http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, Early Online: 1–8 ! 2015 Informa UK Ltd. DOI: 10.3109/02652048.2015.1057250

RESEARCH ARTICLE

Preparation and characterisation of nanoliposomes containing winged bean seeds bioactive peptides Shyan Yea Chay, Wei Kiat Tan, and Nazamid Saari

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Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, Serdang Selangor, Malaysia

Abstract

Keywords

The aim of this study was to produce and characterise nanosize liposomes containing bioactive peptides with antioxidative and ACE-inhibitory properties, derived from winged bean seeds (WBS) protein. WBS powder was papain-proteolysed, at 70  C and pH 6.5 for six hours, followed by encapsulation via a solvent-free heating method. The results showed that the WBS proteolysate was successfully incorporated into spherical, unilamellar liposomal particles, with particle diameter, polydispersity index, zeta potential and encapsulation efficiency of 193.3 ± 0.12 nm, 0.4 ± 0.02 (unit less), 70.5 ± 0.30 mV and 27.6 ± 1.17%, respectively. It also demonstrated good storage stability over eight weeks at 4  C, indicated by slight increment (15.1%) in particle size and a zeta potential only weaker by 17.2% at the end of the study period. These results suggested the feasibility of entrapping water soluble peptides in hydrophobic liposomal system that, upon optimisation, has the potential to act as bioactive food ingredient.

ACE inhibitory activity, antioxidative activity, enzymatic hydrolysis, nanoliposome characterisation, Psophorcarpus tetragonolobus (L.) DC

Introduction Functional food sector has been blossoming in recent years due to the rise in consumer awareness of attaining wellness through diets. This has resulted in constant development of new functional food items through the incorporation of health-promoting ingredients into suitable food systems. One of the newer technologies involves the encapsulation of bioactives or other desired materials within a coating material known as membrane, capsule or shell, to form miniature particles of micrometre or nanometre in size. By encapsulation, sensitive materials can be turned into stable ingredients and thus reduce the amount of material required to exert a specific effect, due to improved stability (Mozafari et al., 2008; Costa et al., 2013). Comparing with other coating agents, such as alginate and chitosan, liposome is among the most favourable coating materials due to its unparalleled advantages, which include the ability to entrap material of different solubilities, cost-effective production from natural ingredients and targetability (Mozafari and Mortazavi, 2005; Mozafari and Khosravi-Darani, 2007). Liposome is defined as ‘‘closed, continuous, bilayered structure made mainly of lipid and/or phospholipid molecules’’ (Mozafari et al., 2002). This suggests that they resemble indigenous molecules in our body and are thus biocompatible and acceptable for human consumption. On the other hand, compared to micronsize carriers, nanocarrier molecules provide larger surface area

Address for correspondence: Prof Dr Nazamid Saari, Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM, Serdang Selangor, Malaysia. Tel: +603 8946 6044. Fax: +603 8942 3552. E-mail: [email protected]

History Received 24 October 2014 Revised 21 April 2015 Accepted 4 May 2015 Published online 16 June 2015

which increase solubility, enhance bioavailability, improve controlled release and enable precise targeting of the encapsulated material to a greater extent (Mozafari et al., 2008; Khare and Vasisht, 2014). Angiotensin-I converting enzyme (ACE) activity, which raises blood pressure by cleaving an inactive decapeptide into vasoconstrictive octapeptide, when coupled with oxidation processes, are responsible for the development of cardiovascular diseases (CVD). Oxidative stress participates in the mechanism of vascular injury, causes a rise in blood pressure and subsequently leads to complications, such as atherosclerosis and CVD (Schiffrin, 2010). Thus, food sources rich in ACE inhibitory and antioxidative properties are gaining more interest as they possess beneficial property that deters the development of CVD. One definite example would be the functional peptides derived from vegetable origin proteins, and legumes are especially promising due to their high protein content (Torruco-Uco et al., 2009). Winged bean [Psophopcarpus tetragonolobus (L.) DC], an underexploited tropical plant, gets its name from the four-sided pods with a characteristic ‘‘wing’’ shape. Practically, all parts of winged bean plant are edible, palatable and rich in vitamins and protein. The seeds, however, have created the greatest interest internationally. They virtually duplicate soyabeans in composition and nutritional value; both contain similar proportions of protein, oil, minerals and vitamins. The exceptional protein content in the seeds may be due to the extensive root systems and large nodules on their roots to conduct efficient nitrogen fixing (National Research Council, 1975). In this study, bioactive peptides were first generated via enzymatic hydrolysis of winged bean seeds (WBS) protein under optimised conditions, incorporated into nanoliposomal particles

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using soya lecithin, then finally characterised for physicochemical properties, morphology, encapsulation efficiency (EE) and shelflife stability. To the authors’ best knowledge, this is one of the few pioneer studies that focus on the encapsulation of dual functionalities biopeptides derived from plant protein, i.e. WBS protein, with ACE inhibitory and antioxidative properties.

Materials and methods

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Chemicals and reagents Mature winged bean seeds [Psophopcarpus tetragonolobus (L.) DC] were purchased from Agriplaza Farm, Selangor, Malaysia. The seeds were ground into fine powder, packed in individual bags and stored at 40  C until further use. Hippuryl-L-histidyl-Lleucine (HHL) substrate and angiotensin-I-converting enzyme were obtained from Sigma Chemical Co. (St. Louis, MO). Soya lecithin was a kind gift from IMCOPA (Parana´, Brazil). All other reagents and chemicals were of analytical grade. Preparation of WBS proteolysate WBS proteolysate was generated according to a method previously described (Chay et al., 2014). Briefly, WBS powder was mixed with distilled water and equilibrated to the desired pH (6.5) and temperature (70  C) before addition of papain, at the enzyme:substrate ratio of 1:50 (w/w). Proteolysis was carried out for 6 h. Upon completion, the proteolysate was boiled for 15 min to inactivate the enzyme, rapidly cooled in an ice bath (to allow easier handling), then subjected to centrifugation at 10 000g, room temperature, for 30 min. Supernatant was collected and either stored at 40  C for analysis or stored at 80  C before being freeze-dried. The freeze-dried sample was stored at 40  C until further use. Determination of ACE inhibitory and antioxidative activities in WBS proteolysate ACE inhibitory activity ACE inhibition assay was performed using a combined method (Cushman and Cheung, 1971; Jimsheena and Gowda, 2009) with some modifications. Firstly, 25 mL of proteolysate was preincubated with 10 mL ACE solution (100 mU/mL, in borate buffer) at 37  C for 10 min. The mixture was then added with 50 mL substrate [5 mM HHL, 100 mM borate buffer (pH 8.3), 300 mM NaCl] and incubated at 37  C for 60 min. The enzymatic reaction was terminated by adding 75 mL of 1.0 N HCl. The released hippuric acid was reacted with 150 mL of pyridine and 75 mL of benzenesulphonyl chloride (BSC), then vortexed and placed in an ice bath. Once cooled, 200 mL solution was transferred to individual wells on a 96-well plate. Absorbance was read at kmax of 410 nm. One unit of ACE activity is defined as 1 lmol hippuric acid released per minute at 37  C and pH ¼ 8.3. The average values from three determinations were used to calculate ACE inhibition using the equation below: ACE inhibition (%) = [(A410 ControlA410 SampleÞ= A410 ControlA410 Blank  100; where A410 represents the absorbance measured at a wavelength of 410 nm, control contained ACE and substrate, blank contained only substrate and sample contained proteolysate, ACE and substrate. DPPH radical scavenging activity 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was measured according to a previously described method

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(Hwang et al., 2009) with some modifications. In each well of a 96-well plate, 50 lL of sample solution was mixed with an equal amount of distilled water, followed by adding 100 lL of DPPH solution (0.15 mM, in ethanol 80%). The mixture was incubated in the dark at room temperature for 45 min. Absorbance was measured at 517 nm. The blank consisted of distilled water and DPPH solution at a 1:1 ratio. The scavenging activity was determined according to the following equation: DPPH radical scavenging activity ð%Þ ¼ ½ðA517 BlankA517 Sample=A517 BlankÞ  100; where A517 indicates the absorbance measured at 517 nm. Metal ion-chelating activity The chelating activity of antioxidants in the sample was estimated according to a previously described method (Dinis et al., 1994) with some modifications. Briefly, 300 lL of sample solution was first diluted with 300 lL distilled water, then mixed with 15 lL of iron (II) chloride solution (2 mM). Afterwards, 30 lL of ferrozine solution (5 mM) was added, and the mixture was vortexed and allowed to stand for 10 min at room temperature before absorbance reading was taken at 562 nm. As a blank, distilled water was used to replace the samples. The inhibition percentage of ferrozine-Fe2+ complex formation was calculated from the following equation: Metal chelating activity ð%Þ ¼ ½ðA562 BlankA562 Sample= A562 BlankÞ  100; where A562 represents the absorbance measured at 562 nm. To make comparisons, the samples in both antioxidative assays were diluted to the same ratio. Preparation of nanocapsules from liposome WBS proteolysate-entrapped nanoliposomes were prepared using heating method described by Mozafari (2010), with some modifications as detailed below. Freeze-dried WBS proteolysate was first dissolved in distilled water at 1 mg/mL. Soya lecithin was then added at a final concentration of 2% (w/w). Solution was heated to 40  C, temperature maintained, and stirred at 8000 rpm using homogenizer DIAX 900 (Heidolph Instruments GmbH & Co., Schwabach, Germany) for a period of 60 min, then bathsonicated in DeltaÕ Ultrasonic Cleaner DC-150H (Delta New Instrument Co., Ltd., Taipei, Taiwan) at 40 kHz for another 60 min. The sample was then filtered through 0.2 lm MinisartÕ RC-15 syringe filter (Sartorius AG, Go¨ttingen, Germany) and kept at room temperature for 60 min to allow for annealing. Liposome suspension was then stored in amber bottle at 4  C until further use. Particle size, polydispersity index and zeta potential analysis All measurements were made using Zetasizer Nano-ZS (Malvern Instruments Ltd., Worcestershire, UK) by non-invasive back-scattering and dynamic light scattering technology. Samples were first diluted with deionised water (1:10) and dispensed into a disposable polystyrene cuvette or gold-plated capillary cell (when PDI was determined), then measured at k ¼ 633 nm at 25  C. The polydispersity index (PDI), a measure of particle size distribution, and correlogram were used simultaneously to indicate liposome quality, of which PDI50.5 and a smooth, consistent correlogram represent good liposomal quality. Measurements were performed in five repetitions right after the liposome preparation.

Nanoliposomes containing WBS bioactive peptides

DOI: 10.3109/02652048.2015.1057250

100.0

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Determination of encapsulation efficiency

EE ð%Þ ¼ ðA340 Total peptide  A340 Free peptideÞ= A340 Total peptide  100 where A340 indicates the absorbance measured at 340 nm. Microscopic assessment of nanoparticles Physical appearance and particle diameter were determined using transmission electron microscopy (TEM) (Hitachi H-7100 TEM, Hitachi Ltd., Chiyoda, Japan). A drop of liposome, diluted at 1:10, was placed onto a copper grid and air-dried for 5 min. The grid was negatively stained with 2% phosphotungstic acid (PTA) solution for 2 min and air dried in an automatic dehumidification storage box, Auto DryBox (Taiwan Dry Tech Corp., Taipei, Taiwan) before assessment under TEM. The particle diameter was determined using a built-in scale bar on the electron micrographs. Storage stability study on nanoliposomal suspension (8 weeks) Storage study was conducted for eight consecutive weeks to observe for liposome particle size and dispersion stability. The size was measured on a weekly basis while zeta potential and visual observation were assessed once in four weeks, on week 0, 4 and 8. Measurements were performed in five repetitions. Throughout the study period, liposome was kept in an air-tight, amber bottle at 4  C for consistency. Statistical analysis Data are presented as the means ± standard deviations from at least triplicate determinations. Statistical analysis was performed using the SAS package (Statistical Analysis Software 9.1) from SAS Institute Inc. (Cary, NC). Tukey’s test was conducted to detect significant differences among the means at p50.05.

Results and discussion Enzymatic proteolysis of WBS and subsequent bioactivities measurement Bioactive peptides that are encrypted within the sequence of food proteins can be released during gastrointestinal digestion, food processing (fermentation and maturation processes) or enzymatic proteolysis. Enzymatic proteolysis is the most common way to produce biopeptides and is preferred over the other processes because of its ability to produce specially tailored peptides with desirable effects (Herna´ndez-Ledesma et al., 2011). In our work, biopeptides with dual functionalities of ACE inhibitory and

90.0 80.0

ACE inhibion DPPH radical scavenging Metal ion chelaon

70.0 Bioacvies (%)

Encapsulation efficiency (EE) was calculated by comparing the total peptide content (after ethanol rupture) and the amount of peptide dispersing freely in solution. Peptide content was measured using OPA method described previously (Chay et al., 2014). Liposomal solution was first diluted (1:25) with deionised water, mixed with heated with absolute ethanol (70  C) at the ratio of 1:2 following the method of Nii et al. (2003) and stirred for 20 min to completely rupture the liposome and release the entrapped content. The ruptured liposome (represents total peptides) was ultracentrifuged using vivaspinÕ concentrator (MWCO: 50 kDa) at room temperature and a speed of 10 000g for 6–10 min, depending on the filtration efficiency of samples, then heated to vaporise ethanol from the sample. Liposome without rupturing (represents free peptides) was subjected to the same ultracentrifugation process. Clear filtrate was collected for both samples (ruptured and unruptured) and their peptide content was measured using OPA method. EE was calculated following the equation, as a mean from triplicate determinations:

3

60.0 50.0 40.0 30.0 20.0 10.0 0.0 0

2

4

6

Proteolysis hour (h)

Figure 1. Bioactivities (%) of winged bean seeds proteolysate, expressed as ACE inhibitory, DPPH radical scavenging and metal ion chelating activities, respectively. Notes: The proteolysis was carried out using papain, at pH 6.5 and 70  C for 6 h. Bars represent standard deviations from triplicate determinations.

antioxidative properties were generated from mature WBS. Proteolysis was conducted for 6 h, at pH 6.5 and temperature 70  C using papain enzyme. Papain, a cysteine proteinase, was chosen because it is a food grade enzyme with broad specificity and commercially available at an economical cost. More importantly, papain had been demonstrated as the most efficient protease to produce WBS proteolysate with potent ACE inhibitory activity, compared with other proteases including alcalase, bromelain and flavourzyme, based on the results previously obtained (Wan et al., 2013). Bioactivities from WBS proteolysate were measured for ACE inhibitory, DPPH radical scavenging and metal ion chelating activities respectively. Figure 1 shows the bioactivities throughout 6 h proteolysis, which range between 40 and 70%. The antioxidative activity in the samples before enzymatic proteolysis displayed 46.0% DPPH radical scavenging activity and 18.3% metal ion-chelating activity, indicating there was still a minute amount of antioxidative substances in the sample prior to proteolysis. However, these antioxidant activities were significantly lower than the levels observed after proteolysis. Thus, proteolysis is considered as a way to improve the antioxidative activity of WBS. Preparation of nanoliposome using heating method Most of the nanoliposome-producing techniques involve the utilisation of potentially harmful solvents (such as methanol, acetone and chloroform) or high shear force procedures. Furthermore, methods to remove residual toxic solvents are practically time-consuming and expensive. In order to overcome the problems arise from existing preparation methods, alternative such as heating method has been developed, by which nanoliposomes can be prepared in a single apparatus in the absence of volatile solvents (Mozafari, 2010). In our work, nanoliposomes were produced via heating method. Soya lecithin and WBS proteolysate were heated to 40  C [well above the phase transition temperature of soya lecithin that is below 0  C (Lautenschla¨ger, 2006)] and stirred vigorously. Heating provides energy to ensure all phospholipids dissolved homogenously in solution and have sufficient flexibility to align themselves during the formation of nanoliposomes, while stirring facilitates the homogenous distribution of the ingredients (Mozafari, 2005). Energy input results in the arrangements of soya phospholipid molecules to form an orderly bilayer structure, enclosing water-soluble WBS proteolysate in the core.

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This alignment would help the molecules to achieve thermodynamic equilibrium, of which fatty acyl chains are sequestered within hydrophobic bilayer interior, while polar heads are exposed to the aqueous media. Successful encapsulation of WBS proteolysate with liposomes indicates the feasibility of incorporating water-soluble peptides into a hydrophobic system. The purpose of incorporating bioactive peptides into liposome is to maintain the integrity of peptides before they reach the target sites. Protection of the peptides from enzymatic degradation in gastrointestinal (GI) tract and in blood is particularly important when the biopeptides are used as food ingredients, as there are many proteases present in the human GI tract and blood, which may cleave vulnerable peptides into inactive fragments. Encapsulation ensures that the peptides reach their target sites in intact form to exert maximum desirable biological effect. Also, encapsulation would improve the stability of peptides. As a consequence, the amount of peptides required to produce a specific level of effect when encapsulated is much lesser compared to the amount required when left unencapsulated, thus reduces the cost for raw material (WBS) and peptide production. Characterisation of nanoliposomal particles entrapped with WBS proteolysate Following successful preparation of nanoliposome loaded with WBS proteolysate, characterisation is required to assess the product quality. Methods of characterisation need to be meaningful and rapid. The most important parameters include visual appearance, particle size, stability (zeta potential), size distribution and encapsulation efficiency (Mozafari, 2010). Particle size, PDI and zeta potential measurements The measurement of particle size lies on the principle of dynamic light scattering, which is based on the fact that particles move randomly under Brownian motion. When a light source hits the moving particles, the light is scattered in all directions. The movement of molecules causes a fluctuation in the light scattering intensity and the rate is dependent on particle size. Large particles will fluctuate slower and small particles will fluctuate quicker. This property is then utilised to dictate the particle size. The size distribution curve of WBS proteolysate-loaded liposome appears as normalised curve, revealing the size variation among particles, ranging from5100 nm to41000 nm, with a peak around 200 nm (on a logarithmic scale, figure not shown). This curve pattern indicates that the solution contains a mixture of different sizes instead of constant size particles. The liposome size depends largely on the preparation conditions, such as sonication time, number of extrusion cycles and pore size used for extrusion. Particle size is reduced by energy input in the form of sonic energy (sonication) and mechanical energy (extrusion). The processing parameters in the current work yield the mean particle size of liposome loaded with WBS proteolysate to be 193.30 ± 0.12 nm, as shown in Table 1. This value is larger than the previously reported liposome containing sea bream scale proteolysate, using the same ingredient of soya lecithin as the current work, which records an average particle diameter of 90.30 nm (Mosquera et al., 2014). However, the previous work

used film hydration method, which utilised chloroform during production whereas current work uses much simpler, direct heating that eliminates the use of harmful solvents. Also, the particle size obtained in the current work is comparable to the particle size between 195 and 201 nm reported by Colas et al. (2007), who conducted the work using same ingredient and method. Bouarab et al. (2014) suggested that the partition of cinnamic acid (a hydrophobic compound) into lipid bilayer increases the size of final nanoliposome product. In the current work, authors postulated that when peptides are incorporated into lipid bilayers during encapsulation, they would form chemical bonds and interact with acyl chains on the lipids, altering the chain structure and membrane fluidity, which then cause volume expansion and size increment. Particle size plays an important role in the absorption rate into cell. The liposome having size around 200 nm can promote membrane fusion with target cells and deliver the encapsulated components into cells efficiently. Encapsulation of curcumin with liposome prepared from lecithin has a diameter of approximately 263 nm and was found to have a high bioavailability of curcumin and plasma antioxidant activity in rats through oral administration (Fricker et al., 2010). Therefore, liposome with the mean size of 193.30 nm may also be suggested as a nutrient delivery system through oral administration. Polydispersity index is a dimensionless measure of the broadness of particle size distribution as well as the tendency of particles to aggregate in the liposomal suspension system. The index ranges from 0.0, indicating a monodisperse system, to an arbitrary maximum value of 1.0, which indicates a polydisperse system and may contain large particles that could be slowly sedementing. Thus, PDI value is preferred to be as low as possible. Our work reported a mean PDI of 0.37 ± 0.02, whereas Mosquera et al. (2014) reported a mean PDI of 0.25 for liposome loaded with sea bream scale proteolysate. This indicates our liposome is less monodisperse than the previously reported work, which utilised similar ingredient (a peptide mixture) but a different source as the core material. The broad PDI reported in the current work possibly arises from the fact that liposome solution was only filtered through 200 nm pore size without passing through 100 nm at the final step of extrusion, which would otherwise improve the homogeneity of size distribution. Thus, the product has lower homogeneity and broader particle size distribution. Also, the higher PDI value may be due to coalescence of molecules whereby liposomes of different sizes merge from smaller particles into larger, single droplets and caused a wide size distribution (Barba et al., 2014). However, a PDI value 50.7 is considered acceptable for measurements using Zetasizer (Shaw, n.d.). Figure 2 shows the correlation of the scattered light signal intensity at time ¼ t and that at different times later (t + qt). Several informations can be extracted from the correlogram. Due to constant, random Brownian movement of the liposomal particles, the correlation reduces with time, eventually reaching zero correlation at high delay times at the end of analysis. The smooth baseline at the end also indicates there is no sedimentation in the sample. The correlogram also shows that the particles have relatively large sizes because the signal changes slowly and the correlation persist for a longer time (levelling off period) before the decay starts. Lastly, the correlation coefficient at the

Table 1. Average size, polydispersity index and zeta potential of WBS proteolysate-loaded liposome.

Parameters

Average diameter (nm)

Average PDI

Average zeta potential (mV)

Encapsulation efficiency (%)

WBS proteolysate-loaded liposome

193.30 ± 0.12

0.37 ± 0.02

70.5 ± 0.30

27.6 ± 1.17

Nanoliposomes containing WBS bioactive peptides

y-intercept of 0.9 (which is51.0) indicates the particle size data is of high confidence because the sample concentration during measurements is sufficient to prevent number fluctuation. Zeta potential gives information about the electrostatic potential of particles in a solution by measuring the charge difference between the electrical double layer of a charged particle (nanoliposome) and the bulk solution at the slipping plane (a notional boundary that separates ions associated to nanoliposomes from bulk solution). It aids to predict the long term stability of a colloidal system, whereby large positive and negative values (more than ± 30 mV) denote sufficient repulsion to prevent aggregation and achieve stability. Two major types of phospholipids found in soya lecithin, namely phosphatidylcholine and phosphatidylethanolamine, are zwitterionic molecules without net charge while phosphotidylinositol has a net charge of 3. Zeta potential defines the overall charge of a lipid vesicle suspended in the medium (Mozafari, 2010). Thus, liposome molecules that contain phosphatidylinositol are negatively charged in nature. The liposomes would attract positively charged particles and polar molecules from the solution to form Stern layer and diffusion layer. The build up of electrical charges around the liposomes would then contribute to the formation of zeta potential. Table 1 shows the zeta potential for WBS proteolysate-loaded nanoliposome to be 70.5 ± 0.30 mV. This value is much lower 1 0.9 Correlogram coefficient

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DOI: 10.3109/02652048.2015.1057250

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0.1

10

1000

100000

10000000

Time (µs)

Figure 2. Correlation of the scattered light intensity in nanoliposome solution as a function of time.

5

than the zeta potential for nanoliposome containing sea bream scale and antimicrobial peptide P34, which recorded 40.8 and 27.4 mV, respectively (Malheiros et al., 2011; Mosquera et al., 2014). Our liposome has much lower tendency to precipitate and higher ability to maintain the integrity of suspension. The high stability may be due to two factors: low solution viscosity and electrostatic stabilisation. Low viscosity indicates that the nanoparticles are separated at significantly far distance that prevent Van der Waals interaction between molecules, which would otherwise draws them closer and causes precipitation under the influence of gravity. On the other hand, electrostatic repulsion is caused by charges of the same type that build-up around the nanoparticles. In the suspension, the negatively charged liposomes attract thick layers of positive charges from solution, which then create a strong repulsive force around the liposomes to prevent the molecules from approaching and adhering to each other, thus preventing flocculation and achieving high stability. Encapsulation efficiency Encapsulation efficiency was determined by comparing the peptide content between intact, unruptured liposomes (free peptide in solution) and ethanol-ruptured liposomes (total peptide), measured using o-phthaldialdehyde that detects peptide bond. The difference between two absorbances represents the amount of peptide entrapped within liposomes. Table 1 displays the EE for WBS proteolysate-loaded nanoliposomes to be 27.6 ± 1.17%. This value is relatively low compared to sea bream scale proteolysate and nisin-calcein loaded liposomes, which recorded EE of 74.6 and 63.0%, respectively. Poor encapsulation may be due to the negative interaction between peptides and liposome membranes, which disrupt the bilayer structure and cause pores formation, eventually result in leakage of the content. WBS proteolysate is a heterogenous mixture of free amino acids and peptide chains of short and long fragments. The proteolysate is suggested to consist of mainly hydrophilic and short chain peptides, which cause poor entrapment. Hydrophilic peptides tend to exert weak binding with neutral soya phosphatidylcholine molecules at the oil-water interface, limiting the product ability to retain the content. On the other hand, short peptides are less likely to possess both hydrophobic and hydrophilic residues on the same molecules. During formation of nanocapsules, short peptides are lacking of hydrophobic side chains to interact with oil droplets (lecithin) and

Figure 3. Microscopic assessment of WBS proteolysate-loaded liposomes. Notes: Images were captured using transmission electron microscope (model: Hitachi H-7100) on a scale bar of 100 nm (A) and 200 nm (B), respectively.

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hydrophilic residues to interact with aqueous phase simultaneously. This would lead to the formation of uneven, noncontinuous interfacial barrier with weak mechanical stability, causing the liposomes to lose the entrapped content easily and results in low EE values (Singh and Dalgleish, 1998; Tirok et al., 2001). Nevertheless, the EE value obtained in our work is comparable to that of 24.2 and 26.5% for nisin-loaded PC liposomes, reported by Colas et al. (2007), who used similar ingredient and procedures to the current work. In order to increase the entrapment efficiency, cholesterol can be added to the lecithin ingredient during mixing, as it reduces the permeability of the phospholipid bilayers and increases the rigidity of the fluidal membrane system.

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TEM visualisation The morphology of particles was examined by imaging the airdried, negatively stained liposome on TEM. TEM was chosen over scanning electron microscopy (SEM) because it provides more powerful magnification that suits for nanosize particles investigation. Viewing under SEM would possibly yield images of clumpy molecules resulting from spontaneous aggregation of the molecules (Zhong and Jin, 2009). As SEM scans the entire sample, it makes the estimation of particle diameter to be difficult. Thus, TEM was preferred. Figure 3 shows the TEM images obtained for WBS proteolysate-loaded nanoliposomes. They appeared as single, discrete particles with completely closed entity of either spherical or elliptical shape. This indicates the successful encapsulation of hypdrophilic WBS proteolysate within a hydrophobic system.

The darker ring dictates the membranous barrier made up of phospholipid bilayers that separate the proteolysate from surrounding media. As we can see, the liposomes exist as unilamellar molecules with single lipidic bilayer. Using the built-in scale bar, the particle size was measured to be 219.02 nm (Figure 3A) and 218.64 nm (Figure 3B), respectively. These figures validate the data obtained from dynamic light scattering measurement which predicts the liposome diameter to be 193.30 nm. Storage stability study (8 weeks) Shelf-life stability is a major characteristic that predicts the liposomal quality product. Liposomes tend to aggregate, fuse and grow into bigger vesicles to achieve a more thermodynamically favourable state. Therefore, a constant size maintained for a long period of time is an indication of liposome stability. In this study, the particle size of liposomes was monitored once a week for a total of eight weeks and its zeta potential was measured in every four weeks. Table 2 shows the measured average diameter (nm) of liposomes. As presented, the liposomes exhibited some significant differences (p50.05) in the increment

Table 3. Zeta potential of liposomes containing WBS proteolysate measured at day 0, 28 and 56 days. Day of measurement Zeta potential (mV)

1

28 a

70.50 ± 0.30

56

52.93 ± 0.31

c

58.37 ± 0.32b

Note: Values with different letters indicate significant difference (p50.05).

Table 2. The average diameter (particle size) of WBS proteolysate-loaded liposome measured over eight weeks consecutively. Weeks

1

193.33 ± 0.12 Average Diameter (nm)

2 a

3 bc

216.37 ± 0.40

213.20 ± 1.15

4 b

5 b

213.37 ± 1.63

6 de

227.60 ± 3.13

7 e

231.67 ± 0.57

8 e

233.80 ± 0.50

222.40 ± 8.80cd

Note: Values with different letters indicate significant difference (p50.05).

Figure 4. Visual observation of liposomal suspension at initial (week 0) and final week of storage (week 8). Note: No separation was observed throughout the study period.

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DOI: 10.3109/02652048.2015.1057250

of average diameter over eight weeks of storage at 4  C, from 193.33 ± 0.12 to 222.40 ± 8.80 nm, which is equivalent to a 15.1% increase. It is considered relatively stable compared to liposomes loaded with medium-chain fatty acids and vitamin C, which recorded a size increment of 33.0% over 60 days storage at the same condition (4  C), as reported by Yang et al. (2013). The zeta potential of our liposomes has also increased over the study period, giving a value of 58.40 ± 0.32 mV at week 8 (Table 3). Even though the zeta potential has increased, the liposomes were still considered to have sufficient repulsive interaction in between particles to achieve a physically stable suspension. The stability of liposomes has also been confirmed by visual observation. As shown in Figure 4, there was no occurrence of phase separation in the solution after eight weeks of storage. The good stability exhibited by liposomes may be due to the positive interaction between phospholipids and peptides. According to Gu¨lseren and Corredig (2013), the barrier property of milk phospholipids was not adversely affected by the presence of tryptic peptides, indicating the ability of phospholipids to retain stability in the presence of peptides. In addition, Hammes and Schullery (1970) proposed that the phospholipid-polypeptide complex is stabilised by both electrostatic and hydrophobic interactions, which reduces the mobility of the fatty acid groups on phospholipids, contributing to a more stabilised structure of the complex. In our study, WBS proteolysate-loaded liposomes are suspended in a peptide-rich solution; the surrounding peptide molecules are freely dispersed in the solution and may interact with the phospholipid bilayers and contribute to the liposome long-term stability.

Conclusions The present work reflects a primitive study on the feasibility to incorporate hydrophilic WBS proteolysate into hydrophobic liposome, followed by subsequent characterisation of the product. Our work demonstrated successful production of nano-sized particles with reasonable particle size, low zeta potential, and good storage stability but poor encapsulation efficiency. This indicates that the liposome is weak in entrapping peptides at a high concentration but it is able to maintain particle size and suspension stability during long-term storage. Further development to improve the nanoliposome entrapment efficiency is deemed necessary to produce high performance bioactive peptides carrier vehicles that retain functionality in food product, while maintaining desirable biological effects upon ingestion.

Acknowledgements The authors acknowledge the help from Prof. Dr Tan Chin Ping (Faculty of Food Science and Technology, Universiti Putra Malaysia) for permitting the usage of Zetasizer during particle size and zeta potential measurements.

Declaration of interest This work has been financially supported by the Ministry of Science, Technology and Innovation, Malaysia under ABI-MOSTI grant (project no: 10-05-ABI-FB 037). The authors report no competing interest in this work and are solely responsible for the content of this article.

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