The addition of oxidized tea polyphenols enhances the physical and oxidative stability of rice bran protein hydrolysate-stabilized oil-in-water emulsions

Abstract

This study investigated the effects of rice bran protein hydrolysate (RBPH) coupled with oxidized tea polyphenols (OTP) on the physical and oxidative stability of oil-in-water emulsions. Emulsions stabilized with RBPH alone were less stable than those stabilized with both RBPH and OTP. The OTP not only improved the emulsification stability, flocculation stability, coagulation stability, and ζ-potential of the emulsions, OTP also decreased the particle size, increased the adsorption capacity of the oil–water interface for RBPH, and effectively reduced the generation of peroxide and malondialdehyde in the emulsion during storage. The stability of the emulsion during the early stage of storage was maximized when the OTP content was 1.5%; at greater contents, the OTP competed with RBPH for adsorption at the interface, which affected the distribution of RBPH on the interface and decreased the emulsion stability. The results indicate that OTP can enhance emulsion stability, providing a reference for the application of oxidized polyphenols in food emulsions.

Introduction

Oil-in-water (O/W) emulsions are important components of many commercial products. An O/W emulsion is a thermodynamically unstable colloidal dispersion that consists of small oil droplets dispersed in a continuous water phase (Wang et al., 2011). Over time, due to various physical and chemical mechanisms including gravity separation, flocculation, coalescence, particle coalescence, austenite ripening, and phase separation, O/W emulsions tend to break, resulting in instability (Shahidi and Zhong, 2010). Therefore, stabilizers include emulsifiers, fabric modifiers, mature inhibitors, and weighting agents are added to emulsions to improve the long-term stability (Yi et al., 2014). In addition to affecting the stability of the emulsion, the properties of the stabilizer also affect the emulsion formation and the functional properties of the final product (Ogawa et al., 2003). Therefore, selecting a suitable stabilizer is one of the most important factors in preparing emulsion products. Emulsifiers, which are usually amphiphilic molecules with hydrophilic and hydrophobic groups, including small-molecule surfactants, phospholipids, proteins, polysaccharides, and other surface-active polymers (Intarasirisawat et al., 2014). In recent years, protein hydrolysates, which are natural antioxidants that also have emulsification effects, have attracted considerable attention in academia. Kong et al. (2010) showed that the restricted hydrolyzed protein can reduced the particle size of emulsion droplets, and the hydrolyzed protein can prevent the lipid oxidation by terminating the free radical chain reaction and chelating metal ions, so as to change the physical properties of food and inhibiting lipid oxidation (Mosca et al., 2013). However, as hydrophilic antioxidants, protein hydrolysates are generally inefficient emulsifiers in O/W emulsions because they are mainly distributed in the water phase and at the oil–water interface (Mao et al., 2010).

Due to the concerns related to synthetic antioxidants, natural extracts have been widely studied. Phenolic compounds are compounds containing hydroxyl and aromatic nucleus. They have high reactivity of hydroxyl substitution and their ability to swallow free radicals. These compounds have the potential of antioxidant activity. Tea polyphenols are commonly used flavor compounds that have been widely researched because of their significant immunomodulatory, antioxidant, antitumor, antiviral, and antibacterial activities (Gomez-Mascaraque and Lopez-Rubio, 2016). A chemical analysis of tea polyphenols indicated a high content of phenolic compounds with antioxidant capacity, including catechins, flavonoids, anthocyanins, and phenolic acids (Pentaramos and Xiong, 2002). The interactions between polyphenols and proteins during food processing can impart food products with special properties. For example, Strauss and Gibson (2004) found that oxidized polyphenols can react with proteins to form a stable molecular structure, thereby improving the functional properties of proteins. Chen et al. (2018) indicated that oxidized chlorogenic acid can facilitate the cross-linking of rice bran protein hydrolysate (RBPH) to form a dense interfacial film around the oil droplets in O/W emulsions. Hence, the addition of oxidized chlorogenic acid to RBPH-stabilized O/W emulsions can improve the emulsification and oxidative stability. However, under the real-world processing conditions, polyphenols are easily oxidized to form quinones. Tea polyphenols can be used as cross-linking agents to improve the functional properties of proteins. In addition, tea polyphenols can react with the nucleophilic groups in lysine, methionine, cysteine, and tryptophan residues to induce cross-linking and improve the emulsification ability of hydrolysates (Erdmann et al., 2017). A large number of studies have proved the biological activities of protein hydrolysates and tea polyphenols, but there are few studies on the joint application of oxidized polyphenols and protein hydrolysates in emulsion. The study of the effect of oxidized tea polyphenols on the stability of emulsion and the interfacial properties of emulsion droplets can not only provide a new idea for the preparation of more stable emulsion, but also increase the application of tea polyphenols in food. In the present study, we evaluated how the addition of oxidized tea polyphenols (OTP) to RBPH-stabilized O/W emulsions affected the physical and chemical stability of the emulsion. We also investigated the mechanism of OTP in the emulsification system to provide a theoretical basis for the application of OTP to produce healthy and safe food products.

Materials and Methods

Materials    Rice bran protein concentrate containing 85% protein (on a total weight basis) was obtained from Avebe A.U Corporation (Amsterdam, Netherlands). Alcalase 2.4L (6×10⁴ U/g) was obtained from Novozymes (Bagsvaerd, Denmark). Soybean oil was purchased from Jinhai Grain and Oil Industry Co., Ltd. (Hebei, China). All other chemicals and reagents were analytically pure.

Preparation of RBPH    RBPH was prepared according to Wang et al. (2017). Briefly, the rice bran protein concentrate was prepared into a solution with a substrate concentration of 4% (w/w). The ratio of enzyme to substrate was 2:100 (w/w), and the pH was kept constant by adding 1 mol/L NaOH. After hydrolysis, the solution was heated in a water bath at 95 °C for 5 min. After adjusting the pH to neutral with 1 mol/L HCl and centrifuging at 9000 ×g for 10 min, the supernatant was dialyzed in a dialysis bag with a molecular weight of 100 Da for 12 h. Subsequently, the salt was removed, and the RBPH was freeze-dried and stored at 4 °C.

Preparation of OTP    OTP were prepared as reported by Balange and Benjamin (2009). Briefly, tea polyphenols were thinned in distilled water at a concentration of 2.0% (w/v) and adjusted to pH 9.0. The phenolic compounds were transformed into quinones by bubbling the solution with high-purity oxygen (99.0%) for 1 h at 40 °C. Finally, the pH of the solution was adjusted to pH 7.0.

Emulsion preparation    Emulsions were prepared by mixing 10 wt% soybean oil and 90 wt% RBPH solution and homogenizing with a high-speed homogenizer (DeBEE2000, Weiliu nano Biotechnology Co., Ltd, Suzhou, China) at 13500 r/min for 2 min. OTP were then added in different mass fractions (0.5%, 1%, 1.5%, and 2%) followed by homogenizing twice under high pressure at 40 MPa. Finally, sodium azide (3 mM) was added to inhibit the growth of microorganisms, and as shown in equation 2.

  

  

where D is the dilution factor, A is the absorbance of the diluted emulsions, C is the initial protein, (g/mL), φ is the optical path (0.01 m), and θ is the oil volume fraction used to form the emulsion. A₀ is the light absorption value of the emulsion after standing for 0 h, A₁₀ is the light absorption value of the emulsion after standing for 10 h.

Emulsion physical stability    Emulsion physical stability was assessed according to the method of Ramirez-Suarez and Xiong (2003). In summary, 50 µL of each emulsion was taken from a distance of 0.5 cm from the bottom of the centrifuge tube and added to a centrifuge tube containing 5 mL of 0.1% sodium dodecyl sulfonate (SDS) followed by mixed. The optical density was then measured at a wavelength of 500 nm. The emulsifying activity (EAI) was determined immediately after emulsion formation. Emulsification stability (ESI) was determined as the ratio of the turbidity of the emulsion after 10 h to that of the newly formed emulsion multiplied by 100%.

Measurement of ζ-potential and droplet size    Fifty microliters of each emulsion was added into 5 mL of ultrapure water and mixed well followed by injection into a curved capillary. The droplet size (d_4,3) and ζ-potential of the emulsion particles were measured at room temperature using a laser droplet sizer (Malvern Instruments Ltd., Worcestershire, UK).

Measurement of flocculation index and coagulation index    The flocculation index (FI) and coagulation index (CI) were measured according to the method of Castellani et al. (2006). The emulsions were diluted with distilled water with or without of 1% (w/v) SDS. FI and CI were calculated by equations 3 and 4, respectively.

  

  

where d_4,3-water and d_4,3-1% SDS are the particle sizes of emulsions dispersed in ultrapure water and 1% SDS, respectively, and d_{4,3-24 h} and d_{4,3-0 h} are the particle sizes of the emulsions at 0 and 24 h after preparation, respectively.

Partitioning of RBPH in emulsions    The emulsion was centrifuged at 15 000 × g for 45 min. The lower layer of the emulsion was then aspirated and centrifuged at 15 000 × g for 30 min and filtered through a 0.22-µm membrane. The protein content of the filtrate was determined by the biuret method.

The RBPH partition coefficient (PC) between the two phases was defined by equation 5 according to Huang et al. (1997).

  

where V_W is the volume of water (mL); V₁ is the volume of oil (mL); W_t is the total protein content (mg/mL); and W_w is the protein content in the water phase (mg/mL). The density (kg/m³) of the oil was calculated to be 0.922. The ratio of protein to total protein in the water phase was calculated as (W_w/W_t) × 100%; the protein content at the interface was determined as the total protein content minus the protein content in the water phase.

Creaming index    The newly prepared emulsion was placed in a 25-mL graduated stoppered glass tube. At room temperature, the emulsion was placed vertically in the dark for 0, 2, 5, 9 and 14 days to observe the stratification of the emulsion. The upper layer was the emulsion layer, while the lower layer was the whey layer. The creaming index was calculated by equation 6.

  

where H_s is the height of the whey layer, and H_t is the total height of the emulsion.

Peroxidation value (POV)    POV determination was conducted using the American Oil Chemists' Society sodium thiosulfate titration method (Mei et al., 1998). The sample (0.5 mL) was added to 10 mL of acetic acid/chloroform solution (3:2, v/v). After mixing, 0.5 mL of saturated potassium iodide solution was added. After mixing, the reaction was allowed to proceed in the dark for 3 min and then terminated with ultrapure water. Subsequently, 0.5 mL of 0.5% starch solution was added followed by titrating with 2 mmol/L Na₂S₂O₃ until the blue color disappeared. POV was calculated by equation 7.

  

where A and B are the volumes of Na₂S₂O₃ consumed by the sample and blank, respectively (mL); C is the concentration of Na₂S₂O₃ (mol/L); and m is the mass of the sample (g).

Thiobarbituric acid-reactive substances (TBARS)    Preparation of thiobarbituric acid solution: mix 0.375 g of thiobarbituric acid, 15 g of trichloroacetic acid, 1.76 mL of 12 mol/L hydrochloric acid with 82.9 mL of water, and then add 3 mL of 2% BHA solution for mixing. The 4 mL of the above solution was mixed with 1 mL of sample and 1 mL of water, put it into a water bath and heat it with boiling water for 15 min. After cooling to room temperature, it will pass 0.45-µM filter membrane, and the filtrate was compared at 532 nm. Take 1,1,3,3-tetraethoxypropane as the standard curve to determine the content of malondialdehyde in the sample.

Statistical analysis    Each test was repeated at least three times, and the results are expressed as mean ± standard deviation (SD). The mean and standard deviation were analyzed by Statistix 8.0 software, and statistical significance was assessed by one-way analysis of variance with Tukey's test (p < 0.05).

Results and Discussion

Emulsion physical stability    EAI is related to the ability of the emulsifier to form and stabilize the emulsion. EAI can reflect the ability of a protein to adsorb at the oil–water interface. In contrast, ESI reflects the ability of emulsifier to maintain the stability of the emulsion over a certain period of time. The electrostatic interaction between protein and oxidized polyphenols forms a stable structure at the oil-water interface, which provides additional interactions between adjacent droplets, such as electrostatic repulsion and spatial repulsion, and improves the emulsifying performance of the emulsion. As shown in Fig. 1, the EAI and ESI values of the emulsions containing OTP were significantly higher than those of the control (i.e., the emulsion without OTP; p < 0.05). This shows that the addition of OTP significantly improved the emulsion stability. Within a certain OTP concentration range, the OTP covered the surfaces of the oil droplets, resulting in good dispersion. Chen et al. (2019) studied the emulsification of porcine plasma protein hydrolysates modified by oxidized tannic acid (OTA) and oxidized chlorogenic acid. Complex formation was characterized by an increase in emulsifying activity. Both tannic acid and chlorogenic acid bind covalently to porcine plasma protein hydrolysates and adsorb on the droplet surfaces, effectively reducing the interfacial tension and stabilizing the emulsion. Compared to RBPH alone, RBPH and OTA or oxidized chlorogenic acid results in higher emulsification stability. The EAI and ESI values of the emulsions first increased and then decreased with increasing OTP concentration (p < 0.05). Among the OTP concentrations, 1.5% produced the highest emulsifying activity and stability; increasing the OTP concentration beyond 1.5% reduced the emulsifying activity and stability of the emulsions. Polyphenols may accumulate in the hydrophobic side chains of proteins, making it difficult for hydrophobic regions to penetrate the interface (Raikos, 2017). In addition, the conformational changes of proteins caused by polyphenols often create energy barriers to adsorption, resulting in adverse effects on the emulsification process. Moderately oxidized polyphenols can also reduce the structural flexibility of RBPH (Aewsiri et al., 2009).

Fig. 1.

Emulsifying activity index (EAI) and emulsifying stability index (ESI) of the O/W emulsions prepared with RBPH and different concentrations of OTP. The different uppercase letters (A–D) indicate significant differences (p < 0.05).

ζ-potential    Droplet surface potential plays an important role in clarifying the stability of emulsion and electrostatic interaction in food emulsion. The ζ-potential reflects the physical properties of the particles in an emulsion along with the electrostatic force on the particle surface. A ζ-potential with a large magnitude (either negative or positive) corresponds to a large electrostatic repulsion force and a large separation distance between particles in the emulsion, resulting in high droplet stability (Chevallier et al., 2018). In contrast, emulsions with low-magnitude ζ-potentials tend to coagulate or flocculate. The OTA can stabilize oil droplets, prevent coalescence or flocculation, reduce oil–water interfacial tension, and help to form a dense interfacial structure. Therefore, the ζ-potential can be used to explain any changes in surface structure. As shown in Fig. 2, the ζ-potential values of the emulsions ranged from −19.93 to −39.01 mV. The ζ-potential values of the emulsions containing OTP were significantly higher compared to the control (p < 0.05). The ζ-potential can reflect the repulsive force between surface-active molecules adsorbed at the oil–water interface (Temenouga et al., 2016). The higher absolute ζ-potential values of the emulsions containing OTP may indicate that OTP have a higher the adsorption rate at the interface. Tea polyphenols are oxidized to form quinone compounds. Quinone is a reactive electrophilic intermediate, which can be used as a protein crosslinker to form a rigid molecular structure. At the same time, the polyphenol ring can complex with the hydrophobic surface of the polypeptide through covalent or non covalent interaction to increase the electrostatic adsorption of the emulsion. With the increase of OTP content, the absolute value of the potential of the emulsion increased significantly and then decreased. The addition of OTP caused the cross-linking of peptide chain molecules between proteins, which increased the negative charge of the entire emulsion. The addition of OTP increased the interfacial activity and adsorption rate of RBPH, thus improving the emulsion stability. However, compared to the emulsion containing 1.5% OTP, the ζ-potential of the emulsion containing 2.0% OTP was significantly lower. This indicates that the electrostatic attraction between the protein-stabilized emulsion droplets was sensitive, and the interfacial structure may have been affected by competitive adsorption, resulting in the decrease in ζ-potential (Duan et al., 2016). This phenomenon also explains the change trend of EAI and ESI.

Fig. 2.

ζ-potential values of RBPH-stabilized O/W emulsions containing different concentrations of OTP. Different uppercase letters (A–D) indicate significant differences (p < 0.05).

Droplet diameter    Particle size is an important parameter affecting the physical properties (color, viscosity and texture) and shelf life of emulsion food. The droplet diameter can determine the interactions between protein and polyphenol. The particle sizes of the emulsions containing different OTP contents are shown in Table 1. Among the emulsions, the emulsion containing 1.5% OTP had the smallest particle size. The droplet size can affect the stability of the emulsion against oxidation; oxidation stability increases with decreasing droplet diameter. Duan et al. (2016) reviewed the effect of vitellin resveratrol complex on the antioxidant capacity along with the physical and chemical properties of emulsions; they found that emulsions with smaller particle sizes had higher emulsification and oxidation stability. As the concentration of resveratrol decreased, the adsorption capacity of the complex at the interface along with the interfacial area decreased, causing the droplet size to increase. Adjonu et al. (2014) reported that protein hydrolysate had poor spatial stability and was prone to aggregation, which may accelerate the flocculation of oil droplets. When only RBPH was added to the emulsion, the protein formed a thin interfacial layer that breaks easily during storage, resulting in poor emulsion stability (Balange and Benjakul, 2009). However, the addition of OTP significantly improved the activity of RBPH at the interface, and the enhanced interfacial adsorption of RBPH significantly reduced the particle size of the emulsion. Therefore, the smaller droplet diameter in the presence of OTP indicates that the addition of OTP enhanced the accumulation of protein and polyphenol compounds at the oil–water interface. Chen et al. (2018) showed that the interaction between protein hydrolysate and oxidation would form protein hydrolysate oxidized polyphenol complex. Modification of oxidized polyphenols can increase the interfacial activity of protein hydrolysates, increase the speed of interface adsorption, and reduce the particle size of the emulsion.

Table 1. Droplet sizes of the RBPH-stabilized O/W emulsions prepared with different concentrations of OTP.

Concentration of OTP (%)	Relative span factor		Droplet size, d4,3 (µm)
	Day 1	Day 14	Day 1	Day 14
0	0.83 ± 0.008Ab	0.95 ± 0.003Aa	2.32 ± 0.02Ab	3.08 ± 0.03Aa
0.5	0.75 ± 0.006Bb	0.80 ± 0.001Ba	2.11 ± 0.02Bb	2.87 ± 0.11Ba
1.0	0.73 ± 0.003Bb	0.77 ± 0.003Ca	1.98 ± 0.07Cb	2.37 ± 0.03Da
1.5	0.62 ± 0.005Cb	0.71 ± 0.005Da	1.47 ± 0.03Eb	2.12 ± 0.05Ea
2.0	0.51 ± 0.002Db	0.59 ± 0.003Ea	1.85 ± 0.02Db	2.55 ± 0.08Ca

Values are given as the mean ± SD from triplicate determinations. A–E in the same column of letters, A–B in the same column of letters means that the difference is not significant, and different means that the difference is significant (p < 0.05)

Flocculation and coagulation    Particle size evaluation is one of the most commonly used methods to evaluate the flocculation and coalescence of emulsions during storage. The flocculation state can be affected by different factors, including proteins adsorbed at the interface, the protein/oil ratio, pH, temperature, and ionic strength (Li et al., 2017). The effects of OTP on the flocculation index (FI) and agglomeration index (CI) of the RBPH-stabilized emulsions are shown in Table 2. Compared to the control, FI and CI were significantly lower in the emulsions containing OTP (p < 0.05). Thus, the emulsion stability was improved by adding OTP. Among the emulsions, the emulsion containing 1.5% OTP had the highest physical stability. The physical stability of emulsion usually depends on the electrostatic stability and spatial stability as well as the protein content at the oi-water interface. Electrostatic stability primarily depends on the charge on the outer layer of the droplet, while spatial stability is related to the barrier to polymer adhesion on the droplet surface (Qiu et al., 2015). Wooster and Augustin (2007) reported that increasing spatial stability helps slow flocculation and coalescence. Our results show that the addition of OTP may form a boundary film around the interface, thereby increasing spatial stability. Therefore, the addition of OTP to the RBPH-stabilized emulsion can greatly inhibit the accumulation of RBPH on the surfaces of oil droplets. However, when the concentration of OTP exceeded 1.5%, the stability of the emulsion against flocculation and coagulation decreased to some extent, which can be explained as follows. The structure of protein hydrolysate is relatively loose, and protein hydrolysate competes with OTP for adsorption at the interface, which destroys the interactions between the protein and polyphenol compounds (Uluata et al., 2015).

Table 2. FI, CI, and Viscosity values of the RBPH-stabilized O/W emulsions containing different OTP concentrations.

Concentration of OTP/%	FI /%	CI /%	Interfacial tension /(mN/m)	Viscosity / Pa·s
0	29.91±0.14A	101.87±0.49A	0.104±0.001A	1.36±0.03E
0.5	28.61±0.19B	101.07±0.51B	0.082±0.002B	5.33±0.14D
1.0	27.87±0.32C	100.96±0.14C	0.047±0.001C	12.84±0.52C
1.5	26.06±0.28E	99.02±0.27E	0.011±0.001D	23.85±0.49B
2.0	27.23±0.45D	100.16±0.15D	0.008±0.0003E	34.71±0.50A

Partitioning of RBPH in emulsions    The specific position or distribution of protein hydrolysates between the interface and the water phase is considered as an important factor affecting emulsion stability and lipid oxidation rate in emulsion systems (Zhu et al., 2017). As shown in Table 3, in this emulsion prepared with only RBPH in this study, most of RBPH (about 89.6%) was distributed in the water phase, while only 10.4% was located at the interface. In addition, as the OTP concentration increased, less RBPH was adsorbed at the oil–water interface. It is possible that OTP can desorb RBPH from the interface in a competitive way. However, the resolved RBPH can still scavenge free radicals and chelate metal ions in the aqueous phase to combat lipid oxidation (Owens et al., 2018). Faraji et al. (2004) found that proteins or hydrolysates with antioxidant activity located at the interface and in the aqueous phase can delay lipid oxidation during emulsion storage. Moreover, oxidation occurs mainly at the oil–water interface, where lipid hydroperoxides are concentrated. The interactions between RBPH and OTP at the interface can form a physical barrier on the surface of the oil drop, preventing the infiltration of free radicals and stabilizing the emulsion (Xu et al., 2017).

Table 3. RBPH distributions between the aqueous phase and the interface and corresponding partition coefficients for RBPH-stabilized O/W emulsions with different concentrations of OTP.

Concentration of OTP/%	RBPH in the aqueous phase/%	RBPH at the interface/%	Partition coefficient (aqueous phase)
0	89.2 ± 0.5A	10.8 ± 0.3E	1.09
0.5	88.6 ± 0.4B	12.4 ± 0.7D	1.16
1.0	85.9 ± 0.2D	14.1 ± 0.4B	1.48
1.5	84.1 ± 0.6E	16.9 ± 0.5A	1.70
2.0	87.0 ± 0.2C	13.0 ± 0.3C	1.35

Creaming index    The density of the water phase in an O/W emulsion is higher than that of the oil phase. Due to the movement of oil molecules during emulsion storage, the lipid droplets float upward (Gomezmascaraque and Lopezrubio, 2016), resulting in phase separation (i.e., stratification). To observe emulsion stratification, the freshly prepared emulsions were stored at room temperature for 14 d, and the state of the emulsions were observed regularly. As shown in Fig. 3, no stratification was observed in the freshly prepared emulsion. However, as the storage time increased, phase separation gradually occurred, leading to stratification. During storage, the emulsion components are attracted to each other, resulting in droplet flocculation and aggregation and eventually phase separation (Seta et al., 2013). When stored for 1 day, the emulsion without OTP quickly appeared the phenomenon of oil-water stratification. The addition of OTP to the RBPH-stabilized emulsion increase the interfacial activity and diffusion ability of the emulsion, which effectively prevented aggregation and weakened stratification. Compared with the protein hydrolysate in the dispersed phase, oxidized polyphenols have better ability to cover the water / oil interface. Oxidized polyphenols are adsorbed on the droplet surface, increase covalent connection, and prevent droplet flocculation or coalescence by increasing spatial exclusion. At the same time, the oxidized polyphenols produce some advanced components with stronger antioxidant activity. These compounds can be used as hydrogen donors in the process of scavenging free radicals and improve the stratification stability of the emulsion.

Fig. 3.

Creaming index values of the RBPH-stabilized O/W emulsions prepared with different concentrations of OTP during storage at 37 °C for 14 days.

Oxidative stability    POV and TBARS were evaluated after 14 days of storage. The effect of OTP on the lipid oxidation of the RBPH-stabilized emulsion was evaluated. Oil is rich in a variety of polyunsaturated fatty acids, which are especially sensitive to oxygen and temperature. As displayed in Fig. 4, the POV values of all emulsions increased significantly during storage, and the rate of increase depended on the OTP concentration, whereas the POV values of the oil without the emulsification were 53 meq/kg oil. Among the emulsions, the POV value of the emulsion prepared with RBPH alone was the highest throughout the storage period. This indicates that RBPH produced by restricted hydrolysis had stronger reduction ability and free radical scavenging activity than intact rice bran protein, and the RBPH-stabilized emulsion was easy to oxidize. Compared to RBPH alone, OTP and RBPH inhibited lipid oxidation in the emulsion more effectively. In addition, the adhesion of OTP induced more hydroxyl groups, thus increasing the hydrogen supply capacity of RBPH in the aqueous phase and protecting the emulsion from oxidation (Hemung et al., 2013). In conclusion, the synergistic effect of OTP and RBPH at the interface provides a physical barrier that improves emulsion stability. Moreover, the OTP has a higher antioxidant activity in the aqueous phase, which likely promotes the oxidative stability of emulsion during storage.

Fig. 4.

POV (A) and TBARS (B) values of the RBPH-stabilized O/W emulsions prepared with different concentrations of OTP during storage at 37°C for 14 days.

The trend in TBARS was similar to that observed for POV. In the emulsion without OTP, TBARS increased significantly during storage. The TBARS values of the emulsions containing OTP were lower than that of the emulsion without OTP. As the OTP content increased, the TBARS value of the emulsion decreased, which reflects the ability of OTP to inhibit the oxidation of oil. Charlton et al. (2002) found that the interactions between polyphenols and proteins can affect the antioxidant activity of polyphenols. They reported that milk inhibited the antioxidant activity of tea polyphenols in solution and solid–liquid systems. However, milk can also improve the antioxidant capacity of tea polyphenols; the different effects are related to the positions of the protein and polyphenol in milk. In conclusion, OTP can significantly reduce the formation of O/W emulsion oxidation products. Intarasirisawat et al. (2014) reported that OTA increased the content of protein located at the oil–water interface in skipjack roe protein hydrolysate (SRPH)-stabilized emulsions. The OTA-SRPH complex inhibited lipid oxidation in the emulsion system and significantly enhanced the oxidation stability of the emulsion, consistent with the experimental results of the current study.

Conclusions

This study demonstrated that RBPH obtained via limited hydrolysis (60 min) can be applied along with OTP to improve the stability of O/W emulsions. The addition of OTP improves the emulsification stability, flocculation stability, coagulation stability, and ζ-potential while reducing the particle size. Polyphenols can form covalent bonds with proteins, exposing hydrophobic groups and polar groups in the protein to the surface, which facilitates intermolecular interaction and improves the physical stability of the emulsion. Polyphenols can improve the adsorption capacity of rbph at the interface and effectively reduce the formation of peroxide and malondialdehyde during emulsion storage. The synergistic effect of OTP and RBPH on the interface can produce physical barrier, improve emulsion stability, and have higher antioxidant activity in water phase, thereby promoting the oxidation stability of emulsion during storage. In the emulsion studied in this paper, the emulsion containing 1.5% OTP is most stable during storage. Among the emulsions studied herein, the emulsion containing 1.5% OTP was the most stable during storage. When the OTP content exceeded 1.5%, OTP competed with RBPH for adsorption at the interface, which affected the distribution of RBPH at the interface and led to reduced stability.

Acknowledgements    This study was supported by Science and technology research planning project of Jilin Provincial Department of Education (JJKH20220394KJ).

Conflict of interest    There are no conflicts of interest to declare.

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