Towards generation of bioactive peptides from meat industry waste proteins: Generation of peptides using commercial microbial proteases

Food Chemistry 208 (2016) 42–50

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Towards generation of bioactive peptides from meat industry waste proteins: Generation of peptides using commercial microbial proteases Kate Ryder a,⇑, Alaa El-Din Bekhit b, Michelle McConnell c, Alan Carne a a

Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand Department of Food Science, University of Otago, Dunedin, New Zealand c Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand b

a r t i c l e

i n f o

Article history: Received 25 January 2016 Received in revised form 30 March 2016 Accepted 30 March 2016 Available online 1 April 2016 Chemical compounds studied in this article: Hippuric acid (PubChem CID: 464) Hippuryl-L-histidyl-L-leucine (PubChem CID 94418) Trolox (PubChem CID: 40634) Captopril (PubChem CID: 44093) Fluorescein (PubChem CID: 16850) 2,20 -Azobis(2-amidinopropane) dihydrochloride (AAPH) (PubChem CID: 1969)

a b s t r a c t Five commercially available food-grade microbial protease preparations were evaluated for their ability to hydrolyse meat myofibrillar and connective tissue protein extracts to produce bioactive peptides. A bacterial-derived protease (HT) extensively hydrolysed both meat protein extracts, producing peptide hydrolysates with significant in vitro antioxidant and ACE inhibitor activities. The hydrolysates retained bioactivity after simulated gastrointestinal hydrolysis challenge. Gel permeation chromatography sub-fractionation of the crude protein hydrolysates showed that the smaller peptide fractions exhibited the highest antioxidant and ACE inhibitor activities. OFFGEL electrophoresis of the small peptides of both hydrolysates showed that low isoelectric point peptides had antioxidant activity; however, no consistent relationship was observed between isoelectric point and ACE inhibition. Cell-based assays indicated that the hydrolysates present no significant cytotoxicity towards Vero cells. The results indicate that HT protease hydrolysis of meat myofibrillar and connective tissue protein extracts produces bioactive peptides that are non-cytotoxic, should be stable in the gastrointestinal tract and may contain novel bioactive peptide sequences. Ó 2016 Elsevier Ltd. All rights reserved.

Keywords: Bovine Meat hydrolysates Bioactive peptides Antioxidant ACE inhibitor

1. Introduction Traditionally, protein in the diet has been considered mainly as a source of essential amino acids for cellular maintenance, growth and energy. More recently, dietary proteins have been recognised additionally for their bioactive properties once hydrolysed by proteases in the gastrointestinal tract following consumption. Bioactive peptides are defined as being short amino acid sequences that possess one or more biologically significant activities when taken up into the body (Fitzgerald, Murray, & Walsh, 2004; Lafarga & Hayes, 2014). These peptides usually range from two to 30 amino acids in length (Clare & Swaisgood, 2000; Di Bernardini et al., 2011). Their small size makes the peptide sequences less ⇑ Corresponding author. E-mail addresses: [email protected] (K. Ryder), aladin.bekhit@ otago.ac.nz (A.E.-D. Bekhit), [email protected] (M. McConnell), alan. [email protected] (A. Carne). http://dx.doi.org/10.1016/j.foodchem.2016.03.121 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.

prone to further degradation in the gut and increases their bioavailability, particularly when higher numbers of hydrophobic residues are present in the peptide sequence (Segura-Campos, Chel-Guerrero, Betancur-Ancona, & Hernandez-Escalante, 2011). These peptide sequences do not display any bioactivity when contained in the intact parent protein sequence and so first must be excised, generally by hydrolysis catalysed by the incubation with one or more proteases, by fermentation, or by a combination of both (Korhonen & Pihlanto, 2006). The uptake of these peptides contributes to health promotion in addition to nutrition. Protein-containing waste materials are becoming increasingly attractive for the production of bioactive peptides. This contributes to utilisation and an increase in value of the waste stream (Harnedy & FitzGerald, 2012). Within the meat industry, considerable waste material is produced from the slaughter of animals for human consumption, such as the organs, limbs, bones, meat trimmings, blood and fatty tissues. This can present an array of both environmental and economic problems and ways to dispose

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of waste material or repurpose it cleanly and efficiently are increasingly being sought (Lafarga & Hayes, 2014). Whilst to date there has been a number of studies focusing on the production of bioactive peptides from milk, gelatin and plant material, there are only limited studies exploring the production of bioactive peptides from bovine meat proteins using exogenous proteases (De Gobba, Tompa, & Otte, 2014; Korhonen & Pihlanto, 2006; Nagpal et al., 2011; Singh, Vij, & Hati, 2014). Angiotensin I-converting enzyme inhibitory peptides are one class of bioactive peptides that have been commonly found originating from hydrolysis of skeletal muscle proteins and connective tissues, from animals such as chicken and pig (Arihara, Nakashima, Mukaai, Ishikawa, & Itoh, 2001; Atsuta, Himizu, Amada, & Ishimura, 2003; Gomez-Guillen, Gimenez, Lopez-Caballero, & Montero, 2011). Angiotensin I-converting enzyme (ACE) (EC 3.4.15.1) is a membrane-bound, heavily glycosylated, zinc metalloprotease whose role is to catalyse the proteolysis of the 10 amino acid peptide angiotensin I to the eight amino acid peptide angiotensin II, a potent vasoconstrictor, and to degrade bradykinin, a vasodilator (Bernstein et al., 2013). Due to the vasoconstriction caused by the production of angiotensin II and the loss of the vasodilation effect of bradykinin, ACE has been implicated as playing a central role in hypertension (Gobbetti, Minervini, & Rizzello, 2004), a major risk factor for the development of cardiovascular disease. It has been suggested that ACE inhibitor peptides from natural product sources may impart less side effects compared to their synthetic drug counterparts such as captopril (Kim & Wijesekara, 2010). Studies have indicated that peptides which possess ACE inhibitor activity may also act as antioxidants, due to shared requirements for both activities in terms of the length and structure of the peptide (De Gobba et al., 2014; Hernández-Ledesma, Miralles, Amigo, Ramos, & Recio, 2005). Reactive oxygen species (ROS) are chemical substances that are independent in existence and, at the subatomic level, contain unpaired electrons in their outer shell (Di Bernardini et al., 2011). When maintained in homeostasis, endogenous enzymatic antioxidants, such as glutathione peroxidase and superoxide dismutase, and non-enzymatic antioxidants, such as vitamin C, act as radical scavengers, reducing the effect of ROS on the cells (Lafarga & Hayes, 2014). When this balance is no longer maintained by endogenous antioxidants, an excessive build-up of ROS can send a cell into oxidative stress. On average, antioxidant peptides are reported to be larger than ACE inhibitory peptides, typically 5–15 amino acids in length (Clare & Swaisgood, 2000; Korhonen & Pihlanto, 2006). In a previous report we evaluated the hydrolysis capability of several commercial protease preparations for application in meat tenderising (Ryder, Ha, Bekhit, & Carne, 2015). It was demonstrated that of the proteases evaluated, HT, a Bacillus-derived protease, was the least suitable for application in meat tenderisation, due to its excessive hydrolysis of meat myofibrillar and connective tissue proteins. However, it was recognised that the extensive protein hydrolysis capability of HT could have application in the generation of bioactive peptides from meat industry waste and may produce novel peptides, due to differences in hydrolytic specificity compared to gastrointestinal proteases. Therefore, the objective of the present study was to evaluate the potential use of peptides derived from bovine myofibrillar and connective tissue in the production of antioxidant and ACE-inhibitory bioactive peptides. 2. Materials and methods 2.1. Materials All chemicals used were of analytical reagent grade or higher. All protease preparations were supplied as powders by Enzyme Solutions Pty. Ltd. (Croydon South, Victoria, Australia).

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2.2. Bovine Musculus semimembranosus meat myofibril extraction preparation Meat myofibrils were extracted from bovine M. semimembranosus from cull dairy cattle using the protocol described by Goll, Young, and Stromer (1974). Extracted myofibrils were then resuspended in 70 mM phosphate buffer (pH 6.0). Aliquots were stored at 20 °C until use. 2.3. Connective tissue collagen protein extraction from bovine Achilles tendon Collagen was extracted from Achilles tendon obtained from cull dairy cattle, according to the procedure described by Davis and Mackle (1981). The collagen protein extract was diluted 1 in 10 with 70 mM phosphate buffer (pH 6.0) and the pH was readjusted to 6.0 using 1 M KOH as required. Aliquots were stored at 4 °C for short periods of time until use. 2.4. Hydrolysis of meat myofibrillar and connective tissue extracts Aliquots (1 mL) of meat myofibrillar extract were incubated with 40 lL of either acidic fungal protease (AFP; 20 mg mL1), fungal protease II (FPII; 4 mg mL1), fungal protease 31,000 (F31K; 10 mg mL1), Fungal protease 60,000 (F60K 4 mg mL1) or HT proteolytic (HT; 2 mg mL1) protease preparations. Meat connective tissue hydrolysis was conducted under the same conditions but with the concentrations of AFP, FPII, F31K, F60K and HT protease preparations being 100, 30, 20, 10 and 4 mg mL–1, respectively. Samples were incubated for 24 h at 45 °C for initial investigation into the extent of hydrolysis and were tumbled continuously. For subsequent investigation into bioactive peptide production, the incubation was increased to 48 h, in order to ensure maximal degradation of the meat myofibrillar and connective tissue proteins. The concentration of each protease used was based on a 20-fold increase, compared to the preliminary two-hour time course experiments previously reported (Ryder et al., 2015), as the objective was to achieve extensive hydrolysis of the meat protein material. 2.5. Simulated gastrointestinal hydrolysis challenge Gastrointestinal hydrolysis of the meat myofibrillar and connective tissue hydrolysates was simulated based on the method by Hernández-Ledesma, Quirós, Amigo, and Recio (2007). The protein concentration of the hydrolysates was estimated using a NanoDrop 2000 (Thermo Scientific) based on the absorbance at 215 nm. The pH of the hydrolysates was lowered to 3.0 using 1 M HCl; the hydrolysates were then incubated at 37 °C for 30 min with pepsin (1750 U mg1 protein). The pH was then raised to 7.0 using 1 M NaHCO3 and incubated for a further hour with pancreatin (1750 U mg1 protein). Further hydrolysis was stopped by heating to 95 °C for ten minutes and products were then stored immediately at 20 °C until use. 2.6. 1D-SDS–PAGE analysis of meat myofibrillar and connective tissue hydrolysates Meat myofibrillar and connective tissue hydrolysates were displayed by one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS–PAGE) using Bolt gradient (4–12%) Bis-Tris gels (Life Technologies (Thermo Fisher), #BG0122BOX). An aliquot of each protein hydrolysate was added to an appropriate volume of Bolt LDS sample buffer (Life Technologies, #B0007) and Bolt sample reducing agent (Life Technologies, #B0004) and incubated at 90 °C for five minutes prior to being loaded on to the

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gel. Protein standards (Novex Sharp Pre-stained Protein Standard; Life Technologies, #LC5800) were included on the gel as a molecular marker. Electrophoresis was conducted at room temperature in 1  Bolt MES SDS running buffer (Life Technologies, #B0002) at 164 V for 34 min. Following electrophoresis, the gel was washed three times for five minutes each in Milli-Q water and stained in SimplyBlueTM SafeStain solution (#LC6060; Invitrogen, Carlsbad, CA) overnight. The gel was destained in Milli-Q water and an image was obtained with a Canon CanoScan LiDE 600F scanner. 2.7. Gel permeation Superdex pg30 chromatography of peptide hydrolysates Peptide hydrolysates were size fractionated on a Superdex 30 pg column (2.6  60 cm) equilibrated in Milli-Q water on an AKTA Purifier FPLC system. Chromatography was conducted at 2 mL min1 using Milli-Q water, collecting 4-mL fractions. The column effluent was monitored at 215 and 280 nm. Fractions were then pooled to generate a nominally 15–30 amino acid fraction and a less than 15 amino acid peptide pool fraction, which were concentrated by freeze drying and re-dissolving in 2 mL Milli-Q water. 2.8. Desalting of gel permeation peptide hydrolysate pools A C18 Sep-Pak cartridge (Waters Corp., Milford, MA) was used to desalt a 1-mL aliquot of the Superdex 30 pg pooled peptide fractions prior to sub fractionation, according to the manufacturer’s instructions. The sample (1 mL) was loaded onto a cartridge equilibrated in 0.1% trifluoroacetic acid (TFA) and then washed with 10 mL 0.1% TFA. . The bound material was then eluted with 5 mL acetonitrile and dried in a Speed Vac (Savant, France) before being resuspended in 3 mL Milli-Q water. 2.9. OFFGEL isoelectric focusing of peptide pools Desalted samples were further fractionated based on their isoelectric point (pI) using an OFFGEL isoelectric focusing system. Milli-Q water (350 lL) and 50% (v/v) glycerol (350 lL) was added to 3.0 mL of desalted peptide sample. A pH 3–10 L IPG Immobiline IEF strip (GE Healthcare) was set up with a 24-well hopper strip in an OFFGEL tray and rehydrated with the addition of 40 lL 4.8% (v/v) glycerol for 15 min. Aliquots (150 lL) of the desalted peptide sample were added to each of the 24 wells, wicks were applied to the ends of the IPG strip and mineral oil was added to prevent evaporation prior to electrophoresis. Electrophoresis was conducted according to the manufacturer’s instructions in the absence in of ampholine with a 50 lA current limit and a maximum of 4500 V until 100 kV h had been accumulated. 2.10. Antioxidant assay of bovine meat myofibrillar and connective tissue protein hydrolysates A fluorescence-based oxygen-radical absorbance capacity (ORAC-FL) assay was used to assess the antioxidant capacity of the meat myofibrillar and connective tissue hydrolysates, as reported by Dávalos, Gómez-Cordovés, and Bartolomé (2004). Potential antioxidant-containing samples (20 lL) were incubated at 37 °C in the presence of a free radical generator (AAPH, 60 lL) and their protective activity towards fluorescein (120 lL) was measured based on the decay of fluorescent signal over 80 min. Samples were diluted as required in 75 mM Na2HPO4 buffer, pH 7.0. The results are presented as antioxidant capacity referenced to Trolox, a vitamin E analogue that has an established antioxidant activity (Forrest, Kang, McClain, Robinson, & Ramakrishnan, 1994).

2.11. Angiotensin I-converting enzyme inhibitor assay of bovine meat myofibrillar and connective tissue hydrolysates The ability of the meat myofibrillar and meat connective tissue hydrolysates to inhibit ACE was measured using a microtitre plate protocol reported by Jimsheena and Gowda (2009). No dilutions were used for the measurement of ACE inhibition by each of the peptide hydrolysates. The amount of hippuric acid (HA) produced in the absence of any potential inhibitor was taken to represent 100% ACE activity or 0% ACE inhibition and the amount of HA produced in the presence of a peptide-containing hydrolysate was compared to this, to give a percentage of ACE inhibition obtained. Captopril (10 nM) was used as a positive control. 2.12. Cytotoxicity of meat myofibrillar and connective tissue hydrolysates The effect of meat myofibrillar and connective tissue extract hydrolysate fractions on mammalian cell viability was assessed according to the method of Saotome, Morita, and Umeda (1989) with some modifications. Vero cells were chosen due to their quick growth and susceptibility to a wide range of external challenges, making them suitable for cytotoxicity assays. All cell culture was conducted in Dulbecco’s Modified Eagle Medium, High Glucose (DMEM) containing 10% (v/v) foetal calf serum (FCS), 1% (w/v) penicillin and 1% (w/v) streptomycin. Vero cells were seeded in 96-well plates at a volume of 100 lL per well with approximately 3.0  105 cells mL1. Following 48 h of initial incubation at 37 °C in 5% CO2, the DMEM media was replaced with an equal volume and cells were treated with 50 lL of meat myofibrillar or connective tissue hydrolysates, or one of the subsequent fractionated pools. The plates were then incubated for a further 48 h under the same conditions. Following the second incubation the wells were washed twice with 100 lL PBS each time. The cells contained in each well were then fixed by incubating in 100 lL of 100% methanol for one minute and stained for five minutes in 100 lL of 1% (w/v) crystal violet. Excess stain was removed by washing in Milli-Q water before the cells were rehydrated in 100 lL of 33% (v/v) acetic acid. In order to calculate the percentage of viable cells present following exposure to protein hydrolysate material, the optical density at 595 nm was measured for each well using a microtitre plate reader and the following calculation was used:

% uninfected cells ¼ 100 

  ODc  ODt  100 ODc

where ODc is equal to the average OD of the six media-only control wells and ODt is equal to the average OD of the six test wells at the dilution used. 2.13. Statistical analysis All the analyses were carried out in triplicate and mean values were used for the statistical analysis. The reported values are mean ± standard deviation. The data were subjected to one-way analysis of variance (ANOVA), followed by Tukey’s test to determine the significant differences between samples at p < 0.05 using Minitab statistical software (Version 16; Minitab Inc., State College, PA). 3. Results and discussion 3.1. Hydrolysis of meat myofibrillar and connective tissue extracts In a previous study it was shown that of the four commercially available microbial-derived protease preparations evaluated, the

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bacterial-derived HT protease was the most effective in hydrolysing meat myofibrillar and connective tissue extracts under the conditions tested (Ryder et al., 2015). In order to ascertain if HT was the most effective protease preparation for the generation of small peptides that may potentially yield bioactive properties, meat myofibrillar and connective tissue extracts were incubated with each of the four protease preparations previously studied (AFP, FPII, F60K and HT) as well as an additional Aspergillus oryzae derived fungal protease preparation (F31K) for 24 h. Qualitative analysis with 1D-SDS–PAGE showed HT to be the only protease preparation capable of completely hydrolysing meat myofibrillar extract proteins over this time course (Fig. 1A). For the connective tissue protein extract, both FPII and HT showed extensive hydrolysis of the substrate (Fig. 1B). In order to determine if any of these hydrolysates resulted in a potential increase in bioactive properties, the hydrolysates were screened using the ORAC assay to obtain an indication of their antioxidant capacity. This assay was selected for initial screening of hydrolysates, as it provides a low cost, high throughput method to obtain an indication of potential bioactivity. The results of the ORAC assay are presented as Trolox equivalents. The greatest increase in antioxidant activity for meat myofibrillar hydrolysates was seen with HT (Fig. 1C). Hydrolysis of the connective tissue extract with FPII showed no significant increase in antioxidant activity based on the ORAC assay compared to the starting material; however hydrolysis with HT showed a signifi-

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cant increase in Trolox equivalence of 45 mmol L1 after 24 h (Fig. 1D). F60K hydrolysis of connective tissue extract did not appear to hydrolyse particularly well, based on 1D-SDS–PAGE (Fig. 1B) but did exhibit substantial antioxidant activity. The bioactive properties of peptides generated by hydrolysis with HT were further investigated. This was due to the extensive hydrolysis and significant increase in antioxidant activity of both the myofibrillar and connective tissue protein extracts after 24 h. 3.2. Antioxidant properties of meat myofibrillar and connective tissue hydrolysates As it is established in the literature that both antioxidant and ACE inhibitor effects of peptides are associated with short amino acid sequences (Clare & Swaisgood, 2000; Di Bernardini et al., 2011), the crude hydrolysates were first fractionated using gel permeation Superdex 30 pg chromatography, in order to generate a small peptide fraction of nominally less than 15 amino acids. The 30 pg column size fractionates polypeptides below 10 kDa and hence is suitable for initial size fractionation of the peptide hydrolysates to obtain peptide pooled fractions of nominally less than 15 amino acids and between 15 and 30 amino acids in length. Furthermore, peptides with significant antioxidant activity characteristically contain hydrophobic amino acid residues. Histidine, cysteine and methionine are commonly found within antioxidant sequences (Yea et al., 2014). Histidine and cysteine are able to

Fig. 1. Time course hydrolysis of meat myofibrillar and connective tissue protein extracts with protease preparations. A: 1D-SDS–PAGE analysis of meat myofibrillar extract hydrolysates. B: 1D-SDS–PAGE analysis of meat connective tissue extract hydrolysates. Aliquots of meat protein extract were incubated at 45 °C for 4, 8 or 24 h with one of either AFP, FPII, F31K, F60K or HT protease preparations. C: Increase in Trolox equivalence of meat myofibrillar hydrolysates from each protease preparation compared to the unhydrolysed starting material. D: Increase in Trolox equivalence of meat connective tissue hydrolysates from each protease preparation compared to the unhydrolysed starting material. Error bars indicate the mean (n = 3) ± 1 SD. Different letters above bars within a set indicate that the mean is statistically significant as determined by ANOVA with post hoc Tukey’s test.

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directly interact with the reactive oxygen species (ROS) through their respective imidazole and thiol side-chains whilst hydrophobic and methionine residues have the ability to donate protons to the negatively-charged ROS (Sarmadi & Ismail, 2010). The myofibrillar extract gel permeation chromatography (Fig. 2A) showed that a major proportion of the A215 nm and most of the A280 nm absorbing peptide material eluted from the column in the lower mass range, consistent with what was observed on 1D-SDS–PAGE (Fig. 1A). Gel permeation chromatography of the connective tissue hydrolysate (Fig. 2B) showed that although there was a significant amount of small peptide material, there were also a lot of peptides up to 10 kDa, which likely reflects the unique characteristics of the collagen amino acid sequence. The gel permeation chromatography was consistent with the 1D-SDS–PAGE analysis (Fig. 1B). Every third fraction from gel permeation chromatography was screened for antioxidant activity. This allowed for an initial screening of the fractions to determine the area of high antioxidant activity for pooling of fractions. The antioxidant activity of both the size-fractionated meat myofibrillar (Fig. 2A) and connective tissue (Fig. 2B) hydrolysate was found to be highest in the later eluting fractions, correlating with the presence of smaller peptide sequences. The A280 nm trace of the connective tissue extract was found to be fivefold smaller than that of the myofibrillar extract, despite the A215 nm traces of both the hydrolysed extracts being of similar magnitude. This is likely due to the low proportion of aromatic amino acid residues present in collagen, which is comprised predominantly of Gly-Ala-Pro repeats (Kar et al., 2009). For both the meat myofibrillar (Fig. 2C) and connective tissue (Fig. 2D) hydrolysates, the small peptide pool corresponding to peptides of nominally less than 15 amino acids showed significantly more antioxidant activity than the large peptide pools. This is in agreement with numerous studies which have shown antioxidant activity to be associated with peptides between 5 and 16 amino acids in length (Clare & Swaisgood, 2000; Korhonen &

Pihlanto, 2006; Sarmadi & Ismail, 2010). However the antioxidant activity in the connective tissue small peptide pool was significantly lower compared to the total hydrolysate. The meat myofibrillar and connective tissue small peptide pools underwent a gastrointestinal hydrolysis challenge using pepsin and pancreatin to determine if there was any change in activity following digestion by gastrointestinal proteases (Fig. 2). The further hydrolysis by gastrointestinal proteases resulted in no significant decrease in antioxidant activity (p > 0.05) for both small peptide pools. The pools created following gel permeation chromatography were expected to contain a complex mixture of peptide sequences. OFFGEL electrophoresis was employed to sub-fractionate the small peptides further based on their isoelectric point (pI), to reduce the complexity of the sample and to provide information about bioactivity in relation to pI. It has been shown previously that a relationship may exist between antioxidant activity and the pI of a peptide. In a study by Eun, Morimae, Matsumura, Nakamura, and Sato (2008), an isoelectric focusing (IEF) system was used to fractionate gluten and soy protein hydrolysates. The authors showed that the highest antioxidant activity was in fractions containing peptides with a pI of 2–5 for the gluten hydrolysates and a pI of 3–6 for the soy protein hydrolysates. This indicated that acidic peptides have a higher level of antioxidant activity compared to their basic and neutral counterparts. The authors hypothesised that the relationship between pI and antioxidant activity was due to low pI amino acid residues having the ability to become proton donors with the ability to neutralise the charge on reactive oxygen species (ROS). It was shown that for the meat myofibrillar extract most of the antioxidant activity was located in fractions 6–10 based on the ORAC assay, with fractions 7–9 displaying significant activity above 12 mmol L1 TE (Fig. 3A). As the 24 OFFGEL fractions covered a linear pH (pI) range of 3–10, this suggested that peptides with an acidic pI had the highest antioxidant activity, in line with

Fig. 2. Antioxidant activity of size-fractionated bovine meat myofibrillar and connective tissue protein extract HT hydrolysates and simulated gastrointestinal hydrolysis challenge. A: 30 pg gel permeation chromatography of meat myofibrillar protein extract HT hydrolysate. B: 30 pg gel permeation chromatography of meat connective tissue protein extract HT hydrolysate. Dashed line indicates A215 nm of column effluent; dotted line indicates A280 nm of column effluent; bars indicate the Trolox equivalence (TE) of every third fraction as measured using the ORAC assay; vertical lines indicate fractions combined to form pools of peptides nominally between 15 and 30 amino acids (large peptides) and less than 15 amino acids (small peptides), termed the ‘large’ and ‘small’ peptide pools respectively. C: Antioxidant capacity of myofibrillar protein extract, unhydrolysed starting material, myofibrillar protein extract hydrolysed with HT protease for 24 h and peptide pools generated from gel permeation chromatography. D: Antioxidant capacity of connective tissue protein extract unhydrolysed starting material, connective tissue protein extract hydrolysed with HT protease and peptide pools generated from gel permeation chromatography. Error bars indicate the mean (n = 3) ± 1 SD. Statistical significance between the starting and hydrolysed material is indicated above the relevant bars (⁄⁄ = p 6 0.01, ⁄⁄⁄ = p 6 0.001).

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the results previously reported by Eun et al. (2008). Subsequent fractions in the basic pH range showed lower activity between 2.1 and 3.7 mmol L1 TE. Sub-fractionation of the meat connective tissue hydrolysate small peptide pool showed elevated antioxidant activity around fractions 7–10 (Fig. 3B), in a similar location as the antioxidant activity observed for the meat myofibrillar hydrolysate OFFGEL sub fractionation; however, the activity was lower (5.1 mmol L1 compared to 16.5 mmol L1). Overall the results showed that extensive hydrolysis of bovine meat myofibrillar and connective tissue protein extracts with the bacterial protease preparation HT produces peptides with antioxidant activity and that this activity is associated with the shorter peptide sequences. Whilst there seems to be some correlation between low pI and antioxidant activity, the trend appeared to be inconsistent for the two protein hydrolysates examined. 3.3. Angiotensin I-converting enzyme inhibitor assay of bovine meat myofibrillar and connective tissue protein hydrolysates Potent ACE inhibitor peptides are generally short di- or tripeptides in order to insert themselves and bind to the buried active site of ACE; however, sequences up to 14 amino acids have been reported to possess ACE inhibitory activity (Ferreira et al., 1998). It has been reported that rats consuming a diet containing 5% hydrolysed meat had significantly lower levels of angiotensin II after two weeks, compared to rats receiving only intact meat (Ahhmed & Muguruma, 2010). ACE inhibitors normally contain hydrophobic or large aromatic peptides at their ultimate and penultimate positions, with proline being the most favoured residue in both of these positions (Gobbetti et al., 2004; Wu, Aluko, & Nakai, 2006). Synthetic inhibitors of ACE such as captopril are widely used to treat patients with hypertension. Captopril has been demonstrated to be a non-competitive and highly specific inhibitor of ACE that has been used in the treatment of hypertension since 1975 (Antonaccio, Rubin, & Horovitz, 1980). The negative side effects reported by some patients surrounding the use of captopril and other synthetic ACE inhibitors have prompted the development and a search for other treatment options (Pitt et al., 2000). In this study, 10 nM captopril was found to inhibit 82% of ACE activity compared to the uninhibited negative control. Whilst it is difficult to compare this percentage of ACE inhibition with other studies, due to differences in concentrations of captopril and sources of ACE that were used, the IC50 of captopril towards somatic ACE reported in the literature ranges from 0.75 to 22 nM (Cushman & Ondetti, 1999; Vermeirssen, Van Camp, & Verstraete, 2002; Williams, Yamamoto, Walsh, & Allsop, 1993). In this study, the crude HT-hydrolysed myofibrillar extract was determined to inhibit 69% of ACE activity (Fig. 4A) whilst the

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HT-hydrolysed connective tissue extract showed 72% inhibition (Fig. 4B). These hydrolysates were again fractionated by gel permeation chromatography and pooled as previously described (Fig. 2A and B). This allowed for isolation of the smaller (nominally less than 15 amino acids) and potentially more bioactive peptides from the complex protein hydrolysate. The pool of myofibrillar hydrolysate small peptides was shown to exhibit most of the ACE inhibitory activity (89.0 ± 2.9% inhibition) (Fig. 4A). The ACE inhibition observed with the larger peptide pool was determined to be negligible (7.1 ± 1.2%). The pool of connective tissue hydrolysate small peptides obtained from gel permeation chromatography was also found to be the better inhibitor of ACE activity (90.9 ± 1.0%) compared to the large peptide counterpart (9.7 ± 1.1%) (Fig. 2B). As these hydrolysate samples are crude hydrolysates containing a complex mixture of peptides and the peptide concentration was not known, it was not possible to compare the level of ACE inhibition activity obtained by the two starting materials; however, the data presented are consistent with the literature that suggests that peptide sequence length plays an important role in the ACE-inhibition ability of a peptide (Gobbetti et al., 2004; Sarmadi & Ismail, 2010; Wu et al., 2006). As the majority of ACE activity was found to be in the small peptide pool for both hydrolysates, this fraction became the focus of further analysis. Only fractions pooled from the 30 pg gel permeation were screened for ACE inhibitory activity, due to the cost of performing this assay. In addition to the size of the peptide, the potency of ACE inhibitors is reported to be dictated by the three C-terminal residues, with aromatic residues being particularly favourable (Gobbetti et al., 2004). For both the meat myofibrillar and connective tissue hydrolysates the majority of the aromatic amino acids were found in the small peptide pool based on the A280 nm monitoring of the Superdex 30 pg gel permeation column effluent (Fig. 2A and B). The small peptide pools from the myofibrillar and connective tissue extracts were subjected to a further gastrointestinal challenge (Fig. 4). Following hydrolysis with pepsin and pancreatin to simulate gastrointestinal digestion, there was no significant change in ACE inhibition from the small peptides of myofibrillar origin (p > 0.05). Simulated gastrointestinal digest of the small peptides of connective tissue origin resulted in a significant reduction of ACE inhibition (p < 0.05), although the extent of ACE inhibition still remained high at 84.9 ± 8.9%. There is little published in the literature that presents a definitive link between ACE inhibitors and the isoelectric point of the peptide. A recent study, however, used a protein extract from winged bean (Psophocarpus tetragonolobus) seeds as a protein source and showed that peptides from both the acidic (pH 4.16– 5.89) and basic regions (pH 9.38–10.0) had the greatest ACE inhibitor activity, whilst neutral pI peptides were not very active (Yea et al., 2014).

Fig. 3. Antioxidant activity of OFFGEL isoelectric focusing fractions from meat myofibrillar and connective tissue protein extract HT hydrolysate small peptide pools. A: ORAC activity of meat myofibrillar small peptide OFFGEL electrophoresis fractions. B: ORAC activity of meat connective tissue small peptide OFFGEL electrophoresis fractions. Fraction 1 contains fractions with a pI of nominally 3 whilst fraction 24 contains fractions with a pI of nominally 10. Error bars indicate the mean (n = 3) ± 1 SD.

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Fig. 4. ACE inhibition by bovine meat myofibrillar and connective tissue protein extract HT hydrolysates. A: ACE inhibition of meat myofibrillar protein extract pools hydrolysed with HT as generated by gel permeation chromatography as compared to captopril. B: ACE inhibition of meat connective tissue extract pools hydrolysed with HT as generated by gel permeation chromatography as compared to captopril. C: ACE inhibition of OFFGEL electrophoresis pooled fractions from the small peptide meat myofibrillar pool. D: ACE inhibition of OFFGEL electrophoresis pooled fractions from the small peptide meat connective tissue pool. Pools of OFFGEL electrophoresis fractions were created by combining four consecutive fractions to generate six pools. Error bars indicate the mean (n = 3) ± 1 SD.

OFFGEL sub-fractionation of the meat myofibrillar hydrolysate small peptide pool produced 24 fractions from which six pools containing four consecutive fractions each were constructed. Following sub-fractionation of the small myofibrillar peptides, the fourth OFFGEL pool (fractions 13–16) was found to result in the greatest percentage of ACE inhibition (48.7 ± 9.0%) compared to the remaining pools (Fig. 4C). OFFGEL pools 1 and 6 corresponding to the acidic and basic pI extremes, respectively, were the least effective at inhibiting ACE, with the acidic pool producing minimal inhibition of ACE (1.7 ± 0.8%). The fractions that were produced by OFFGEL sub-fractionation of the connective tissue small peptide pool showed elevated ACE inhibition in the acidic pool (53.6 ± 0.9% for pool 1) and again in the basic pool (15.6 ± 10.0% for pool 5) (Fig. 4D). However, elevated ACE inhibition was also observed in pool 3 (48.1 ± 6.3%), in partial agreement with Yea et al. (2014). However the result presented here for the subfractionated myofibrillar peptides was the inverse. Yea et al. (2014) first separated the hydrolysates they generated using reverse-phase high-performance liquid chromatography (RPHPLC) prior to sub-fractionation using OFFGEL electrophoresis, in addition to using a different protein source. Two RP-HPLC fractions which eluted early in the acetonitrile gradient were selected for sub-fractionation, eliminating highly hydrophobic peptide sequences from the mixture. The prior elimination of significantly hydrophobic peptides would have resulted in fractions containing a different mixture of peptides following electrophoresis and therefore likely produce different activity profiles compared to that reported here. 3.4. Cytotoxicity of meat myofibrillar and connective tissue hydrolysates A potential concern surrounding the use of bioactive molecules such as peptides is the possibility that they may prove to be cytotoxic. The meat myofibrillar and connective tissue hydrolysates

were therefore evaluated for cytotoxicity. A peptide designed to act as an antioxidant or ACE inhibitor should not be cytotoxic, in order to avoid producing unwanted cell death towards normal cells. However, it has been proposed that a peptide displaying cytotoxic effects towards selected cells may still find a use, for example, in the treatment of cancer where cell death is the target outcome (Korhonen & Pihlanto, 2006). A study by Jang, Jo, Kang, and Lee (2008) investigated the cytotoxic effects of four synthetic peptides originally identified from beef sarcoplasmic protein hydrolysates. All of these peptides had previously been shown to have significant ACE inhibitor capability. One of the peptides, GLSDGEWQ, was shown to decrease the viability of the stomach cancer cell line AGS by 80%. Conversely, the peptide DFHINQ had the opposite effect, increasing viability of the same cell line, leading to the suggestion that it was acting as a nutrient for the cell. From this, Jang et al. (2008) concluded that there was the consideration that potential nutritional effects may also have negative effects towards cell viability. The Vero cells are an immortalised mammalian cell line originally derived from kidney epithelial cells of the African green monkey (Chlorocebus sabaeus). Vero cells grow quickly and have been shown to be susceptible to a wide range of external challenges, including viral and bacterial toxins, making them suitable for assay of cytotoxicity, in addition to the cells being amenable for laboratory experimental work (Konowalchuk, Speirs, & Stavric, 1977; Miyamura, Tajiri, Ito, Murata, & Kono, 1974). All peptide-containing hydrolysates from both meat myofibrillar and connective tissue extracts showed no significant reduction in cell viability following incubation at 37 °C for 48 h (Fig. 5). The assays were all conducted using undiluted peptide-containing hydrolysates. As all of the pooled OFFGEL fractions tested from both the protein extracts showed high cell viability it was deemed unnecessary to further investigate the individual fractions. This result indicated that as complex mixtures none of the samples appear to cause significant cytotoxicity.

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Fig. 5. Cytotoxicity of meat myofibrillar and connective tissue hydrolysate fractions towards Vero cells. A. Cell viability following incubation with meat myofibrillar extract, small peptide gel permeation pool and OFFGEL electrophoresis pooled fractions compared to media only control. B: Cell viability following incubation with meat connective tissue extract, small peptide gel permeation pool and OFFGEL electrophoresis pooled fractions compared to media-only control. OFFGEL electrophoresis pools were created by combining six consecutive fractions to generate four pools. Error bars indicate the mean (n = 3) ± 1 SD.

4. Conclusions HT, derived from Bacillus, was shown to be the most proficient protease preparation for the hydrolysis of meat myofibrillar and connective tissue extracts, when compared to four commercially available fungal-derived protease preparations. The extensive hydrolysis of the protein substrates achieved by HT indicated that the protease preparation may be suitable for the generation of bioactive peptides and have potential for production of novel bioactive peptides, due to the different hydrolytic specificity of HT compared to proteases in the gastrointestinal tract. It was shown that the majority of the antioxidant and ACE inhibitor activity was associated with this pool of smaller peptides that were generated by the hydrolysis of both meat myofibrillar and connective tissue. The resultant fractions showed that the majority of the antioxidant activity was associated with acidic pI peptides from both meat protein extracts whilst the hydrolysates showed a more variable profile of ACE inhibition. Further separation methods would need to be employed to reduce the complexity of the fractions further, in order to identify the peptide sequences responsible for the observed bioactivity. Finally, none of the peptide fractions were shown to reduce cell viability below 90% in cytotoxicity assays. The results presented here demonstrate the potential for the generation of peptide hydrolysates containing significant bioactivity from bovine meat myofibrillar and connective tissue using the HT protease preparation. This may be seen as having a potential application in the production of health-promoting products. Conflict of interest The authors have no conflicts of interest to disclose. Acknowledgements K. Ryder acknowledges the receipt of a University of Otago Masters Research Scholarship and a University of Otago Masters Publishing Bursary. We thank Enzyme Solutions Pty. Ltd. (Croydon South, Victoria, Australia 3136) for the supply of the protease preparations used. References Ahhmed, A. M., & Muguruma, M. (2010). A review of meat protein hydrolysates and hypertension. Meat Science, 86(1), 110–118. http://dx.doi.org/10.1016/j. meatsci.2010.04.032. Antonaccio, M. J., Rubin, B., & Horovitz, Z. P. (1980). Effects of captopril in animal models of hypertension. Clinical and Experimental Hypertension, 2, 613–637. Arihara, K., Nakashima, Y., Mukaai, T., Ishikawa, S., & Itoh, M. (2001). Peptide inhibitors for angiotensin I-converting enzyme from enzymatic hydrolysates. Meat Science, 57, 319–324.

Atsuta, S. H. K., Himizu, T. S. S., Amada, R. Y. Y., & Ishimura, T. O. N. (2003). Angiotensin I-converting enzyme inhibitory peptides in a hydrolyzed chicken breast muscle extract. Blood Pressure, 18(18), 1741–1745. Bernstein, K. E., Ong, F. S., Blackwell, W.-L. B., Shah, K. H., Giani, J. F., GonzalezVillalobos, R., ... Touyz, R. M. (2013). A modern understanding of the traditional and nontraditional biological functions of angiotensin-converting enzyme. Pharmacological Reviews, 65(1), 1–46. http://dx.doi.org/10.1124/pr.112.006809. Clare, D. A., & Swaisgood, H. E. (2000). Bioactive milk peptides: A prospectus. Journal of Dairy Science, 83(6), 1187–1195. http://dx.doi.org/10.3168/jds.S0022-0302 (00)74983-6. Cushman, D. W., & Ondetti, M. A. (1999). Design of angiotensin converting enzyme inhibitors. Nature, 5(10), 1110–1112. Dávalos, A., Gómez-Cordovés, C., & Bartolomé, B. (2004). Extending applicability of the oxygen radical absorbance capacity (ORACfluorescein) assay. Journal of Agricultural and Food Chemistry, 52(52), 48–54. Davis, P. F., & Mackle, Z. M. (1981). A simple procedure for the separation of insoluble collagen and elastin. Analytical Biochemistry, 115(1), 11–17. http://dx. doi.org/10.1016/0003-2697(81)90514-5. De Gobba, C., Tompa, G., & Otte, J. (2014). Bioactive peptides from caseins released by cold active proteolytic enzymes from Arsukibacterium ikkense. Food Chemistry, 165, 205–215. http://dx.doi.org/10.1016/j.foodchem.2014.05.082. Di Bernardini, R., Harnedy, P., Bolton, D., Kerry, J., O’Neill, E., Mullen, A. M., & Hayes, M. (2011). Antioxidant and antimicrobial peptidic hydrolysates from muscle protein sources and by-products. Food Chemistry, 124(4), 1296–1307. http://dx. doi.org/10.1016/j.foodchem.2010.07.004. Eun, Y. P., Morimae, M., Matsumura, Y., Nakamura, Y., & Sato, K. (2008). Antioxidant activity of some protein hydrolysates and their fractions with different isoelectric points. Journal of Agricultural and Food Chemistry, 56(19), 9246–9251. http://dx.doi.org/10.1021/jf801836u. Ferreira, L. A. F., Galle, A., Raida, M., Schrader, M., Lebrun, I., & Habermehl, G. (1998). Analysis and properties of three bradykinin-potentiating peptides (BPP-II, BPPIII, and BPP-V) From Bothrops neuwiedi venom. Journal of Protein Chemistry, 17 (3), 285–289. Fitzgerald, R. J., Murray, B. A., & Walsh, D. J. (2004). The emerging role of dairy proteins and bioactive peptides in nutrition and health. The Journal of Nutrition, 134, 980–988. Forrest, V. J., Kang, Y., McClain, D. E., Robinson, D. H., & Ramakrishnan, N. (1994). Oxidative stress-induced apoptosis prevented by trolox. Free Radical Biology and Medicine, 16(6), 675–684. Gobbetti, M., Minervini, F., & Rizzello, C. G. (2004). Angiotensin I-convertingenzyme-inhibitory and antimicrobial bioactive peptides. International Journal of Dairy Technology, 57(2–3), 173–188. http://dx.doi.org/10.1111/j.14710307.2004.00139.x. Goll, D. E., Young, R. B., & Stromer, M. H. (1974). Separation of subcellular organelles by differential and density gradient centrifugation. In Proceedings of the 27th Annual Reciprocal Meat Conference, Chicago, IL (pp. 250–254). Gomez-Guillen, M. C., Gimenez, B., Lopez-Caballero, M. E., & Montero, M. P. (2011). Functional and bioactive properties of collagen and gelatin from alternative sources: A review. Food Hydrocolloids, 25(8), 1813–1827. http://dx.doi.org/ 10.1016/j.foodhyd.2011.02.007. Harnedy, P. A., & FitzGerald, R. J. (2012). Bioactive peptides from marine processing waste and shellfish: A review. Journal of Functional Foods, 4(1), 6–24. http://dx. doi.org/10.1016/j.jff.2011.09.001. Hernández-Ledesma, B., Miralles, B., Amigo, L., Ramos, M., & Recio, I. (2005). Identification of antioxidant and ACE-inhibitory peptides in fermented milk. Journal of the Science of Food and Agriculture, 85(6), 1041–1048. http://dx.doi. org/10.1002/jsfa.2063. Hernández-Ledesma, B., Quirós, A., Amigo, L., & Recio, I. (2007). Identification of bioactive peptides after digestion of human milk and infant formula with pepsin and pancreatin. International Dairy Journal, 17(1), 42–49. http://dx.doi. org/10.1016/j.idairyj.2005.12.012. Jang, A., Jo, C., Kang, K.-S., & Lee, M. (2008). Antimicrobial and human cancer cell cytotoxic effect of synthetic angiotensin-converting enzyme (ACE) inhibitory peptides. Food Chemistry, 107(1), 327–336. http://dx.doi.org/10.1016/ j.foodchem.2007.08.036.

50

K. Ryder et al. / Food Chemistry 208 (2016) 42–50

Jimsheena, V. K., & Gowda, L. R. (2009). Colorimetric high-throughput assay for screening angiotensin I-converting enzyme inhibitors. Analytical Chemistry, 81 (22), 9388–9394. http://dx.doi.org/10.1021/ac901775h. Kar, K., Ibrar, S., Nanda, V., Getz, T. M., Kunapuli, S. P., & Brodsky, B. (2009). Aromatic interactions promote self-association of collagen triple-helical peptides to higher-order structures. Biochemistry, 48(33), 7959–7968. http://dx.doi.org/ 10.1021/bi900496m. Kim, S. K., & Wijesekara, I. (2010). Development and biological activities of marinederived bioactive peptides: A review. Journal of Functional Foods, 2(1), 1–9. http://dx.doi.org/10.1016/j.jff.2010.01.003. Konowalchuk, J., Speirs, J. I., & Stavric, S. (1977). Vero response to a cytotoxin of Escherichia coli. Infection and Immunity, 18(3), 775–779. Korhonen, H., & Pihlanto, A. (2006). Bioactive peptides: Production and functionality. International Dairy Journal, 16(9), 945–960. http://dx.doi.org/ 10.1016/j.idairyj.2005.10.012. Lafarga, T., & Hayes, M. (2014). Bioactive peptides from meat muscle and byproducts: Generation, functionality and application as functional ingredients. Meat Science, 98(2), 227–239. http://dx.doi.org/10.1016/j.meatsci.2014.05.036. Miyamura, K., Tajiri, E., Ito, A., Murata, R., & Kono, R. (1974). Micro cell culture method for determination of diphtheria toxin and antitoxin titres using VERO cells. II. Comparison with the rabbit skin method and practical application for seroepidemiological studies. Journal of Biological Standardization, 2(3), 203–209. Nagpal, R., Behare, P., Rana, R., Kumar, A., Kumar, M., Arora, S., ... Yadav, H. (2011). Bioactive peptides derived from milk proteins and their health beneficial potentials: An update. Food & Function, 2(1), 18–27. http://dx.doi.org/10.1039/ c0fo00016g. Pitt, B., Poole-Wilson, P. A., Segal, R., Martinez, F. A., Dickstein, K., Camm, A. J., ... Thiyagarajan, B. (2000). Effect of losartan compared with captopril on mortality in patients with symptomatic heart failure: Randomised trial–the Losartan Heart Failure Survival Study ELITE II. Lancet, 355(9215), 1582–1587. http://dx. doi.org/10.1016/S0140-6736(00)02213-3. Ryder, K., Ha, M., Bekhit, A. E.-D., & Carne, A. (2015). Characterisation of novel fungal and bacterial protease preparations and evaluation of their ability to hydrolyse

meat myofibrillar and connective tissue proteins. Food Chemistry, 172, 197–206. http://dx.doi.org/10.1016/j.foodchem.2014.09.061. Saotome, K., Morita, H., & Umeda, M. (1989). Cytotoxicity test with simplified crystal violet staining method using microtitre plates and its application to injection drugs. Toxicology in Vitro, 3(4), 317–321. http://dx.doi.org/10.1016/ 0887-2333(89)90039-8. Sarmadi, B. H., & Ismail, A. (2010). Antioxidative peptides from food proteins: A review. Peptides, 31(10), 1949–1956. http://dx.doi.org/10.1016/j.peptides.2010. 06.020. Segura-Campos, M., Chel-Guerrero, L., Betancur-Ancona, D., & Hernandez-Escalante, V. M. (2011). Bioavailability of bioactive peptides. Food Reviews International, 27 (3), 213–226. http://dx.doi.org/10.1080/87559129.2011.563395. Singh, B. P., Vij, S., & Hati, S. (2014). Functional significance of bioactive peptides derived from soybean. Peptides, 54, 171–179. http://dx.doi.org/10.1016/j. peptides.2014.01.022. Vermeirssen, V., Van Camp, J., & Verstraete, W. (2002). Optimisation and validation of an angiotensin-converting enzyme inhibition assay for the screening of bioactive peptides. Journal of Biochemical and Biophysical Methods, 51(1), 75–87. http://dx.doi.org/10.1016/S0165-022X(02)00006-4. Williams, C. H., Yamamoto, T., Walsh, D. M., & Allsop, D. (1993). Endopeptidase 3.4.24.11 converts N-1-(R, S)carboxy-3-phenylpropyl-Ala-Ala-Phe-pcarboxyanilide into a potent inhibitor of angiotensin-converting enzyme. Biochemical Journal, 684, 681–684. Wu, J., Aluko, R. E., & Nakai, S. (2006). Structural requirements of angiotensin Iconverting enzyme inhibitory peptides: Quantitative structure  Activity relationship study of di- and tripeptides. Journal of Agricultural and Food Chemistry, 54, 732–738. Yea, C. S., Ebrahimpour, A., Hamid, A. A., Bakar, J., Muhammad, K., & Saari, N. (2014). Winged bean [Psophocarpus tetragonolobus (L.) DC] seeds as an underutilised plant source of bifunctional proteolysate and biopeptides. Food & Function, 5(5), 1007–1016. http://dx.doi.org/10.1039/c3fo60667h.