Effects and mechanisms of ultrasonic-assisted emulsification on the properties of rennet casein emulsions under low salt conditions

Abstract

The physical instability of rennet casein emulsions in natural systems limits their applications in low-salt products. This study was designed to investigate the effects and mechanisms of ultrasonic-assisted emulsification on the properties of rennet casein emulsion under simulated milk ultrafiltrate conditions. Results showed that ultrasonic treatment at 500 W for 7 min can significantly improve the stability of a rennet casein-corn oil emulsion. Compared with rennet casein emulsions prepared by high-speed homogenization, the emulsions prepared by ultrasonics had a higher emulsifying activity index (0.17–1.00 m²/g) and better storage stability. The apparent viscosity of rennet casein emulsions was also increased after ultrasonic treatment. Ultrasonic treatment significantly reduced the droplet size (D_3,2 decreased from 8.10 to 1.66 μm) and protein aggregates in the rennet casein emulsions. The concentration of rennet casein at the water–oil interface and the absolute zeta potential of emulsions were increased after ultrasonic treatment. The oil contact angle and surface hydrophobicity of rennet casein after ultrasonic treatment were also higher than those of the high-speed shear homogenization-treated samples. In summary, ultrasonic-assisted emulsification treatment can significantly improve the stability of rennet casein emulsions under natural conditions.

1. Introduction

Rennet casein powder is obtained by coagulating pasteurized milk with rennet, then separating, drying, and pulverizing [1], [2]. It exhibits diverse functions in the production of cheese, yogurt, bakery, meat analogues, and protein-fortified foods [3]. Rennet casein retains a more closely structured and nutritional profile than casein obtained by other methods, such as acid precipitation [4]. Meanwhile, compared with acid casein, rennet casein contains higher levels of protein and minerals and tends to produce firmer, more elastic textures in its products [5]. However, a large amount of calcium paracaseinate is formed during rennet casein production, resulting in low water solubility and emulsifying properties [6]. To overcome these limitations of rennet casein, different amounts and types of emulsifying salts are often added during the production of rennet casein-based products such as processed cheese [7]. The addition of emulsifying salt converts the insoluble calcium paracaseinate in rennet casein into soluble sodium paracaseinate, making it easier to distribute in an aqueous solution and at the oil–water interface [6]. However, adding emulsifying salt might lead consumers to ingest more Na⁺, thereby increasing the risk of health problems, including high blood pressure, kidney damage, and heart disease [8]. Therefore, improving the emulsifying properties of rennet casein without adding emulsifying salts is of great significance for broadening the applications of rennet casein.

Low-frequency ultrasound treatment (lower than 100 kHz) has been widely reported to enhance the emulsifying properties of different food proteins, including myofibrillar protein, soy protein isolate, and sodium caseinate [9], [10], [11], [12]. Unlike using low-frequency ultrasound to treat protein and then adding oil to prepare emulsion systems, ultrasonic-assisted emulsification directly utilizes the cavitation effect of ultrasound to treat the mixtures of protein and oil [13], [14]. During the ultrasonic-assisted emulsification process, protein molecule modification and emulsion formation occur simultaneously, which can greatly improve the emulsion stability of protein emulsions and the emulsification activity of protein [15]. Ultrasonic-assisted emulsification has been used to fabricate different protein-based emulsion systems, including soy protein isolate [16], myofibrillar protein [17], and skim milk protein [18], to increase their emulsion stability and decrease the emulsion droplet size. The effects of ultrasound treatments on the functional properties of rennet proteins have also been reported [19], [20]. However, these studies mainly focus on the effects of ultrasound treatment on the formation of rennet casein [21], [22] or on the functional properties, such as gel properties of rennet casein [23], [24]. The changes in the emulsifying properties of rennet casein during ultrasonic-assisted emulsification, especially in the absence of emulsifying salt addition, are currently unclear.

Therefore, this study was designed to investigate whether ultrasonic-assisted emulsification could improve the emulsion stability of rennet casein emulsions in simulated milk ultrafiltrate conditions (without added emulsifying salts). Meanwhile, the mechanism of ultrasonic-assisted emulsification affecting the properties of rennet casein emulsions under simulated milk ultrafiltrate conditions was also investigated. First, the effects of different ultrasonic powers and times on the stability of rennet casein emulsions were compared to choose the optimal ultrasonic treatment conditions. The effects of ultrasonic-assisted emulsification on emulsion stability were then analyzed by comparing the stability, emulsifying activity, rheological properties, and average size and distribution of emulsion droplets of rennet-casein emulsions by different emulsification methods. The mechanism by which ultrasonic-assisted emulsification affects the stability of rennet-casein emulsions was then elucidated by analyzing the interfacial and protein molecular properties of the different emulsions. The findings of this study contribute to expanding the application of ultrasound and broadening the applications for rennet casein.

2. Materials and methods

2.1. Materials and chemicals

Rennet casein was purchased from the EURIAL (NANTES, France) and corn oil was sampled from a local supermarket (Yihai Kerry, Shanghai, China). The other chemicals are analytical grade or better.

2.2. Fabrication of pre-prepared rennet casein emulsion

The 30 g of rennet caseins were diluted in 1 L of simulated milk ultrafiltrate (1.58 g of KH₂PO₄, 1.2 g of C₆H₅K₃O₇·H₂O, 1.8 g of Na₃C₆H₅O₇·2H₂O, 0.18 g of K₂SO₄, 1.00 g of CaCl₂, 0.65 g MgCl₂·6H₂O, 0.3 g of K₂CO₃, 0.6 g KCl, pH 6.6) for 12 h [25], and then the pH was adjusted to the selected range with 0.1 M lactic acid. The 100 mL of the above mixture was then mixed with 5 mL of corn oil at 10000 rpm for 1 min to obtain a pre-prepared emulsion (PSE).

2.3. Preparation and characterization of ultrasonically prepared emulsion

The PSE samples were subjected to different ultrasonic powers to fabricate ultrasonic-prepared emulsions. Briefly, 50 mL of PSE sample was placed into a glass beaker and then treated at different ultrasonic powers (100 W, 200 W, 300 W, 400 W, 500 W, and 600 W) for 7 min to obtain fine emulsions. The ultrasonic device was purchased from Ningbo Xinzhi Biotechnology Co., Ltd. (Jiangsu, China) and equipped with a 10 mm probe, and the probe was inserted 2 cm into the emulsion during ultrasonic-assisted emulsification treatment. The ultrasonic treatment conditions were 2 s on and 3 s off, and the sample temperature was controlled below 20 °C. The fine rennet casein emulsion at 500 W under different treatment times (1 min, 3 min, 5 min, 7 min, and 9 min) was also prepared followed the above methods.

The emulsion instability properties of rennet casein emulsions were tested with a Turbiscan multiple light scattering analyzer (Formulaction, Toulouse, France). The backscattered light (ΔBS) curves and Turbiscan instability index (TSI) of different emulsions were obtained during 6 h of storage.

2.4. Preparation of normal and ultrasonic fine emulsions

The normal fine rennet casein emulsions were fabricated with high-speed shear homogenization (IKA, Staufen, Germany) at 20,500 rpm for 6 min. The ultrasonic fine emulsions were fabricated at 500 W for 7 min based on the above results. The high-speed shear homogenization prepared rennet casein emulsion was marked as HSE, the ultrasonic-fabricated rennet casein emulsion was abbreviated as UAE, and the pre-prepared rennet casein emulsion was recorded as PSE.

2.5. Characterization of emulsion activity and stability

The emulsifying activity index (EAI) was tested according to the methods of Zhou et al. [26] with little modifications. The 50 μL of fresh emulsions (PSE, HSH, UAE, and rennet casein emulsions with different ultrasonic powers and times) were mixed with 5 mL of 0.1% sodium dodecyl sulfate. The absorbances of the diluted solutions at 500 nm were recorded to calculate the EAI values as follows (Eq. (1)):

$$\mathit{EAI}(m^2/g)=2\times 2.303\times \frac{A\times N}{1000\times C\times (1-\phi )}$$ (1)

where A is the absorbance of the diluted solution, N is the dilution factor of 100 in this work, C is the protein concentration of 30 mg/mL in this work, and φ is the oil concentration of 0.05 in this work.

The physical stabilities of PSE, HSE, and UAE emulsions were determined with a Turbiscan multiple light scattering analyzer. The TSI and ΔBS values of PSE, HSH, and UAE samples were recorded during a 6-hour test.

2.6. Rheological properties test

The viscosity properties of different emulsions were measured using a rheometer attached with a 40 mm flat plate within the shear rate between 0.01 and 100 s⁻¹. The viscosity coefficient (K, Pa·sⁿ) and flow index (n) were calculated with the power law equation (Equation (2) based on the change in viscosity (η, Pa·s) and shear rate (γ, s⁻¹) [27].

$$\mathrm{\eta }=\mathrm{K}\times {\gamma }^{n-1}$$ (2)

2.7. Changes in the emulsion droplet size

2.7.1. Droplet size

The droplet sizes of PSE, HSE, and UAE emulsions were determined with a Malvern 3000 laser particle size analyzer (Malvern Panalytical Ltd., Worcestershire, UK). The absorption rate is 0.001, the refractive index is 1.436, and the light blocking degree ranges from 10% to 20% [26]. The droplet distribution curve and average droplet size (D_4,3 and D_3,2) of the emulsion were obtained.

2.7.2. Optical microscope

The morphology and size distribution of emulsion droplets of different emulsions were observed using an ECLIPSE E100 biological microscope (Nikon Corporation, Tokyo, Japan) under bright field and 40 × magnification [28].

2.7.3. Laser confocal scanning microscopy (LCSM)

The distributions of the oil droplet and protein in PSE, HSE, and UAE emulsions were obtained using an LCSM (ZEISS, Oberkochen, Germany) under 63 × magnification. The 0.2% Nile red and 0.2% fast green were used for staining the oil phase and protein, respectively. The samples were then placed on a glass slide and observed using LCSM at excitation wavelengths of 543 nm and 633 nm [29].

2.8. Protein content at the oil–water interface

The protein contents at the oil–water interface of different emulsions were determined with centrifuge methods as described by Xie et al. [30]. Briefly, emulsions were centrifuged at 10,000 g and 4 °C for 30 min, and then three phases, including creaming phase, aqueous solution, and sediment, were obtained. The undissolved proteins in the precipitate were washed three times with phosphate buffer before determining the protein content. Then, the protein content was determined using the Coomassie brilliant blue method with bovine serum albumin as the standard protein. The protein contents at the oil–water interface were calculated as follows (Equation (3):

$$\mathit{Interficalprotein}(\%)=(1-\frac{m_a+m_b}{m_t})\times 100\%$$ (3)

where m_a is the protein content in the aqueous solution, m_b is the protein content in the precipitate, and m_t is the total protein content.

2.9. Zeta potential

The zeta potential values of different emulsions were measured with a Zetasizer (Malvern Panalytical Ltd., Worcestershire, UK). Before the measurement, the emulsion was diluted 500 times with simulated milk ultrafiltrate.

2.10. Protein properties of emulsions

2.10.1. Protein surface hydrophobicity

The surface hydrophobicity curves of rennet casein in different emulsions were measured using a multifunctional microplate reader (Spark, Tecan Group Ltd., Männedorf, Switzerland) [26]. The 10 μL of 15 mM 8-anilino-1-naphthalenesulfonic acid (10 mM KH₂PO₄, pH 7.0) was mixed with 2 mL of emulsions and then placed in a dark place for 25 min before the test. The excitation wavelength is 374 nm, and the emission wavelength ranges from 420 nm to 600 nm.

2.10.2. Contact angle measurement

The water and oil contact angles of different samples after compressing into a disc with a 13 mm diameter and 2 mm thick were measured with a goniometer (DSA25, KRÜSS, Hamburg, Germany) using the hanging drop method. The water contact angles were obtained using the following methods: 2 μL of ultrapure water was added to the bottom of different sample discs after immersing them in corn oil for 24 h via a high-precision syringe. Then the water drop was photographed with a high-speed camera and fitted using the Laplace-Young equation after adding ultrapure water for 10 s [31].

The oil contact angles were obtained using the following methods: 2 μL of corn oil was added to the bottom of the sample discs after immersing them in ultrapure water for 12 h via a high-precision syringe. After 10 s, the water or oil drop was photographed with a high-speed camera and fitted using the Laplace-Young equation [31].

2.11. Data analysis

All tests were repeated three times, and the results are expressed as mean ± standard error. Data were analyzed using one-way ANOVA using SPSS software (Version 24, IBM, Chicago, USA). Significant differences were considered at the P < 0.05 level using the Duncan test.

3. Results and discussion

3.1. Effects of ultrasonic powers on the properties of rennet casein emulsions

The sedimentation, flocculation/coalescence, clarification, and creaming phenomena in rennet casein emulsions could be observed using ΔBS curves [32]. Fig. 1a–f show the ΔBS curves for rennet-casein emulsions prepared using ultrasound at 100 W to 600 W. As the storage time increases, rennet casein emulsion gradually transforms into the sediment layer, clarifying layer, and creaming layer (the pictures in Fig. 1a–f). The ΔBS curves show that the height of the sedimentation layer of rennet casein emulsions was generally 0 to 5 mm. Typically, the ΔBS value at the bottom of the emulsion is greater than 0 and gradually increases with storage time, indicating that the sediment content of the solution gradually increases [33]. The ΔBS values (around 0 to 5 mm) of rennet casein emulsions after 100 W to 400 W treatment indicate that sedimentation occurred in these emulsions, while it disappeared when the ultrasound power reached 500 W. The above results show that ultrasonic-assisted emulsification treatment at higher than 500 W can significantly reduce the spontaneous precipitation of rennet casein in the emulsion [33]. The ΔBS value of the clarifying layer (around 5 mm to 38 mm) gradually decreased as the storage time increased, indicating that the rennet casein emulsion gradually aggregated to form sediments or creaming. The ΔBS value of the creaming layer (around > 38 mm) gradually increased with storage time, indicating that the emulsion droplets gradually aggregated and floated upward [32].

Fig. 1.

The properties of rennet casein emulsion prepared by different ultrasound powers (100–600 W). Images a-f are the backscattered light (ΔBS) curves of rennet casein emulsions prepared by ultrasonic-assisted emulsification under different powers, image g is the TSI values of different emulsions, and image h is the EAI values of different emulsions. The pictures in images a-f are the rennet casein emulsions after storage for 6 h. Different letters (a–e) in image h represent significant differences among different samples at the P < 0.05 level.

The changes in ΔBS values (Fig. 1a–f) show that the clarified layer of rennet casein emulsion gradually decreased while the content of the creaming layer increased when the power reached 400 W. The total TSI values of rennet casein emulsions with different ultrasound power treatments were obtained as shown in Fig. 1g. The TSI values of the emulsion gradually increased with ultrasonic power from 100 W to 300 W, then decreased and reached the lowest values (measured at 6 h) after 500 W of ultrasonic treatment. When the power was further increased to 600 W, the TSI values of the sample increased again. These changes indicate that ultrasonic treatment below 400 W may reduce the overall stability of rennet casein emulsions under simulated milk ultrafiltrate conditions [26], [32]. This change might be explained by the fact that most proteins in the emulsion treated with ultrasound below 400 W did not participate in the emulsion formation, but instead form insoluble aggregates and precipitate during storage. The changes in TSI values at the bottom and middle might support the above illustration (Fig. S1). As the ultrasonic power increases, the EAI values of rennet casein gradually increased and then decreased, reaching a maximum at 500 W (Fig. 1h).

The characteristics of rennet casein determine that it is difficult to dissolve in simulated milk ultrafiltrate and difficult to adsorb around oil droplets to form a stable emulsion [34]. Fig. 1a shows that a lot of rennet casein in 100 W to 300 W treated samples was not adsorbed around the oil droplets, but remained in the solution and gradually formed sediment as the storage time increased. As the ultrasonic power increased further, more protein molecules were adsorbed around the oil droplets to form an emulsion, which led to an increase in the emulsion layer (Fig. 1e–f) [35]. When the ultrasonic power reaches 600 W, the height of the emulsion layer decreases, and the sedimentation layer increases. Among them, when the ultrasonic power was 500 W, the height of the sedimentation layer of the emulsion was the smallest, while the height of the emulsion layer was the highest. Studying the effects of ultrasonic-assisted emulsification on the stability of rice bran protein-chlorogenic acid and myofibrillar protein emulsions, Wang et al. [36] and Zhou et al. [37] found that appropriate ultrasound treatment improved the stability of the emulsion, while excessively high ultrasound power would lead to a decrease in the stability of the emulsion. The results in Fig. S2 also show that the stability and EAI of the rennet casein emulsion reached their optimal values when treated at 500 W for 7 min. Therefore, in the subsequent experiment to prepare the rennet casein emulsion under simulated milk ultrafiltration conditions, ultrasonic treatment at 500 W for 7 min is selected.

3.2. Emulsion activity indexes and emulsion stabilities of different emulsions

EAI is the amount of oil that can be emulsified per unit weight of protein and is widely used to characterize the ability of protein molecules to form stable emulsions [38]. Fig. 2a shows that the PSE sample had the lowest EAI values of 0.06 m²/g, then increased to 0.17 m²/g with high-speed shear homogenization treatment (HSE sample), and the highest EAI value of 1.00 m²/g was obtained after ultrasonic-assisted emulsification treatment (UAE sample). The above changes indicate that ultrasonic-assisted emulsification treatment can significantly improve the emulsifying activity of rennet casein compared with high-speed homogenization treatment. Rennet casein is an aggregated casein micelle held together by calcium-induced cross-linking, with high hydrophobicity and calcium content [35]. The aggregated protein particle and high hydrophobicity of rennet protein make it low solubility and emulsifying properties in the absence of emulsifying salts. Therefore, different emulsifying salts are usually added to break calcium-induced cross-linking and enhance the functional properties of rennet casein when using rennet casein in different food products [34]. Previous studies have shown that ultrasound treatment could disrupt the insoluble aggregates to reduce the particle size of rennet casein [20], [24]. Smaller protein particles have a better emulsifying activity index and can form a more stable emulsion system [13], [39]. Therefore, the EAI values of the UAE sample were significantly higher than those of the HSH and PSE samples. Zhou et al. [26] also found that myofibrillar protein emulsion prepared by ultrasonic-assisted emulsification had the highest EAI values when they applied different emulsification methods to fabricate the emulsion. The cavitation effect of ultrasonic-assisted emulsification treatment could also modify protein molecules and lead to more adsorption to the oil–water interface, thereby increasing the EAI value of rennet casein [13], [14].

Fig. 2.

The emulsifying activity index (a), the TSI values during 6 h (b), and the emulsion stabilities of rennet casein emulsions (c to e). The pictures in images c-e are the rennet casein emulsions after storage for 6 h. PSE represents the pre-prepared rennet casein emulsion, HSH represents the rennet casein emulsion prepared by high-speed shear homogenization, and UAE represents the rennet casein emulsion prepared by ultrasonic-assisted emulsification. Different letters (a–c) in image a represent significant differences among different samples at the P < 0.05 level.

Fig. 2b shows the TSI values of PSE, HSE, and UAE samples over a 6 h storage. Higher TSI values indicate poorer emulsion stability. The TSI values of all samples gradually increased with increasing storage time, with the UAE sample exhibiting the lowest TSI value after 6 h, consistent with the trend of the EAI. These changes indicate that the UAE emulsion exhibited the highest stability during storage. The higher EAI values and emulsion stability of the UAE sample indicate that rennet casein can emulsify more oil and form a dense, highly viscoelastic interfacial film after ultrasonic-assisted emulsification treatment. A stable interfacial film between oil droplets and the water phase could inhibit the aggregation of oil droplets during storage (Fig. 4), thereby endowing the emulsion with higher stability. Meanwhile, the UAE sample had a higher content of interfacial proteins (Fig. 5a) and a higher absolute value of zeta potential (Fig. 5b), which was the main reason for their higher emulsion stability [26], [39].

Fig. 4.

The droplet distribution (a), optical microscopy (b to d), and LCSM (e to g) results of different emulsions. PSE represents the pre-prepared rennet casein emulsion, HSH represents the rennet casein emulsion prepared by high-speed shear homogenization, and UAE represents the rennet casein emulsion prepared by ultrasonic-assisted emulsification.

Fig. 5.

The protein properties of rennet casein emulsions with different fabrication methods. PSE represents the pre-prepared rennet casein emulsion, HSH represents the rennet casein emulsion prepared by high-speed shear homogenization, and UAE represents the rennet casein emulsion prepared by ultrasonic-assisted emulsification. Different letters (a to c) indicate a significant difference at the P < 0.05 level among different emulsions.

Fig. 2c–e shows that the PSE, HSE, and UAE samples exhibited different ΔBS curves during the 6-hour storage test, with the PSE and HSE samples exhibiting similar ΔBS curves. Similar curves for PSE and HSE samples indicate that both samples exhibited protein sedimentation, middle clarification, and top creaming during 6 h of storage. This may be because these two emulsification methods struggle to form a fine emulsified system, and most protein molecules are not adsorbed around the oil droplets (Fig. 5a), but gradually precipitate during storage. In particular, corn oil appeared at the top of the HSE sample after 6 h of storage, indicating that the HSE emulsion was extremely unstable [40]. After ultrasonic treatment, the sedimentation layer in the ΔBS curve decreased, and the emulsified and clear layers did not completely separate, indicating that the storage stability of the rennet casein emulsion was significantly improved [41]. Similar changes were also reported by Zhou et al. [26] when they applied ultrasonic-assisted emulsification to fabricate myofibrillar protein-soybean oil emulsion.

3.3. Rheological properties of different emulsions

Fig. 3 shows the shear stress and apparent viscosity of different emulsions within the shear rate of 0.01 to 100 s⁻¹. The shear stress gradually increased while the apparent viscosity gradually decreased with the shear rate. Fig. 3b shows that the UAE emulsion had the highest apparent viscosity, followed by the HSE sample, while the PSE sample had the lowest apparent viscosity. The power law equation is used to fit the apparent viscosity curves to further explore the changes in the properties of different emulsions, and the results are shown in Table 1. The coefficients of determination (R²) of all curves were 0.99, indicating that all curves could be well fitted using the power law equation. The UAE sample had the highest K and n values, followed by the HSE sample, and the PSE sample had the lowest K and n values. All the n values are lower than 1, indicating that all the rennet casein emulsions were pseudoplastic fluids [27]. The K value shows a similar trend to the apparent viscosity, and the UAE sample has the highest viscosity value. Rheology tests indicate that ultrasonic treatment can effectively increase the viscosity of rennet casein emulsion. The increased viscosity after ultrasonic-assisted emulsification was also reported in soy protein fibril and myofibrillar protein emulsions [26], [40]. However, due to the differences in protein and oil phase properties, it has been reported that ultrasonic-assisted emulsification treatment can significantly reduce the viscosity of the emulsion [42], [43]. A higher viscosity emulsion system can inhibit the aggregation of emulsion droplets during storage [13], [39], which might explain the higher stability of the UAE sample (Fig. 2).

Fig. 3.

The rheological properties of different emulsions. Image a is the apparent viscosity and b is the shear stress of PSE, HSH, and UAE samples during the 0.01–100 s⁻¹ test. PSE represents the pre-prepared rennet casein emulsion, HSH represents the rennet casein emulsion prepared by high-speed shear homogenization, and UAE represents the rennet casein emulsion prepared by ultrasonic-assisted emulsification.

Table 1.

The rheological properties and average droplet size of different emulsions.

Properties	PSE	HSE	UAE
K (Pa·sn)	0.05 ± 0.01c	0.08 ± 0.01b	0.21 ± 0.02a
n	0.03 ± 0.01c	0.15 ± 0.02b	0.33 ± 0.02a
R2	0.99	0.99	0.99
D3,2 (μm)	32.60 ± 0.10a	8.10 ± 0.03b	1.66 ± 0.01c
D4,3 (μm)	57.10 ± 0.30a	29.93 ± 0.81b	19.37 ± 0.35c

Different letters (a to c) in the same row indicate a significant difference at the P < 0.05 level among PSE, HSE, and UAE samples. PSE represents the pre-prepared rennet casein emulsion, HSH represents the rennet casein emulsion prepared by high-speed shear homogenization, and UAE represents the rennet casein emulsion prepared by ultrasonic-assisted emulsification.

3.4. Droplet size characteristics of different emulsions

Fig. 4a shows that the volume distribution curve of the PSE sample is to the right. Then it shifts toward smaller droplet sizes (left) after high-speed shear homogenization and ultrasonic treatment, especially after ultrasonic treatment. Table 1 illustrates that the D_3,2 and D_4,3 values of PSE sample 32.60 and 57.10 μm (PSE sample), then decreased to 8.10 and 29.93 μm after high-speed shear homogenization treatment (HSE sample), and decreased to 1.66 and 19.37 μm after ultrasonic treatment (UAE sample). The above changes indicate that ultrasonic-assisted emulsification treatment can effectively reduce the droplet size of rennet casein emulsions more than high-speed shear homogenization treatment. Comparing the effects of different emulsification methods on the droplet size of myofibrillar protein-soybean oil emulsions, Zhou et al. [26] also found that the average droplet size of the emulsion prepared by ultrasonic-assisted emulsification was lower than that of the emulsion prepared by high-speed shear homogenization. The smaller droplet size of the UAE sample is an important reason for its higher storage stability (Fig. 2) [13], [42].

Fig. 4b–d are the emulsion droplet distributions of PSE, HSE, and UAE samples obtained from the optical microscope. The PSE emulsion exhibited an extremely uneven droplet size distribution, characterized by a higher concentration of large droplets. The small droplet content of the emulsion increases after high-speed shear homogenization treatment (HSE sample), but some large droplets remain. In contrast, the PSE emulsion exhibits no large droplets and a more uniform droplet size distribution. Emulsions with large droplets are extremely unstable and tend to aggregate or coagulate during storage, which might be an important factor in the instability of PSE and HSE emulsions [39]. The small and uniform distribution of emulsion droplets will make them difficult to aggregate into large droplets, thereby increasing the stability of the UAE samples [13], [39].

Fig. 4e–g are the LCSM images of the PSE, HSE, and UAE samples, and the green and red colors represent the distribution of proteins and oil droplets, respectively. The protein distribution pictures show that rennet casein in the PSE sample aggregated into sediments, and gradually dispersed into small particles after high-speed shear homogenization treatment (HSE sample) and ultrasonic treatment (UAE sample). Smaller protein particles can be adsorbed around the oil droplets more quickly and stably, thereby forming a stable rennet casein emulsion [44]. More protein molecules are dispersed around the oil droplets in the UAE sample to form a stronger interfacial protein film, thus enhancing emulsion stability, which is consistent with the results of the protein distribution coefficient (Fig. 5a). At the same time, ultrasonic treatment can also significantly reduce the droplet size, which is consistent with the results of droplet size distribution (Fig. 4a) and optical microscopy (Fig. b–d).

3.5. Protein properties of different emulsions

Fig. 5a shows that the protein distribution coefficient of the PSE sample at the oil–water interface is lowest, at 40.56%. This increases to 49.10% and 62.98% after high-speed shear homogenization and ultrasonication, respectively. The increased protein distribution coefficient indicates that high-speed shear homogenization and ultrasonic treatment, especially ultrasonic treatment, could allow more protein molecules to adsorb around the oil droplets, leading to a stable rennet casein-coated oil droplet. This result is consistent with the LCSM images, which show that more protein molecules were adsorbed around the oil droplets in the UAE sample (Fig. 4e–g). A higher protein content around the oil droplet can effectively inhibit the aggregation of emulsion droplets caused by the external environment, such as gravity, thereby improving the stability of rennet casein emulsion [45], [46]. Similar results were also reported by Gul et al. [47] when they applied ultrasound to form hazelnut meal protein emulsion.

Fig. 5b shows that the zeta potential of all samples was negative because the pH 5.8 is higher than the isoelectric point of the rennet casein [35]. Among the three emulsions, the UAE sample has the highest absolute value of zeta potential (41.10 mV), followed by the HSE sample (39.17 mV), while the PSE sample has the lowest absolute value of zeta potential (33.97 mV). This change indicates that both high-speed homogenization and ultrasonic treatment significantly increase the exposure of charged groups in the rennet casein emulsion. Generally, the higher the absolute value of the zeta potential of an emulsion, the higher the emulsion stability [13], [39]. Therefore, the high absolute value of the zeta potential induced by ultrasonic-assisted emulsification may be an important factor in the high emulsion stability (Fig. 2).

Surface hydrophobicity is widely used to characterize the exposure of hydrophobic groups of protein molecules in solution. Fig. 5c shows the surface hydrophobicity curves of PSE, HSE, and UAE samples. The PSE sample had the highest surface hydrophobicity, and then decreased after high-speed shear homogenization and ultrasonic treatment. The high surface hydrophobicity of the PSE sample may be due to a large amount of rennet casein not participating in emulsion formation but existing in the emulsion as aggregates (Fig. 2) [48]. Most protein in the PSE sample exists in the form of insoluble aggregates exhibiting high hydrophobicity [35], [49]. The insoluble aggregates gradually transform into an interface film after high-speed shear homogenization and ultrasound treatment, resulting in a significant decrease in the surface hydrophobicity of the HSH and UAE samples [46]. Compared with high-speed shear homogenization treatment, ultrasonic treatment allows more protein molecules to adsorb at the oil–water interface (Fig. 4), leading to their unfolding at the oil–water interface. Therefore, the surface hydrophobicity of the UAE sample is higher than that of the HSH sample. Studying the effects of ultrasonic-assisted emulsification and high-speed shear homogenization on the surface hydrophobicity of myofibrillar protein emulsion, Zhou et al. [24] also found that the emulsion prepared by ultrasonic-assisted emulsification exhibited higher surface hydrophobicity.

Fig. 6 shows the contact angles between rennet casein and water droplets of different emulsions. The PSE sample exhibited the highest internal contact angles between the water droplet and the protein, which decreased after both high-speed homogenization and ultrasonic treatment (Fig. 6a–c). A high internal contact angle indicates a low hydrophilicity of the protein molecule [50]. These changes suggest that both high-speed homogenization and ultrasonic treatment can enhance the hydrophilicity of rennet casein. The higher internal contact angles of the UAE sample may be due to exposure of hydrophobic groups on the protein, leading to increased lipophilicity and decreased hydrophilicity [51]. Fig. 6d–f show that the PSE sample has a higher internal contact angle between the oil droplet and the rennet casein molecule, which decreases after high-speed homogenization and ultrasonic treatment, particularly to 145.7° after ultrasonic treatment. This result indicates that ultrasonic treatment significantly enhances the contact between the rennet casein molecules and corn oil, thereby forming a more stable protein-encapsulated oil droplet structure [51]. The above results showed that ultrasonic-assisted emulsification treatment could balance the hydrophilicity and lipophilicity of rennet casein, thereby obtaining a more stable emulsion. Cheng et al. [52] also observed that ultrasonic treatment can change the amphiphilicity of yeast protein molecules, thereby improving the stability of emulsions.

Fig. 6.

The water and oil contact angles of rennet casein with different treatment methods. PSE represents the pre-prepared rennet casein emulsion, HSH represents the rennet casein emulsion prepared by high-speed shear homogenization, and UAE represents the rennet casein emulsion prepared by ultrasonic-assisted emulsification. Images a-c are water contact angles of rennet casein and images d-f are the oil contact angles of rennet casein.

Rennet casein is formed through calcium-induced cross-linking [1], [53]. Adding emulsifying salts disrupts these cross-linking, thus imparting excellent solubility and emulsifying properties to rennet casein [35], [54], [55]. While some studies showed that ultrasonic treatment alone was insufficient to fully disrupt calcium-induced cross-linking and achieve complete casein dissolution without emulsifying salts[19], [20]. This work proved that ultrasonic-assisted emulsification allows casein molecules to adsorb onto the oil–water interface because emulsification and protein dissociation occur simultaneously (Fig. 2, Fig. 3) [13], [26]. Furthermore, the superior emulsifying properties of ultrasonic-assisted emulsification result in high hydrophobic interactions (Fig. 5c) and electrostatic repulsion (Fig. 5b) between rennet casein molecules, endowing higher emulsion stability. Simultaneously, ultrasonic-assisted emulsification modifies the hydrophilic-lipophilic balance of protein molecules (Fig. 6), forming emulsions with smaller droplet sizes, thereby contributing to better emulsion stability of rennet casein even without emulsifying salts.

4. Conclusions

Ultrasonic-assisted emulsification treatment at 500 W for 7 min could significantly increase the stability of rennet casein emulsions under natural conditions. Compared to high-speed homogenization-treated rennet casein emulsion, ultrasonically treated emulsions exhibited higher emulsifying activity, emulsion stability, absolute zeta potential, as well as smaller emulsion droplets and protein aggregates. Ultrasonic-assisted emulsification may enhance the binding capacity of rennet casein molecules to the oil phase and the protein content at the oil–water interface, and improve the hydrophobic interactions and electrostatic repulsion between casein molecules, thereby producing more stable rennet casein emulsions. Future research should explore the application of these emulsions in low-emulsified salt dairy products.

CRediT authorship contribution statement

Jie Luo: Supervision, Resources, Funding acquisition, Conceptualization. Miaolin Wang: Visualization, Validation, Resources, Investigation, Conceptualization. Peiling Li: Visualization, Validation, Resources. Yang Liu: Writing – review & editing, Resources. Caiyun Wang: Resources, Funding acquisition. Chao Liang: Resources, Investigation. Jing Zhang: Supervision, Software, Resources. Ying Zhang: Supervision, Software, Resources. Jian He: Supervision, Resources, Funding acquisition. Ying Xu: Writing – review & editing, Supervision, Resources, Investigation. Lei Zhou: Writing – review & editing, Writing – original draft, Resources.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: