Functional properties of Australian blue lupin (Lupinus angustifolius) protein and biological activities of protein hydrolysates

Lupin is an undervalued legume despite its high protein content with known health benefits. In this research, Australian blue lupin protein was isolated and hydrolysed enzymatically to produce bioactive peptides with a view to assess their potential for nutraceutical and therapeutic applications. Pepsin, pancreatin and flavourzyme were used to enzymatically hydrolyse blue lupin protein, and the hydrolysates were subjected to molecular weight cut‐off (MWCO) fractionation. Measurement of biological activities led to the identification of angiotensin converting enzyme (ACE) inhibitory fractions in the molecular weight range of 2–3 and 3–5 kDa. For the most active fractions in this range, the ACE inhibitory activities were very significant with IC50 values from 450 to 600 μg/ml. Blue lupin protein‐derived MWCO fractions were significantly active against Gram‐positive bacteria and only a little inhibition was observed against Gram‐negative bacteria. Pancreatin hydrolysed fractions showed the best antimicrobial activities with several fractions exhibiting ≥85% inhibition against Bacillus cereus and Staphylococcus aureus. These properties reveal the potential of lupin protein hydrolysates for developing antihypertensive and host defence agents. In order to demonstrate the potential of isolated blue lupin protein in food industry, functional properties including water and oil absorption capacity, gelling properties, solubility and emulsifying properties were evaluated and found to be extremely suitable for developing functional foods with enhanced health benefits.

Lupin seeds have gained popularity as health food items and research on their application as ingredients in food production continues to emerge. Special focus has been on lupin seed protein that exhibits beneficial functional and nutraceutical properties when incorporated into food products such as bread, pasta, biscuits, soups and salad dressing (Johnson, McQuillan, Sin, & Ball, 2003;Lee et al., 2009;Torres, Frias, Granito, Guerra, & Vidal-Valverde, 2007). For instance, incorporation of lupin flour into wheat flour increases the protein and fibre content and improves the amino acid profile of food products (Jayasena, Leung, & Nasar-Abbas, 2010;Jayasena & Nasar-Abbas, 2011;Villarino, Jayasena, Coorey, Chakrabarti-Bell, & Johnson, 2015). Lupin protein has been shown to have good solubility, foaming, gelling and emulsifying properties that are comparable with other legumes and soybean and hence has been used to develop various types of food products (Bader, Oviedo, Pickardt, & Eisner, 2011).
Antimicrobial peptides (AMPs) have been the focus of research around the world due to their potential application to combat the emergence of antibiotic resistant pathogenic microorganisms. The AMPs can exist naturally and are derived from food protein substrates and known as nature's antibiotics (Hancock & Chapple, 1999;Tang et al., 2015). Research on AMPs from blue lupin protein is scarce in literature. Yeo, Lee, Cha, and Hahm (2011) have identified a thermally stable AMP, AMP IC-1, from Korean traditional fermented soybean paste. Its activity was comparable with the previously reported peptide (BSAP-254) against Bacillus cereus (Sanjukta & Rai, 2016;Yeo, Lee, Cha, & Hahm, 2011). The purified AMP IC-1 is a 33 amino acid sequence with 13 different residues (namely, Cys, Asn or Asp, Gln or Glu, Ser, Ala, Pro, Gly, Arg, Thr, Val, Ile, Leu and Lys). The amino acids in both of these AMPs are very similar except for some minor differences (Sanjukta & Rai, 2016;Yeo, Lee, Cha, & Hahm, 2011). A novel inhibitor mungoin, derived from mung bean (Phaseolus mungo) seeds, has displayed significant antifungal and antibacterial activities (S. Wang et al., 2006). It exerted a potent inhibitory action toward a variety of fungal species including Physalospora piricola, Mycosphaerella arachidicola, Botrytis cinerea, Pythium aphanidermatum, Sclerotium rolfsii and Fusarium oxysporum, as well as an antibacterial action against Staphylococcus aureus (S. Wang et al., 2006;S. Y. Wang, Wu, Ng, Ye, & Rao, 2004). In addition, this novel plant protease inhibitor displayed anti-proliferative activity towards tumour cells (S. Wang et al., 2006).
In this paper, the term lupin protein refers to the protein isolated from blue lupin seeds. To the best of our knowledge, there is no report on the discovery of AMPs from lupin protein hydrolysates.
There is no literature involving any attempts to study antimicrobial and antifungal potentials of lupin protein-derived peptides. Also, very limited information on the production of bioactive peptides with ACE inhibitory activity is available in the literature. Therefore, this research has two major aims: (i) isolation of blue lupin protein and evaluation of their functional properties and (ii) enzymatic hydrolysis of blue lupin protein and the investigation of ACE inhibitory and antimicrobial properties of these hydrolysates.

| Chemicals and consumables
Angiotensin I-converting enzyme from rabbit lung and the ACE syn- The culture was diluted in the same broth to attain a final concentration of 10 7 CFU/mL that corresponded to an optical density reading of about 1.0 at 450 nm.

| Extraction of lupin protein
Australian sweet lupin (L. angustifolius) flour was obtained from Curtin University, Western Australia, and was prepared from de-hulled seeds. Lupin protein was isolated by alkaline water extraction and isoelectric precipitation by the method of Sironi, Sessa, and Duranti (2005) with some modifications. After defatting with 2-propanol (1:4 w/v), lupin flour was suspended in distilled water (1:10 w/v) and the pH of the suspension was adjusted to 9.0 using 1 M NaOH. The suspension was stirred for 1 h at room temperature and centrifuged at 10,000g for 30 min. The extraction steps were repeated twice for maximum yield. The supernatants were collected and acidified to pH 4.5, using 1 M HCl. The precipitate was recovered, neutralized and freeze dried for further studies (Sironi et al., 2005

| Protein solubility
The solubility of lupin protein isolate was measured by the method of King, Aguirre, and De Pablo (1985) with some modifications. Protein suspensions (0.5% w/v) were prepared at different pH values ranging from 2 to 10 by using 1 M HCl and 1 M NaOH and stirred for 60 min.
Centrifuged the mixtures at 16,000g for 15 min to remove solid content and observed the percentage protein by using Bradford assay at 595 nm. Solubility was calculated by the ratio of protein dissolved in supernatant to total protein in the initial sample. To study the effect of ionic strength on protein solubility, the above process was repeated by preparing suspensions with 1 M NaCl.

| Gelling property
The gelling property of lupin protein isolate was determined by the method of Rodriguez-Ambriz, Martinez-Ayala, Millan, and Davila-Ortiz (2005). The protein suspensions of 4%, 6%, 8%, 10% and 12% were prepared with MQW (5 ml each) and the test tubes were heated in boiling water bath for 1 h. The test tubes were then rapidly cooled under running tap water and then for up to 2 h at 4 C. The least gelation concentration was determined from the sample that did not fall out when test tube inverted.

| Enzymatic hydrolysis of lupin protein
Lupin protein isolates were digested with pepsin and pancreatin (enzyme/substrate ratio = 1:200) at 37 C and at pH 2 and 7, respectively. Hydrolysed samples were collected at hourly intervals for 4 h (Yoshie-Stark, Bez, Wada, & Waesche, 2004). For the digestion with flavourzyme the conditions were enzyme/substrate ratio of 1:10, pH 8 and 50 C. Samples were collected at hourly intervals for 4 h (Barbana & Boye, 2011). The hydrolysates were subjected to 10, 5, 3 and 2 kDa VivaSpin molecular weight cut-off (MWCO) membranes to separate peptide fractions of different molecular weights. A total of 48 MWCO fractions were obtained and were further analysed for their bioactivities.

| Determination of ACE inhibitory activity
The ACE inhibitory activity was measured in vitro by following the The organic phase was aliquoted and transferred to a glass tube to be heat evaporated. The residue was dissolved with 800 μl of distilled water and measured spectrophotometrically at 228 nm.
The activity of each sample was tested in triplicate. The assay mixture without protein hydrolysate was used as a blank. The ACE inhibitory activity was expressed as a percentage using the formula: The activity of lupin hydrolysates was expressed as the concentration of protein needed to inhibit 50% of ACE activity (IC 50 ).
Captopril (1 mM, Sigma-Aldrich) was used as a positive ACE inhibitor control in this assay.

| Functional properties of lupin protein
Protein is an important ingredient in food product development. Its composition has significant influence on consumer health and acceptability. Functional properties of food protein such as solubility, water holding, oil binding, foaming, emulsifying and gelation capacities have significant impact on the food product quality. These properties of blue lupin protein are presented and discussed in this section.

| Water and oil absorption capacities
Isolated blue lupin protein was estimated for water and oil absorption capacities. It showed 1.16 ml/g water absorption and 3.57 ml/g oil absorption capacities (Table 1). Sathe, Deshpande, and Salunkhe (1982) have reported 1.37 ml/g water absorption and 3.25 ml/g oil absorption capacities for lupin protein which are comparable with the results reported in this paper. Rodriguez-Ambriz, Martinez-Ayala, Millan, and Davila-Ortiz (2005) isolated Lupinus campestris protein by isoelectric method and demonstrated that both water and oil absorption capacities of lupin protein isolate was 1.7 mL/g whereas for soybean protein, these properties were 2.2 and 1.5 mL/g of protein, respectively. These properties of lupin are comparable with that of soybean protein.
Water absorption capacity depends on polarity, size and shape of amino acid residues which determine the extent of interaction of protein molecules with polar water molecules, whereas the fat absorption capacity depends on nonpolar amino acid side chains within the protein molecules that interact with hydrocarbon chains of fat molecules.

| Protein solubility
Blue lupin protein solubility is shown in Table 1. The results showed that the solubility of isolated protein is highest at pH 10 (95%) and it is lowest at pH 4 (7.25%) in 1 M NaCl (  Foam formation depends on the ability of protein to reduce surface tension between two phases (air-water) and retain the film formed around the air bubble. Foaming property is important to make cake, ice cream, mousses, whipped cream, and so on (Chao, Jung, & Aluko, 2018;Smith, 2010).

| Emulsifying property
The emulsifying property of blue lupin protein was determined, and the emulsifying activity index (EAI) was calculated to be 41.78 m 2 /g, and the emulsifying stability index (ESI) was 15.34 min (

| Gelling property
The gelling property of blue lupin protein was measured, and the least gelation capacity was found with 8% protein solution ( Table 1) networks that exhibit elasticity and provide textural strength to gels (Damodaran, 1996). Differential scanning calorimetry (DSC) analysis performed to compare the gelling properties of lupin and soy protein isolates indicated that the soy protein is superior in this aspect (Berghout, Boom, & van der Goot, 2015). It is concluded that even though the lupin protein isolate forms weaker gelling networks, it is very appropriate for high protein foods that require low viscosity after heating (Berghout, Boom, & van der Goot, 2015).
These results indicate that blue lupin protein isolate has great potential as a value-added ingredient in food industry.

| Biological activities of blue lupin protein hydrolysates
As described in Section 2, blue lupin protein was subjected to enzymatic hydrolysis using three enzymes, namely, pepsin, pancreatin and flavourzyme. Hydrolysates collected at hourly intervals (1 to 4 h) were subjected to separation by MWCO membranes. A total of 48 MWCO fractions were isolated, and each of these fractions were named based on the enzyme used, hydrolysis time and the molecular weight range. For instance, "Pep.5k.1h" indicates the fraction obtained by 1-h pepsin hydrolysis and has a molecular weight range of 3-5 kDa (Table 4).
Enzymes chosen for this study were based on their specificity and their significance in human digestive system. A brief description of their activity is provided below.
Pepsin is a principal enzyme of the stomach and is active in acidic environment in the pH range of 1.3 to 2.0. Pepsin's cleavage is more specific at pH 1.3 and its specificity is lost above pH 2. As

| ACE inhibitory activities
The enzymatic hydrolysis of blue lupin protein followed by MWCO fractionation produced 48 fractions. ACE inhibitory properties of these MWCO fractions have been determined and the results are presented in Figures 1-3 and Table 2.
The lowest IC 50 value was obtained from 2 to 3 kDa fraction of 4-h hydrolysis (450 ± 11 μg/ml). The IC 50 values obtained from flavourzyme hydrolysis ranged from 600 ± 18 to 1210 ± 27 μg/ml (Table 2). The lowest IC 50 value was obtained from 3 to 5 kDa fraction of 1-h hydrolysis (600 ± 18 μg/ml). The ACE inhibitory activity of lupin protein hydrolysates varied widely and was significantly different (p < 0.05) by the enzymes used, hydrolysis times and MWCO fractions. The 2-3 kDa fraction of 4-h pancreatin hydrolysis exhibited the lowest IC 50 value (450 ± 11 μg/ml). As discussed before, pancreatin is a mixture of the three main digestive enzymes: pepsin, trypsin and chymotrypsin. It is, therefore, expected that this combination of the three enzymes will produce smaller peptides due to the cumulative specificities these enzymes possess. Herrera Chalé, Ruiz Ruiz, Acevedo Fernández, Betancur Ancona, and Segura Campos (2014) have reported the use of pepsin-pancreatin mixture to hydrolyse Mucuna pruriens proteins.

Results from ACE inhibitory
Their ACE inhibitory activities were significant with the best IC 50 value of 19.5 μg/ml. In other research with peanut proteins, Quist, Phillips, and Saalia (2009) reported highly significant ACE inhibitory activities with IC 50 values of 7.9-65.9 μg/mL, and 11-36 μg/mL for raw and roasted peanut, respectively, with pepsin-pancreatin hydrolysis. These reported findings are better than the results from the present study on the ACE inhibitory activities of blue lupin-derived peptides with pancreatin.
In general, pepsin produced a number of active peptide fractions (Table 2) Due to its broad specificity, flavourzyme was expected to produce peptides with different affinities towards ACE. Flavourzyme has been shown to produce ACEIPs with lower IC 50 values due to its broad specificity and hence may cleave the active peptides from either C-or N-terminal ends (Chiang, Tsou, Tsai, & Tsai, 2006). Suh, Whang, Kim, Bae, and Noh (2003) have indeed reported an increase in ACE inhibitory activity of corn gluten protein hydrolysed by flavourzyme. In this study, flavourzyme hydrolysates of blue lupin proteins showed lower ACE inhibitory activity similar to soybean proteins (Chiang, Tsou, Tsai, & Tsai, 2006). These results suggest that soybean and lupin proteins share some similarities in their amino acid sequences as these two plants belong to the same Fabaceae family.

| CONCLUSION
The results on functional properties of lupin seed protein and biologi- potential for the preparation of functional foods and nutraceutical formulations.
Pancreatin hydrolysed fractions displayed best antimicrobial activities with seven fractions exhibiting excellent inhibition against B. cereus and S. aureus. Pepsin hydrolysed fractions were found to be more active against Gram-negative bacterial growth, and pancreatin and flavourzyme derived fractions were better for their Gram-positive bacterial inhibition. To the best of our knowledge, this is the first study on antimicrobial activities of lupin seed protein hydrolysates.
Overall, the results on ACE inhibitory and antimicrobial activities highlight the potential of incorporating lupin seed protein and protein hydrolysates into food products as preventative agents towards hypertension and microbial diseases. It is concluded that, lupin is an affordable and competitive ingredient for the preparation of nutraceutical and functional foods.

CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest with respect to the research, authorship and/or publication of this article.

ETHICS STATEMENT
This research did not involve any human or animal ethics issues to be considered.

DATA AVAILABILITY STATEMENT
Complete raw data and the processed data related to this publication are available with authors.