muro Urista, 42 Álvarez Fernández, Riera Rodriguez, Arana Cuenca, & Téllez Jurado, 2011). 43 Antihypertensive activity is one of the most relevant bioactivities since hypertension is the main 44 risk factor for cardiovascular diseases. The antihypertensive effect of hydrolysates is usually 45 studied in vitro by the capacity of inhibiting angiotensin converting enzyme (ACE). In the 46 organism, this enzyme is a key component of the blood pressure regulation process (Johnston, 47 1992) and its inhibition would help to control high blood pressure. Contrary to commercial ACE 48 inhibitory drugs, food derived peptides have not shown any side effects, even at high doses 49 3 (Ishida et al., 2011). The ACE inhibitory potential of goat milk protein hydrolysates has been 50 reported in previous studies employing subtilisin or trypsin as enzymes. (Espejo-Carpio et al., 51 2013; Manso & López-Fandiño, 2003). Additionally, some of the peptides released from goat 52 protein have shown in vivo effects. For example, a goat milk hydrolysate originated a significant 53 decreased in systolic blood pressure in spontaneously hypertensive rats. Moreover it reduced 54 the ACE activity in aorta, left ventricle, and kidney in a similar way that a commercial drug 55 (Geerlings et al., 2006). 56 Antioxidants compounds, apart from inhibiting or retard lipid oxidation in food, might also 57 prevent oxidation at physiological level (Hajieva & Behl, 2006). Because of the potentially 58 hazardous effects of synthetic antioxidants (Ito, Fukushima, & Tsuda, 1985), there is an 59 increasing interest in finding natural antioxidants with reduced side effects. In this context, goat 60 milk proteins have shown its potential as raw material for the production of hydrolysates 61 exhibiting antioxidant activity. Recently, De Gobba, Espejo-Carpio, Skibsted and Otte (2014) 62 produced antioxidant peptides by enzymatic hydrolysis of goat milk microfiltration fractions 63 using subtilisin and trypsin. Likewise, Li et al. (2013) identified antioxidant peptides in goat milk 64 casein hydrolysates produced by a combination of neutral and alkaline proteases. Bezerra et al. 65 (2013) also reported antioxidant activity for the permeate of goat casein hydrolysates produced 66 by using papain. 67 In addition, some protein fractions present in milk have also been reported to exert cholesterol 68 lowering activity. The lowering activity of a given compound depends on its ability to inhibit the 69 intestinal absorption of dietary cholesterol or to sequester bile acids, which are not reabsorbed 70 but excreted. In response to a lower rate of bile recycling, the liver increases bile acid synthesis, 71 which is done at the expense of removing cholesterol from bloodstream. Several studies have 72 reported the cholesterol lowering ability of cow milk proteins but none of goat milk proteins. For 73 instance, Lanzini, Fitzpatrick, Pigozzi, and Northfield (1987) conducted in vivo studies on ileal 74 resection patients demonstrating the bile binding capacity of caseins. Furthermore, Nagaoka et 75 al. (2001) identified an active peptide in tryptic -lactoglobulin hydrolysates able to inhibit the 76 absorption of dietary cholesterol through the intestine wall. 77 4 In any case, to exert their biological effect, bioactive peptides should reach their target organs 78 and tissues in an active form. In this regard, gastrointestinal digestion is a key process 79 determining the bioavailability of the peptides. In vitro digestion processes, which employ 80 gastrointestinal proteases, could be employed for evaluating the effect of digestive enzymes 81 over bioactive hydrolysates and peptides. To the best of the authors' knowledge, there are no 82 published studies dealing with the effect of gastrointestinal digestion on antioxidant or 83 cholesterol lowering activity of milk protein-derived peptides. With respect to ACE inhibitory 84 activity, the few studies carried out are focused on the changes originated on isolated peptides 85 instead of on the whole hydrolysate (Contreras, Sanchez, Sevilla, Recio, & Amigo, 2013; 86 Quirós, Contreras, Ramos, Amigo, & Recio, 2009; Tavares et al., 2011). Although the 87 evaluation of the effect of digestive enzymes over isolated peptides could be interesting, as far 88 as nutraceuticals are concern, large scale purification or synthesis of individual bioactive 89 peptides would be very costly. Furthermore, bioactivities such as antihypertensive could be 90 influenced by apparent inactive peptides which could interact with subsites different from the 91 active center of ACE (López-Fandiño, Otte, & van Camp, 2006; Meisel, 1997). Additionally, 92 because of the different conformational requirements of the catalytic sites of ACE, a mixture of 93 peptides with different conformational features would be necessary for inhibiting ACE (Gobbetti, 94 Stepaniak, De Angelis, Corsetti, & Di Cagno, 2002). Therefore, studying the effect of the 95 digestion over the complete hydrolysate seems a better approach to evaluate the global 96 bioactive capacity.The aim of this work was to evaluate the effect of gastrointestinal enzymes in 97 the ACE inhibitory activity, antioxidant activity and cholesterol lowering activity of hydrolysates 98 of goat milk caseins. 99 2. MATERIALS AND METHODS 100 2.1. ENZYME HYDROLYSIS 101 Goat milk caseins were hydrolysed according to the procedure described in Espejo-Carpio et al. 102 (2013). Firstly, commercial UHT goat milk was microfiltered through a 0.14 m ceramic 103 membrane, where the casein fraction was obtained in the retentate side. Subtilisin 104 (EC.3.4.21.62) and trypsin (EC.3.4.21.4), both from Novozymes A/S (Bagsværd, Denmark), 105 were used as enzymes for producing three different hydrolysates: subtilisin hydrolysate (SH), 106 5 trypsin hydrolysate (TH) and a hydrolysate produced with a mixture of both enzymes (STH). In 107 all the cases, the reaction was carried out in a 0.2 L stirrer tank reactor at 50 ºC and pH 8. The 108 degree of hydrolysis (DH) was monitored throughout the reaction by the pH-stat method (Adler109 Nissen, 1986). After 3 h of reaction, enzyme was thermally deactivated in boiling water for 15 110 min. The final hydrolysates were freeze dried for further analysis. 111 2.2. PROTEIN DETERMINATION 112 The protein content of both the hydrolysates and the digested hydrolysates were determined by 113 the Total Protein Kit, Micro Lowry, Peterson’s Modification (Sigma-Aldrich, St. Louis, MO, USA). 114 The dried samples were re-dissolved in Milli-Q water and analysed in triplicate according to the 115 kit instructions. The absorbance was measured at 650 nm. 116 2.3. GASTROINTESTINAL DIGESTION 117 The method proposed by Lo and Li Chan (2005) with some modifications was used for 118 evaluating the susceptibility of active peptides to digestive enzymes. In this work, the effects of 119 both the gastric digestion alone and the gastrointestinal digestion over the bioactivities of 120 hydrolysates were evaluated. To this end, each hydrolysate sample was subjected, in triplicate, 121 to two digestion procedures: gastric and gastrointestinal digestion. 122 The digestion process was carried out in a jacketed stirred reactor at 37 ºC. Firstly, the freeze 123 dried samples were reconstituted in water until obtaining a protein concentration of 10 mg mL-1. 124 Then, the pH was decreased to pH 2 by adding 1 M HCl. Subsequently, the gastric digestion 125 was initiated by adding pepsin (EC 3.4.23.1, Merck, Darmstadt, Germany) at enzyme-substrate 126 ratio of 4 % (w/w). The reaction was stopped after 1 h raising the pH to 5.3 with 0.9 M NaHCO3. 127 For gastric digested samples, the digestion process was finished at this point by inactivating 128 thermally the enzyme (100 ºC for 15 min). However, the digestion process continued for the 129 gastrointestinal digested samples by the incorporation of pancreatin to the reaction media. 130 Pancreatin (Sigma-Aldrich) is a mixture of porcine pancreas enzymes containing proteases 131 such as trypsin, chymotrypsin, elastase, carboxy-peptidase A and carboxypeptidase B. The 132 enzyme mixture was prepared at 2 mg mL-1 in NaHCO3 0.1 M and added to the reactor to obtain 133 an enzyme-substrate ratio of 4% (w/w). Immediately, the pH was raised to pH 7.5 with 1 M 134 NaOH. The intestinal digestion was performed for 2 h at 37 ºC, then, the enzymes were 135 6 thermally deactivated (100 ºC for 15 min). Once deactivated, both gastric samples and 136 gastrointestinal samples were freeze-dried until further analysis. 137 2.4. SIZE EXCLUSION CHROMATOGRAPHY 138 The molecular size distribution of the hydrolysates and their digests was determined by size 139 exclusion chromatography (SEC). A fast protein liquid chromatography system (Pharmacia LKB 140 Biotechnology AB, Uppsala, Sweden) mounted with Superdex Peptide 10/300GL column (GE 141 Healthcare, Uppsala, Sweden) was used. The column was operated at 25 ºC with a mobile 142 phase composed of 70:30 water/acetonitrile with 0.1 % trifluoroacetic acid (TFA). The 143 absorbance was measured at 280 nm. 144 Hydrolysates and digested hydrolysates were diluted in distilled water (5 mg protein mL-1) and 145 filtered through a 0.2 m filter. An aliquot of 100 L was injected and eluted at 0.5 mL min-1. 146 Glycine (75 Da), alanine (89 Da), Phe-Gly-Gly (279 Da), (Gly)6 (360 Da), vitamin B12 (1355 147 Da), insulin (5733 Da), aprotinin (6511 Da) and ribonuclease (13700 Da) were used as 148 standards. 149 2.5. ACE-INHIBITORY ACTIVITY 150 A spectrophotometric assay (Shalaby, Zakora, & Otte, 2006) was selected to measure the 151 capacity of the hydrolysates and their digested samples to inhibit ACE. The method is based on 152 the decrease in absorbance observed when the synthetic tripeptide N-[3-(2-furyl)acryloyl]-L153 phenylalanyl-glycyl-glycine (FAPGG) is hydrolysed by ACE (EC 3.4.15.1). Both reagents were 154 purchased from Sigma-Aldrich. The assay was carried out in a 96-well microplate at 37 ºC 155 measuring the absorbance at 340 nm for 30 min in a Multiskan FC microplate photometer 156 (Thermo Scientific, Vantaa, Finland). Inhibition percentage was determined according to Eq. 1. 157 Samples were analysed in triplicate. 158 [1] 159 Where pi was the slope in the presence of inhibitor and p0 the slope in the absence of inhibitor 160 (pure water). These slopes were calculated in the interval from 10 to 25 min, when a better 161 linearity was registered. The IC50 value, defined as the concentration of sample which reduces 162 7 the ACE activity to 50%, was calculated for each sample in triplicate using five different protein 163 concentrations. 164 2.6. ANTIOXIDANT ACTIVITY 165 The antioxidant activity of the hydrolysates produced and their digested samples was 166 determined in vitro by testing their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) 167 radical. The method described by Picot et al. (2010) was used. In short, a volume of 1 mL of 168 each sample having different protein concentrations (0.5 - 6 mg mL-1) was added to 1 mL of 0.1 169 mM DPPH in methanol. The mixture was shaken and left for 30 min at room temperature in the 170 dark. Then, the absorbance of the reaction mixture was measured at 517 nm. A blank was run 171 in the same way by using distilled water instead of sample, and sample control was also made 172 for each sample by adding methanol instead of DPPH solution. Triplicate measurements were 173 carried out for each sample and DPPH scavenging activity was calculated by Eq. 2: 174 [2] 175 The IC50 value, which defines the concentration of hydrolysate (mg protein mL-1) needed to 176 inhibit DPPH activity by half, was also calculated. 177 2.7. BILE ACID BINDING CAPACITY 178 Both the hydrolysates and their digested samples were tested for their in vitro bile acid-binding 179 capacity. A bile preparation was formulated containing 75 % of glycocholic bile acids and 25 % 180 of taurine bile acids, simulating human bile (Lin, Tsai, Hung, & Pan, 2010). The hydrolysate 181 powder (100 - 110 mg) was mixed with 4 mL of 0.72 mM bile acid solution. This suspension was 182 incubated under agitation at 1 h and 37 ºC. It was then centrifuged at 60,000 g for 30 min, and 183 the supernatant was recovered. Bile acid binding was related to the amount of bile retained by 184 the hydrolysate (i.e. present in the sludges after centrifugation). The unbound bile acids in the 185 supernatant were determined using a total bile acids determination kit (Diazyme, Poway, CA, 186 USA), employing cholic acid (Sigma Aldrich) as standard. This method is based on the 187 enzymatic conversion of bile acids to 3-ketosteroids, which can be followed 188 spectrophotometrically at 405 nm. The bile acid-binding capacity is commonly expressed as the 189 8 μmol of bile acids bound by 100 mg of dry matter (i.e. retained in the sludge after 190 centrifugation). A sample of 24 - 26 mg of cholestyramine (Sigma-Aldrich) was employed as 191 positive control. In our case, 100 mg of cholestyramine sample bound on average 9.42 ± 0.02 192 μmol of the bile acids, which represents 90 % of bile acid binding capacity. Considering that 193 proteins were the major compounds responsible for the sequestration of bile acids, the bile acid194 binding capacity of the hydrolysates was related to cholestyramine on equal protein basis. 195 2.8. STATISTICAL ANALYSIS 196 Statgraphics Centurion XV (Statistical Graphics Corp., Rockville, MD, USA) was used for data 197 analysis. Data were expressed as mean ± standard deviation (SD) with three replications. Firstly 198 a one-way analysis of variance (ANOVA) was performed to identify significant differences 199 between original and digested samples. Secondly, mean values were compared by using the 200 Fisher’s least significant difference (LSD) test. Differences between means were considered 201 significant at p values ≤ 0.05. 202 3. RESULTS AND DISCUSSION 203 3.1. PRODUCTION OF HYDROLYSATES 204 The variation of the DH along the reaction was similar for the three treatments. As observed in 205 Fig. 1, an initial period of constant reaction rate was followed by a period of decreasing rate 206 where the curve became asymptotic. The lowest DH was achieved for TH, which might be due 207 to the higher specificity of this enzyme and the relatively high temperature employed in the 208 hydrolysis. Nevertheless, in all the treatments, the final DH obtained was above 20 %, which 209 assures hydrolysates containing small peptides. Indeed, the average peptide chain length 210 (PCL), calculated as the inverse of DH (Adler-Nissen, 1986), would be 5 residues. Short length 211 chain peptides are highly desirable since they usually exhibit better antihypertensive and 212 antioxidant activities than larger peptides (Contreras, Carrón, Montero, Ramos, & Recio, 2009; 213 Di Pierro, O’Keeffe, Poyarkov, Lomolino, & FitzGerald, 2014; Pihlanto-Leppälä, Rokka, & 214 Korhonen, 1998) and also have better resistance to degradation by digestive enzymes (Cinq215 Mars, Hu, Kitts, & Li-Chan, 2007; Roberts, Burney, Black, & Zaloga, 1999). The final DH 216 depends mainly on the enzyme employed, its concentration and the reaction time. The high 217 9 enzyme-substrate ratio employed in this work allowed to reach high DH level in a relatively short 218 reaction time. Similar final DH values were obtained in the hydrolysis of goat caseins by papain 219 when employing lower E:S ratios (0.5-1.5 %) but longer reactions times (5 h) (Bezerra et al., 220 2013). 221 3.2. MOLECULAR SIZE DISTRIBUTION 222 Fig. 2 shows the SEC chromatograms of the original hydrolysates and their digests. The original 223 hydrolysates and their gastric digests presented similar molecular size distributions in all cases. 224 This would indicate that pepsin did not modify the original hydrolysates. In contrast, 225 gastrointestinal digests presented important differences compared to the original and gastric 226 digested hydrolysates. Particularly, the fraction of long chain peptides (29 - 0.9 kDa) which 227 eluted before 32 min, showed an increase. This raise could be attributed to large protein 228 fragments released from the previously added enzymes by the action of pancreatin. As a 229 consequence, this initial fraction was not considered for studying the variation between the 230 original hydrolysates and their digested samples. Then, three fractions were selected from each 231 chromatogram for comparison. These fractions, which are delimited by vertical lines in Fig. 2, 232 were noted as A, B and C, and had a molecular size ranging between 900 – 330 Da, 330 – 160 233 and <160 Da, respectively. According to the values of area percentage of each fraction shown 234 in Table 1, in almost all the cases there were significant differences between the original 235 hydrolysate and their gastric and/or gastrointestinal digested samples. 236 As shown Table 1, the original SH and STH presented a very similar molecular size distribution. 237 In contrast, TH presented a higher proportion of large peptides (fraction A) and lower of smaller 238 fractions (B and C). These differences are consistent with the fact that TH had a considerably 239 lower degree of hydrolysis than SH and STH (Fig. 1), and then the proportion of long chain 240 peptides should be higher. 241 Gastric digestion produced small changes in the proportion of fractions A and B in the 242 hydrolysates produced with subtilisin (SH and STH), while no significant changes were 243 observed for TH. On the other hand, the smallest peptides (fraction C) seemed to remain 244 unmodified by gastric digestion in any case. With respect to gastrointestinal digestion, it 245 generated changes in the fractions of peptides larger than 160 Da (A and B fractions) for all the 246 16 Geerlings, A., Villar, I. C., Zarco, F. H., Sanchez, M., Vera, R., Gomez, A. Z., Boza, J., Duarte, 420 J. (2006). identification and characterization of novel Angiotensin-converting enzyme 421 inhibitors obtained from goat milk. Journal of Dairy Science, 89, 3326–3335. 422 Gobbetti, M., Stepaniak, L., De Angelis, M., Corsetti, A., & Di Cagno, R. (2002). Latent bioactive 423 peptides in milk proteins: proteolytic activation and significance in dairy processing. 424 Critical Reviews in Food Science and Nutrition, 42, 223–239. 425 Hajieva, P., & Behl, C. (2006). Antioxidants as a potential therapy against age-related 426 neurodegenerative diseases: amyloid Beta toxicity and Alzheimer’s disease. Current 427 Pharmaceutical Design, 12, 699–704. 428 Hernández-Ledesma, B., Del Mar Contreras, M., & Recio, I. (2011). Antihypertensive peptides: 429 Production, bioavailability and incorporation into foods. Advances in Colloid and 430 Interface Science, 165, 23–35. 431 Hwang, J.-S. (2010). Impact of processing on stability of angiotensin I-converting enzyme (ACE) 432 inhibitory peptides obtained from tuna cooking juice. Food Research International, 43, 433 902–906. 434 Ishida, Y., Shibata, Y., Fukuhara, I., Yano, Y., Takehara, I., & Kaneko, K. (2011). Effect of an 435 excess intake of casein hydrolysate containing Val-Pro-Pro and Ile-Pro-Pro in subjects 436 with normal blood pressure, High-normal blood pressure, or mild hypertension. 437 Bioscience, Biotechnology, and Biochemistry, 75, 427–433. 438 Ito, N., Fukushima, S., & Tsuda, H. (1985). Carcinogenicity and modification of the carcinogenic 439 response by bha, Bht, and other antioxidants. Critical Reviews in Toxicology, 15(2), 440 109–150. 441 Iwami, K., Sakakibara, K., & Ibuki, F. (1986). Involvement of post-digestion “hydrophobia” 442 peptides in plasma cholesterol-lowering Effect of dietary plant proteins. Agricultural and 443 Biological Chemistry, 50, 1217–1222. 444 Jao, C.-L., & Ko, W.-C. (2002). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging by 445 protein hydrolyzates from tuna cooking juice. Fisheries Science, 68, 430–435. 446 Jiang, J., Chen, S., Ren, F., Luo, Z., & Zeng, S. S. (2007). Yak milk casein as a functional 447 ingredient: Preparation and identification of angiotensin-I-converting enzyme inhibitory 448 peptides. Journal of Dairy Research, 74, 18–25. 449 17 Jiang, S.-J., Qian, F., Shen, X., & Mu, G. (2013). Separation and purification of angiotensin 450 converting enzyme inhibitory peptides derived from bovine casein. Journal of Pure and 451 Applied Microbiology, 7, 789–793. 452 Johnston, C. I. (1992). Renin-angiotensin system: A dual tissue and hormonal system for 453 cardiovascular control. Journal of Hypertension, 10, S13–S26. 454 Kahlon, T. S., & Smith, G. E. (2007). In vitro binding of bile acids by blueberries (Vaccinium 455 spp.), plums (Prunus spp.), prunes (Prunus spp.), strawberries (Fragaria X ananassa), 456 cherries (Malpighia punicifolia), cranberries (Vaccinium macrocarpon) and apples 457 (Malus sylvestris). Food Chemistry, 100, 1182–1187. 458 Korhonen, H., & Pihlanto, A. (2006). Bioactive peptides: Production and functionality. 459 International Dairy Journal, 16(9), 945–960. http://doi.org/10.1016/j.idairyj.2005.10.012 460 Lanzini, A., Fitzpatrick, W. J. F., Pigozzi, M. G., & Northfield, T. C. (1987). Bile acid binding to 461 dietary casein: A study in vitro and in vivo. Clinical Science, 73, 343–350. 462 Lin, Y.-H., Tsai, J.-S., Hung, L.-B., & Pan, B. S. (2010). Hypocholesterolemic effect of 463 compounded freshwater clam protein hydrolysate and Gracilaria. Food Chemistry, 123, 464 395–399. 465 Li, Z., Jiang, A., Yue, T., Wang, J., Wang, Y., & Su, J. (2013). Purification and identification of 466 five novel antioxidant peptides from goat milk casein hydrolysates. Journal of Dairy 467 Science, 96, 4242–4251. 468 López-Fandiño, R., Otte, J., & van Camp, J. (2006). Physiological, chemical and technological 469 aspects of milk-protein-derived peptides with antihypertensive and ACE-inhibitory 470 activity. International Dairy Journal, 16, 1277–1293. 471 Lo, W. M. Y., & Li-Chan, E. C. Y. (2005). Angiotensin I converting Enzyme Inhibitory Peptides 472 from in vitro pepsin−pancreatin digestion of soy protein. Journal of Agricultural and 473 Food Chemistry, 53, 3369–3376. 474 Manso, M. A., & López-Fandiño, R. (2003). Angiotensin I converting enzyme-inhibitory activity 475 of bovine, ovine, and caprine k-casein macropeptides and their tryptic hydrolysates. 476 Journal of Food Protection, 66, 1686–1692. 477 18 Mao, X.-Y., Cheng, X., Wang, X., & Wu, S.-J. (2011). Free-radical-scavenging and anti478 inflammatory effect of yak milk casein before and after enzymatic hydrolysis. Food 479 Chemistry, 126, 484–490. 480 Meisel, H. (1997). Biochemical properties of regulatory peptides derived from mil proteins. 481 Peptide Science, 43, 119–128. 482 Möller, N. P., Scholz-Ahrens, K. E., Roos, N., & Schrezenmeir, J. (2008). Bioactive peptides and 483 proteins from foods: indication for health effects. European Journal of Nutrition, 47, 484 171–182. 485 Muro Urista, C., Álvarez Fernández, R., Riera Rodriguez, F., Arana Cuenca, A., & Téllez 486 Jurado, A. (2011). Review: Production and functionality of active peptides from milk. 487 Food Science and Technology International, 17, 293–317. 488 Nagaoka, S., Futamura, Y., Miwa, K., Awano, T., Yamauchi, K., Kanamaru, Y., Tadashi, K., 489 Kuwata, T. (2001). Identification of novel hypocholesterolemic peptides derived from 490 bovine milk β-lactoglobulin. Biochemical and Biophysical Research Communications, 491 281, 11–17. 492 Nagpal, R., Behare, P., Rana, R., Kumar, A., Kumar, M., Arora, S., Morotta, F., Jain, S., Yadav, 493 H. (2011). Bioactive peptides derived from milk proteins and their health beneficial 494 potentials: an update. Food & Function, 2, 18–27. 495 Park, Y. W. (2009). Bioactive components in goat milk. In Y.W. Park (Ed), Bioactive 496 components in milk and dairy products (pp. 43–81). Ames, IA, USA: Wiley-Blackwell. 497 Peña-Ramos, E. a., & Xiong, Y. l. (2002). Antioxidant activity of soy protein hydrolysates in a 498 liposomal system. Journal of Food Science, 67, 2952–2956. 499 Picot, L., Ravallec, R., Fouchereau-Péron, M., Vandanjon, L., Jaouen, P., Chaplain-Derouiniot, 500 M., et al. (2010). Impact of ultrafiltration and nanofiltration of an industrial fish protein 501 hydrolysate on its bioactive properties. Journal of the Science of Food and Agriculture, 502 90, 1819–1826. 503 Pihlanto-Leppälä, A., Rokka, T., & Korhonen, H. (1998). Angiotensin I converting enzyme 504 inhibitory peptides derived from bovine milk proteins. International Dairy Journal, 8, 505 325–331. 506 19 Power, O., Jakeman, P., & FitzGerald, R. J. (2013). Antioxidative peptides: enzymatic 507 production, in vitro and in vivo antioxidant activity and potential applications of milk508 derived antioxidative peptides. Amino Acids, 44, 797–820. 509 Quirós, A., Contreras, M. del M., Ramos, M., Amigo, L., & Recio, I. (2009). Stability to 510 gastrointestinal enzymes and structure–activity relationship of β-casein-peptides with 511 antihypertensive properties. Peptides, 30, 1848–1853. 512 Rajapakse, N., Mendis, E., Byun, H.-G., & Kim, S.-K. (2005). Purification and in vitro 513 antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative 514 systems. The Journal of Nutritional Biochemistry, 16, 562–569. 515 Roberts, P. R., Burney, J. D., Black, K. W., & Zaloga, G. P. (1999). Effect of chain length on 516 absorption of biologically active peptides from the gastrointestinal tract. Digestion, 60, 517 332–337. 518 Samaranayaka, A. G. P., Kitts, D. D., & Li-Chan, E. C. Y. (2010). Antioxidative and angiotensin519 i-converting enzyme inhibitory potential of a pacific hake (Merluccius productus) fish 520 protein hydrolysate subjected to simulated gastrointestinal digestion and caco-2 cell 521 permeation. Journal of Agricultural and Food Chemistry, 58, 1535–1542. 522 Shalaby, S. M., Zakora, M., & Otte, J. (2006). Performance of two commonly used angiotensin523 converting enzyme inhibition assays using FA-PGG and HHL as substrates. Journal of 524 Dairy Research, 73, 178–186. 525 Sugano, M., Goto, S., Yamada, Y., Yoshida, K., Hashimoto, Y., Matsuo, T., & Kimoto, M. 526 (1990). Cholesterol-lowering activity of various undigested fractions of soybean protein 527 in rats. Journal of Nutrition, 120, 977–985. 528 Tavares, T., Contreras, M. del M., Amorim, M., Pintado, M., Recio, I., & Malcata, F. X. (2011). 529 Novel whey-derived peptides with inhibitory effect against angiotensin-converting 530 enzyme: In vitro effect and stability to gastrointestinal enzymes. Peptides, 32, 1013– 531 1019. 532 Vaštag, Ž., Popović, L., Popović, S., Peričin-Starčević, I., & Krimer-Malešević, V. (2013). In vitro 533 study on digestion of pumpkin oil cake protein hydrolysate: Evaluation of impact on 534 bioactive properties. International Journal of Food Sciences and Nutrition, 64, 452–460. 535 20 Wu, H.-C., Chen, H.-M., & Shiau, C.-Y. (2003). Free amino acids and peptides as related to 536 antioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus). Food 537 Research International, 36, 949–957. 538 539 21 FIGURE CAPTIONS 540 Figure 1. Evolution of degree of hydrolysis (DH) during the hydrolysis of caseins with subtilisin 541 (dashed line), trypsin (dotted line) and the combination of subtilisin plus trypsin (solid line). 542 543 Figure 2. Molecular size distribution of the hydrolysates produced with subtilisin (a), trypsin (b) 544 and the combination of subtilisin plus trypsin (c). Each chart shown the original hydrolysate 545 (solid line), the gastric digested hydrolysate (gray solid line) and the gastrointestinal digested 546 hydrolysate (dotted line) 547 548 22 FIGURE 1 549 0 10 20 30 40 0 1 2 3 DH (%) Reaction time (h) 550 551 552 23 FIGURE 2 553 554 a) 0 10 20 30 40 50 60 70 80 90 15 20 25 30 35 40 45 50 55 Absorbance Elution time (min) A B C b) 0 10 20 30 40 50 60 70 80 90 15 20 25 30 35 40 45 50 55 Absorbance Elution time (min) c) 0 10 20 30 40 50 60 70 80 90 15 20 25 30 35 40 45 50 55 Absorbance Elution time (min) ABC 555 556 24 557 Table 1. Area percentages of the fractions from the goat caseins hydrolysates and their digests. 558 Hydrolysate Sample Percentage of Area Fraction A 900-330 Da Fraction B 330-160 Da Fraction C < 160 Da SH (subtilisin) Original 44.9 ± 0.5ab 26.7 ± 0.1a 28.4 ± 0.6ab Gastric Digested 50.0 ± 0.2cd 23.9 ± 0.1b 26.1 ± 0.3ac Gastrointestinal Digested 50.4 ± 1.1cd 25.1 ± 0.5cd 26.9 ± 4.0ac TH (trypsin) Original 56.3 ± 0.6e 19.8 ± 0.5e 23.9 ± 0.2ac Gastric Digested 58.2 ± 0.4e 18.8 ± 0.7e 23.2 ± 1.0c Gastrointestinal Digested 46.9 ± 3.9bc 26.8 ± 0.6a 26.3 ± 4.1ac STH (subtilisin +trypsin) Original 40.9 ± 0.1a 26.0 ± 0.4ad 33.1 ± 0.3b Gastric Digested 45.6 ± 2.1b 22.8 ± 0.6f 32.6 ± 2.0b Gastrointestinal Digested 51.0 ± 2.2d 24.8 ± 0.6c 24.2 ± 2.1ac Data are means of triplicate determinations ± standard deviation. Equal super index in the same column 559 indicates no statistical differences between the area percentages (p<0.05). 560 561 562 25 Table 2. ACE inhibitory, antioxidant and bile acid-binding capacities of the original goat caseins 563 hydrolysates and their digests. 564 Hydrolysate Sample ACE inhibitory activity Antioxidant activity Bile acidbinding capacity IC50 (g mL-1) IC50 (mg mL-1) (%) SH (subtilisin) Original 243.1 ± 12.6ab 4.62 ± 0.15ab 22.03 ± 1.76a Gastric Digested 215.2 ± 10.7c 3.26 ± 0.16ac 27.18 ± 4.37ab Gastrointestinal Digested 249.3 ± 13.7abd 1.57 ± 0.62d 41.74 ± 4.18c TH (trypsin) Original 269.0 ± 3.2d 3.78 ± 0.07ac 26.23 ± 5.21ab Gastric Digested 259.1 ± 17.9bd 3.04 ± 0.14c 40.73 ± 1.16c Gastrointestinal Digested 399.8 ± 15.3e 1.39 ± 0.56d 31.47 ± 1.16b STH (subtilisin +trypsin) Original 230.7 ± 19.2ac 5.51 ± 0.08b 21.86 ± 4.36a Gastric Digested 212.0 ± 12.7c 4.33 ± 0.24ac 41.43 ± 10.20c Gastrointestinal Digested 266.9 ± 5.7d 1.30 ± 0.22d 52.74 ± 6.35d Data are means of triplicate determinations ± standard deviation. Equal super index in the same column 565 indicates no statistical differences (p<0.05) 566 567