Application of whey protein-pectin nano-complex carriers for loading of lactoferrin

Accepted Manuscript Title: Application of whey protein-pectin nano-complex carriers for loading of lactoferrin Authors: Masoomeh Raei, Fakhri Shahidi, Majid Farhoodi, Seid Mahdi Jafari, Ali Rafe PII: DOI: Reference:

S0141-8130(17)31953-0 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.07.037 BIOMAC 7845

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

31-5-2017 5-7-2017 6-7-2017

Please cite this article as: Masoomeh Raei, Fakhri Shahidi, Majid Farhoodi, Seid Mahdi Jafari, Ali Rafe, Application of whey protein-pectin nanocomplex carriers for loading of lactoferrin, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.07.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Application of whey protein-pectin nano-complex carriers for loading of lactoferrin Running title: Whey protein-pectin nano-complex for lactoferrin Masoomeh Raei1,2, Fakhri Shahidi1, Majid Farhoodi3, Seid Mahdi Jafari4*, Ali Rafe2 1

Department of Food Science, Ferdowsi University of Mashhad, Mashhad, Iran

2

Research Institute of Food Science and Technology, Mashhad, Iran

3

Razi Vaccine & Serum Research Institute, Mashhad, Iran

4

Department of Food Materials and Process Design Engineering, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran Corresponding author: [email protected]

Research Highlights: 

The complex of whey protein and pectin can be formed at pH values of 3-4.



Lactoferrin was successfully loaded into WPI-pectin complex nano-particles.

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Complex formation was influenced by pH, WPI-HMP ratio, and acidifying method.

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WPI-HMP ratio of 2:1 via pre-blending at pH= 3.5 resulted in optimum LF carriers.

Abstract Our aim was to entrap lactoferrin (LF) in complex nano-particles of whey protein isolate (WPI)-high methoxyl pectin (HMP) with the ratios of 2:1, 1:1, and 1:2 through acidification at pH values of 3, 3.5, and 4. The zeta-potential, size, sedimentable-complex yield, LF content, encapsulation efficiency, SEM, AFM, FTIR, and DSC of nano-particles were investigated. Our results revealed that almost all 1

analyzed parameters of the final nano-particles were related to preparation pH value, WPI/HMP ratios, and acidification methods. In both methods of pre- and post-acidification, the zeta potential was decreased via decreasing of pH from 4 to 3 and particle size was increased at higher HMP ratios to WPI. In general, the pre-blending acidification provided a larger mass of complexes compared with post-blending counterparts. Also, the nano-particles produced by WPI/HMP with the ratio of 2:1 at pH= 3.5 had the smallest sizes. The highest LF content of the complexes as well as the optimal entrapment efficiency was observed at pH= 3.5, in both methods of post and pre-blending. Finally, the pre-blending by a ratio of 2:1 for WPI/HMP was chosen as the optimal treatment for producing nanoparticles containing LF. This was confirmed by SEM, AFM, FTIR, and DSC studies.

Keywords: Lactoferrin; Nano-particles; Complexation. 1. Introduction Lactoferrin (LF) is a critical bioactive component which has a major contribution in the healthy foods, baby formula, and pharmaceutical industry [1, 2]. LF is a non-heme iron binding glycoprotein that belongs to the transferrin family. It has a molecular weight of 80 kDa containing an iron atom in its interior and amino acid residues along its surface, such as histidine and tryptophan [2]. The main sources of LF are milk, blood, and neutrophils. LF is a whey protein and mostly exists in milk from a number of species. For example, the concentration of LF in camel, human, and bovine milks is 2.00– 6.00, 1.00–5.00, and 0.10–0.40 mg/mL, respectively [3]. It has a key role in breast-fed infants as a source of iron because of its iron-binding properties. Also, there are many important bioactive activities of LF including antioxidant, antitumor, antimicrobial, antifungal, antiviral, antibacterial, immunomodulatory and osteogenic activities [3]. But, these activities of LF are limited due to the sensitivity of LF against environmental or process stresses. Therefore, the stabilization of LF is necessary for optimal utilization and delivery of LF into body organs. 2

In the food and pharmaceutical sectors, encapsulation is a popular method for protection and delivery of sensitive bioactive compounds. The encapsulation process consists entrapping of bioactive ingredients into wall materials to from capsules. The produced capsules are classified into macro (>5000 mm), micro (1.0–5000 mm), and nano (<1.0 mm) [4]. Recently, nano-encapsulation systems have been incestigated in protecting and delivery of bioactive compounds due to many advantages such as improving bioavailability, higher solubility, and better permeability [5, 6]. Among nanoencapsulation systems, fabrication of nano-particles based on complex formation between biopolymers as an alternative method for the delivery of peptides and proteins is taken into consideration by researchers in the last few years [7, 8]. The bio-polymer complex nano-particles can be produced by the electrostatic interaction of proteins and polysaccharides. At pH values below the isoelectric point (pI) of proteins, the net-positive charge of proteins interacts with the anionic groups on the polysaccharides. The bio-polymer complexation is initiated by adsorption of ionic polysaccharides onto pre-formed protein nanoparticles (layer-by-layer method) or by direct interaction of protein/polysaccharide complexes at a specific pH value [9]. These interactions can be influenced by many factors including pH value, ionic strength, order of biopolymer addition, ratio of the biopolymers, conformation, charge density and the concentration of both proteins and polysaccharides [10]. The formation of biopolymer complexes can potentially lead to different functional properties such as solubility, surface activity, conformational stability, gel-forming ability, emulsifying and foaming properties, compared to the biopolymers taken individually [11, 12]. Therefore, encapsulation of bioactive compounds through nano-particles produced by association of two different biopolymers (e.g., protein-polysaccharide complex) can provide improved functional properties [10, 13-18]. Application of the appropriate wall materials is a critical step in producing nano-particles. The main biopolymers which can be used for production of complexed nano-particle carriers are proteins such as 3

β-lactoglobulin, zein, gelatin, soy protein, collagen, and albumin, and polysaccharides including pectin, alginate and chitosan [19, 20]. Whey protein is a natural emulsifier and stabilizer with the ability to produce thermodynamically and kinetically stable systems and its association with polysaccharides creates a complex with thickening and steric stabilizing behaviors [21]. For example, pectin is one of the most popular polysaccharides which has been widely used in a complexed form with whey proteins for producing multilayer encapsulation systems [22-24]. Pectin has been applied as a gelling, thickening and stabilizing agent in the food and pharmaceutical industries for many years. It is extracted from plant cell walls, especially from apple pomace, citrus fruits, and sugar beet root. Pectin is a family of galacturonic acid-rich polysaccharides including, rhamnogalacturonan I, homogalacturonan, and the substituted galacturonan rhamnogalacturonan II, and xylogalacturonan [25]. Therefore, the nano-particle complex of whey protein/pectin can be a good candidate for encapsulation of bioactive compounds. Our main goal of this study was to develop an optimized nano-particle formulation by whey protein-pectin complexes for entrapment and protection of LF. For this purpose, the main factors which could affect the properties of nano-particle weres investigated including the ratios of whey protein-pectin, pH value, and preparation method in two forms of pH decrease before (pre-blending) and after (post-blending) mixing the biopolymer solutions. 2. Materials and methods Raw camel milk was provided from dromedary camels (Camelus dromedarius) in Torkaman Sahra (Golestan, Northeast of Iran) and was kept refrigerated until further experiments. Camel lactoferrin was extracted and purified according to our previous work [26]. Whey protein isolate (96.2% protein, 1.54% ash and 2.01% moisture) was purchased from Davisco (Davisco Foods International Inc., Le Sueur, MN, USA). High methoxyl pectin was purchased from Merck (Darmstadt, Germany) (36% esterified, the molar mass of 100 kDa). Other used chemicals, reagents and solvents were all analytical grade and purchased from Merck (Darmstadt, Germany). 4

2.1. Biopolymer solution preparation The 2% (w/v) solutions of whey protein isolate (WPI) and high methoxyl pectin (HMP) were prepared, separately. For this work, the pectin and whey protein powders were dispersed in distilled water and stirred by a magnetic stirrer for 2 hours at 400 rpm at ambient and kept overnight in the refrigerator to ensure full hydration. WPI and HMP solutions were stirred for 15 minutes at 400 rpm by a magnetic stirrer before experiments. 2.2. Preparation of complex WPI/pectin nano-particles According to the literature, a total biopolymer concentration of 0.4% (w/w) in acidic pH values (3, 3.5 and 4) was used [27]. For complexion by post blending acidification, WPI and HMP stock solutions were initially diluted to concentrations corresponding to WPI: HMP ratios of 2:1, 1:1, and 1:2 for a total concentration of 0.4% (w/w). The diluted WPI and HMP solutions were adjusted to pH= 7.0, mixed at the predetermined ratios and the pH of the final mixture (pH= 7.0) was adjusted gradually to 4.0, 3.5, and 3.0 by addition of HCl (0.5 N) with gentle magnetic stirring for 3 min at each pH before decreasing the pH to the next value. For complexion by pre-blending acidification, diluted WPI and HMP solutions were individually acidified to pH= 4.0, 3.5, and 3.0 by the addition of HCl (0.5 N), then mixed at ratios of 2:1, 1:1 and 1:2 and gently stirred for 3 min. Lactoferrin entrapment was done by combining (0.4% w/w) lactoferrin into HMP solution followed by adding WPI solutions at determined ratios; for instance in a 2:1 ratio of WPI:HMP, the final suspension was containing 0.27% WPI, 0.13% HMP and 0.1% lactoferrin. A 30 g portion of this solution was centrifuged (HERMLE, Korea) (8000g/30 min at 22C) and the pellet was freeze-dried (Operon, Germany). 2.3. Zeta potential and particle size measurement The zeta potential and particle size at the liquid shear plane of the complexes was measured using a Zetasizer 2000 (Malvern Instrument Ltd., Malvern, Worcestershire, UK) with 10 ml samples. The size

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analysis of complex nanoparticles produced by WPI-HMP via pre-acidification method at pH= 3.5 is shown in Fig. 1. Fig. 1 2.4. Sedimentable-complex yield After 3 min stirring of WPI–HMP mixtures at a specific pH, 30 g samples were centrifuged at 8000g for 30 min and the pellets were freeze-dried (HERMLE, Korea). The dry pellets were kept at -18C until use. The sedimentable-complex yield, or precipitation rate of the biopolymers in complex formation, was calculated as follows: Sedimentable-complex yield=

Weight of dry pellets Weight of total biopolymers in 30 g of mixture

×100

2.5. Entrapment and encapsulation efficiency Complex-entrapped LF was determined by HPLC [28]. A freeze-dried pellet was suspended in 0.6 M TCA solution in order to reduce the pH value to 1.0 and release LF as WPI-HMP complexes dissociate at this pH value (the positive charge of WPI molecules and the neutral charge of HMP molecules prevents their binding to LF molecules). The suspension was stirred for 1 h and filtered on 0.2 mm nylon membrane to a final volume of 30 ml with 0.6 M TCA. A 2 ml portion of the filtrate was centrifuged at 16,000g for 10 min (HERMLE, Korea) to obtain samples for injection into the HPLC column. An HPLC (Knauer., Berlin, Germany), equipped with LiChrospher 100 RP-18 column (250 mm × 4 mm, 5.0 mm) and a C 15 column (250 × 4.5 mm, 5 µm) was used for LF quantification. Isocratic mobile phases of A (92.5 % water, 5% methanol, and 2.5% formic acid) and B (92.5 % methanol, 5% water, and 2.5% formic acid) at a flow rate of 0.5 ml/min were applied. LF was detected by a UV-visible detector at 280 and 320 nm and compared to a calibration curve for pure LF. The LF loading of the complexes was calculated as follows: LF loading (%) =

Weight of lactoferrin in dry pellet Weight of dry pellet

×100

The entrapment (encapsulation) efficiency was also calculated as below: 6

Encapsulation efficiency (%) =

lactoferrin content in 30 g of dissolved dry pellet lactoferrin content in 30 g of initial complex suspension

×100

2.6. Morphology study Selected samples were chosen for microstructural analysis using scanning electron microscopy (SEM) and atomic force microscopy (AFM). In the case of SEM study, some nano-particles were sprinkled onto a two-sided adhesive tape and then coated with a thin layer of gold. Morphological features of particles were then observed by a field emission scanning electron microscope (VP 1450., LEO, Germany). For AFM study, some nano-particles were dispersed in distilled water and then exposed to ultrasonic bath (30 kHz) for 90 min. Then, samples were mounted on a newly cleaved mica slide by injecting 5 µL of the dilute sample onto the surface. Mica slides were fixed to magnetic steel wafers using adhesive strips. The sample-mica assemblies were scanned using a Bio AFM (Ara-research, Iran). 2.7. FTIR study The microstructure analysis of the samples was examined by Fourier transform infrared spectroscopy (FT-IR). The IR spectra of the samples were recorded by a FT-IR spectrophotometer (Avatar, Thermo Nicolet, USA) using the attenuated total reflection technique. The spectrum was scanned in transmission mode from 400 to 4000 cm-1 wavenumber range. The dry samples were blended with KBr powder and pressed into a disk before spectrum acquisition. 2.8. DSC study Thermal behavior of WPI-HMP nano-particles was investigated by a differential scanning calorimeter (DSC-100., China). Samples were scanned at the temperature range of 0 – 400oC with the rate of 30oC/min. 2.9. Statistical analysis

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The experiments were all carried out in triplicate. The collected data were analyzed by randomized factorial design (ANOVA); the means were compared by Duncan's multiple range tests at the 95% level through SPSS version 21 (IBM, USA). 3. Results and discussion 3.1. Zeta potential and size of WPI-HMP nanocomplexes The zeta-potential and z-average of complex nano-particles is shown in Fig. 2. Our results revealed a decreasing zeta potential from pH= 4 to 3 in the nano-particles produced by both methods of pre- and post-acidification (Fig. 2a). In the nano-complexed formed via pre-acidification method, the lowest zeta potential was related to WPI/HMP formulation with a ratio of 1:2 at pH= 3. On the other hand, for nano-particles produced by the post-acidification method, the ratios of WPI-HMP did not have a significant influence (P<0.05) on the zeta potential of formed complexes at pH= 4; but, in pH values of of 3.5 and 3, a significant effect (P>0.05) was observed (Fig. 2b). Also, the lowest zeta potential in all treatments was found for the nano-particles produced by WPI-HMP with the ratio of 2:1 at pH= 3 (Fig. 2a). Fig. 2 In fact, the higher concentrations of HMP can provide a more negatively charged of z-potential. That’s why when there is an increase in HMP content, it leads to an increase in the negative surface charge of nano-particles confirmed by increasing the negatively charged z-potential. Similar observation of increasing z-potential at higher concentrations of gums was reported by Mirhosseini, Tan, Hamid and Yusof [29] in the formulation of emulsions. A high zeta potential (either positive or negative), maintains a stable system. Indeed, a higher electric charge on the surface of emulsion droplets will prevent aggregation of the droplets in buffer solution because of the strong repulsive forces among droplets [30].

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As can be seen in Fig. 3, in both methods of post and pre-acidification, the size of nano-particles was increased via decreasing the ratio of WPI to HMP. In the case of pre-acidification, WPI-HMP with the ratio of 2:1 at pH= 3.5 provided nano-particles with small sizes, while the biggest particle sizes were seen by WPI-HMP ratio of 1:2 at pH= 3.5 (Fig. 3a). In the produced complex nano-particles by postacidification method, the biggest and smallest particle sizes was observed in WPI-HMP ratios of 1:2 and 2:1 at pH= 3, respectively (Fig. 3b). Fig. 3 A lower pH value results in an increase in the positive charge of whey proteins and as a result, the negatively charged molecules of pectin interact with the highly positive charge surface of whey protein which then, creates complexed particles with a smaller size. On the other hand, the size of nanoparticles was increased at higher ratios of HMP to WPI. It may be due to increasing the thickness of outer layer with the application of higher biopolymer concentrations (WPI and pectin). In agreement with the results obtained in the present study, Peinado, Lesmes, Andrés and McClements [31] reported that addition of polysaccharides to the protein nanoparticle suspension caused an appreciable increase in the measured mean particle diameter, which can be attributed to the formation of a polysaccharide coating around the protein particles and/or to some aggregation of the protein particles due to bridging flocculation. In general, post-acidification method provided larger particles compared to the pre-acidification method (Fig. 3b). This can be explained by the gradual acidification in a post-acidification method which allows more time for complexes to rearrange in a compact structure. In comparison, no time is allowed to the pre-blend system to arrange because the complex formation is spontaneous at the combination of biopolymer solutions [32]. 3.2. The sedimentable-complex yield and encapsulation efficiency of WPI-HMP nanocomplexes

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In both post- and pre-acidification methods, the sedimentable-complex yield was increased at higher ratios of WPI and decreased by increasing pH value from 3 to 4 (Fig. 4). This can be explained by better interaction of negative surface charges of pectin and positive surface charge of whey proteins when decreasing the pH value [32]. Also, as shown in Fig. 4, the sedimentable-complex yield in the prepared complex nano-particles by pre-acidification method was higher than post-acidification. Fig. 4 The LF loading and its encapsulation efficiency in WPI/HMP complex nano-particles prepared by different treatments is shown in Fig. 5. The highest and lowest LF content of nano-particles was observed in WPI-HMP complex with a ratio of 2:1 through post-acidification at pH= 4, and those prepared by both acidification methods at pH= 3, respectively (Fig. 5a). Also, at pH= 3.5, the preblending method provided nano-particles with a significantly higher content of LF compared to the post-acidification method (P<0.005). In the case of encapsulation efficiency, the pre-acidification method significantly (P<0.005) resulted in a higher encapsulation efficiency than the post-acidification one (Fig. 5a). Fig. 5 As an example, the HPLC analysis of WPI-HMP (2:1) complex nano-particle produced by the preblending method at pH= 3.5 revealed the loading of LF in this nanocomplex (Fig. 6). Fig. 6 The results of section 3.1 and 3.2 showed that the nano-particles prepared by WPI-HMP with the ratio of 2:1 through pre-acidification method at pH= 3.5 was the optimum carrier for LF. So, for further analysis, this treatment was selected and its results for morphology, FTIR, and DSC are given in the following sections. 3.3. Morphology and FTIR analysis of nanocomplexes loaded with LF

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The morphology of WPI-HMP nanocomplexes (with a ratio of 2:1) prepared by pre-acidification was studied through SEM and AFM (Fig. 7). It was revealed that the nano-particles had spheroid shapes, nano-scale size, and exhibited a broad range of particle sizes. Also, there was an interesting similarity in size distribution of nanoparticles observed in both SEM and AFM images. Fig. 7 At the next step, the complexation of WPI and HMP and the nature of bonds in the final complex nanoparticles was analyzed by the FTIR data. The infrared absorption spectrum of pure LF, WPI, HMP, free and LF-loaded WPI/HMP nano-particles is shown in Fig. 8. Fig. 8 The pure LF showed the characteristic protein absorption band peaks; two peaks were observed in 1645.26 cm-1 and 1544.15 cm-1 related to C=O stretching vibration of the peptide group and N–H bending with contribution of C–N stretching vibrations, respectively (Fig. 8a) [33, 34]. The peaks at 1747.33 cm-1 and 1638.97 cm-1 correspond to the esterified and non-esterified carboxyl groups, respectively (Fig. 8b) [35]. Also, the peaks at 1655.74 cm-1, 1532.47 cm-1, and 1392.55 cm-1 which correspond to the C=O, C-H bond deformation vibrations of Amide II, and C-N stretching of Amid I structures, respectively (Fig. 8c) [36]. The FTIR of complexed biopolymers in Fig. 8d and 8e showed peaks in 1646.64 cm-1 and 1655.74 cm-1 for complex nano-particles loaded with LF and without it, respectively. These peaks are related to the non-esterified carboxyl groups of pectin in particles spectra. Also, the FTIR of free and LF-carrying complex nano-particles showed the peaks at 1530.55 cm-1 and 1532.47 cm-1, respectively; these are related to the N-H bond deformation vibrations of proteins Amide II. Finally, the peaks observed in 1392.55 cm-1 or 1397.87 cm-1 are contributed by the C-N stretching of proteins Amid I. Our results showed that there was no notable difference between FTIR spectra of free and LF-carrying complex-

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nano-particles suggesting that LF encapsulation within nanocomplexes was mostly a physical entrapment process. 3.4. Thermal analysis of WPI-HPM nanocomplexes The DSC thermograms of WPI, HMP, pure form of LF, and LF-loaded WPI-HMP nanocomplexes prepared by pre-acidification method is shown in Fig. 9. The WPI exhibited an endothermic peak in the range of 81-123oC, which indicates a great conformity with the results of Gauche, Barreto and Bordignon-Luiz [37] (Fig. 9a). The DSC of pectin showed an endothermic peak in the range of 79-119oC, which is corresponding to the glass transition temperature of pectin; it can be also related to the removal of certain water molecules from the pectin sample (Fig. 9b) [38]. In the pure LF, there was a sharp endothermic peak around 100oC, which has been previously attributed to the denaturation temperatures (Tm) of LF protein (Fig. 9c) [39]. In the case of WPI-HMP complex nano-particles, there was also a peak around 100oC possibly due to the denaturation temperatures (Tm) of LF (Fig. 9d). The peak of LF in complex nano-particles was smaller than the pure LF that can be related to entrapping of LF inside nano-particles. Fig. 9 4. Conclusion The results of this study showed that LF can be loaded in the biopolymer complexes of whey protein isolate and high methoxyl pectin at pH values of 3 to 4. This interaction was influenced by pH, the ratio of WPI-HMP, and the preparation method (including post- and pre-acidification). The smallest particle size was provided by applying WPI-HMP in a ratio of 2:1 through post-acidification at pH= 3.5. Production of large particles by post acidification could be related to the gradual acidification which allows more time for complexes to rearrange in a compact structure. The sedimentable-complex yield of nano-particles was increased at lower pH values due to better interaction of negative HMP molecules with the positive WPI surface. Also, the pre-acidification method provided higher sedimentable-complex yield, more LF loading, and higher encapsulation efficiency. In general, our results introduced the nano-particles prepared by WPI-HMP complex at a ratio of 2:1 through preacidification method in pH= 3.5 as an optimal nanocarrier for LF which can be used in the food formulations and pharmaceutical industries.

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Figure Captions: Fig. 1. Droplet size analysis results of nano-particles contains lactoferrin by a dynamic light scattering (DLS). Fig. 2. Zeta potential (mV) of WPI–HMP nano-particles at 0.4% total polymer. Fig. 3. Size (nm) of WPI–HMP nano-particles at 0.4% total polymer. Fig. 4. Sedimentable complex yield (%) of WPI–HMP nano-particles at 0.4% total polymer. Fig. 5. LF content and encapsulation efficiency of WPI–HMP nano-particles at 0.4% total polymer. Fig. 6. HPLC graphs of nano-particles prepared by pre-blending method. Fig. 7. SEM micrographs of nano-particle produced by WPI-HMP complex. Fig. 8. FTIR spectra of LF, WPI, HMP, WPI-HMP complex with and without LF. Fig. 9. DSC thermograms of LF, WPI, HMP, and WPI-HMP complex with and without LF.

17

Fig. 1. Droplet size analysis results (by a dynamic light scattering (DLS) technique) of complex nano-particles loaded with lactoferrin for pre-acidification method of WPI-HMP at pH= 3.5.

18

a 4

0

pH 3

3.5

Zeta potential (mV)

-10

-20

-30

-40

-50

-60

4

0

pH 3

3.5

-5 -10

Zeta potential (mV)

b

-15 -20 -25 -30 -35 -40 -45 -50

Fig. 2. Zeta potential (mV) of WPI–HMP nano-complexes at the ratios of 2:1 ( ), 1:1 (

) and 1:2 ( ):

(a) pre-blending acidification and (b) post-blending acidification.

19

a

1000 900 800

Size (nm)

700 600 500 400 300 200 100 0 4

3.5

3

pH

b

1000 900 800

Size (nm)

700 600 500 400 300 200 100 0 4

3.5

3

pH Fig. 3. Size (nm) of WPI–HMP nano-complexes at the ratios of 2:1 (

), 1:1 (

) and 1:2 (

):

(a) pre-blending acidification and (b) post-blending acidification.

20

a Sediment. Comp. yield

25%

20%

15%

10%

5%

0%

4

3

3.5 pH

b

14%

Sediment. Comp. yield

12% 10% 8% 6% 4% 2%

4

0%

3.5

3 pH

Fig. 4. Sedimentable complex yield (%) of WPI–HMP nano-complexes at ratios of 2:1 (

), 1:1 (

) and 1:2 (

):

(a) pre-blending acidification and (b) post-blending acidification.

21

3%

a

LF Loading

2%

2%

1%

1%

0% 2:1-post

2:1-Pre

Ratio (WPI-HMP)-acidification method

b Encapsulation efecciency

30% 25% 20% 15% 10% 5% 0% 2:1-POST

2:1-PRE

Ratio (WPI-HMP)-acidification method Fig. 5. (a) LF loading (%) and (b) encapsulation efficiency (%) of WPI–HMP nano-complexes at the pH values of: 4(

), 3.5 (

) and 3 ( ).

a

22

b a

Fig. 6. The HPLC graphs of the (a) pure LF and (b) WPI–HMP (2:1) nano-complex loaded with LF and prepared by preblending acidification method at pH = 3.5 before centrifugation (complex form).

23

a

b

c

Fig. 7. (a) and (b) SEM, and (c) AFM micrographs of nano-particles produced by WPI-HMP complex at a ratio of 2:1 through preblending acidification in pH= 3.

24

a

b

c

d (a) pure LF, (b) HMP, (c) WPI, (d) WPI-HMP-complex without LF, and (e) WPI-HMP complex Fig. 8. FTIR spectra of: loaded with LF.

e

25

a

b

c

d d

Fig. 9. DSC thermograms of (a) WPI, (b) HMP, (c) pure LF, and (d) WPI-HMP complex nano-particles contains LF.

26