Preparation of Corn Starch Nanoparticles by Wet-Stirred Media Milling for Chia Oil Vehiculization ^†

Abstract

An organic-free method was applied to produce corn starch nanoparticles, which were designed to stabilize Pickering emulsions containing chia oil, the richest vegetable source of omega-3 fatty acids. The liquid stream resulting from a laboratory-scale mill assisted by zirconia beads was filtered, centrifuged and homogenized to prepare the continuous phase of the emulsions. Experiments were performed as follows: 24 h (milling time), 0.1–0.2 mm (beads’ diameter), 1600 rpm (impeller speed), 25% (volume occupied by the grinding media), 1–7% w/v (starch concentration) and 0–1% w/v of sodium dodecyl sulfate (SDS). Particle sizes in the obtained nanosuspensions were reduced from 376–432 nm to 160–200 nm after centrifugation and homogenization. The product formulated with 0.01% w/v of SDS showed the most stable particle size during storage. Hence, this latter formulation was selected to prepare Pickering emulsions. Oil droplets showed surface mean diameters and polydispersity indexes of 283.33 ± 1.53 nm and 1.36 ± 0.03, respectively, with no significant variations during storage for around two weeks. Finally, nanosuspensions containing 7% w/v of starch, and the above three concentrations of SDS, were filtered, centrifuged, homogenized and spray-dried to obtain redispersible powders able to stabilize Pickering emulsions. The most stable particle size after redispersion (385.83 ± 5.85 nm) was obtained with the highest concentration of SDS. Moreover, SEM images revealed the presence of round-shaped particles with sizes below 1 μm. These results highlight that wet-stirred media milling can be applied as a green-method to produce new food-grade starch nanoparticles, which are able to deliver bioactive compounds from chia oil.

1. Introduction

Chia (Salvia hispanica L.) oil represents the most abundant vegetable source of omega-3 fatty acids. Despite the health benefits associated with a regular consumption of omega-3-rich oils, the polyunsaturated structure of fatty acids makes this oil highly susceptible to oxidation. Several technological strategies have been applied to stabilize chia oil [1]. Within this context, Pickering emulsions are rising as a very promising alternative for the vehiculization of omega-6 and omega-3 fatty acids in foods [2].

Pickering emulsions are stabilized by micro- or nanoparticles, which are located in the interfacial area of immiscible liquids. For some years, they have attracted the attention of many researchers due to their special characteristics: greater stability, less toxicity, better response to stimuli and resistance to coalescence phenomena [3]. Starch, a very abundant compound in nature, is positioned as a strong candidate for obtaining nanoparticles, which are well recognized within the food sector as novel stabilizing agents for dispersed systems [4].

Nanomilling in the stirred media mills (a top-down technique) is an efficient process for the preparation of ultrafine materials, owing to its advantageous features, viz., ease of operation, simple construction, high size reduction rate, ability to run continuously, and absence of organic solvents [5,6].

The objective of this work was to prepare stable corn starch nanoparticles via wet-stirred media milling, which were further used to stabilize Pickering emulsions containing chia oil. In addition, redispersible powders were obtained by spray-drying of the nanosuspensions due to the ease of storage and transport of solid materials compared with their fluid counterparts.

2. Materials and Methods

2.1. Materials

Chia seed oil (CSO) was extracted from seeds coming from the Salta province (Nutracéutica Sturla SRL, Salta, Argentina), as described by Martínez et al. [7], in a pilot plant screw press (Komet Model CA 59 G, IBG Monforts, Mönchengladbach, Germany). Corn starch was purchased to a local distributor (Distribuidora NICCO, Córdoba, Argentina); sodium dodecyl sulfate (SDS) (Sigma–Aldrich, San Luis, MO, USA) was used as stabilizer during milling experiments. Other reagents were HPLC or analytical grade.

2.2. Milling Experiments and Post-Processing of the Obtained Nanosuspensions

Corn starch nanoparticles (CSN) were prepared by media milling using a NanoDisp^® laboratory-scale mill (NanoDisp^®, Córdoba, Argentina). The temperature of the process was fixed at 15 °C by circulation of cold water with a Thermo Haake^® compact refrigerated circulator (Thermo Fisher Scientific, Waltham, MA, USA) [8].

First, suspensions containing starch (1% w/v) and SDS (0, 0.1 and 1% w/v) were prepared. The resultant mixtures were stirred for 10 min. Afterwards, suspensions and zirconia beads (0.1–0.2 mm and 25% v/v) were placed in the milling chamber and processed at 1600 rpm for 24 h. The obtained nanosuspensions were filtered through a 200 ASTM screen (Zonytest, Buenos Aires, Argentina) (74 μm), and the effects of centrifugation (13,000× g 40 min) and homogenization (18,000 rpm, 2 min, Ultraturrax homogenizer IKA T18, Janke & Kunkel GmbH, Staufen, Germany; followed by 1 cycle at 700 bar, in a high-pressure valve homogenizer, EmulsiFlex C5, Avestin, Ottawa, ON, Canada) on the stability of particle size were analyzed.

2.3. Characterization of the Obtained Nanosuspensions

The average particle size (Z_av) and the polydispersity index (PDI) immediately after milling and post-processing, and after storage for one week, were determined by dynamic light scattering at 25 °C (Nano Zetasizer, Malvern Instruments, Worcestershire, UK). The Zeta potential of nanosuspensions were also determined by dynamic light scattering [8].

2.4. Preparation and Characterization of Pickering Emulsions

The filtered and centrifuged nanosuspensions (1% w/v starch and 0.01% w/v SDS) obtained as described in Section 2.2 were blended with CSO (solids/CSO ratio: 5/1 w/w) by high-speed homogenization (18,000 rpm, 2 min). Subsequently, the coarse emulsions were processed in a high-pressure valve homogenizer (2 cycles, 700 bar, EmulsiFlex C5, Avestin, Ottawa, ON, Canada) to obtain fine emulsions. The oil droplet size distribution and polydispersity index were determined according to [1] with a LA 950V2 Horiba (Kyoto, Japan) analyzer, and the Zeta potential was measured according to Section 2.3.

2.5. Spray-Drying of Nanosuspensions and Characterization of Powders

Nanosuspensions containing 7% w/v of starch, and the three concentrations of SDS (Section 2.2), were filtered, centrifuged, homogenized (Section 2.2 and Section 2.4) and spray-dried to obtain redispersible powders able to stabilize Pickering emulsions. The spray-drying process was carried out in a laboratory-scale spray-dryer, Büchi B-290 (Büchi Labortechnik AG, Flawil, Switzerland) equipped with a two-fluid nozzle atomizer. The drying conditions given by Fu et al. [9] were followed. The obtained powders were redispersed in Milli-Q water to determine the Zav the and PDI as described in Section 2.3. Finally, the morphology of powders was evaluated by scanning electron microscopy (SEM, LSM5 Pascal; Zeiss, Oberkochen, Germany).

3. Results and Discussion

3.1. Characterization of the Obtained Nanosuspensions and Pickering Emulsions

Nanomilling refers to the reduction in particle size below 1000 nm by wet media-milling, and the intermediate product is a nanoparticle suspension or nanosuspension [5]. In order to obtain a stable product, the selection of the stabilizer formulation is a resource-demanding task, with potentially serious consequences such as aggregation, Ostwald ripening, sedimentation of particles, and cake formation during milling and storage [10]. In the present study, a widely-used amphiphilic surfactant such as SDS was used to provide electrostatic stabilization.

Milling could modify the granular morphology and micro molecular structure in starch, leading to improved physicochemical properties such as low gelatinization temperature and paste viscosity, enhanced redispersion, and large specific surface area, thus widening the scope of starch applications [6].

Table 1. Average particle size and polydispersity index of starch nanoparticles.

Formulation-Process	Zav,0 (nm)	Zav,f (nm)	PDI0	PDIf
1% S-M	416.13gh	2194.33f	0.332def	0.275ab
1% S-C	212.43c	300.73bc	0.233ab	0.383de
1% S-H	315.40e	502.87e	0.184a	0.504f
1% S-C+H	199.93bc	274.53b	0.260bc	0.352cd
1% S + 0.01% SDS-M	444.00h	416.90d	0.314cde	0.397de
1% S + 0.01% SDS-C	182.87ab	182.37a	0.247abc	0.242a
1% S + 0.01% SDS-H	373.60f	341.93c	0.365efg	0.276ab
1% S + 0.01% SDS-C+H	161.27a	158.10a	0.239ab	0.228a
1% S + 1% SDS-M	397.57fg	468.73de	0.400g	0.448ef
1% S + 1% SDS-C	265.23d	247.47b	0.288bcd	0.273ab
1% S + 1% SDS-H	407.97g	341.97c	0.387fg	0.291abc
1% S + 1% SDS-C+H	265.43d	253.33b	0.380efg	0.328bcd

S: starch; M: milling; C: centrifugation; H: homogenization; “0” and “f” subscripts indicate initial and final values after one week, respectively; different letters in each column indicate statistically significant differences (p < 0.05) among samples.

shows the average particle size and polydispersity index of fresh nanosuspensions and after storage for one week. The values for different concentrations of SDS, and through different processing steps (milling, centrifugation and homogenization), are included. The initial average particle sizes of starch suspensions, before milling, were around 15 μm (1% starch), 14 μm (1% starch + 0.01% SDS) and 90.6 μm (1% starch + 1% SDS). After a similar milling time (25 h), with native corn starch granules ranging from 2–20 μm, Lu et al. [6] observed Z_av values around 700 nm, above those found in Table 1.

The significantly higher (p < 0.05) average particle size, observed with the highest concentration of surfactant, may be attributed to the formation and aggregation of SDS micelles above its critical micelle concentration (0.23% w/v, 25 °C in the absence of any other additive). Moreover, it has been suggested that beyond a certain level, the use of a higher surfactant concentration may not prevent particle aggregation, but promote Ostwald ripening [10]. Multiple range tests by formulation show significantly (p < 0.05) higher Z_av and PDI values with 1% SDS. On the other hand, the lowest values were observed with 0.01% SDS, suggesting a proper stabilization of nanoparticles despite the lesser surface charge given by the Zeta potential (

Table 2. Zeta potential of starch nanoparticles.

Formulation-Process	Zeta Potential (mV)
1% S-M	−8.70ef
1% S-C	−7.68f
1% S-H	−9.59ef
1% S-C+H	−16.70d
1% S + 0.01% SDS-M	−9.23ef
1% S + 0.01% SDS-C	−12.43e
1% S + 0.01% SDS-H	−20.97c
1% S + 0.01% SDS-C+H	−19.00cd
1% S + 1% SDS-M	−62.27a
1% S + 1% SDS-C	−41.47b
1% S + 1% SDS-H	−38.70b
1% S + 1% SDS-C+H	−39.40b

S: starch; M: milling; C: centrifugation; H: homogenization; different letters in each column indicate statistically significant differences (p < 0.05) among samples.

). Therefore, this latter surfactant concentration was selected for the forthcoming formulation of Pickering emulsions.

Additional multiple range tests showed a significant (p < 0.05) reduction in Z_av and PDI after centrifugation, highlighting a necessary removal of large suspended particles. The homogenization of milled suspensions may further promote the disruption of particles [1], as can be seen from Z_av values. However, no additional reduction in Z_av and PDI values were observed (p > 0.05) after homogenization of the centrifuged nanosuspensions, evidencing a high mechanical input necessary to disrupt such nanoparticles. The homogenization step is key during emulsification of oil droplets, though. Hence, it was included as part of the preparation of Pickering emulsions.

The oil droplet size distribution of fresh emulsions, and after storage for one week, is given in

Table 3. Oil droplet size distribution and zeta potential of emulsions.

Emulsion	Zeta Potential (mV)	D32.0 (μm)	D32.f (μm)	PDI0	PDIf
Native starch	−8.35c	8.83a	12.29a	3.84a	60.86a
1% milled starch + 0.01% SDS	−18.43b	0.28c	0.47c	1.36b	1.73b
0.01% SDS	−48.93a	0.74b	4.29b	1.23c	1.22c

D₃₂: Sauter mean diameter; different letters in each column indicate statistically significant differences (p < 0.05) among samples.

and shown in Figure 1. The Zeta potential values are shown in Table 3. As can be seen, emulsions containing only native starch or SDS were also prepared. It has been informed that native starch granules may not form stable Pickering emulsions [2]. Indeed, sedimentation of solid particles was observed during storage (images not shown). Decreasing starch particle size tends to decrease the oil droplet size, while increasing the storage stability [2]. Moreover, the adsorption behavior of solid particles is different from that of small-molecule surfactants. Slow adsorption of soluble starch particles enables their rearrangement at the oil/water interface. Once these particles are adsorbed, irreversible dense interfacial layers may be formed due to the high-energy barrier against desorption [4], which might explain the stability observed in the emulsions of the present study, despite their low surface charge (−18.43 mV). Finally, the highest surface charge (−48.93 mV) was recorded for the SDS-stabilized emulsions, accounting for their stability during storage.

3.2. Characterization of the Powders Obtained by Spray-Drying

Redispersible powders, meant to stabilize Pickering emulsions, were obtained by spray-drying of nanosuspensions containing 7% w/v of starch, and the three concentrations of SDS. The average particle size and polydispersity index, before and after spray-drying, are given in

Table 4. Average particle size and polydispersity index of nanosuspensions and powders obtained after spray-drying (7% w/v starch).

Formulation-Process	Zav,0 (nm)	Zav, powder (nm)	PDI0	PDIpowder
7% S-C+H	469.37c	881.93b	0.31b	1.00b
7%S + 0.07%SDS-C+H	329.40a	200.50a	0.25a	0.29a
7%S + 1%SDS-C+H	359.30b	282.67a	0.24a	0.27a

S: starch; C: centrifugation; H: homogenization; different letters in each column indicate statistically significant differences (p < 0.05) among samples.

. Moreover, SEM micrographs of powders (including native starch and SDS) are shown in Figure 2. According to Table 4, SDS greatly improves (p < 0.05) the dispersion of powders, as reflected by the highest Z_av (881.93 nm) and PDI (1.00) values when no surfactant was used. When a suspension is atomized, a new air/water interface is formed [1] and rapidly stabilized by small-molecule surfactants such as SDS. Regarding the particles’ morphology, corn starch granules (Figure 1A,B) showed the characteristic truncated shape of native starch [9]. As a consequence of the milling operation, the internal structure of granules was modified and the resulting particles were swollen considerably [2,9]. With subsequent spray-drying, the volumetric shrinkage was remarkable (Figure 2E–J). Finally, it should be pointed out that the development of wrinkled surface morphologies in the gelatinized starch particles after milling might follow the same mechanisms found in solutions of polymeric macromolecules, such as maltodextrins and whey proteins. The effective moisture diffusivity in such solutions is very low, which results in high moisture gradients between drying air and the interior of droplets/particles. In order to reduce the diffusion path, and to provide a greater interfacial area for moisture evaporation, the drying of droplets try various ways to reduce the distance between the particle surface and its core. This ultimately leads to the deviation from sphericity, and to the formation of surface folds and troughs that facilitates the outward diffusion of water [9].

4. Conclusions

The stability of particle size in the starch nanosuspensions proved to be greatly affected by the presence of surfactants and post-processing after milling (mainly centrifugation and homogenization). Subsequently, the stability of oil droplets during storage for one week in Pickering emulsions could be confirmed, and might be attributed to the slow adsorption and rearrangement of soluble starch particles at the oil/water interface. This study highlights that wet-stirred media milling can be applied as a green-method to produce new food-grade starch nanoparticles, which are able to deliver bioactive compounds from chia oil.