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Continuous preparation of zein colloidal particles by Flash NanoPrecipitation (FNP)
Journal of Food Engineering 127 (2014) 103–110
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Continuous preparation of zein colloidal particles by Flash NanoPrecipitation (FNP) Kang-Kang Li a, Xi Zhang a, Qin Huang b, Shou-Wei Yin a,⇑, Xiao-Quan Yang a,⇑, Qi-Biao Wen a, Chuan-He Tang a, Fu-Rao Lai a a b
Research and Development Center of Food Proteins, Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, PR China School of Business Administration, South China University of Technology, Guangzhou 510640, PR China
a r t i c l e
i n f o
Article history: Received 15 July 2013 Received in revised form 27 November 2013 Accepted 3 December 2013 Available online 11 December 2013 Keywords: Flash NanoPrecipitation Confined Impinge Jet Zein colloidal particle Sodium caseinate
a b s t r a c t This study utilized a facile and continuous technique termed Flash NanoPrecipitation (FNP) to produce zein colloidal particles (ZP) without or with sodium caseinate (NaCas) as the stabilizer. The colloidal particle preparations were performed through a self-made Confined Impinge Jet (CIJ) mixer, and compared with traditional antisolvent processes. The influences of FNP processing parameters on the size of zein colloid particles, including flow rates, outlet configurations, solute concentrations and ethanol concentrations, were extensively discussed. The influences of viscous properties, buffering capacity and self-assembly of zein or NaCas on particle size and size distributions were also discussed. Particle sizes of ZP produced by the FNP technique are below 350 nm, even at high zein concentrations (2.5– 7.5% w/v). Solvent systems with different ethanol concentrations yielded zein colloid particles with similar size, showing an attractive feature for industrial applications and encapsulating actives with different solubility in ethanol–water binary solvent. This study opens a promising pathway for continuously producing ZP via the FNP procedure. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Protein colloidal particles with generally regarded as safe (GRAS) status are promising as delivery system in food industry. The inherent biodegradability and biocompatibility of protein colloid particles make them more effective in maintaining the functions of actives in extracellular matrix than synthetic polymers (Chan and Mooney, 2008; Stegemann et al., 2007). The amino and carboxyl groups of amino acid residues provide protein molecules with different charges depending on the pH environments while synthetic polymers usually lack such functional groups. Therefore, proteins can be easily modulated to fabricate vehicles for charged actives via electrostatic interactions (Chen et al., 2006). Protein colloidal particles are favourable to load uncharged actives via hydrophobic interactions since proteins also have notable hydrophobic domains (Chen et al., 2006). Zein, a prolamin, is abundantly available and utilized for various industrial applications (Lawton, 2002; Shukla and Cheryan, 2001). Zein is an ideal material for preparing colloidal particles due to its unique solubility and self-assembly property. Many works were successfully carried out to prepare zein colloidal particle vehicles
⇑ Corresponding authors. Tel.: +86 20 87114262; fax: +86 20 87114263. E-mail addresses: [email protected] (S.-W. Yin), [email protected] (X.-Q. Yang). 0260-8774/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2013.12.001
for drug delivery (Patel et al., 2010a; Muthuselvi and Dhathathreyan, 2006; Lai and Guo, 2011), nutrition protection (Luo et al., 2011, 2012), encapsulating antimicrobials (Xiao et al., 2011; Wu et al., 2012) and antioxidants (Parris et al., 2005; Wu et al., 2012). However, the zein colloidal particles are usually not water-soluble, and are easy to aggregate during the drying process of zein particle dispersions. When incorporated in liquid food matrices they may aggregate to form the flocculates or precipitates, which limit to some extent their usages in food industries. A general way to break these limitations is by coating a layer of stabilizers around zein colloid particles, such as chitosan (Luo et al., 2011), NaCas (Patel et al., 2010b) and polyvinylpyrrolidone (PVP) (Hurtado-López and Murdan, 2006). NaCas, a milk protein, has been proved to be an ideal stabilizer for ZP (Patel et al., 2010b; Li et al., 2012, 2013) against aggregation in neutral condition and drying process, due to the combination of electrostatic and steric stabilization (Dickinson, 1997). Antisolvent process is an approach to produce colloidal particles via creating supersaturation by mixing a solvent with an antisolvent. It is a low-cost and easy to prepare ZP. However, traditional antisolvent processes are discontinuous and difficult to scale up. Spray drying and supercritical anti-solvent (SAS) processes are continuous and scalable methods. However, ZP produced by spray drying is microscale (Xiao et al., 2011; Xiao and Zhong, 2011), and spray drying technique is not suitable for encapsulating
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temperature-sensitive actives. SAS processes produce micro-scale zein colloidal particles, and toxic solvents such as methanol, acetone and dimethyl sulfoxide (DMSO) were usually included in the SAS procedure (Zhong et al., 2008; Hu et al., 2012). In addition, it is costly and need complex equipments. Based on the advantages and problems of above-mentioned methods, there is no ideal method to produce colloidal ZP. In this work, an FNP approach that was firstly investigated by Johnson and Prud’homme (Johnson, 2003; Gindy et al., 2008), was utilized to solve those problems in the preparation of zein colloid particles. In the FNP process, an antisolvent stream with or without stabilizers and a solvent stream containing actives collide in a confined mixing chamber which limits bypassing of high mixing intensity (Johnson, 2003). There are 3 main advantages for the FNP approach in producing ZP particles when compared with classical antisolvent procedures: (1) flow rate itself provides driving force for efficient mixing; (2) particle size can be quantitatively controlled by processing parameters; (3) the scale-up is possible from laboratory apparatus to industrial continuous production (Johnson and Prud’homme, 2003a). The FNP procedure had been utilized to produce colloidal particles of active small molecules such as carotenoid (Johnson and Prud’homme, 2003b), and synthetic polymers such as polystyrene (Zhang et al., 2012). However, biopolymerbased colloidal particles had not been produced via an FNP procedure, and the properties of the formed colloidal particles were unknown. Therefore, the synthesis of ZP by the FNP process using a selfmade CIJ mixer was performed in this research. Several antisolvent processes under magnetic agitation were performed and compared with the FNP approach. To systematically study the FNP process for producing ZP, zein colloidal particles were produced at a variation of flow rates, outlet configurations, solute concentrations and ethanol concentrations. We also explored the effects of protein properties on zein particle size and size distributions, including viscous property, buffering capacity and self-assembly.
Fig. 1. Actual laboratory setup of the CIJ mixer for FNP process.
2. Materials and methods 2.1. Materials Zein was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Food-grade NaCas was obtained from Murray Goulburn Cooperative Co., Limited, Australia. All other chemicals used were of analytical grade. 2.2. Confined impingement jets Fig. 1 shows the actual setup of a self-made confined impinging jet (CIJ) mixer. The CIJ mixer included two separate streams. Here, a syringe containing zein solutions was placed at the inlet of Stream 1 (left), and a syringe containing Milli-Q water with or without NaCas was placed at the inlet of Stream 2 (right). The FNP process was carried out via hand operation since previous research proved that turbulent flow could be achieved motion by small, low-friction syringes with hand (Han et al., 2012). In addition, hand motion can provide the solvent and antisolvent with high pressure which was needed in the FNP process. The schematic image (Fig. 2a) shows that the solvent and the antisolvent streams come into the CIJ device from left and right site, respectively, collide in the centre cavity. The resultant particle dispersions come out from the downside outlet nozzle. Fig. 2b shows the dimension of the CIJ chamber in detail. The main cavity was cylindrical in an upright position, with two inlets and adapters fitted with the syringes and an outlet nozzle. The two nozzles with diameters of 3 mm (O1) and/or 0.5 mm (O2) were used as the
Fig. 2. Schematic representations of the FNP process (a) and the dimensions of the self-made CIJ mixer (b).
outlet nozzle to investigate the effects of outlet configurations on the formation zein colloid particle. Sealed tape was used in each joint to prevent leakage of the dispersions during FNP process. 2.3. Particle synthesis via the FNP approach The mixing efficiency in the FNP process is fully dependent on collision of fluids from each side, and the driving force depends on flow rate and diameter of outlet nozzle. Some other parameters, including protein concentrations, solvent types and solventto-antisolvent ratios in the FNP process also affect the formation of zein colloid particles. Therefore, the influences of selected FNP parameters on the particle formations were extensively investigated to optimize the preparation procedure of zein colloid particles via FNP process. 2.3.1. Influence of flow rate and outlet configurations An aliquot of 4 mL of 2.5% w/v zein in 80% v/v ethanol–water binary solution was placed in the syringe of Stream 1 (left), and 10 mL of deionized water with or without 1% w/v NaCas was placed in the syringe of Stream 2 (right). The trails were performed
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using O1 or O2 outlet. The mixing times are 4, 5, 10, 20, 40, 80 s, respectively. The flow rates of zein solution are about 5.0, 4.0, 2.0, 1.0, 0.5, and 0.25 m/s, respectively, and the corresponding flow rates of water are about 12.7, 10.2, 5.0, 2.5, 1.3, and 0.64 m/s, calculated by Eq. (1): 2
Flow rate ¼ V=ðt p r Þ
ð1Þ
where V is the volume of mixed solution, t is the time for this procedure and r is the radium of the inlet nozzle. 2.3.2. Influence of zein concentration Ten millilitres of deionized water with or without NaCas was mixed with 4 mL of 80% v/v ethanol solution containing zein via the CIJ mixer. The mixing time was 10 s and O2 outlet was used. Zein concentrations were set as 0.1, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0% w/v to investigate the influences of solute concentrations on particle size. For NaCas-stabilized ZP, the correspond NaCas concentrations were 0.04, 0.2, 0.4, 1.0, 2.0, 3.0 and 4.0% w/v, with a zein/NaCas ratio of 1:1. To investigate the influence of viscosity on particle size, 0.5% w/v zein solution and 0.2, 0.4, 1.0, 2.0, and 3.0% w/v NaCas solutions were mixed at the same conditions through the CIJ setup. 2.3.3. Influence of ethanol concentration Ten millilitres of deionized water with or without NaCas was mixed with 4 mL of ethanol solution containing zein via the CIJ setup. The mixing time was 10 s and O2 outlet was used in this FNP process. The ethanol concentrations were 60, 70, 80 and 90% v/v. 2.3.4. Influence of solvent-to-antisolvent ratio Deionized water with or without NaCas was mixed with 4 mL of ethanol solution containing zein via the CIJ setup. The mixing time was 10 s and O2 outlet was used in this FNP process. The volumes of deionized water were 4, 8, 12, 16 and 20 mL. 2.4. Comparison of each mixing processes of solvent and antisolvent In the FNP process, 10 mL of deionized water (as an antisolvent) with or without 1% w/v NaCas were mixed with 4 mL of 80% v/v ethanol solution (solvent) containing 2.5% w/v zein using the CIJ device. The trails were performed with O2 via the CIJ device and the mix time was 10 s. ZP dispersions were directly collected at the end of the outlet of the CIJ device. Three classic antisolvent procedures, including dropwise addition of solvent into antisolvent, rapid addition of antisolvent into solvent, rapid addition of solvent into antisolvent were performed and compared with the FNP process. In the dropwise addition approach, 4 mL of 2.5% zein solution was dropwise added into 10 mL of de-ionized water with or without NaCas to yield ZP particles. In the rapid addition approaches, the solvents/antisolvents were poured into antisolvents/solvents to produce ZP according to the procedure of Li et al. (2013), other experimental parameters coincided with the dropwise addition approach. In traditional antisolvent processes, vigorous agitation or shearing is effective in producing colloidal ZP, but it has strong limitation in spatial variations of fluid, and it is even tougher to maintain performance upon scale up (Johnson, 2003). Therefore, a moderate agitation (500 rpm) was adopted in this study. 2.5. Analysis of particle size and surface charge The particle size and zeta-potential of ZP dispersions was measured using a Zetasizer Nano (Malvern Instruments Ltd., UK). Before measurements, all the dispersions were subjected to the centrifugation at 4000 rpm for 10 min to separate out the minor
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part of larger aggregates. The dispersions were measured for particle size at within 30 min at 25 °C. The results reported are an average of three readings. 2.6. Morphology observation Morphological structures of plain and NaCas ZP were obtained by a Quanta 200 Field emission scanning electron microscope (FEI company, America). The freeze-dried powers were utilized to investigate morphology character of zein colloidal particles. To observe surface morphology, powdered samples were loosely glued onto a conductive adhesive mounted on a stainless steel stage. Subsequently, they were coated with a thin (<20 nm) conductive gold and platinum layer using a sputter coater (Hummer XP, Anatech, CA). Representative SEM images were reported. 2.7. Statistics Statistical analyses were performed using an analysis of variance (ANOVA) procedure of the SPSS 13.0 statistic analysis program, and the differences between means of the trails were detected by a least Duncan test (P < 0.05). 3. Results and discussion The Flash NanoPrecipitation process is shown schematically in Fig. 2a. The Stream 1 (zein solution) was rapidly mixed with an incoming Stream 2 (water or NaCas solution as the antisolvent). The kinetic energy of each jet stream is converted into a turbulent-like motion through a collision and redirection of the flow in this small cavity of the CIJ device (Johnson and Prud’homme, 2003a), resulting in a high energy dissipation which occurs along the jet shear layers and in the impingement plane (Yeo, 1993). Finally, zein colloid particle dispersions drained directly out of the outlet after the solvent and antisolvent were efficiently mixed in the chamber of the CIJ setup. 3.1. The influences of FNP parameters on the formation of zein particles 3.1.1. Effects of flow rates and outlet configurations It is important to note that the precipitation process could be divided into two stages: the first one is the particle formation, which is modelled as Brownian aggregation and occurs on much faster time scales than the mixing process; the second one is that particles slowly grow in mass (Cheng et al., 2010). As mentioned earlier, both plain and NaCas ZP have electrostatic repulsion against size growth by positive and negative charges, respectively. The particle size of fresh ZP was measured, reflecting the mixing efficiency of various procedures. To investigate the influences of mixing conditions of the FNP process on the formation of zein particles, the colloid particles were produced via the CIJ setup at a series of flow rates and two outlet nozzles. Particle sizes of zein colloid particles are shown in Fig. 3. Sizes of plain ZP produced using O1 and O2 outlet decreased with the increases of flow rates from 0.25 to 2.0 m/s (Fig. 3a). Johnson and Prud’homme built a theory to explain the relationship between flow rates and particle sizes, and the basic premise behind Flash NanoPrecipitation is that high flow rate produced rapid micromixing in a short time scale (Johnson, 2003; Zhang et al., 2012). Particle growth is controlled by the micromixing time, and the micromixing time decrease with an increase of flow rate (Gindy et al., 2008; Johnson and Prud’homme, 2003a). In addition, sizes of plain ZP stopped decreasing after the flow rate reached a critical point (Fig. 3a). At that time, mixing time is equivalent to
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200
200
Size (nm)
(b) 250
Size (nm)
(a) 250
150
150 O1 O2
O1 O2 100 0.1
1
10 -1
Flow rate (m s )
100 0.1
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Flow rate (m s )
Fig. 3. Change in particle size as a function of flow rate of Stream 1 and outlet configuration for ZP without (a) and with NaCas (b).
the characteristic time of particle formation, and further increases in flow rate and decrease in mixing time no longer affected the particle size (Han et al., 2012; Cheng et al., 2010; Johnson and Prud’homme, 2003a; Zhu et al., 2010). A critical point of plain ZP produced by O1 and O2 appeared at the same flow rate, i.e., 2.0 m/s. As expected, plain ZP produced by O1 outlet was roughly bigger than that by O2 outlet except the particles produced at the flow rate of 1.0 m/s. (Fig. 3a). This phenomenon was attributed to different stretching history of the fluids, including ethanol solution (the solvent) and de-ionized water (the antisolvent). The outlet area of O1 was much bigger than the whole inlet area, so mixed solutions dripped from CIJ mixer due to gravity without stretching. While the area of O2 was half of the whole inlet area, changing the deformation history of the fluid and stretched the fluid within the CIJ setup (Johnson and Prud’homme, 2003a), providing the stretched fluid with an additional pressure and a high exiting flow rate equals to the sum of 2 inlet streams and finally improving the mixing efficiency. In all, mixing efficiency with both O1 and O2 increased upon the increase in flow rate of inlet streams, resulting in smaller zein colloid particles. However, the size gap of ZP produced using O1 and O2 outlet nozzles was narrowed with the increase in flow rate (Fig. 3a). A similar result was reported that outlet configuration has a significant effect at low flow rate and a limited effect at a higher one (Johnson and Prud’homme, 2003a). On the other hand, NaCas stabilized ZP was produced at the same condition with plain ZP except the addition of NaCas, and the result is shown in Fig. 3b. NaCas ZP showed a similar trend at the variation of flow rates and outlet configurations when compared with plain ZP. The particle size decreases from about 240 nm to about 140 nm until the flow rate reached the critical point which also appeared at 2.0 m/s. In addition, the FNP approach with O2 outlet produced slightly smaller particles than that with O1 outlet, and size gap of ZP produced by O1 and O2 outlet nozzle also decreased upon increasing low flow rate (Fig. 3b). However, the difference between the two size curves is narrower than plain ZP (Fig. 3). Although the detailed understanding on the formation mechanism of zein colloid particles is outside the scope of this paper, the reasons may be that the fluid stretching and deformation effects of NaCas solution is weak or the effects of the alteration in size of the outlet were limited by the absorption of NaCas onto zein colloidal particles. 3.1.2. Effects of zein concentrations Generally, zein concentrations are directly proportional to the yield of zein particles. Thus, the influence of zein concentrations on the FNP particles was investigated. Zein concentrations in Stream 2 varied from 0.1% w/v to 10% w/v. Particle size distribu-
tions of plain ZP produced by 0.1–7.5% w/v zein solutions are shown in Fig. 4a, whereas large precipitates occurred when zein concentration is 10% w/v. The zein concentrations had marked effects on zein self-assembly during the FNP process by which zein self-assembled to form colloid particles at a concentration lower than 7.5% w/v. After that, zein molecules tended to form formed sheets in ethanol solution and zein particles fused together after the solvent evaporation process (Wang and Padua, 2010). A lower concentration of solute in organic solution generally corresponds to a shorter time for nucleation and growth, and stabilization resulted in a smaller particle size in FNP process (Johnson and Prud’homme, 2003b). Zein particles also had a bigger size at higher zein concentration upon solvent evaporation because the increase of zein concentration turns more zein monomers or dimers into aggregation (Wang and Padua, 2010). Interestingly, Fig. 4a shows a different result that big particles were produced at 0.1% w/v. In contrast, particle sizes remained 130–140 nm at the zein concentrations of 0.5–5.0% w/v and increased to about 205 nm at the zein concentration of 7.5% w/v. The phenomenon may be attributed to the nature of zein material. Proteins usually have buffering capacity. In acid condition, basic amino acid residues mainly account for the capacity. Variations of pH value by changing protein concentration are usually not taken into consideration. However, zein ethanol solution is weak acid (pH 5.61 for 0.5% w/v) and the pH value of zein dispersion decreases with the increase of zein concentrations, because zein is deficient in basic amino acid residues (Shukla and Cheryan, 2001). Zein solutions (0.1% w/v) had a pH of 6.54 which is close to the isoelectric points of zein (Lawton, 2002). Therefore, the zein particles produced at the isoelectric regions during the CIJ setup were labile to aggregate to yield larger particles with particle seize about 700 nm. Particles in the range of 135–205 nm were produced at higher zein concentrations because the pH values of these zein solutions were below 4.5. When zein concentrations increased from 0.5 to 7.5% w/v, the pH further decreased from 4.5 to 3.6, limiting the increase of particle size. There are two possible explanations why the pH of zein solution affects particle size of the resulted nanoparticles: the pH during mixing affected the selfassembly of zein and particle formation, or the pH after the mixing decided the charge of ZP and then affected the particle size. Thus, we adjusted pH value of all mixed dispersion to 3.6 to rule out the difference of final pH, and the results shows in Fig. 4a. Plain ZP produced by 0.1% w/v had a bigger particle size about 750 nm after the pH adjustment because the big particle was unstable and grew over time. Apart from that, there were no significant changes after adjusting pH value. Therefore, the pH values during the formation of ZP rather than the final pH significantly influenced the final particle sizes.
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6
8
Concentration of zein (wt%)
(c) 400 Size (nm)
300 200 100 0
pH
7
5
3 0
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Concentration of NaCas (wt%) Fig. 4. Dependence of size on zein concentration in Stream 1 of the FNP process for ZP without (a) and with NaCas (b). (c) Change in particle size as a function of NaCas concentration.
For NaCas ZP, particles were also produced at zein concentrations of 0.1–10% w/v and NaCas concentrations of 0.04–4% w/v with a zein/NaCas ratio of 1:1. The sizes of NaCas ZP significantly increased from 127 to 327 nm upon increasing zein concentrations from 0.1 to 7.5% w/v (P < 0.05), as shown in Fig. 4b. Concomitantly, large precipitates also formed at 10% w/v. As expected, zein concentrations had a different influence on sizes of NaCas ZP. The addition of NaCas changed two factors in the mixing process, viscosity and pH value. Zein ethanol solution is acid and zein is deficient in buffering capacity, but the addition of NaCas turned the acid condition to neutral pH values (pH 6.4–6.95). The phenomenon showed strong buffering capacity of NaCas, due to its basic amino acid residuals, as well as the presence of phosphoserine (Dickinson, 1997). On the other hand, the viscosity of Stream 2 increased directly proportional to the exponent of concentrations of NaCas (Hermansson, 1975), which may affect mixing efficiency. The relationship between viscosity and mixing efficacy is complex. The mixing time generally scales with viscosity, but it has no relationship with viscosity when turbulent diffusion or inertial convective mixing were active (Johnson and Prud’homme, 2003a). To investigate whether the viscosity affects particle size, 0.5% w/v zein solution was mixing with NaCas solutions at a variety of concentrations from 0.2 to 3% w/v, and the result shows in Fig. 4c. The particle size remained 130–140 nm with the increase of NaCas concentration, indicating that in this case the viscosity of NaCas solution has little influence on particle size.
We concluded that the pH during mixing is the key factor of particle formation. As the solvent gradually became hydrophilic, the self-assembly is mainly driven by amphiphilicity (Wang and Padua, 2012). It was believed that zein particles formed by a layer-by-layer by adsorption to a central core or nucleus, and the radial growth occurred by hydrophobic associations (Wang and Padua, 2010). Comparing to the neutral condition, the acid condition charged zein molecules with +23.6 mV which limits hydrophobic interactions. Therefore, plain ZP has small particle sizes at 0.5% w/v, and when the zein concentration increases to 7.5% w/v, the further decrease of pH limits the growth of the particle size. As for the NaCas ZP, all dispersions produced at a variety of zein concentrations are in neutral condition, so then zein molecules carries a few charge, and the sizes of NaCas ZP gradually increase with the increase of zein concentrations. The schematic illustration of plain and NaCas ZP produced by FNP shows in Fig. 5. In previous study, the size of polystyrene particle (Zhang et al., 2012) significantly increases from less than 100 nm to about 140 nm when polystyrene concentration increased from 0.1 to 1% w/v, which was similar with NaCas ZP. This phenomenon was due to that polystyrene has no charged groups to limit the hydrophobic interaction at high concentrations. While the aggregation of charged zein molecules at acid pH was to a great extent limited by electrostatic repulsion, especially at high zein concentrations (2.5– 7.5% w/v). Therefore, the charged groups of amino acid residuals endowed proteins with better controlled behaviour at high concentrations than many synthetic polymers.
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in a narrow range from 120 to 160 nm. Ethanol concentrations can be set in a wide range to modulate to encapsulate actives with different solubility in an ethanol–water binary system without big changes of particle size, which is an attractive feature of formation of ZP via the FNP procedure. 3.2. Comparison between classic antisolvent procedures and FNP approach
Fig. 5. A schematic illustration of Plain (left) and NaCas (right) ZP produced via FNP process. For Plain ZP, ethanol solution (red) and water (blue) were impinged into CIJ mixer. Then pH of mixed solution is below 4.0, so positive charges shows around zein. For NaCas ZP, ethanol solution (red) and NaCas solution (green) were impinged into CIJ mixer. The pH is neutral, so zein is uncharged. Particle sizes produced by 5% zein solution show at last. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.1.3. Effect of ethanol proportion The aim of this work is to build a colloid particle formation system in which actives with different solubility in ethanol–water binary solvent may also is included in the practical application. Thus, the effects of ethanol concentration on the zein particle formation via the CIJ setup were investigated. The mean diameters of colloidal particles produced with stock solutions containing 60– 90% v/v ethanol are plotted in Fig. 6. Plain ZP showed smaller size than NaCas ZP, as discussed before. For both plain ZP and NaCas ZP, 80 & 90% v/v ethanol led to a slightly bigger size than 60 & 70% v/v. This phenomenon may be associated with the supersaturation level of zein in various ethanol/water solutions. The supersaturation level played a key role in governing the size of particle. The nucleation rate was quickened with increased supersaturation level because more nucleation sites were generated at a higher supersaturation, thus the aggregations were dissipated to more nucleation sites, and the resultant particles were smaller (Shen et al., 2011). Zein well dissolves in each ethanol solution because zein solubility occurs between 50% and 90% ethanol. During the mixing, a lower ethanol proportion led to a higher supersaturation of zein and slightly smaller particle size, confirmed by Fig. S1 . In addition, sizes of all ZP produced by each ethanol proportion were
Size (nm)
250
Plain ZP NaCas ZP
200
150
100 60
70
80
90
Ethanol concentration (v/v%) Fig. 6. Dependence of size on ethanol concentration in Stream 1 of the FNP process for ZP without and with NaCas.
Zein colloidal particles were synthesized by three classical antisolvent processes in comparison with those from FNP procedure. The zein concentrations is 2.5% w/v, and the ethanol to water ratio is 1:2.5 for all processes, which was consistent with our previous research (Li et al., 2013). The pH values of plain ZP dispersions (without NaCas) were 3.7–3.8, and the plain ZP was positively charged, as evidenced by the zeta-potential test (data not shown). Size distributions of ZP produced by each mixing process are shown in Fig. 7ac. Dropwise addition of zein solution into an antisolvent is a common method to preparing ZP (Luo et al., 2010, 2011). In this process, the zein stream was separated into drops prior to mixing with the antisolvent to avoid aggregation behaviour from each zein droplet. As a result, the spatial concentration distributions of zein remained uniform during the antisolvent procedure. Therefore, uniform and small colloidal particles were obtained by the dropwise addition process (Fig. 7a). However, this approach was time-consuming and restricted usually in lab scale. The understanding of rapid mixing by the solvent (zein stream) and antisolvent (water or NaCas dispersion) was very prerequisite in view of practical applications of ZP. For rapid addition approaches, large precipitates occurred after adding the solvent into the agitated antisolvent (data not shown), whereas the opposite addition process (adding the antisolvent into the agitated solvent) produced monodisperse colloidal particles distribution (Fig. 7b). The main difference between two procedures was whether the zein solution was provided with high flow rate. In the former case, the zein stream was at a low flow rate, zein drops aggregated to yield precipitates before dispersing into uniform dispersion during the mixing process. In the latter case, the zein stream was stirred at a high flow rate, and each zein drop was dispersed into a uniform dispersion as soon as the antisolvent was added. Therefore, the flow rate of the zein stream placed a key role in the formation of zein colloid particle in rapid mixing approach, and the sizes of ZP produced by this process can be modulated by providing zein stream with high flow rate. In FNP process, ZP was also successfully produced by rapidly injecting the zein solution (stream) and deionized water with or without NaCas (antisolvent) into the cylindrical cavity at high flow rate. The energy dissipation and turbulent-like motion well dissipated the zein stream into mixed solution. The major part of the ZP colloidal particles was below 100 nm (Fig. 7c), which was much smaller than the ZP colloidal particles produced by adding antisolvent into solvent approach (Fig. 7b), indicating the higher mixing efficiency of FNP than previous processes. However, Fig. 7c shows polydispersity because the energy density in collision was not evenly distributed that it gradually decreased from the centre to the wall of the CIJ setup (Liu and Fox, 2006). To investigate effects of stabilizer on the formation of zein colloidal particles, the NaCas solution was used as the antisolvent. The NaCas concentration was 1% w/v, corresponding to a zein/NaCas ratio of 1:1 which was sufficient for stabilizing ZP (Li et al., 2013). The formation mechanism of NaCas-stabilized ZP is well established (Zhong and Jin, 2009; Patel et al., 2010b): the emergence of zein nanoparticles or precipitates, absorption of sodium caseinate (NaCas) around zein nanoparticles. NaCas stabilized ZP was stable against rotary evaporation and redispersible after
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Fig. 7. Representative size distributions of ZP without (a–c) and with NaCas (d–f). Particles synthesized by dropwise addition of solvent into antisolvent, rapid addition of antisolvent into solvent, and FNP process shows in (a)&(d), (b)&(e) and (c)&(f), respectively.
freeze-drying due to electrostatic and steric stabilization of NaCas. In addition, the addition of NaCas maintained pH values in neutral (6.5–6.9) and shifted the surface charge of zein colloidal particles from positive to negative. NaCas ZP produced by each process showed similar phenomena. Zeta potential of NaCas ZP ranged from 34 to 41 mV, which was coordinate with a previous study (Patel et al., 2010b). Size distributions of NaCas ZP produced by each mixing process are shown in Fig. 7df for quantitative comparison. Generally, stabilizers were utilized to avoid aggregation behaviour during an antisolvent procedure. Unexpectedly, NaCas stabilized ZP showed similar or even slightly bigger average sizes than the counterpart of plain ZP produced by three synthesis
processes. This phenomenon may be explained by the nature of NaCas and zein molecules rather than the synthesis approaches. The comparison of the particle sizes between plain ZP and NaCas ZP were systematically discussed in 3.1.2 and this section focused on the comparison among three synthesis methods. By dropwise addition of solvent into agitated antisolvent, zein drops were uniformly dispersed in the NaCas solution, resulting in monodisperse particles (Fig. 7d). In rapid mixing approach, large zein precipitates were formed (data not shown) through rapid addition of solvent into agitated NaCas solution, whereas the addition of antisolvent into zein solution approach yielded monodisperse particles (Fig. 7e). Therefore, the flow rate of zein is still the key factor of zein–NaCas (the stabilizer) system in rapid mixing. The size
Fig. 8. Representative SEM images of ZP without (a–c) and with NaCas (d–f). Particles synthesized by dropwise addition of solvent into antisolvent, rapid addition of antisolvent into solvent, and FNP process shows in (a)&(d), (b)&(e) and (c)&(f), respectively.
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distribution of NaCas stabilized ZP produced by the FNP process is showed in Fig. 7f. The average size of NaCas stabilized ZP from the FNP procedure was slightly bigger than the counterpart of rapid mixing approaches (Fig. 7de). However, Fig. 7f shows a comparable distribution and most particles are smaller than 1 lm. Fig. 8af shows SEM images of ZP produced by each process, roughly corresponding to sizes of particles determined by DSL (Fig. 8af). The sizes of most plain ZP produced by the aforementioned 4 processes were below 100 nm, and the FNP procedure led to smaller particle sizes in Fig. 8c than Fig. 8ab, confirming the size distribution of Fig. 8ac. The sizes of NaCas ZP were bigger than plain ZP that much more particles are bigger than 100 nm. Although Fig. 8f shows an obvious large distribution than Fig. 8de, there is no clear difference between Fig. 8f and Fig. 8de. In addition, plain ZP had a whole spherical shape and partially connected with each particle in Fig. 8ac. While NaCas ZP showed a different morphology that the NaCas fuses ZP into a continuous structure, with partial spheres left in the surface of the structure. 4. Conclusion We introduced a facile method termed FNP to successfully generate ZP with and without stabilizer. Comparing to ZP synthesized by traditional antisolvent processes, the colloidal particles prepared by FNP processes showed comparable size-distributions. Using this technology, the sizes of ZP were well controlled by the flow rate of the zein solution and outlet configuration of CIJ mixer. The sizes of plain and NaCas ZP show different trend with an increase of zein concentration, due to proteinic nature of buffering capacity and self-assembly. The viscous property of NaCas has little influence on particle size. In addition, we can produce ZP a high zein concentration by the FNP procedure, which largely reduces cost for the FNP process. The particle size of ZP is stable with the variation of ethanol proportion, showing an attractive feature to encapsulate different actives in future studies. Compared with synthetic material, proteins are more suitable for preparing colloid particles at high concentration due to its charged groups of amino acid residuals. In summary, the FNP process is practical and applicable method to produce ZP in large scale. Acknowledgments This work was partially supported by The Project Supported by Guangdong Natural Science Foundation (S2013010012097). We also appreciate the financial support the Fundamental Research Funds for the Central Universities (SCUT 2012ZZ0082, 2014ZG0021, 2013ZM0065), Key Projects in the National Science & Technology Program during the Twelfth Five-year Plan Period (2013BAD18B10-4). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jfoodeng.2013. 12.001. References Chan, G., Mooney, D.J., 2008. New materials for tissue engineering: towards greater control over the biological response. Trends Biotechnol. 26, 382–392. Chen, L., Remondetto, G.E., Subirade, M., 2006. Food protein-based materials as nutraceutical delivery systems. Trends Food Sci. Technol. 17, 272–283. Cheng, J.C., Vigil, R.D., Fox, R.O., 2010. A competitive aggregation model for Flash NanoPrecipitation. J. Colloid Interface Sci. 351, 330–342. Dickinson, E., 1997. Properties of emulsions stabilized with milk proteins: overview of some recent developments. J. Dairy Sci. 80, 2607–2619.
Gindy, M.E., Panagiotopoulos, A.Z., Prud’homme, R.K., 2008. Composite block copolymer stabilized nanoparticles: simultaneous encapsulation of organic actives and inorganic nanostructures. Langmuir 24, 83–90. Han, J., Zhu, Z., Qian, H., Wohl, A.R., Beaman, C.J., Hoye, T.R., Macosko, C.W., 2012. A simple confined impingement jets mixer for Flash NanoPrecipitation. J. Pharm. Sci. 101, 4018–4023. Hermansson, A.M., 1975. Functional properties of proteins for foods-flow properties. J. Texture Stud. 5, 425–439. Hu, D., Lin, C., Liu, L., Li, S., Zhao, Y., 2012. Preparation, characterization, and in vitro release investigation of lutein/zein nanoparticles via solution enhanced dispersion by supercritical fluids. J. Food Eng. 109, 545–552. Hurtado-López, P., Murdan, S., 2006. An investigation into the adjuvanticity and immunogenicity of zein microspheres being researched as drug and vaccine carriers. J. Pharm. Pharmacol. 58, 769–774. Johnson, B.K., 2003. Flash NanoPrecipitation of Organic Actives via Confined Micromixing and Block Copolymer Stabilization. Ph.D. Thesis, Prinseton University. Johnson, B.K., Prud’homme, R.K., 2003a. Chemical processing and micromixing in confined impinging jets. AIChE J. 49, 2264–2282. Johnson, B.K., Prud’homme, R.K., 2003b. Flash nanoprecipitation of organic actives and block copolymers using a confined impinging jets mixer. Aust. J. Chem. 56, 1021–1024. Lai, L.F., Guo, H.X., 2011. Preparation of new 5-fluorouracil-loaded zein nanoparticles for liver targeting. Int. J. Pharm. 404, 317–323. Lawton, J.W., 2002. Zein: a history of processing and use. Cereal Chem. 79, 1– 18. Li, K.K., Yin, S.W., Yang, X.Q., Tang, C.H., Wei, Z.H., 2012. Fabrication and characterization of novel antimicrobial films derived from thymol-loaded zein-sodium caseinate (SC) nanoparticles. J. Agric. Food. Chem. 60, 11592– 11600. Li, K.K., Yin, S.W., Yin, Y.C., Tang, C.H., Yang, X.Q., Wen, S.H., 2013. Preparation of water-soluble antimicrobial zein nanoparticles by a modified antisolvent approach and their characterization. J. Food Eng. 119, 343–352. Liu, Y., Fox, R.O., 2006. CFD predictions for chemical processing in a confined impinging-jets reactor. AIChE J. 52, 731–744. Luo, Y.C., Zhang, B., Cheng, W.H., Wang, Q., 2010. Preparation, characterization and evaluation of selenite-loaded chitosan/TPP nanoparticles with or without zein coating. Carbohydr. Polym. 82, 942–951. Luo, Y.C., Zhang, B., Whent, M., Yu, L.L., Wang, Q., 2011. Preparation and characterization of zein/chitosan complex for encapsulation of a-tocopherol, and its in vitro controlled release study. Colloids Surf., B 85, 145–152. Luo, Y.C., Teng, Z., Wang, Q., 2012. Development of zein nanoparticles coated with carboxymethyl chitosan for encapsulation and controlled release of vitamin D3. J. Agric. Food. Chem. 60, 836–843. Muthuselvi, L., Dhathathreyan, A., 2006. Simple coacervates of zein to encapsulate Gitoxin. Colloids Surf., B 51, 39–43. Parris, N., Cooke, P.H., Hicks, K.B., 2005. Encapsulation of essential oils in zein nanospherical particles. J. Agric. Food. Chem. 53, 4788–4792. Patel, A., Hu, Y., Tiwari, J.K., Velikov, K.P., 2010a. Synthesis and characterisation of zein–curcumin colloidal particles. Soft Matter 6, 6192–6199. Patel, A.R., Bouwens, E.C.M., Velikov, K.P., 2010b. Sodium caseinate stabilized zein colloidal particles. J. Agric. Food. Chem. 58, 12497–12503. Shen, H., Hong, S., Prud’homme, R.K., Liu, Y., 2011. Self-assembling process of flash nanoprecipitation in a multi-inlet vortex mixer to produce drug-loaded polymeric nanoparticles. J. Nanopart. Res. 13, 4109–4120. Shukla, R., Cheryan, M., 2001. Zein: the industrial protein from corn. Ind. Crops Prod. 13, 171–192. Stegemann, J.P., Kaszuba, S.N., Rowe, S.L., 2007. Review: advances in vascular tissue engineering using protein-based biomaterials. Tissue Eng. 13, 2601–2613. Wang, Y., Padua, G.W., 2010. Formation of zein microphases in ethanol-water. Langmuir 26, 12897–12901. Wang, Y., Padua, G.W., 2012. Nanoscale characterization of zein self-assembly. Langmuir 28, 2429–2435. Wu, Y., Luo, Y.G., Wang, Q., 2012. Antioxidant and antimicrobial properties of essential oils encapsulated in zein nanoparticles prepared by liquid–liquid dispersion method. LWT – Food Sci. Technol. 48, 283–290. Xiao, D., Zhong, Q., 2011. In vitro release kinetics of nisin as affected by Tween 20 and glycerol co-encapsulated in spray-dried zein capsules. J. Food Eng. 106, 65– 73. Xiao, D., Davidson, P.M., Zhong, Q., 2011. Spray-dried zein capsules with coencapsulated nisin and thymol as antimicrobial delivery system for enhanced antilisterial properties. J. Agric. Food. Chem. 59, 7393–7404. Yeo, R.W., 1993. A Numerical Study of Jet-to-Jet Impingement in a Mixing Head. Ph.D. Thesis, McMaster University. Zhang, C., Pansare, V.J., Prud’homme, R.K., Priestley, R.D., 2012. 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Contents lists available at ScienceDirect
Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Continuous preparation of zein colloidal particles by Flash NanoPrecipitation (FNP) Kang-Kang Li a, Xi Zhang a, Qin Huang b, Shou-Wei Yin a,⇑, Xiao-Quan Yang a,⇑, Qi-Biao Wen a, Chuan-He Tang a, Fu-Rao Lai a a b
Research and Development Center of Food Proteins, Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, PR China School of Business Administration, South China University of Technology, Guangzhou 510640, PR China
a r t i c l e
i n f o
Article history: Received 15 July 2013 Received in revised form 27 November 2013 Accepted 3 December 2013 Available online 11 December 2013 Keywords: Flash NanoPrecipitation Confined Impinge Jet Zein colloidal particle Sodium caseinate
a b s t r a c t This study utilized a facile and continuous technique termed Flash NanoPrecipitation (FNP) to produce zein colloidal particles (ZP) without or with sodium caseinate (NaCas) as the stabilizer. The colloidal particle preparations were performed through a self-made Confined Impinge Jet (CIJ) mixer, and compared with traditional antisolvent processes. The influences of FNP processing parameters on the size of zein colloid particles, including flow rates, outlet configurations, solute concentrations and ethanol concentrations, were extensively discussed. The influences of viscous properties, buffering capacity and self-assembly of zein or NaCas on particle size and size distributions were also discussed. Particle sizes of ZP produced by the FNP technique are below 350 nm, even at high zein concentrations (2.5– 7.5% w/v). Solvent systems with different ethanol concentrations yielded zein colloid particles with similar size, showing an attractive feature for industrial applications and encapsulating actives with different solubility in ethanol–water binary solvent. This study opens a promising pathway for continuously producing ZP via the FNP procedure. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Protein colloidal particles with generally regarded as safe (GRAS) status are promising as delivery system in food industry. The inherent biodegradability and biocompatibility of protein colloid particles make them more effective in maintaining the functions of actives in extracellular matrix than synthetic polymers (Chan and Mooney, 2008; Stegemann et al., 2007). The amino and carboxyl groups of amino acid residues provide protein molecules with different charges depending on the pH environments while synthetic polymers usually lack such functional groups. Therefore, proteins can be easily modulated to fabricate vehicles for charged actives via electrostatic interactions (Chen et al., 2006). Protein colloidal particles are favourable to load uncharged actives via hydrophobic interactions since proteins also have notable hydrophobic domains (Chen et al., 2006). Zein, a prolamin, is abundantly available and utilized for various industrial applications (Lawton, 2002; Shukla and Cheryan, 2001). Zein is an ideal material for preparing colloidal particles due to its unique solubility and self-assembly property. Many works were successfully carried out to prepare zein colloidal particle vehicles
⇑ Corresponding authors. Tel.: +86 20 87114262; fax: +86 20 87114263. E-mail addresses: [email protected] (S.-W. Yin), [email protected] (X.-Q. Yang). 0260-8774/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jfoodeng.2013.12.001
for drug delivery (Patel et al., 2010a; Muthuselvi and Dhathathreyan, 2006; Lai and Guo, 2011), nutrition protection (Luo et al., 2011, 2012), encapsulating antimicrobials (Xiao et al., 2011; Wu et al., 2012) and antioxidants (Parris et al., 2005; Wu et al., 2012). However, the zein colloidal particles are usually not water-soluble, and are easy to aggregate during the drying process of zein particle dispersions. When incorporated in liquid food matrices they may aggregate to form the flocculates or precipitates, which limit to some extent their usages in food industries. A general way to break these limitations is by coating a layer of stabilizers around zein colloid particles, such as chitosan (Luo et al., 2011), NaCas (Patel et al., 2010b) and polyvinylpyrrolidone (PVP) (Hurtado-López and Murdan, 2006). NaCas, a milk protein, has been proved to be an ideal stabilizer for ZP (Patel et al., 2010b; Li et al., 2012, 2013) against aggregation in neutral condition and drying process, due to the combination of electrostatic and steric stabilization (Dickinson, 1997). Antisolvent process is an approach to produce colloidal particles via creating supersaturation by mixing a solvent with an antisolvent. It is a low-cost and easy to prepare ZP. However, traditional antisolvent processes are discontinuous and difficult to scale up. Spray drying and supercritical anti-solvent (SAS) processes are continuous and scalable methods. However, ZP produced by spray drying is microscale (Xiao et al., 2011; Xiao and Zhong, 2011), and spray drying technique is not suitable for encapsulating
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temperature-sensitive actives. SAS processes produce micro-scale zein colloidal particles, and toxic solvents such as methanol, acetone and dimethyl sulfoxide (DMSO) were usually included in the SAS procedure (Zhong et al., 2008; Hu et al., 2012). In addition, it is costly and need complex equipments. Based on the advantages and problems of above-mentioned methods, there is no ideal method to produce colloidal ZP. In this work, an FNP approach that was firstly investigated by Johnson and Prud’homme (Johnson, 2003; Gindy et al., 2008), was utilized to solve those problems in the preparation of zein colloid particles. In the FNP process, an antisolvent stream with or without stabilizers and a solvent stream containing actives collide in a confined mixing chamber which limits bypassing of high mixing intensity (Johnson, 2003). There are 3 main advantages for the FNP approach in producing ZP particles when compared with classical antisolvent procedures: (1) flow rate itself provides driving force for efficient mixing; (2) particle size can be quantitatively controlled by processing parameters; (3) the scale-up is possible from laboratory apparatus to industrial continuous production (Johnson and Prud’homme, 2003a). The FNP procedure had been utilized to produce colloidal particles of active small molecules such as carotenoid (Johnson and Prud’homme, 2003b), and synthetic polymers such as polystyrene (Zhang et al., 2012). However, biopolymerbased colloidal particles had not been produced via an FNP procedure, and the properties of the formed colloidal particles were unknown. Therefore, the synthesis of ZP by the FNP process using a selfmade CIJ mixer was performed in this research. Several antisolvent processes under magnetic agitation were performed and compared with the FNP approach. To systematically study the FNP process for producing ZP, zein colloidal particles were produced at a variation of flow rates, outlet configurations, solute concentrations and ethanol concentrations. We also explored the effects of protein properties on zein particle size and size distributions, including viscous property, buffering capacity and self-assembly.
Fig. 1. Actual laboratory setup of the CIJ mixer for FNP process.
2. Materials and methods 2.1. Materials Zein was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Food-grade NaCas was obtained from Murray Goulburn Cooperative Co., Limited, Australia. All other chemicals used were of analytical grade. 2.2. Confined impingement jets Fig. 1 shows the actual setup of a self-made confined impinging jet (CIJ) mixer. The CIJ mixer included two separate streams. Here, a syringe containing zein solutions was placed at the inlet of Stream 1 (left), and a syringe containing Milli-Q water with or without NaCas was placed at the inlet of Stream 2 (right). The FNP process was carried out via hand operation since previous research proved that turbulent flow could be achieved motion by small, low-friction syringes with hand (Han et al., 2012). In addition, hand motion can provide the solvent and antisolvent with high pressure which was needed in the FNP process. The schematic image (Fig. 2a) shows that the solvent and the antisolvent streams come into the CIJ device from left and right site, respectively, collide in the centre cavity. The resultant particle dispersions come out from the downside outlet nozzle. Fig. 2b shows the dimension of the CIJ chamber in detail. The main cavity was cylindrical in an upright position, with two inlets and adapters fitted with the syringes and an outlet nozzle. The two nozzles with diameters of 3 mm (O1) and/or 0.5 mm (O2) were used as the
Fig. 2. Schematic representations of the FNP process (a) and the dimensions of the self-made CIJ mixer (b).
outlet nozzle to investigate the effects of outlet configurations on the formation zein colloid particle. Sealed tape was used in each joint to prevent leakage of the dispersions during FNP process. 2.3. Particle synthesis via the FNP approach The mixing efficiency in the FNP process is fully dependent on collision of fluids from each side, and the driving force depends on flow rate and diameter of outlet nozzle. Some other parameters, including protein concentrations, solvent types and solventto-antisolvent ratios in the FNP process also affect the formation of zein colloid particles. Therefore, the influences of selected FNP parameters on the particle formations were extensively investigated to optimize the preparation procedure of zein colloid particles via FNP process. 2.3.1. Influence of flow rate and outlet configurations An aliquot of 4 mL of 2.5% w/v zein in 80% v/v ethanol–water binary solution was placed in the syringe of Stream 1 (left), and 10 mL of deionized water with or without 1% w/v NaCas was placed in the syringe of Stream 2 (right). The trails were performed
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using O1 or O2 outlet. The mixing times are 4, 5, 10, 20, 40, 80 s, respectively. The flow rates of zein solution are about 5.0, 4.0, 2.0, 1.0, 0.5, and 0.25 m/s, respectively, and the corresponding flow rates of water are about 12.7, 10.2, 5.0, 2.5, 1.3, and 0.64 m/s, calculated by Eq. (1): 2
Flow rate ¼ V=ðt p r Þ
ð1Þ
where V is the volume of mixed solution, t is the time for this procedure and r is the radium of the inlet nozzle. 2.3.2. Influence of zein concentration Ten millilitres of deionized water with or without NaCas was mixed with 4 mL of 80% v/v ethanol solution containing zein via the CIJ mixer. The mixing time was 10 s and O2 outlet was used. Zein concentrations were set as 0.1, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0% w/v to investigate the influences of solute concentrations on particle size. For NaCas-stabilized ZP, the correspond NaCas concentrations were 0.04, 0.2, 0.4, 1.0, 2.0, 3.0 and 4.0% w/v, with a zein/NaCas ratio of 1:1. To investigate the influence of viscosity on particle size, 0.5% w/v zein solution and 0.2, 0.4, 1.0, 2.0, and 3.0% w/v NaCas solutions were mixed at the same conditions through the CIJ setup. 2.3.3. Influence of ethanol concentration Ten millilitres of deionized water with or without NaCas was mixed with 4 mL of ethanol solution containing zein via the CIJ setup. The mixing time was 10 s and O2 outlet was used in this FNP process. The ethanol concentrations were 60, 70, 80 and 90% v/v. 2.3.4. Influence of solvent-to-antisolvent ratio Deionized water with or without NaCas was mixed with 4 mL of ethanol solution containing zein via the CIJ setup. The mixing time was 10 s and O2 outlet was used in this FNP process. The volumes of deionized water were 4, 8, 12, 16 and 20 mL. 2.4. Comparison of each mixing processes of solvent and antisolvent In the FNP process, 10 mL of deionized water (as an antisolvent) with or without 1% w/v NaCas were mixed with 4 mL of 80% v/v ethanol solution (solvent) containing 2.5% w/v zein using the CIJ device. The trails were performed with O2 via the CIJ device and the mix time was 10 s. ZP dispersions were directly collected at the end of the outlet of the CIJ device. Three classic antisolvent procedures, including dropwise addition of solvent into antisolvent, rapid addition of antisolvent into solvent, rapid addition of solvent into antisolvent were performed and compared with the FNP process. In the dropwise addition approach, 4 mL of 2.5% zein solution was dropwise added into 10 mL of de-ionized water with or without NaCas to yield ZP particles. In the rapid addition approaches, the solvents/antisolvents were poured into antisolvents/solvents to produce ZP according to the procedure of Li et al. (2013), other experimental parameters coincided with the dropwise addition approach. In traditional antisolvent processes, vigorous agitation or shearing is effective in producing colloidal ZP, but it has strong limitation in spatial variations of fluid, and it is even tougher to maintain performance upon scale up (Johnson, 2003). Therefore, a moderate agitation (500 rpm) was adopted in this study. 2.5. Analysis of particle size and surface charge The particle size and zeta-potential of ZP dispersions was measured using a Zetasizer Nano (Malvern Instruments Ltd., UK). Before measurements, all the dispersions were subjected to the centrifugation at 4000 rpm for 10 min to separate out the minor
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part of larger aggregates. The dispersions were measured for particle size at within 30 min at 25 °C. The results reported are an average of three readings. 2.6. Morphology observation Morphological structures of plain and NaCas ZP were obtained by a Quanta 200 Field emission scanning electron microscope (FEI company, America). The freeze-dried powers were utilized to investigate morphology character of zein colloidal particles. To observe surface morphology, powdered samples were loosely glued onto a conductive adhesive mounted on a stainless steel stage. Subsequently, they were coated with a thin (<20 nm) conductive gold and platinum layer using a sputter coater (Hummer XP, Anatech, CA). Representative SEM images were reported. 2.7. Statistics Statistical analyses were performed using an analysis of variance (ANOVA) procedure of the SPSS 13.0 statistic analysis program, and the differences between means of the trails were detected by a least Duncan test (P < 0.05). 3. Results and discussion The Flash NanoPrecipitation process is shown schematically in Fig. 2a. The Stream 1 (zein solution) was rapidly mixed with an incoming Stream 2 (water or NaCas solution as the antisolvent). The kinetic energy of each jet stream is converted into a turbulent-like motion through a collision and redirection of the flow in this small cavity of the CIJ device (Johnson and Prud’homme, 2003a), resulting in a high energy dissipation which occurs along the jet shear layers and in the impingement plane (Yeo, 1993). Finally, zein colloid particle dispersions drained directly out of the outlet after the solvent and antisolvent were efficiently mixed in the chamber of the CIJ setup. 3.1. The influences of FNP parameters on the formation of zein particles 3.1.1. Effects of flow rates and outlet configurations It is important to note that the precipitation process could be divided into two stages: the first one is the particle formation, which is modelled as Brownian aggregation and occurs on much faster time scales than the mixing process; the second one is that particles slowly grow in mass (Cheng et al., 2010). As mentioned earlier, both plain and NaCas ZP have electrostatic repulsion against size growth by positive and negative charges, respectively. The particle size of fresh ZP was measured, reflecting the mixing efficiency of various procedures. To investigate the influences of mixing conditions of the FNP process on the formation of zein particles, the colloid particles were produced via the CIJ setup at a series of flow rates and two outlet nozzles. Particle sizes of zein colloid particles are shown in Fig. 3. Sizes of plain ZP produced using O1 and O2 outlet decreased with the increases of flow rates from 0.25 to 2.0 m/s (Fig. 3a). Johnson and Prud’homme built a theory to explain the relationship between flow rates and particle sizes, and the basic premise behind Flash NanoPrecipitation is that high flow rate produced rapid micromixing in a short time scale (Johnson, 2003; Zhang et al., 2012). Particle growth is controlled by the micromixing time, and the micromixing time decrease with an increase of flow rate (Gindy et al., 2008; Johnson and Prud’homme, 2003a). In addition, sizes of plain ZP stopped decreasing after the flow rate reached a critical point (Fig. 3a). At that time, mixing time is equivalent to
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200
200
Size (nm)
(b) 250
Size (nm)
(a) 250
150
150 O1 O2
O1 O2 100 0.1
1
10 -1
Flow rate (m s )
100 0.1
1
10 -1
Flow rate (m s )
Fig. 3. Change in particle size as a function of flow rate of Stream 1 and outlet configuration for ZP without (a) and with NaCas (b).
the characteristic time of particle formation, and further increases in flow rate and decrease in mixing time no longer affected the particle size (Han et al., 2012; Cheng et al., 2010; Johnson and Prud’homme, 2003a; Zhu et al., 2010). A critical point of plain ZP produced by O1 and O2 appeared at the same flow rate, i.e., 2.0 m/s. As expected, plain ZP produced by O1 outlet was roughly bigger than that by O2 outlet except the particles produced at the flow rate of 1.0 m/s. (Fig. 3a). This phenomenon was attributed to different stretching history of the fluids, including ethanol solution (the solvent) and de-ionized water (the antisolvent). The outlet area of O1 was much bigger than the whole inlet area, so mixed solutions dripped from CIJ mixer due to gravity without stretching. While the area of O2 was half of the whole inlet area, changing the deformation history of the fluid and stretched the fluid within the CIJ setup (Johnson and Prud’homme, 2003a), providing the stretched fluid with an additional pressure and a high exiting flow rate equals to the sum of 2 inlet streams and finally improving the mixing efficiency. In all, mixing efficiency with both O1 and O2 increased upon the increase in flow rate of inlet streams, resulting in smaller zein colloid particles. However, the size gap of ZP produced using O1 and O2 outlet nozzles was narrowed with the increase in flow rate (Fig. 3a). A similar result was reported that outlet configuration has a significant effect at low flow rate and a limited effect at a higher one (Johnson and Prud’homme, 2003a). On the other hand, NaCas stabilized ZP was produced at the same condition with plain ZP except the addition of NaCas, and the result is shown in Fig. 3b. NaCas ZP showed a similar trend at the variation of flow rates and outlet configurations when compared with plain ZP. The particle size decreases from about 240 nm to about 140 nm until the flow rate reached the critical point which also appeared at 2.0 m/s. In addition, the FNP approach with O2 outlet produced slightly smaller particles than that with O1 outlet, and size gap of ZP produced by O1 and O2 outlet nozzle also decreased upon increasing low flow rate (Fig. 3b). However, the difference between the two size curves is narrower than plain ZP (Fig. 3). Although the detailed understanding on the formation mechanism of zein colloid particles is outside the scope of this paper, the reasons may be that the fluid stretching and deformation effects of NaCas solution is weak or the effects of the alteration in size of the outlet were limited by the absorption of NaCas onto zein colloidal particles. 3.1.2. Effects of zein concentrations Generally, zein concentrations are directly proportional to the yield of zein particles. Thus, the influence of zein concentrations on the FNP particles was investigated. Zein concentrations in Stream 2 varied from 0.1% w/v to 10% w/v. Particle size distribu-
tions of plain ZP produced by 0.1–7.5% w/v zein solutions are shown in Fig. 4a, whereas large precipitates occurred when zein concentration is 10% w/v. The zein concentrations had marked effects on zein self-assembly during the FNP process by which zein self-assembled to form colloid particles at a concentration lower than 7.5% w/v. After that, zein molecules tended to form formed sheets in ethanol solution and zein particles fused together after the solvent evaporation process (Wang and Padua, 2010). A lower concentration of solute in organic solution generally corresponds to a shorter time for nucleation and growth, and stabilization resulted in a smaller particle size in FNP process (Johnson and Prud’homme, 2003b). Zein particles also had a bigger size at higher zein concentration upon solvent evaporation because the increase of zein concentration turns more zein monomers or dimers into aggregation (Wang and Padua, 2010). Interestingly, Fig. 4a shows a different result that big particles were produced at 0.1% w/v. In contrast, particle sizes remained 130–140 nm at the zein concentrations of 0.5–5.0% w/v and increased to about 205 nm at the zein concentration of 7.5% w/v. The phenomenon may be attributed to the nature of zein material. Proteins usually have buffering capacity. In acid condition, basic amino acid residues mainly account for the capacity. Variations of pH value by changing protein concentration are usually not taken into consideration. However, zein ethanol solution is weak acid (pH 5.61 for 0.5% w/v) and the pH value of zein dispersion decreases with the increase of zein concentrations, because zein is deficient in basic amino acid residues (Shukla and Cheryan, 2001). Zein solutions (0.1% w/v) had a pH of 6.54 which is close to the isoelectric points of zein (Lawton, 2002). Therefore, the zein particles produced at the isoelectric regions during the CIJ setup were labile to aggregate to yield larger particles with particle seize about 700 nm. Particles in the range of 135–205 nm were produced at higher zein concentrations because the pH values of these zein solutions were below 4.5. When zein concentrations increased from 0.5 to 7.5% w/v, the pH further decreased from 4.5 to 3.6, limiting the increase of particle size. There are two possible explanations why the pH of zein solution affects particle size of the resulted nanoparticles: the pH during mixing affected the selfassembly of zein and particle formation, or the pH after the mixing decided the charge of ZP and then affected the particle size. Thus, we adjusted pH value of all mixed dispersion to 3.6 to rule out the difference of final pH, and the results shows in Fig. 4a. Plain ZP produced by 0.1% w/v had a bigger particle size about 750 nm after the pH adjustment because the big particle was unstable and grew over time. Apart from that, there were no significant changes after adjusting pH value. Therefore, the pH values during the formation of ZP rather than the final pH significantly influenced the final particle sizes.
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For NaCas ZP, particles were also produced at zein concentrations of 0.1–10% w/v and NaCas concentrations of 0.04–4% w/v with a zein/NaCas ratio of 1:1. The sizes of NaCas ZP significantly increased from 127 to 327 nm upon increasing zein concentrations from 0.1 to 7.5% w/v (P < 0.05), as shown in Fig. 4b. Concomitantly, large precipitates also formed at 10% w/v. As expected, zein concentrations had a different influence on sizes of NaCas ZP. The addition of NaCas changed two factors in the mixing process, viscosity and pH value. Zein ethanol solution is acid and zein is deficient in buffering capacity, but the addition of NaCas turned the acid condition to neutral pH values (pH 6.4–6.95). The phenomenon showed strong buffering capacity of NaCas, due to its basic amino acid residuals, as well as the presence of phosphoserine (Dickinson, 1997). On the other hand, the viscosity of Stream 2 increased directly proportional to the exponent of concentrations of NaCas (Hermansson, 1975), which may affect mixing efficiency. The relationship between viscosity and mixing efficacy is complex. The mixing time generally scales with viscosity, but it has no relationship with viscosity when turbulent diffusion or inertial convective mixing were active (Johnson and Prud’homme, 2003a). To investigate whether the viscosity affects particle size, 0.5% w/v zein solution was mixing with NaCas solutions at a variety of concentrations from 0.2 to 3% w/v, and the result shows in Fig. 4c. The particle size remained 130–140 nm with the increase of NaCas concentration, indicating that in this case the viscosity of NaCas solution has little influence on particle size.
We concluded that the pH during mixing is the key factor of particle formation. As the solvent gradually became hydrophilic, the self-assembly is mainly driven by amphiphilicity (Wang and Padua, 2012). It was believed that zein particles formed by a layer-by-layer by adsorption to a central core or nucleus, and the radial growth occurred by hydrophobic associations (Wang and Padua, 2010). Comparing to the neutral condition, the acid condition charged zein molecules with +23.6 mV which limits hydrophobic interactions. Therefore, plain ZP has small particle sizes at 0.5% w/v, and when the zein concentration increases to 7.5% w/v, the further decrease of pH limits the growth of the particle size. As for the NaCas ZP, all dispersions produced at a variety of zein concentrations are in neutral condition, so then zein molecules carries a few charge, and the sizes of NaCas ZP gradually increase with the increase of zein concentrations. The schematic illustration of plain and NaCas ZP produced by FNP shows in Fig. 5. In previous study, the size of polystyrene particle (Zhang et al., 2012) significantly increases from less than 100 nm to about 140 nm when polystyrene concentration increased from 0.1 to 1% w/v, which was similar with NaCas ZP. This phenomenon was due to that polystyrene has no charged groups to limit the hydrophobic interaction at high concentrations. While the aggregation of charged zein molecules at acid pH was to a great extent limited by electrostatic repulsion, especially at high zein concentrations (2.5– 7.5% w/v). Therefore, the charged groups of amino acid residuals endowed proteins with better controlled behaviour at high concentrations than many synthetic polymers.
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in a narrow range from 120 to 160 nm. Ethanol concentrations can be set in a wide range to modulate to encapsulate actives with different solubility in an ethanol–water binary system without big changes of particle size, which is an attractive feature of formation of ZP via the FNP procedure. 3.2. Comparison between classic antisolvent procedures and FNP approach
Fig. 5. A schematic illustration of Plain (left) and NaCas (right) ZP produced via FNP process. For Plain ZP, ethanol solution (red) and water (blue) were impinged into CIJ mixer. Then pH of mixed solution is below 4.0, so positive charges shows around zein. For NaCas ZP, ethanol solution (red) and NaCas solution (green) were impinged into CIJ mixer. The pH is neutral, so zein is uncharged. Particle sizes produced by 5% zein solution show at last. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.1.3. Effect of ethanol proportion The aim of this work is to build a colloid particle formation system in which actives with different solubility in ethanol–water binary solvent may also is included in the practical application. Thus, the effects of ethanol concentration on the zein particle formation via the CIJ setup were investigated. The mean diameters of colloidal particles produced with stock solutions containing 60– 90% v/v ethanol are plotted in Fig. 6. Plain ZP showed smaller size than NaCas ZP, as discussed before. For both plain ZP and NaCas ZP, 80 & 90% v/v ethanol led to a slightly bigger size than 60 & 70% v/v. This phenomenon may be associated with the supersaturation level of zein in various ethanol/water solutions. The supersaturation level played a key role in governing the size of particle. The nucleation rate was quickened with increased supersaturation level because more nucleation sites were generated at a higher supersaturation, thus the aggregations were dissipated to more nucleation sites, and the resultant particles were smaller (Shen et al., 2011). Zein well dissolves in each ethanol solution because zein solubility occurs between 50% and 90% ethanol. During the mixing, a lower ethanol proportion led to a higher supersaturation of zein and slightly smaller particle size, confirmed by Fig. S1 . In addition, sizes of all ZP produced by each ethanol proportion were
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Zein colloidal particles were synthesized by three classical antisolvent processes in comparison with those from FNP procedure. The zein concentrations is 2.5% w/v, and the ethanol to water ratio is 1:2.5 for all processes, which was consistent with our previous research (Li et al., 2013). The pH values of plain ZP dispersions (without NaCas) were 3.7–3.8, and the plain ZP was positively charged, as evidenced by the zeta-potential test (data not shown). Size distributions of ZP produced by each mixing process are shown in Fig. 7ac. Dropwise addition of zein solution into an antisolvent is a common method to preparing ZP (Luo et al., 2010, 2011). In this process, the zein stream was separated into drops prior to mixing with the antisolvent to avoid aggregation behaviour from each zein droplet. As a result, the spatial concentration distributions of zein remained uniform during the antisolvent procedure. Therefore, uniform and small colloidal particles were obtained by the dropwise addition process (Fig. 7a). However, this approach was time-consuming and restricted usually in lab scale. The understanding of rapid mixing by the solvent (zein stream) and antisolvent (water or NaCas dispersion) was very prerequisite in view of practical applications of ZP. For rapid addition approaches, large precipitates occurred after adding the solvent into the agitated antisolvent (data not shown), whereas the opposite addition process (adding the antisolvent into the agitated solvent) produced monodisperse colloidal particles distribution (Fig. 7b). The main difference between two procedures was whether the zein solution was provided with high flow rate. In the former case, the zein stream was at a low flow rate, zein drops aggregated to yield precipitates before dispersing into uniform dispersion during the mixing process. In the latter case, the zein stream was stirred at a high flow rate, and each zein drop was dispersed into a uniform dispersion as soon as the antisolvent was added. Therefore, the flow rate of the zein stream placed a key role in the formation of zein colloid particle in rapid mixing approach, and the sizes of ZP produced by this process can be modulated by providing zein stream with high flow rate. In FNP process, ZP was also successfully produced by rapidly injecting the zein solution (stream) and deionized water with or without NaCas (antisolvent) into the cylindrical cavity at high flow rate. The energy dissipation and turbulent-like motion well dissipated the zein stream into mixed solution. The major part of the ZP colloidal particles was below 100 nm (Fig. 7c), which was much smaller than the ZP colloidal particles produced by adding antisolvent into solvent approach (Fig. 7b), indicating the higher mixing efficiency of FNP than previous processes. However, Fig. 7c shows polydispersity because the energy density in collision was not evenly distributed that it gradually decreased from the centre to the wall of the CIJ setup (Liu and Fox, 2006). To investigate effects of stabilizer on the formation of zein colloidal particles, the NaCas solution was used as the antisolvent. The NaCas concentration was 1% w/v, corresponding to a zein/NaCas ratio of 1:1 which was sufficient for stabilizing ZP (Li et al., 2013). The formation mechanism of NaCas-stabilized ZP is well established (Zhong and Jin, 2009; Patel et al., 2010b): the emergence of zein nanoparticles or precipitates, absorption of sodium caseinate (NaCas) around zein nanoparticles. NaCas stabilized ZP was stable against rotary evaporation and redispersible after
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Fig. 7. Representative size distributions of ZP without (a–c) and with NaCas (d–f). Particles synthesized by dropwise addition of solvent into antisolvent, rapid addition of antisolvent into solvent, and FNP process shows in (a)&(d), (b)&(e) and (c)&(f), respectively.
freeze-drying due to electrostatic and steric stabilization of NaCas. In addition, the addition of NaCas maintained pH values in neutral (6.5–6.9) and shifted the surface charge of zein colloidal particles from positive to negative. NaCas ZP produced by each process showed similar phenomena. Zeta potential of NaCas ZP ranged from 34 to 41 mV, which was coordinate with a previous study (Patel et al., 2010b). Size distributions of NaCas ZP produced by each mixing process are shown in Fig. 7df for quantitative comparison. Generally, stabilizers were utilized to avoid aggregation behaviour during an antisolvent procedure. Unexpectedly, NaCas stabilized ZP showed similar or even slightly bigger average sizes than the counterpart of plain ZP produced by three synthesis
processes. This phenomenon may be explained by the nature of NaCas and zein molecules rather than the synthesis approaches. The comparison of the particle sizes between plain ZP and NaCas ZP were systematically discussed in 3.1.2 and this section focused on the comparison among three synthesis methods. By dropwise addition of solvent into agitated antisolvent, zein drops were uniformly dispersed in the NaCas solution, resulting in monodisperse particles (Fig. 7d). In rapid mixing approach, large zein precipitates were formed (data not shown) through rapid addition of solvent into agitated NaCas solution, whereas the addition of antisolvent into zein solution approach yielded monodisperse particles (Fig. 7e). Therefore, the flow rate of zein is still the key factor of zein–NaCas (the stabilizer) system in rapid mixing. The size
Fig. 8. Representative SEM images of ZP without (a–c) and with NaCas (d–f). Particles synthesized by dropwise addition of solvent into antisolvent, rapid addition of antisolvent into solvent, and FNP process shows in (a)&(d), (b)&(e) and (c)&(f), respectively.
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distribution of NaCas stabilized ZP produced by the FNP process is showed in Fig. 7f. The average size of NaCas stabilized ZP from the FNP procedure was slightly bigger than the counterpart of rapid mixing approaches (Fig. 7de). However, Fig. 7f shows a comparable distribution and most particles are smaller than 1 lm. Fig. 8af shows SEM images of ZP produced by each process, roughly corresponding to sizes of particles determined by DSL (Fig. 8af). The sizes of most plain ZP produced by the aforementioned 4 processes were below 100 nm, and the FNP procedure led to smaller particle sizes in Fig. 8c than Fig. 8ab, confirming the size distribution of Fig. 8ac. The sizes of NaCas ZP were bigger than plain ZP that much more particles are bigger than 100 nm. Although Fig. 8f shows an obvious large distribution than Fig. 8de, there is no clear difference between Fig. 8f and Fig. 8de. In addition, plain ZP had a whole spherical shape and partially connected with each particle in Fig. 8ac. While NaCas ZP showed a different morphology that the NaCas fuses ZP into a continuous structure, with partial spheres left in the surface of the structure. 4. Conclusion We introduced a facile method termed FNP to successfully generate ZP with and without stabilizer. Comparing to ZP synthesized by traditional antisolvent processes, the colloidal particles prepared by FNP processes showed comparable size-distributions. Using this technology, the sizes of ZP were well controlled by the flow rate of the zein solution and outlet configuration of CIJ mixer. The sizes of plain and NaCas ZP show different trend with an increase of zein concentration, due to proteinic nature of buffering capacity and self-assembly. The viscous property of NaCas has little influence on particle size. In addition, we can produce ZP a high zein concentration by the FNP procedure, which largely reduces cost for the FNP process. The particle size of ZP is stable with the variation of ethanol proportion, showing an attractive feature to encapsulate different actives in future studies. Compared with synthetic material, proteins are more suitable for preparing colloid particles at high concentration due to its charged groups of amino acid residuals. In summary, the FNP process is practical and applicable method to produce ZP in large scale. Acknowledgments This work was partially supported by The Project Supported by Guangdong Natural Science Foundation (S2013010012097). We also appreciate the financial support the Fundamental Research Funds for the Central Universities (SCUT 2012ZZ0082, 2014ZG0021, 2013ZM0065), Key Projects in the National Science & Technology Program during the Twelfth Five-year Plan Period (2013BAD18B10-4). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jfoodeng.2013. 12.001. References Chan, G., Mooney, D.J., 2008. New materials for tissue engineering: towards greater control over the biological response. Trends Biotechnol. 26, 382–392. Chen, L., Remondetto, G.E., Subirade, M., 2006. Food protein-based materials as nutraceutical delivery systems. Trends Food Sci. Technol. 17, 272–283. Cheng, J.C., Vigil, R.D., Fox, R.O., 2010. A competitive aggregation model for Flash NanoPrecipitation. J. Colloid Interface Sci. 351, 330–342. Dickinson, E., 1997. Properties of emulsions stabilized with milk proteins: overview of some recent developments. J. Dairy Sci. 80, 2607–2619.
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