Peptide identification in a salmon gelatin hydrolysate with antihypertensive, dipeptidyl peptidase IV inhibitory and antioxidant activities

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Peptide identification in a salmon gelatin hydrolysate with antihypertensive, dipeptidyl peptidase IV inhibitory and antioxidant activities Adriana C. Nevesa, Pádraigín A. Harnedya, Martina B. O'Keeffea, Monisola A. Alashib, Rotimi E. Alukob, Richard J. FitzGeralda,⁎ a b

Department of Biological Sciences, University of Limerick, Limerick, Ireland Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada

A R T I C L E I N F O

A B S T R A C T

Keywords: Salmon gelatin Bioactive peptides Angiotensin converting enzyme Dipeptidyl peptidase IV Oxygen radical absorbance capacity Spontaneously hypertensive rats

Salmon gelatin (Salmo salar, SG) enzymatic hydrolysates were generated using Alcalase 2.4 L, Alcalase 2.4 L in combination with Flavourzyme 500 L, Corolase PP, Promod 144MG and Brewer's Clarex. The hydrolysate generated with Corolase PP for 1 h (SG-C1) had the highest angiotensin converting enzyme (ACE, IC50 = 0.13 ± 0.05 mg mL− 1) and dipeptidyl peptidase IV (DPP-IV, IC50 = 0.08 ± 0.01 mg mL− 1) inhibitory activities, and oxygen radical absorbance capacity (ORAC, 540.94 ± 9.57 μmol TE g− 1 d.w.). The in vitro bioactivities of SG-C1 were retained following simulated gastrointestinal digestion. Administration of SG and SGC1 (50 mg kg− 1 body weight) to spontaneously hypertensive rats (SHR) lowered heart rate along with systolic, diastolic and mean arterial blood pressure. The SG-C1 hydrolysate was fractionated using semi-preparative RPHPLC and the fraction with highest overall in vitro bioactivity (fraction 25) was analysed by UPLC-MS/MS. Four peptide sequences (Gly-Gly-Pro-Ala-Gly-Pro-Ala-Val, Gly-Pro-Val-Ala, Pro-Pro and Gly-Phe) and two free amino acids (Arg and Tyr) were identified in this fraction. These peptides and free amino acids had potent ACE and DPP-IV inhibitory, and ORAC activities. The results show that SG hydrolysates have potential as multifunctional food ingredients particularly for the management of hypertension.

1. Introduction Gelatin is a coiled, partially hydrolysed form of collagen which has significant value in the food and pharmaceutical industries due to its versatility as an enhancer of a range of techno- functional properties. Moreover, gelatin is capable of forming highly stable three-dimensional gels (Gudipati, 2013). Gelatin has traditionally been extracted from bovine and porcine sources, specifically from skin and bone co-products of the meat processing industry. The global gelatin market is expected to grow at an annual rate of 8.25% between 2015 and 2022 (MRC, 2016). Currently, there is an increased interest in the utilisation of marine origin gelatin for religious (for halal and kosher food consumers) and disease risk reduction (specifically bovine spongiform encephalopathy) reasons (Gómez-Guillén et al., 2009; Karim & Bhat, 2008). Food-grade gelatin is usually extracted using the so-called ‘pH shift’ process at temperatures in the region of 60 °C (Jaswir, Mirghani, Mohd Salleh, Hassan, & Yaakob, 2009; Montero & Gómez-Guillén, 2000; Zhou & Regenstein, 2005). Being rich in amino acids such as glycine (Gly), proline (Pro) and hydroxyproline (Hyp), gelatin has significant potential as

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a starting substrate for the generation of bioactive peptides (BAPs) for incorporation into functional foods (Grant, Weilbaecher, & Lichlyter, 2007; Ketnawa, Rungraeng, & Rawdkuen, 2017; Schrieber & Gareis, 2007). Fish gelatin hydrolysates/peptides have been shown to display a range of biological activities. These include antioxidant, anti-anaemia, immunoregulatory, Ca-binding, mineral chelating, ACE and DPP-IV inhibitory, antimicrobial and anti-hypertensive activities. Hydrolysates/ peptides having these in vitro bioactivities have been reported for gelatin extracted from Atlantic salmon, cod, herring, hoki, Pacific whiting, pollack, snapper and sole (Harnedy & FitGerald, 2013a). Bioactive peptides with multifunctional activities are of interest in the management of different diseases. Cardiovascular disease (CVD) and type 2 diabetes are major causes of death in developed countries, globally accounting for 17.5 million and 1.5 million deaths, respectively, in 2012 (World Health Organization, 2016). These diseases are often associated with each other. Furthermore, these diseases are linked with enhanced oxidative stress which can result in cell and tissue damage (Harnedy & FitGerald, 2013a). Studies with SHR have reported that gelatin hydrolysates from sea cucumber (Zhao et al., 2007), skate skin (Ngo et al., 2015), jellyfish (Zhuang, Sun, & Li, 2012) and squid

Corresponding author. E-mail address: dick.fi[email protected] (R.J. FitzGerald).

http://dx.doi.org/10.1016/j.foodres.2017.06.065 Received 25 April 2017; Received in revised form 23 June 2017; Accepted 27 June 2017 0963-9969/ © 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Neves, A.C., Food Research International (2017), http://dx.doi.org/10.1016/j.foodres.2017.06.065

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Herisau, Switzerland) adjusted with 0.1 M NaOH or 0.5 M HCl. Samples were taken at 0, 1, 2 and 4 h during hydrolysis. All samples were inactivated by heating at 90 °C for 20 min, cooled to room temperature and then freeze-dried (FreeZone freeze dryer system, Labconco, Missouri, USA).

skin (Lin, Lv, & Li, 2012) have the ability to reduce blood pressure, a controllable risk factor in the development of CVD. However, no studies with SHRs appear to have been performed with salmon gelatin hydrolysates. Therefore, the objectives of this study were (a) to generate enzymatic hydrolysates from gelatin extracted from salmon (Salmo salar) processing co-products (consisting of skin, bone and residual meat), (b) to characterise the physicochemical and in vitro bioactive properties of the resultant hydrolysates, (c) to assess the hypotensive effects of the most potent hydrolysate and (d) to fractionate and identify the peptides within the most bioactive hydrolysate.

2.4. Characterisation of the hydrolysates The degree of hydrolysis (DH) of the SG hydrolysates was determined using the 2, 4, 6 - trinitrobenzene sulfonic acid (TNBS) method following the procedure of Adler-Nissen (1979) as modified by Spellman, McEvoy, O’cuinn and FitzGerald (2003). Hydrolysate samples (0.125 mL) diluted in 1% (w/v) SDS were mixed with a 0.1% (w/v) TNBS solution (1 mL) and 0.2125 M sodium phosphate buffer at pH 8.21 (1 mL). The samples were incubated at 50 °C for 1 h in the dark before 2 mL of 0.1 N HCl was added to terminate the reaction. The samples were allowed to stand in the dark for 30 min and the absorbance was measured at 340 nm (Shimadzu UV mini 1240, Kyoto, Japan). All samples were analysed in triplicate (n = 3) and the amino nitrogen content of the hydrolysates (mg g− 1 protein) was estimated by reference to an L-leucine standard curve in the range of 0 to 28 mg amino N L− 1. The intact gelatin and its associated enzymatic hydrolysates were assessed using reverse-phase high performance liquid chromatography (RP-HPLC) and gel permeation high performance liquid chromatography (GP-HPLC) as described by Neves et al. (2017).

2. Materials and methods 2.1. Materials Bovine lung was kindly provided by Gaelic Meats and Livestock Ltd. (Limerick, Ireland), Abz-Gly-p-nitro-Phe-Pro-OH, Abz-Gly-OH-HCl, HGly-Pro-7-amino-4-methyl coumarin (AMC), and Diprotin A (Ile-ProIle) were obtained from Bachem (Bubendorf, Switzerland). HPLC-grade acetonitrile (ACN) and water were obtained from VWR (Dublin, Ireland). The synthetic peptides Gly-Gly-Pro-Ala-Gly-Pro-Ala-Val, GlyPro-Val-Ala, Pro-Pro and Gly-Phe and two free amino acids Arg and Tyr were obtained from Thermo Fisher Scientific (Ulm, Germany). Trinitrobenzenesulphonic acid (TNBS) reagent was from Medical Supply Co. Ltd. (Dublin, Ireland). Brewer's Clarex™ (An-PEP specific activity: 37 × 10− 3 U/mg) was supplied as a gift from Dutch State Mines (DSM, Heerlen, Netherlands), Corolase PP was provided by AB Enzymes (Darmstadt, Germany), Alcalase 2.4 L and Flavourzyme 500 L were obtained from Sigma Aldrich Ltd. (Gillingham, UK) and Promod 144MG was kindly provided by Biocatalysts Ltd. (Parc Nantgarw, Wales, UK). Captopril™ and all other reagents were obtained from Sigma Aldrich Ltd. (Gillingham, UK).

2.5. Simulated gastrointestinal digestion (SGID) of the hydrolysate The SG hydrolysate generated with Corolase PP for 1 h (SG-C1) was subjected to SGID using the method of Walsh et al. (2004). Briefly, the freeze-dried hydrolysate was suspended in distilled water at 2% (w/v) protein equivalent and was incubated at 37 °C for 30 min. This suspension was initially incubated with pepsin at 37 °C, pH 2.0 at an E:S of 1% (w/w) for 90 min and then with Corolase PP at pH 7.5 at an E:S of 2.5% (w/w) for 150 min. The enzymes were then inactivated by heating at 90 °C for 20 min. The resulting solution was freeze-dried (FreeZone freeze dryer system, Labconco, Missouri, USA) and stored at − 20 °C.

2.2. Sample preparation Representative samples of salmon trimmings (consisting of skin, bone and residual meat) were obtained from the Irish Seafood Producers Group (ISPG, Kilkieran, Connemara, Co. Galway) and the proteins therein were extracted as per Neves, Harnedy, O'Keeffe, and FitzGerald (2017) involving mincing the salmon trimmings, extracting the alkaline and acid soluble proteins and washing the pellets with distilled water. The pellet obtained following protein extraction from the salmon trimmings was used for gelatin extraction. The pellet obtained from 15 g of trimmings was suspended in 20 mL distilled water and the gelatin therein was extracted during heating at 70 °C over 16 h. The suspension was then centrifuged at 4000 × g for 15 min (Hettich Universal 320R, Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany). The supernatant containing gelatin was then freeze-dried (FreeZone freeze dryer system, Labconco, Missouri, USA) and stored at − 20 °C until required for analysis.

2.6. Semi-preparative RP-HPLC The SG-C1 was fractionated using semi-preparative RP-HPLC according to the protocol described by Nongonierma and FitzGerald (2013) with some modifications. The hydrolysate was resuspended at 10% (w/v) in HPLC grade water. Sample (0.5 mL) was injected onto a C18 semi-preparative column (250 × 15 mm I.D., 10 mm particle size, Phenomenex, Cheshire, UK) attached to a C18 guard column (Phenomenex, Cheshire, UK) coupled to an RP-HPLC system (Waters, Dublin, Ireland). Mobile phase A was HPLC grade water and mobile phase B was 80% (v/v) acetonitrile. Peptide separation was carried out at a flow rate of 5 mL min− 1 using the following linear gradient: 0–10 min: 0% B; 10–20 min: 0–20% B; 20–30 min: 20% B; 30–40 min: 20–80% B; 40–50 min: 80% B; 50–60 min: 80–100% B. The absorbance of the eluent was monitored at 214 nm. Fractions (5 mL) were collected from min 6 to min 60 during the course of 14 separate runs. All fractions were evaporated to dryness using a solvent evaporator (GenVac, EZ2plus, Genevac Ltd., Ipswich, UK).

2.3. Generation of gelatin hydrolysates Hydrolysates were generated according to the protocol outlined by Neves et al. (2017). Briefly, a 5% (w/v) gelatin suspension (600 mL) at 96% protein (w/w) was divided into six 100 mL aliquots. Four aliquots were equilibrated at 50 °C, adjusted to pH 7 and the food-grade enzyme preparations Alcalase 2.4 L, Alcalase 2.4 L in combination with Flavourzyme, Corolase PP or Promod 144MG were added at an enzyme:substrate (E:S) ratio of 1% (w/w or v/w depending on the enzyme format). One aliquot was equilibrated at 50 °C, adjusted to pH 4 with 1.0 M HCl and was supplemented with Brewers Clarex at an E:S of 1% (v/w). The remaining aliquot was used as a no enzyme control sample. During hydrolysis at 50 °C the suspensions were gently stirred and maintained at pH 7 or pH 4 using a pH stat (718 stat Titrino, Metrohm,

2.7. Mass spectrometry analysis of fraction 25 of the SG-C1 hydrolysate Fraction 25 from the semi-preparative RP-HPLC separation of the SGC1 hydrolysate was analysed using UPLC-ESI-MS/MS. The peptide sequences therein were determined as described by Neves et al. (2017). In short, samples (2 μL at 0.1 mg mL− 1 in mobile phase A (0.1% formic acid in H2O)) were separated on an Aeris™ 1.7 μm PEPTIDE XB-C18 column (150 × 2.1 mm, Phenomenex, Cheshire, UK) using an ultra-performance 2

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Fig. 1. Reverse-phase high-performance liquid chromatography of the gelatin extract from salmon trimmings and its associated hydrolysates after 4 h incubation with Alcalase 2.4 L, Alcalase 2.4 L in conjunction with Flavourzyme 500 L, Corolase PP, Promod 144MG and Brewer's Clarex.

1.3 Brewer's Clarex

1.1

Promod 144MG

Detector response @ 214nm

0.9

Corolase PP

0.7 Alcalase 2.4L + Flavourzyme 500L

0.5 Alcalase 2.4L

0.3

0.1

Intact gelatin extract

0

10

-0.1

20

30

40

50

60

Time (min) Captopril™ was used as a positive control. The DPP-IV inhibitory activity assay was performed as described by Harnedy and FitGerald (2013b) using human DPP-IV. One unit (U) of DPP-IV activity was the amount of enzyme necessary for hydrolysis of 1 μmol of H-Gly-Pro-AMC min− 1 at 37 °C. The positive control used was Diprotin A™. Experiments were carried out as independent triplicates assayed in triplicate. IC50 values were calculated using GraphPad® Prism 4.0 from sigmoidal dose response plots of inhibitor concentration (μM) versus % inhibition. The values were expressed as the mean IC50 ± standard deviation (n = 3). The ORAC assay was performed as described by Harnedy and FitGerald (2013b) using a micro plate reader (BioTek Instruments Limited, Bedfordshire, UK). The ORAC results were expressed as μmol of Trolox equivalents per gram of freeze-dried powder (μmol TE g− 1 dw). Experiments were carried out as independent triplicates assayed in triplicate.

liquid chromatography system (UPLC, Ultimate 3000, Thermo Fisher, MA, USA) and analysed using an Impact HD UHR-Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). The MS/MS protocol specifically targeted short peptides (auto MS/MS scans were between 50 and 600 m/z) as previously described (O'Keeffe & FitzGerald, 2015). Peptide sequence identification was carried out by de novo sequencing using PEAKS Studio software, (version 7.0, Bioinformatics Solutions Inc., Waterloo, Canada), Data Analysis (version 4.0, Bruker Daltonics) and Biotools (version 3.2, Bruker Daltonics) software. 2.8. Biological activity assessment ACE inhibitory activity was determined using a fluorometric microtitre assay as described by Sentandreu and Toldrá (2006) and Norris, Casey, FitzGerald, Shields, and Mooney (2012) with an enzyme extract from bovine lung containing 8 mU mL− 1 activity. One unit of ACE activity (U) was defined as the amount of enzyme capable of hydrolysing 1 μmol of Abz-Gly-Phe-(NO2)-Pro per min at 37 °C. The ACE used for the assay was extracted from fresh bovine lung by a modification of the method of Meng, Balcells, Dell'Italia, Durand, and Oparil (1995) as described by Kleekayai et al. (2015). Experiments were carried out as independent triplicates assayed in triplicate. IC50 values (i.e., the concentration of inhibitor inhibiting 50% of the total ACE activity) were calculated using GraphPad® Prism 4.0 from sigmoidal dose response plots of inhibitor concentration (μM) versus % inhibition. The values were expressed as the mean IC50 ± standard deviation (n = 3).

2.9. Animal model and experimental design Twelve 6-week old male spontaneously hypertensive rats (SHR) were purchased from Charles River (Montreal, PQ, Canada) and housed under a 12 h day and night cycle at 21 °C with regular chow feed and tap water provided ad libitum. Animal experiments were carried out following the Canadian Council on Animal Care Ethics guidelines with a protocol approved by the University of Manitoba Fort Garry Campus Animal Care Committee. For each experiment, there were 4 rats per 3

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group: phosphate buffered saline (PBS), Captopril™ (20 mg kg− 1 body weight dissolved in PBS), and gelatin or gelatin hydrolysate (50 mg kg− 1 body weight dissolved in PBS). Each rat was orally gavaged with a 1 mL solution using a disposable plastic syringe; blood pressure was then recorded continuously in free moving rats for 24 h by telemetry. The surgical implantation of the telemetry sensors as well as the recording and signal processing was performed as described by O'Keeffe, Norris, Alashi, Aluko, and FitzGerald (2017).

4 h incubation period (Table 1). As previously mentioned, distinct differences in the peptide profiles of hydrolysates generated with different enzymes were evident (Fig. 1). However, minimal differences were seen in the RP-HPLC profiles for the hydrolysates generated with a given enzyme preparation following incubation for 1, 2 or 4 h (data not shown). Moreover, with each of the different enzymatic preparations there was an increase in low molecular mass peptides (< 500 Da and 500–1000 Da) with a corresponding decrease in peptides having a larger molecular mass (> 1000 Da) shown in the GP-HPLC profiles (Fig. 2). However, although it was possible to observe this decrease of molecular mass as the time of hydrolysis increased, it was minimal after the first hour (Fig. 2). This may be indicative that most of the hydrolysis took place within the first hour of incubation.

2.10. Statistical analysis The results were analysed by one-way analysis of variance (ANOVA) at a significance level equivalent to p ≤ 0.05 and, where applicable, ttest and Tukey's multiple comparison test were performed. The software employed for statistical analysis was Graphpad Prism, version 4.00 for Windows (Graphpad software, San Diego, California, USA).

3.2. Biological activities A number of studies exist in the literature describing the biological activities associated with fish gelatin hydrolysates and their associated peptides. To date, hydrolysates and peptides derived from salmon, cod, hoki, pollack, snapper and sole gelatin have been reported to exhibit ACE and DPP-IV inhibitory, antioxidant and iron-chelating activities (Byun & Kim, 2001; Giménez, Alemán, Montero, & Gómez-Guillén, 2009; Gu, Li, Liu, Yi, & Cai, 2011; Guo et al., 2013; Khantaphant & Benjakul, 2008; Li-Chan et al., 2012; Mendis, Rajapakse, & Kim, 2005; Ngo, Wijesekara, Vo, Van Ta, & Kim, 2011; Phanturat, Benjakul, Visessanguan, & Roytrakul, 2010). The ACE inhibitory properties of the salmon gelatin hydrolysates generated herein with Alcalase 2.4 L, Promod 144MG and Brewer's Clarex (with IC50 values ranging from 0.32 ± 0.11 to 1.16 ± 0.09 mg mL− 1) were comparable to those reported for gelatin hydrolysates from pollock hydrolysed with Pronase E and Flavourzyme, and salmon hydrolysed with Alcalase for 3 h which had IC50 values of 0.49 and 1.17 ± 0.09 mg mL− 1, respectively (Gu et al., 2011; Park, Kang, & Kim, 2009). More potent ACE inhibitory hydrolysates were generated herein following 4 h incubation with Corolase PP and Alcalase 2.4 L in combination with Flavourzyme 500 L with IC50 values of 0.13 ± 0.05 and 0.28 ± 0.13 mg mL− 1, respectively (Table 1). Previous studies used single enzyme preparations such as Alcalase, Flavourzyme, Neutrase and Pronase to hydrolyse pollock derived gelatin (Park et al., 2009) while Alcalase and papain were used to hydrolyse salmon-derived gelatin (Gu et al., 2011). The results herein suggest that the use of a combination of enzymes for hydrolysis increased the possibility of generating potent ACE inhibitory peptides. Moreover, the GP-HPLC results show that the majority of the peptides generated with Alcalase 2.4 L, Alcalase 2.4 L in combination with Flavourzyme 500 L and Corolase PP had molecular masses < 1000 Da (Fig. 2). These hydrolysates

3. Results and discussion 3.1. Characterisation of protein hydrolysates The food-grade proteolytic enzyme preparations used for the generation of peptides from the gelatin extracted from salmon trimmings were: Alcalase 2.4 L (from Bacillus licheniformis), a preparation of 1:1 Alcalase 2.4 L and Flavourzyme 500 L (from Aspergillus oryzae), Corolase PP (derived from porcine pancreas), Promod 144MG (from Carica papaya) and Brewer's Clarex (derived from Aspergillus niger). The broad specificities of the chosen enzymes allowed different peptide preparations to be formed as shown in Fig. 1 with the different RPHPLC profiles obtained for each enzyme preparation. The DH of the hydrolysates is shown in Table 1. Hydrolysates generated with Brewer's Clarex and Alcalase 2.4 L had the lowest DH (16.3 and 24.3%, respectively). While there are no direct comparisons with the hydrolysis of salmon gelatin, Khantaphant and Benjakul (2008) reported DH values < 20% during the hydrolysis of gelatin from brownstripe red snapper (Lutjanus vitta) skin during incubation with endogenous proteolytic activity. The hydrolysates generated with Promod 144MG, Alcalase 2.4 L plus Flavourzyme 500 L and Corolase PP gave DH values of 46.9, 52.8 and 52.8%, respectively. Hydrolysis of salmon skin gelatin with Bromelain and Flavourzyme was reported to yield DH values of 41.0 and 45.2%, respectively (Li-Chan, Hunag, Jao, Ho, & Hsu, 2012). The TNBS analysis showed that hydrolysis occurred throughout the

Table 1 Degree of hydrolysis (DH), angiotensin-converting enzyme (ACE) inhibitory, dipeptidyl peptidase (DPP-IV) inhibitory and oxygen radical absorbance capacity (ORAC) activities of salmon (Salmo salar) trimmings gelatin and associated hydrolysates after 1, 2 and 4 h incubation with different enzyme preparations. Sample

Duration of hydrolysis (h)

Degree of hydrolysis (%)

ACE IC50 (mg mL− 1)

DPP-IV IC50 (mg mL− 1)

ORAC (μmol TE g− 1)

Extracted gelatin Alcalase 2.4 L

0 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4

0 14.81 ± 0.15a 23.16 ± 0.30b 24.33 ± 0.41b 42.03 ± 0.34a 51.29 ± 0.19b 52.82 ± 0.23b 48.27 ± 0.61a 53.10 ± 0.21b 52.82 ± 0.51b 36.77 ± 0.12a 43.99 ± 0.16b 46.86 ± 0.23b 7.01 ± 0.09a 15.68 ± 0.04b 16.28 ± 0.19b

2.07 0.39 0.41 0.36 0.22 0.23 0.28 0.19 0.21 0.13 0.32 0.47 1.12 0.91 1.12 1.16

1.67 0.16 0.18 0.15 0.08 0.08 0.10 0.08 0.08 0.08 0.09 0.18 0.12 0.49 0.62 0.71

114.95 223.75 197.84 174.11 295.29 324.24 234.24 540.94 502.27 387.05 504.27 367.65 358.84 103.00 137.47 198.38

Alcalase 2.4 L + Flavourzyme 500 L

Corolase PP

Promod 144MG

Brewer's Clarex

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.08 0.10a 0.07a 0.07a 0.09a 0.09a 0.13a 0.05a 0.06a 0.05a 0.11a 0.15a 0.10b 0.05a 0.17a 0.09a

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.12 0.02a 0.02a 0.01a 0.01a 0.00a 0.01a 0.01a 0.01a 0.02a 0.01a 0.02a 0.01a 0.07a 0.08a 0.09a

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

8.05 20.73a 20.69a 6.62a 10.97a 44.56a 19.36a 9.57a 34.14a 29.61a 30.53a 26.77a 32.95a 9.02a 20.60a 25.62a

Values represent mean ± SD (n = 3). IC50: inhibitor concentration that inhibits enzyme activity by 50%. For each enzyme and each test/activity different superscript letters are significantly different (p > 0.05). TE: Trolox equivalents.

4

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Fig. 2. Molecular mass distribution profiles of the salmon gelatin and its associated hydrolysates after 1, 2 and 4 h incubation with different enzyme preparations.

1 h with Corolase PP was selected for further analysis. In the first instance this hydrolysate, SG-C1, was subjected to SGID in order to investigate the potential fate of the peptides therein when subjected to hydrolysis with gastrointestinal enzymes. All in vitro bioactivities were maintained when compared with SG-C1 (Table 1) not only following the gastric phase (ACE IC50: 0.19 ± 0.01 mg mL− 1, DPP-IV IC50: 0.07 ± 0.02 mg mL− 1 and ORAC activity: 613.13 ± 10.47 μmol TE g− 1) but also following the intestinal phase of the SGID process (ACE IC50: 0.14 ± 0.02 mg mL− 1, DPP-IV IC50: 0.09 ± 0.02 mg mL− 1 and ORAC activity: 618.46 ± 8.46 μmol TE g− 1). This would indicate that the peptides associated with the observed bioactivity may survive gastrointestinal digestion and that the cardioprotective activity seen in vitro may translate to in vivo.

also displayed the most potent ACE inhibitory activity (Table 1). This is in agreement with previous studies showing that lower molecular mass peptides are associated with higher ACE inhibitory activity (Neves et al., 2017; Suetsuna & Nakano, 2000; Van Platerink, Janssen, & Haverkamp, 2008). The DPP-IV inhibitory activity of the SG hydrolysates generated with Alcalase 2.4 L, Alcalase 2.4 L in combination with Flavourzyme 500 L, Corolase PP and Promod 144MG had IC50 values ranging from 0.08 ± 0.00 to 0.18 ± 0.02 mg mL− 1 (Table 1). Li-Chan et al. (2012) reported IC50 values > 5 mg mL− 1 for salmon skin hydrolysates generated with Alcalase, Bromelain and Flavourzyme. The differences may be due to the differences in starting material as well as differences in the extraction conditions. The DPP-IV inhibitory activity of the hydrolysates generated herein were among the most potent reported to date from a range of marine sources, i.e., 0.14 to > 5 mg mL− 1 (Harnedy & FitGerald, 2013b; Li-Chan et al., 2012; Pascual et al., 2007). Brewer's Clarex hydrolysis of SG led to less potent hydrolysates, with DPP-IV IC50 values ranging from 0.49 ± 0.07 to 0.71 ± 0.09 mg mL− 1 (Table 1). These results indicate that enzyme specificity plays a key role in the release of potent DPP-IV inhibitory peptides. The antioxidant activities of the SG hydrolysates were evaluated using the ORAC assay. The activities ranged from 103.00 ± 9.02 μmol TE g− 1 when hydrolysed with Brewer's Clarex for 1 h to 540.94 ± 9.57 μmol TE g− 1 when hydrolysed with Corolase PP for 1 h (Table 1). Generally, the hydrolysates generated with Corolase PP and Promod 144MG showed the highest antioxidant activity (Table 1). These activities were comparable with papaya latex hydrolysates of shark and seabass skin gelatin (158.92 to 709.42 μmol TE g− 1) as reported by Kittiphattanabawon, Benjakul, Visessanguan, and Shahidi (2012) and Sae-Leaw, O'Callaghan, Benjakul, and O’ Brien, N.M. (2016). Overall, all the SG hydrolysates had higher in vitro bioactivities when compared with intact SG while the SG hydrolysates generated with Corolase PP had the most potent ACE and DPP-IV inhibitory, and antioxidant activities. Furthermore, the values for these in vitro bioactivities in the 1 h hydrolysate were not significantly different (p > 0.05) in comparison to the 2 and 4 h hydrolysates (Table 1). Therefore, due to potential cost savings associated with a shorter hydrolysis time, in an industrial context, the hydrolysate generated after

3.3. In vivo study with SHR The hypotensive effects of SG and the SG-C1 hydrolysate were assessed using SHR. When administrated to SHR by oral gavage the SG and the SG-C1 samples exhibited good antihypertensive properties (Fig. 3) which is consistent with the in vitro results for ACE inhibition. The administration of SG and SG-C1 to SHR gave significant reductions in all the parameters measured, i.e., mean arterial blood pressure (MAP), systolic (SBP) and diastolic blood pressure (DBP) and heart rate (HR). The MAP of SHR was decreased for 24 h after administration of SG and SG-C1. However, the maximum decrease (− 20.50 mm Hg), was observed 8 h after administration of SG-C1 (Fig. 3A). This value is less than that obtained for other marine derived gelatin hydrolysates such as skate skin (Ngo et al., 2015) or squid skin (Lin et al., 2012) which gave MAP decreases of − 94.2 and − 65 mm Hg, respectively. However, these differences may be due to the nature of these marine gelatins and the enzymes used for their hydrolysis. In some cases the dosages of protein/protein hydrolysate given to the SHR were higher than in the study herein which may also explain the higher decreases in MAP. Although the control, Captopril™, has a stronger effect in decreasing SBP compared to SG and SG-C1, its activity began to decrease following the first hour of ingestion, while both SG and SG-C1 reached their 5

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A

B

C

D

Fig. 3. Mean arterial pressure (MAP, A), changes in systolic blood pressure (SBP, B) and in diastolic blood pressure (DBP, C) and heart rate (HR, D), of spontaneously hypertensive rats administered intact salmon (Salmo salar) trimmings gelatin and gelatin hydrolysed with Corolase PP when administered at a dose of 50 mg protein/kg body weight to spontaneously hypertensive rats. Captopril™ (10 mg kg− 1 body weight) was used as a control. Each bar represents the means ± SD (n = 4).

For instance, 2 h after ingestion of SG-C1, the SHR had an HR decrease of − 76.95 ± 10.32 bpm. Following administration of the hydrolysed salmon gelatin there was a decrease in HR up to 6 h. The reduction was minimal at 8 and 12 h but increased again 24 h following administration. This could be explained by further degradation of key peptides and/or the rate of absorption of active peptides. The fact that intact SG also reduced HR indicates that peptides released during gastrointestinal digestion in the SHR may be capable of reducing HR. To the best of our knowledge this is the first time that the telemetric approach has been used to study the influence of marine derived hydrolysates in SHR measuring HR, changes in SBP and DBP, and also MAP. Furthermore, the results herein appear to be the first demonstrating a hypotensive effect of salmon gelatin hydrolysates.

maximum SBP decrease 8 h after ingestion (− 22.07 ± 23.73 and − 22.16 ± 4.34 mm Hg, respectively, Fig. 3B). When compared with the effect of other marine derived gelatin hydrolysates such as sea cucumber (Zhao et al., 2007), skate skin (Ngo et al., 2015) and jellyfish (Zhuang et al., 2012), the SG-C1 hydrolysate led to a lower decrease in SBP. This may again be related to differences in the gelatin sequence and the enzymes used during the hydrolysis process as well as the dosage administrated. However, the SG-C1 decreased SBP to a greater extent compared with squid skin gelatin hydrolysates (− 10.0 mm Hg) when the same quantity of hydrolysate was administrated (Lin et al., 2012). Interestingly, the DBP of SHR decreased to a greater extent when intact SG was administrated in comparison to SG-C1. This decrease reached a maximum 12 h after ingestion of the SG achieving a decrease in DBP of − 39.25 ± 9.95 mm Hg (Fig. 3C). The DBP decrease found in the present study was comparable to the decrease found when jellyfish collagen (Zhuang et al., 2012) and squid skin gelatin hydrolysates (Lin et al., 2012) were administrated to SHR. This decrease in DBP was between −18.0 and −52.6 mm Hg even when the gelatin hydrolysates have been administrated at a high dose (up to 200 mg/kg/day). Interestingly, the improvement in HR was particularly noticeable not only with the hydrolysates but also intact gelatin displayed a higher decrease on HR when compared to Captopril™ administration (Fig. 3D).

3.4. Semi-preparative RP-HPLC In order to identify the peptides potentially responsible for the bioactivities observed in vitro and the hypotensive effects in vivo, the SG-C1 hydrolysate was fractionated using semi-preparative RP-HPLC. Fig. 4 shows that most of the peptides therein eluted between 6 and 47 min with very low levels of peptides eluting between 20 and 23 and 29–30 min. Following solvent removal, the peptide fractions were screened for ACE and DPP-IV inhibitory, and ORAC activities (Fig. 5). 6

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4

100

90

3.5

70 2.5

60 50

2

40

1.5

ACN (%)

Detector response @214 nm

80 3

30 1 20 0.5

10 0

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Retention time (min) Fig. 4. Semi-preparative reverse-phase HPLC profile of salmon (Salmo salar) trimmings gelatin hydrolysed with Corolase PP for 1 h. Samples were collected every min from 6 to 59 min.

activities but had no benefit for enhancement of DPP-IV inhibitory activity. This may be explained by a potential loss of synergistic effects between hydrolysate peptides following fractionation. Synergistic effects between peptides and other components in hydrolysate samples have been reported to affect the antioxidant activity of soybean hydrolysates (Chen, Muramoto, Yamauchi, & Nokihara, 1996). Other studies following fractionation of salmon gelatin hydrolysates reported an improvement in bioactivities such as DPP-IV (Gu et al., 2011) and ACE (Li-Chan et al., 2012) inhibition, and ORAC activity (Wu et al., 2017).

The ACE and DPP-IV IC50 values were subsequently determined for those fractions showing high activity when tested at 0.5 mg mL− 1. The most potent SG-C1 fractions for ACE inhibitory activity were F25 to F27 and F36 to F37 with ACE IC50 values ranging from 0.18 to 0.06 mg mL− 1 (Fig. 5A). The most potent DPP-IV inhibitory fractions were F25 and F31 to F35 with DPP-IV IC50 values ranging from 0.47 to 0.43 mg mL− 1 (Fig. 5B). Furthermore, F25 had the highest ORAC activity: 578.33 μmol TE g− 1 (Fig. 5C). These results show that fractionation of the samples improved their ACE inhibitory and antioxidant

ACE inhibition (%)

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40 30 20 10 0

DPP-IV inhibition (%)

100

(B)

80 60 40 20 0

umol TE g-1 fraction

700

(C)

600 500 400 300 200 100 0 6

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 7

Fig. 5. Bioactivities of semi-preparative reversephase HPLC fractions of salmon (Salmo salar) trimmings gelatin hydrolysed with Corolase PP for 1 h tested at 0.5 mg mL− 1. (A) angiotensinconverting enzyme (ACE) inhibitory activity, (B) dipeptidyl peptidase (DPP-IV) inhibitory activity, and (C) oxygen radical capacity absorbance assay (ORAC). Values represent mean ± SD (n = 3). For ACE and DPP-IV inhibition, IC50 values are presented for those fractions showing highest activity when tested at 0.5 mg mL− 1.

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4. Conclusion

Table 2 Angiotensin-converting enzyme (ACE) inhibitory, dipeptidyl peptidase IV (DPP-IV) inhibitory, and oxygen radical absorbance capacity (ORAC) activities of peptides or free amino acids identified in semi-preparative RP-HPLC fraction 25 of Atlantic salmon (Salmo salar) trimmings gelatin incubated with Corolase PP for 1 h. Amino acids represented using the one letter code. Peptide/ amino acid

ACE IC50 (μM)

DPP-IV IC50 (μM)

ORAC (μmol TE μmol peptide− 1/amino acid− 1)

PP GF GPVA GGPAGPAV R Y

1912.46 ± 63.15a 178.14 ± 24.51 445.61 ± 6.94 673.16 ± 15.03 98.04 ± 0.15 132.84 ± 0.52a

4343.48 ± 29.78a 1547.15 ± 34.15 264.74 ± 1.59 8139.11 ± 134.68 110.44 ± 0.47 75.15 ± 0.84a

12.48 ± 1.47a 19.74 ± 1.01 9.48 ± 0.94 5.47 ± 0.94 4.71 ± 0.09 2.74 ± 0.65a

Incubation of SG with different enzyme preparations resulted in hydrolysates with different DHs. These hydrolysates showed more potent ACE and DPP-IV inhibitory, and ORAC activities than intact SG. Overall, the SG-C1 hydrolysate had the highest multifunctional activity (ACE IC50 = 0.13 ± 0.05 mg mL− 1; DPP-IV IC50 = 0.08 ± 0.01 mg mL− 1; and ORAC = 540.94 ± 9.57 μmol of TE g− 1 d.w.). Moreover, the DPPIV inhibitory activity of this hydrolysate was among the most potent reported in the literature for marine derived gelatin hydrolysates. The SG-C1 hydrolysate retained its bioactivities after SGID. Furthermore, both SG and SG-C1 improved SBP, DBP and MAP in SHR when compared with a saline solution and decreased HR in SHR to an extent equivalent to that of the currently used synthetic drug, Captopril™. When SG-C1 was fractioned using semi-preparative RP-HPLC, four peptides and two free amino acids were identified in a fraction with potent ACE and DPP-IV inhibitory and antioxidant activity. The confirmatory studies with synthetic peptides/amino acids showed that Tyr and Arg had the highest activities overall with some of the lowest reported IC50 values for ACE and DPP-IV inhibition. This study shows that SG may have potential as a starting material for the generation of biofunctional food ingredients for the management of hypertension. However, human studies are required to validate these observations.

TE: Trolox equivalents. Values represent mean ± SD (n = 3). IC50: inhibitor concentration that inhibits enzyme activity by 50%. a Bioactivity values reported in Neves et al. (2017).

Therefore, given that F25 showed the highest antioxidant activity and was among the most potent ACE and DPP-IV inhibitory fractions, this fraction was subjected to UPLC-MS/MS analysis in order to identify the peptides therein.

Acknowledgments 3.5. Peptide identification The Marine Functional Foods Research Initiative (NutraMara project) is a programme for marine-based functional food development. This project (Grant-Aid Agreement No. MFFRI/07/01) was carried out under the Sea Change Strategy with the support of the Marine Institute and the Irish Department of Agriculture, Food and the Marine, funded under the National Development Plan 2007–2013. Pádraigín A. Harnedy and Adriana C. Neves were funded under the Sea Change Strategy with the support of the Marine Institute and the Department of Agriculture, Food and the Marine, funded under the National Development Plan 2007–2013 (Grant-Aid Agreement No. MFFRI/07/ 01) and under the National Development Plan, through the Food Institutional Research Measure, administered by the Department of Agriculture, Food and the Marine, Ireland under grant issue 13/F/467. Martina B. O'Keeffe was funded under the National Development Plan, through the Food Institutional Research Measure, administered by the Irish Department of Agriculture, Food and the Marine, Ireland under grant issues 11/F/063, 11/F/064, 13/F/467, 13/F/536 and 14/F/873. The research was also part funded by the Science Foundation Ireland Infrastructure Fund and the Higher Education Authority under the Programme for Research in Third Level Institutions (cycle 4) as part of the National Development Plan (Ireland) 2007–2013.

MS/MS analysis showed that F25 from SG-C1contained 4 peptides (Pro-Pro, Gly-Phe, Gly-Pro-Val-Ala and Gly-Gly-Pro-Ala-Gly-Pro-AlaVal) and 2 free amino acids (Arg and Tyr, Table 2). The in vitro bioactivities of these compounds were tested and are reported in Table 2. Pro-Pro and Gly-Phe had ACE IC50 values of 1912.46 ± 63.15 and 178.14 ± 24.51 μM, respectively. These values are similar to those reported by Suetsuna and Nakano (2000) and Van Platerink et al. (2008, ACE IC50: 2284.7 and 277.9 μM, respectively) for the same peptides. Previous studies have linked peptides containing C-terminal Pro and Phe residues with potent ACE inhibitory activity (Ruiz, Recio, & Belloque, 2004; Norris & FitzGerald, 2013). ACE IC50 values do not appear to have been reported to date for Gly-Pro-Val-Ala and GlyGly-Pro-Ala-Gly-Pro-Ala-Val. Interestingly, the free amino acid Arg had the lowest ACE IC50 value (98.04 ± 0.15 μM) of the components identified in F25 SG-C1. These results show that F25 has potent ACE inhibitory peptides that may be responsible for the high potency of F25 in vitro. Moreover, the presence of these peptides in SG-C1 may potentially explain the in vivo results. The most potent DPP-IV inhibitory peptide in F25 SG-C1 was Gly-Pro-Val-Ala with an IC50 of 264.74 ± 1.59 μM. To our knowledge this was the first time that GlyPro-Val-Ala was identified as having a high DPP-IV inhibitory activity. The DPP-IV inhibitory potency of Pro-Pro was similar to that previously reported by Hatanaka et al. (2012). This peptide has been reported as a DPP-IV inhibitor with an IC50 of 5860 μM and 4343.48 ± 29.78 μM (Hatanaka et al., 2012, Neves et al., 2017, respectively). Yan, Ho, and Hou (1992) reported a correlation between peptides containing Pro and high DPP-IV inhibitory activity. It was shown herein that Arg and Tyr had low DPP-IV IC50 values (110.44 ± 0.47 and 75.15 ± 0.84 μM, respectively). Moreover, the peptides found in F25 also had high ORAC values (Table 2). Gly-Phe showed the highest value (19.7 ± 1.01 μmol TE μmol of peptide− 1). Of the components identified in F25, only Tyr has previously been reported as having antioxidant activity with an ORAC value of 1.57 μmol TE μmol of amino acid− 1 (HernándezLedesma, Amigo, Recio, & Bartolomé, 2007). In the present study this amino acid had an ORAC activity of 2.74 ± 0.65 μmol TE μmol amino acid− 1. The difference may be associated with the use of amino acids coming from different suppliers. The ORAC values for ProePro and Tyr have been previously reported by Neves et al. (2017) as they were also present in hydrolysates arising from a salmon trimming protein extract.

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