油水界面におけるキトサン-カゼインナトリウム複合層の形成が水中油滴エマルション中の油滴の結晶化挙動に及ぼす影響

Abstract

Oil-in-water (O/W) emulsions containing palm oil or trimyristin as the oil phase were prepared using a membrane emulsification method. A polyelectrolyte complex layer composed of chitosan (CHI) and sodium caseinate (SC) was formed on the droplet surfaces, and its effect on oil droplet crystallization was investigated. The crystallization behavior of the oil droplets under isothermal conditions was observed using polarizing light microscopy, which revealed that the O/W emulsions with the CHI-SC complex layer exhibited slower crystal formation than those without the complex. Small-angle X-ray scattering (SAXS) analysis showed that α-form crystals were initially generated and gradually transformed into β′-form crystals, which then gradually increased during isothermal incubation. Comparison of polarizing microscopy and SAXS results indicated that the CHI-SC complex layer delayed the crystallization of oil droplets, particularly during the initial α-crystal formation stage. Similar effects were observed in emulsions containing trimyristin droplets, suggesting that the CHI-SC complex layer suppresses α-crystal formation regardless of the fatty acid composition of the triacylglycerols.

Translated Abstract

固体油脂を含有するエマルション製品においては，油脂の結晶化が品質に顕著な影響を及ぼすことが多い．本研究では，エマルションの安定化手法として知られる高分子複合層による油滴の被覆処理が，室温より高い融点をもつ油脂からなる水中油滴（O/W）エマルションの液滴結晶化に及ぼす影響を調べることを目的とした．まず，膜乳化法によりパーム油を油相とするO/Wエマルションを作製し，得られたエマルション滴の表層にキトサンとカゼインナトリウムからなる高分子複合層（CHI-SC複合層）を形成させた．得られたパーム油含有O/Wエマルションを60°Cで保温した後，20°Cに急冷・保持しながら偏光顕微鏡で油滴の結晶化の様子を観察したところ，時間経過に伴い油滴が部分的に結晶化し，結晶が生成した油滴の個数が徐々に増加していく様子が観察された．油滴表面におけるCHI-SC複合層の有無によるエマルション油滴の結晶化挙動を比較したところ，いずれも時間経過とともに結晶が生成した油滴の個数は増加したが，その増加速度はCHI-SC複合層を形成したO/Wエマルションの方が遅くなっていることがわかった．また，エマルション油滴の結晶化過程を小角X線散乱測定により調べた結果，保温初期にはパーム油の結晶多形であるα型結晶が主に生成し，これがβ′型結晶に転移するとともに徐々にβ′型結晶が増加していくことがわかった．偏光顕微鏡観察と小角X線散乱測定の結果を比較したところ，結晶化の遅延はα型結晶が生成する保温初期において顕著であり，油滴表面におけるCHI-SC複合層の形成がα型結晶の生成を抑制している可能性が示唆された．また，単一脂肪酸から構成されるトリグリセリドであるトリミリスチンを油相とするO/Wエマルションに対しても同様の実験を行った結果，トリミリスチン含有エマルションにおいてもパーム油含有エマルションと同様に，CHI-SC複合層の形成がα型結晶の生成に抑制的に作用していることが示唆された．

1. Introduction

An emulsion is a liquid-liquid dispersion system composed of two or more immiscible liquids. Emulsions are prepared in various forms, such as oil-in-water (O/W), water-in-oil (W/O), and water-in-oil-in-water (W/O/W), and are widely used in food formulations [1,2]. Changes in the physicochemical state of these emulsion products during formulation, distribution, and storage significantly affect their quality. Therefore, understanding the processes leading to instability, their mechanisms, and controlling the factors that cause instability are crucial for maintaining the long-term quality of emulsion products.

Emulsions prepared using solid fats with melting points above room temperature are widely used in various emulsified foods such as creams, margarines, and fat spreads. Solid fat is emulsified as a liquid at a temperature higher than the melting point. Usually, the resulting emulsion products are distributed and stored at room temperature or under refrigerated conditions, i.e., at temperatures below the melting point of the fat. If the emulsion temperature drops below the crystallization temperature of the fat, fat crystals form, leading to the total or partial solidification of the fat in the oil phase. Fat crystallization in the emulsion can cause partial coalescence between oil droplets, “oiling-off” phase separation, generation of coarse crystals, and solidification of the whole emulsion, resulting in reduced product quality [3-5]. Therefore, controlling the crystallization and inhibiting partial coalescence in solid fat emulsions are critical challenges for maintaining the quality of emulsified products. Additionally, some studies have reported that fat crystallization in oil droplets affects the chemical stability of hydrophobic components added to emulsions [6, 7], highlighting the importance of controlling fat crystallization in emulsions for the efficient delivery of functional nutrients.

Fat crystallization in emulsion droplets is a complex process that is influenced not only by temperature changes, including heating/cooling rates, and the nature of constituent oils, such as polymorphic properties, but also by additional factors distinct from the crystallization process of bulk oils, such as droplet size, emulsifier type and concentration, and inter-droplet interactions [8-11]. In our previous studies, we prepared uniform-sized O/W and W/O/W emulsions using microchannel emulsification with sodium caseinate (SC) as an emulsifier and palm oil as the oil phase [12-14] and investigated the effects of the formation of a chitosan (CHI)-SC electrostatic complex layer on the oil droplet surface (oil-water interface) on the stability of the emulsions [13,14]. We inferred that the CHI-SC complex layer formed on the oil droplet surface enhanced the physical strength of the oil-water interface [15,16], as well as provided steric hindrance and electrostatic repulsion [17-20], thereby suppressing droplet aggregation and partial coalescence during storage at a constant temperature or under temperature-changing conditions and contributing to the long-term stabilization of droplets. Furthermore, we found that the presence or absence of the CHI-SC complex layer not only prevented the aggregation and partial coalescence of crystallized oil droplets but also influenced the oil crystallization process [13,14]. Previous studies have reported that the crystallization processes within the oil droplets in the emulsion influence the subsequent crystal growth process and the formation of coarse crystals [21-23]. Therefore, clarifying the influence of the formation of the CHI-SC complex layer on the fat crystallization in oil droplets is expected to provide meaningful insights into the mechanisms by which the polymer electrolyte complex layers contribute to emulsion stability and effective utilization.

This study aimed to investigate the effect of the formation of a CHI-SC complex layer at the oil-water interface on the fat crystallization in an O/W emulsion with solid/semi-solid fats as the dispersed oil phase. SC was used as the emulsifier, and O/W emulsions containing palm oil or trimyristin as the oil phase were prepared using premix membrane emulsification [24-26]. CHI was added to form a CHI-SC complex layer, and the crystallization process including the crystalline polymorphism was evaluated under isothermal conditions using polarizing light microscopy and small-angle X-ray scattering (SAXS). To the best of our knowledge, although various studies have investigated the effect of edible polymer-coated liquid oil droplets on emulsion stability and properties [18,19, 27-31], few studies have focused on the fat crystallization process in O/W emulsions with dispersed solid/semi-solid fats. Therefore, the findings of this study provide novel insights into improving the stability and enhancing the functionality of emulsified food systems.

2. Materials and methods

2.1 Chemicals

Refined palm oil (RPO, analytical grade) was purchased from Sigma-Aldrich (St. Louis, MO, USA), which mainly consisted of oleic acid (35-50%), palmitic acid (35-50%), linoleic acid (6-13%), stearic acid (3-7%), and myristic acid (0.5-6%), as determined by the supplier. CHI (Chitosan 10, degree of deacetylation: 85%, as determined by the supplier; mean molecular weight: 1.5 × 10⁵, determined by viscometry [32], SC (12.6-15.8% as nitrogen after drying), d-(+)-glucose, sodium chloride (NaCl), acetic acid, and sodium hydroxide (NaOH) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The reported values of isoelectric point (pI) of SC and pKa of CHI were 4.5 [33-35] and 6.3-7.0 [36,37], respectively. Trimyristin (purity > 95%) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Fluorescein isothiocyanate (FITC) isomer I was purchased from Sigma-Aldrich (St. Louis, MO, USA). All the chemicals were used as purchased. Ultrapure water (18.2 MΩ∙cm resistivity) was obtained using a water purification system (Direct-Q UV, Merck Millipore Corporation, Billerica, USA) and was used for all experiments in this study.

2.2 Preparation of O/W emulsions by membrane emulsification

O/W emulsions were prepared using premix membrane emulsification [24-26]. RPO and trimyristin were used as the dispersed oil phases. An aqueous solution containing SC (3 wt%) and NaCl (0.2 M) was used as the continuous aqueous phase after filtration through a filter paper (No. 1; Toyo Roshi Kaisha, Ltd., Tokyo, Japan). In this study, 0.2 M (～1.2%) NaCl was added to the aqueous phase to simulate the ionic strength typically found in food emulsions. To prepare the premix emulsion, 0.5 mL of the oil phase, melted at 80 °C, was mixed with 9.5 mL of the continuous phase heated at 60 °C, then the mixture was hand-shaken (30 oscillations/min) for 1 min to form a coarse premix O/W emulsion. The premix O/W emulsion was hand-pressed through a polycarbonate membrane with a pore diameter of 10 µm (Isopore™ membrane filter, Merck, Darmstadt, Germany) fixed in a perfluoroalkoxy-alkane membrane holder (PFA-25, Advantec Toyo, Co., Ltd., Tokyo, Japan) for ten times using a plastic syringe preheated at 80 °C. The permeation rate of the premixed emulsions was adjusted to approximately 1.4 mL/s. The fine permeated O/W emulsion was recovered in a glass bottle and heated on a hot plate (C-MAG HS 4; IKA Werke GmbH & Co. KG, Staufen, Germany) at 80 °C. The obtained O/W emulsions were kept at 60 °C to prevent solidification of the oil phase until their use.

The droplet diameter distribution of the O/W emulsions was measured using a laser diffraction particle size analyzer (SALD-200V ER, Shimadzu Corporation, Kyoto, Japan). The volume-weighted mean diameter (d_4,3) and span factor (span ＝ (d₉₀ - d₁₀)/d₅₀, where d_i denotes the particle diameter corresponding to the cumulative particle volume of i vol%) were obtained using data analysis software (Wing SALD II, Shimadzu Corporation, Kyoto, Japan). The samples were diluted with water to obtain an appropriate turbidity for measurement. This dilution of emulsion samples did not affect the dispersibility of oil droplets at least during the measurement. Optical photomicrographs of the prepared emulsions were obtained in bright-field mode using an inverted microscope (CKX41, Olympus Corporation, Tokyo, Japan). The dispersibility of oil droplets was maintained at least during the observation.

2.3 Formation of CHI-SC complex layers on the oil-water interface

A glass centrifuge tube containing a mixture of the obtained O/W emulsion (2 mL) and a 0.2 M glucose solution (8 mL) was left at 60 °C for 15 min to separate the oil droplets from the continuous phase by droplet flotation, and 8 mL of the bottom continuous phase was removed from the emulsion sample by suction using a needle-connected syringe. The residual emulsion was mixed with 8 mL of glucose solution to dilute the SC in the continuous phase (final SC concentration: 0.12 wt%). This dilution did not affect the dispersibility of oil droplets. The sample temperature was kept at 60 °C during the above procedures.

CHI was dissolved at various concentrations in acetate buffer (0.1 M, pH 5.7) as previously reported [13]. Five milliliters of the SC-diluted emulsion was added dropwise to 12 mL of the solution containing a certain amount of CHI with magnetic stirring, and the mixture was stirred for 30 min at room temperature (22-26 °C). The dropwise addition of the emulsion to the CHI solution is considered to help minimize droplet collisions until the complexation of CHI with SC at the oil droplet surfaces is complete, while also reducing the difference in CHI concentration between the continuous phase and the vicinity of the oil droplets. Subsequently, an emulsion sample modified with the CHI-SC complex layer was obtained at various CHI/SC ratios [g/g], defined as the weight ratio of CHI to SC in the sample mixture [13,14].

To evaluate the formation of the CHI-SC complex layer, the ζ potential of the droplets in the emulsion was determined from the electrophoretic mobility measured using a ζ potential analyzer (Zetasizer Nano Z; Malvern Instruments Ltd., Worcestershire, UK). One milliliter of the emulsion sample was placed into a capillary cell (DTS1060; Malvern Instruments Ltd., Worcestershire, UK) and electrophoretic mobility was measured at 25 °C using the laser Doppler technique. ζ potential values were calculated in the monomodal mode using the Smoluchowski approximation. The samples were diluted with water to obtain an appropriate scattering intensity for measurement.

To observe the fluorescence of the polyelectrolyte complex layer formed at the oil-water interface, O/W emulsions with a CHI-SC complex layer were prepared using FITC-labeled CHI (FITC-CHI) according to a previous study [38]. Confocal fluorescence images of oil droplets modified with FITC-CHI were obtained using a confocal laser scanning microscope system consisting of an upright microscope (ECLIPSE Ni-E, Nikon Corporation, Tokyo, Japan) and a confocal microscope unit (AX R, Nikon Corporation, Tokyo, Japan). An excitation wavelength of 488 nm was used for observation.

2.4 Small-angle X-ray scattering (SAXS) measurement

The SAXS measurements were conducted using a synchrotron radiation source at beamline 6A at the Photon Factory of the High Energy Accelerator Research Organization (KEK, Tsukuba, Japan). The details of this equipment have been described elsewhere [39,40]. The X-ray wavelength was 0.150 nm. The X-ray scattering data were obtained using a PILATUS3 1M detector (DECTRIS Ltd., Baden, Switzerland).

The O/W emulsion samples were preheated at 60 °C and enclosed within the 1-mm-thick inner space of a plastic ring-shaped cell by sealing with Kapton tape on both sides. The sample cell was placed on the sample stage of a thermocontrol system (Linkam Scientific Instruments Ltd., Surrey, UK). The distance between the sample and the detector was approximately 900 mm. The samples were heated at 60 °C on the sample stage for 2 min and then cooled at a rate of 20 °C/min to the specified temperature, after which X-ray scattering data were collected at a constant temperature. The 2-dimensional SAXS image data were analyzed using the SAngler software (KEK, Tsukuba, Japan) [i]. Silver behenate was used as a standard to calibrate the scattering angle. The length of the periodic structure in sample d was calculated using Bragg’s equation as follows:

  

$d = \lambda /\sin (2\theta /2)$(1)

where λ is the wavelength of the X-ray and 2θ is the scattering angle.

2.5 Differential scanning calorimetry (DSC)

DSC thermograms were obtained using a differential scanning calorimeter (DSC-60 Plus; Shimadzu Corporation, Kyoto, Japan). RPO (18 mg) was enclosed in an aluminum sealed pan using a sample-sealing press (SSCP-1; Shimadzu Corporation, Kyoto, Japan). α-Alumina powder was used as a reference compound for measurement. The sample was preheated at 60 °C for 5 min, cooled at 2.5 °C/min, and maintained at 20 °C during the measurement.

2.6 Observation of crystallization behavior using polarizing microscopy

The crystallization of the oil droplets in the O/W emulsions was observed using a polarizing microscope (ECLIPSE Ci-POL, Nikon Corporation, Tokyo, Japan). The emulsion sample on a glass slide was preheated at 60 °C and then transferred to the top of a water-circulating aluminum stage attached to the microscope to control the sample temperature during observation. The surface temperature of the glass slide equilibrated within several seconds after the sample was transferred to the aluminum stage. Bright-field and crossed-Nicols images were captured using a digital camera (α7S, Sony Corporation, Tokyo, Japan) mounted on a microscope. The dispersibility of oil droplets was maintained at least during the observation. The number of oil droplets showing birefringence owing to crystallization was counted, and the number-based fraction of crystallized droplets (ϕ_c) was calculated using the following equation:

  

$\phi _{\text{c}} = N_{\text{c}}/N_{\text{t}}$(2)

where N_c and N_t are the number of oil droplets showing birefringence and the total number of oil droplets in the observed field of view, respectively.

3. Results and discussion

3.1 Crystallization behavior of bulk RPO under constant temperature

The SAXS patterns of RPO in the bulk state measured at a constant temperature (20 °C) are shown in Fig. 1. When RPO, which had completely melted at 60 °C [12], was rapidly cooled and maintained at 20 °C, a peak at 2θ ＝ 1.84° was observed immediately after rapid cooling. This peak showed a maximum intensity 180 s after the sample temperature was reached, was maintained at 20 °C, and then decayed. Meanwhile, a new peak appeared at 2θ ＝ 2.03° with increasing intensity until 3,600 s. From these scattering angles, the periodic length, d, in the sample could be calculated using Eq. (1) based on Bragg’s law, and it was found that the peaks at 2θ ＝ 1.84° and 2θ ＝ 2.03° correspond to the long-spacing lengths of the α-form (d ＝ 4.7 nm) and β′-form (d ＝ 4.3 nm) crystals of palm oil, respectively [21, 41].

Fig. 1

Typical time course small-angle X-ray scattering pattern of bulk refined palm oil (RPO) at 20 °C. The peaks at 2θ ＝ 1.84° (d ＝ 4.7 nm) and 2θ ＝ 2.03° (d ＝ 4.3 nm) are attributed to the long-spacing lengths of the α and β′polymorphic forms of RPO, respectively.

Figure 2 shows the time courses in the peak intensity derived from α and β′-form crystals determined by SAXS (a) and the DSC thermogram (b) of bulk RPO measured at a constant temperature of 20 °C. In Fig. 2b, an initial exothermic baseline drift was observed, which can be attributed to thermal equilibration within the sample following rapid cooling. The SAXS peak intensity of the α-form crystals initially increased (Fig. 2a), and an endothermic peak (peak 1 in Fig. 2b) was observed in the DSC thermogram during the same period, indicating that the α-form crystals were mainly generated from molten RPO in the initial stage (phase I). Next, the intensity of the SAXS peak derived from the α-form crystal decreased, while the intensity of the peak derived from the β′-form crystal increased (Fig, 2a). A corresponding endothermic peak (peak 2 in Fig. 2b) was observed in the DSC, which is thought to be due to a polymorphic transition from the α-form to the β′-form crystal (phase II). Consequently, the intensity of the peak derived from the β′-form of the crystal increased consistently (phase III).

Fig. 2

Time courses of (a) small-angle X-ray scattering peak intensities corresponding to α (circles) and β′ (squares) polymorphic forms of refined palm oil (RPO) crystal and (b) isothermal differential scanning calorimetry thermogram of bulk RPO at 20 °C. For phases I, II, and III, see the main text.

These results suggest that (I) α-form crystals were formed first during the initial stage of crystallization, (II) α-form crystals transformed into β′-form crystals, and then (III) β′-form crystals are formed in palm oil at a constant temperature of 20 °C. This result is qualitatively consistent with the results of previous studies [42,43].

3.2 Preparation of palm oil-in-water emulsions with polyelectrolyte complex layers

Next, we examined the crystallization behavior of O/W emulsions containing RPO as the dispersed oil phase to investigate the effect of the polyelectrolyte complex layer formed on the surfaces of the oil droplets. Figure 3 shows the typical particle size distribution and a microscopic image of the O/W emulsion used in this experiment. The mean droplet diameter of the O/W emulsion prepared using the premix method with a polycarbonate membrane with a pore size of 10 µm was 22 µm, and oil droplets with diameters of 10-30 µm accounted for 58.7% of the total droplet volume.

Fig. 3

Bright-field photomicrograph (a) and typical droplet diameter distribution (b) of palm oil-in-water emulsions prepared using premix membrane emulsification using a polycarbonate membrane (pore diameter: 10 µm).

Using this emulsion, a polyelectrolyte complex (CHI-SC) layer was formed at the oil-water interface according to the procedure described in Section 2.3. Figure 4 shows the effect of the CHI/SC ratio on the ζ-potential of the oil droplets in the emulsion. The ζ-potential of SC-stabilized oil droplets without complexation with CHI was -36.1 mV, reflecting the negative charge of SC at pH 5.7 (pI ＝ ～4.5 [33-35]). Upon increasing the CHI/SC ratio, the ζ-potential became positive, with a value of approximately +30 mV at a CHI/SC ratio of 10. When the CHI/SC ratio was between 0.05 and 0.3, large aggregates were formed because of the cancellation of the surface charges of the oil droplets. Similar results were also obtained in previous studies [13,14], presumably because the surface of the oil droplets reflects the positive charge of the outermost CHI layer, which forms a complex layer with the negative SC through electrostatic interactions. In addition, when the surface of the oil droplets was modified using FITC-CHI at CHI/SC ＝ 10, green fluorescence was observed along the outline of the oil droplets, as shown in the inset of Fig. 4, confirming that CHI was adsorbed onto the surface of the oil droplets. As reported in previous studies [13,14,29,30], there was no significant difference in the average diameter or span of the emulsions prepared with CHI/SC ＝ 0 and 10, confirming that the formation of CHI-SC did not affect the droplet size distribution of the O/W emulsions. In the following experiments, the crystallization behavior of O/W emulsions prepared under the conditions of CHI/SC ＝ 0 (no complex layer) and 10 (with a complex layer) was investigated.

Fig. 4

ζ-potential of palm oil droplets in the emulsion as a function of chitosan/sodium caseinate (CHI/SC) weight ratio. The inset shows a confocal laser scanning micrograph of an oil droplet with polyelectrolyte complex layers of FITC-CHI and SC at CHI/SC ＝ 10.

3.3 Effect of the polyelectrolyte complex layers on the crystallization behavior of palm oil droplets in O/W emulsions

Figure 5 shows the time courses of the SAXS profiles of the O/W emulsions prepared at CHI/SC ratios of 0 (no complex layer) and 10 (with a complex layer). The emulsion samples were preheated to 60 °C to completely melt the oil phase, and then maintained at 20 °C for 1 h. In both O/W emulsion samples, a peak was observed at approximately 2θ ＝ 1.84° (d ＝ 4.7 nm) in the early periods (10-15 min) and then disappeared, whereas a new peak was observed at approximately 2θ ＝ 2.03° (d ＝ 4.3 nm) in later periods. As a result, regardless of whether or not the CHI-SC complex layer was present, the same crystallization behavior was observed as with the bulk RPO: after rapid cooling the complete melt, α-form crystals were formed during the initial period of isothermal incubation at 20 °C and then they transformed into β′-form crystals, and thereafter β′-form crystals were increased.

Fig. 5

Small-angle X-ray scattering time courses of palm oil-in-water emulsions in the absence (CHI/SC ＝ 0, a) and presence (CHI/SC ＝ 10, b) of polyelectrolyte complex layers at 20 °C. The peaks at 2θ ＝ 1.84° (d ＝ 4.7 nm) and 2θ ＝ 2.03° (d ＝ 4.3 nm) are attributed to the α and β′ polymorphic forms of the RPO crystal, respectively.

Figure 6 shows the time-dependent changes in the polarization microscope images of O/W emulsions with CHI/SC ratios of 0 (no complex layer, left photos) and 10 (with complex layer, right photos), which were preheated to 60 °C and then rapidly cooled to 20 °C and then maintained on a temperature-controlled slide stage. In both emulsion samples, no birefringence was observed in the oil droplets under crossed Nicols at 60 s after the isothermal incubation at 20 °C. However, after some time, white spots were observed in the oil droplets, which resulted from birefringence due to palm oil crystallization. Based on these observations, it was possible to evaluate the gradual increase in the number of crystal-forming oil droplets in real time.

Fig. 6

Crystallization process of palm oil droplets in the absence (CHI/SC ＝ 0, left photos) and presence (CHI/SC ＝ 10, right photos) of polyelectrolyte complex layers at 20 °C. Photomicrographs were obtained under bright-field (BF) and crossed Nicols (CN) conditions. Scale bars: 50 µm.

Figure 7 shows the formation of crystal polymorphs in the O/W emulsions at 20 °C as revealed by SAXS measurements, and the progress of crystallization as observed by real-time polarizing microscopy. Figure 7(a) and (b) show the time courses of the peak intensities derived from the α- and β′-form crystals obtained by SAXS measurements for the O/W emulsions prepared at CHI/SC ratios of 0 and 10, respectively. In addition, Fig. 7(c) shows the time dependence of the fraction of crystallized oil droplets, ϕ_c, calculated using Eq. (2): ln(1- ϕ_c) plotted as a function of time based on pseudo-first order kinetics (Eq. (3)) [10,44].

  

$\ln (1 - \phi _{\text{c}}) = - kt$(3)

where k is the apparent kinetic constant of crystal formation and t is the time.

Fig. 7

Time course of the small-angle X-ray scattering peak intensities of α (circles) and β′ (triangles) polymorphic forms of refined palm oil crystals in oil-in-water emulsions at CHI/SC ＝ 0 (a) and 10 (b). (c) Kinetic plots of time-dependent droplet crystallization of palm oil-in-water emulsions in the absence (CHI/SC ＝ 0, squares) and presence (CHI/SC ＝ 10, circles) of polyelectrolyte complex layers at 20 °C. ϕ_c is the ratio of the number of crystallized droplets to the total number of oil droplets as determined by polarizing microscopy (see Section 2. 6). For phases I, II, and III, see the main text.

Based on the changes in crystal polymorphs observed by SAXS (Fig. 7(a) and (b)), the crystallization process in O/W emulsions can be divided into three phases, as discussed for bulk RPO (Fig. 2): the initial formation of α-form crystals (phase I), the subsequent transition of α-form crystals into β′-form (phase II), and the formation of β′-form crystals (phase III). In Fig. 7(c), the linear time dependence of ln(1-ϕ_c) was observed in the early (phase I) and later (phase III) time regions. Based on Eq. (3), assuming that the slope determined from the linear region of the ln(1-ϕ_c) vs. t curve, k, reflects the apparent crystallization rate constant, the effect of the CHI-SC complex layer on the crystallization rate could be significant in phase I but not in phase III, and an intermediate behavior was observed in phase II. This indicates that the formation of the CHI-SC complex layer may affect the initial formation of α-form crystals and the polymorphic transition to β’-form crystals.

Figure 8 shows the plots of the crystallization process of the emulsions at different temperatures. At 15 °C (Fig. 8(b)), although the rate of droplet crystallization was faster than at 20 °C (Fig. 8(a)), the formation of the CHI-SC complex layer delayed the crystallization rate during the early stage of isothermal incubation, similar to that observed at 20 °C . At 12.5 °C (Fig. 8(c)), the rate of droplet crystallization in the early period was much faster than those at other temperatures, regardless of the presence or absence of the CHI-SC complex layers, and more crystallization was observed in the emulsion without the complex layer during the same period.

Fig. 8

Kinetic plots of time-dependent droplet crystallization of palm oil-in-water emulsions in the absence (CHI/SC ＝ 0, squares) and presence (CHI/SC ＝ 10, circles) of polyelectrolyte complex layers at (a) 20, (b) 15, and (c) 12.5 °C. ϕ_c is the ratio of crystallized droplets to the total number of oil droplets as determined by polarizing microscopy (see Section 2. 6). The data in (a) are replotted from Fig. 7(c).

At all temperatures, the CHI-SC complex layer did not affect the crystallization progress in the later stages of isothermal incubation. SAXS measurements at 12.5 °C showed a similar pattern as that at 20 °C, indicating the formation of α-form crystals followed by a polymorphic transition to β′-form crystals (data not shown). This suggests that the formation of the CHI-SC complex layer had the same effect on the initial stages of isothermal incubation even at different incubation temperatures.

In O/W emulsions stabilized by SC, SC is adsorbed at the oil-water interface. SC molecules can diffuse parallel to the interface [45]. In contrast, when CHI molecules are further adsorbed on the outer layer of the oil droplet, CHI and SC molecules could form a CHI-SC complex layer through multipoint attractive interactions (electrostatic interactions, hydrogen bonds, etc.), and the horizontal diffusion of SC at the interface is suppressed compared to that without CHI. Consequently, the motion of the triacylglycerol molecules near the CHI-SC complex layer may be restricted; thus, the mobility of the triacylglycerol molecules near the oil droplet surface may decrease.

The decrease in molecular mobility may affect the frequency of collisions between triacylglycerol molecules, which inhibits the nucleation of fat crystals and subsequent polymorphic transitions [10,46]. This is consistent with the results in Fig. 7, which shows the possible inhibition of the formation of α-form crystals, which are likely to form in the early stages after rapid cooling, and the subsequent polymorphic transition into β′-form crystals. Considering the possible effects of protein molecules on fat crystallization at the oil-water interface, such as interfacial adsorption effects on nucleation kinetics and crystallization temperature [47-49], the formation of a CHI-SC complex layer on the oil droplet surface may also affect the initial crystallization process of oil droplets in emulsion systems. However, the formation of crystals in fats and oils and their polymorphisms are complex phenomena that are influenced by multiple factors, including the structure and mobility of triacylglycerol molecules, the activation and interfacial energies involved in crystallization, and the properties of coexisting catalytic impurities [10, 50, 51]. Thus, further investigation is needed to clarify the detailed mechanism of the impact of the CHI-SC complex layer on the crystallization in the oil droplets.

3.4 Effect of polyelectrolyte complex layers on crystallization behavior of trimyristin droplets

In this study, we investigated the effect of the CHI-SC complex layer on the crystallization behavior of RPO in the oil droplets composed of a mixture of triacylglycerols with various fatty acid moieties such as palmitic acid, stearic acid, and oleic acid [52-54]. To verify the generality of this phenomenon, we prepared an O/W emulsion with trimyristin, a triacylglycerol composed of a single fatty acid, as the oil phase and investigated the effect of the CHI-SC complex layer on the fat crystallization behavior in the same way as RPO.

Figure 9 shows the kinetic plots of crystal formation when the O/W emulsion with trimyristin was maintained at 25 °C (Fig. 9(a)) and 20 °C (Fig. 9(b)). Similar to RPO, the formation of the CHI-SC complex layer on the trimyristin droplets delayed their crystallization. SAXS measurements using trimyristin in bulk form showed that α-form crystals (d ＝ 4.0-4.1 nm [55]) were formed in the early stages of isothermal incubation at all temperatures examined (data not shown), suggesting that the formation of the CHI-SC complex layer also delays the formation of α-form crystals in the early stages of isothermal incubation in trimyristin droplets. These results indicate that the change in crystallization behavior caused by the formation of the CHI-SC complex layer is a common phenomenon in O/W emulsions containing RPO and trimyristin as the oil phases. Unlike trimyristin, which is a single-component triacylglycerol, RPO is a mixture of triacylglycerols with various fatty acid compositions. Therefore, components with different crystallization tendencies likely coexist at the tested temperatures, and the CHI/SC complex layer may have exerted more pronounced effects on the crystallizable components under these conditions (Fig.8).

Fig. 9

Kinetic plots of time-dependent droplet crystallization of trimyristin-in-water emulsions in the absence (CHI/SC ＝ 0, squares) and presence (CHI/SC ＝ 10, circles) of polyelectrolyte complex layers at (a) 25 °C and (b) 20 °C. ϕ_c is the ratio of the number of crystallized droplets to the total number of oil droplets as determined by polarizing microscopy (see Section 2.6).

4. Conclusion

In this study, we investigated the effect of the formation of a CHI-SC complex layer at the oil-water interface on the fat crystallization in oil droplets in an O/W emulsion with solid/semi-solid oils (RPO) as the dispersed oil phase. SAXS and DSC analyses of bulk RPO at a constant temperature below the crystallization temperature of RPO revealed that a metastable α-form crystal first formed, followed by a transition to a β′-form crystal, accompanied by the gradual formation of new β′-form crystals. Next, O/W emulsions with RPO as the dispersed oil phase were prepared by membrane emulsification and modified with CHI-SC complex layers at the oil-water interface. The effect of the CHI-SC complex layers on the crystallization behavior of the RPO droplets at a constant temperature below the crystallization temperature was investigated. These results suggest that the formation of the CHI-SC complex layer partially inhibited the formation of α-form crystals during the initial stage of isothermal incubation. SC molecules, which are adsorbed at the oil-water interface, form a complex with CHI, with CHI molecules reducing the interfacial mobility of SC and triacylglycerol molecules near the oil-water interface, thereby possibly influencing the crystallization process. However, further investigation is necessary to clarify this mechanism in detail. In addition, the suppressive effects of the CHI-SC complex layers on fat crystallization in oil droplets were observed in an O/W emulsion prepared using a single fatty acid triacylglycerol, trimyristin, as the oil phase. The insights obtained from this study regarding the control of oil droplet crystallization may provide a meaningful technical foundation for improving the quality of food emulsion products containing solid lipid components.

Acknowledgements

This study was supported by JSPS KAKENHI [grant number 24K00361]. This study was approved by the Prioritized Study (Advanced Food Process Research Unit) of Advanced Research Laboratories (Tokyo City University, Japan) and Photon Factory Program Advisory Committee (Proposal Nos. 2021G114 and 2023G121). The authors would like to thank Prof. Satoru Ueno, Prof. Haruhiko Koizumi (Hiroshima University, Japan) and Prof. Sosaku Ichikawa (University of Tsukuba, Japan) for their expert advice on SAXS analysis. We would also like to thank Editage (www.editage.jp) for English language editing.

Nomenclature

d

characteristic length of the periodic structure in the sample, m

k

apparent kinetic constant of crystal formation, s^-1

N_c

number of oil droplets showing birefringence in the observed field of view

N_t

total number of oil droplets in the observed field of view

T

time, s

2θ

scattering angle (θ: incident angle according to Bragg’s law), °

λ

wavelength, m

ϕ_c

number-based fraction of crystallized droplets

URL cited

1. i)    http://pfwww.kek.jp/saxs/SAngler.html (Accessed 16 May 2025)

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