Structure and properties of rice protein digests with high emulsification properties

 Abstract: In order to investigate the key components with better emulsification in rice protein digests, acid protease, papain and trypsin were used to restrictively digest rice proteins, and the surface hydrophobicity, secondary structure, emulsification activity and emulsion stability were analyzed to investigate the relationship between structural and emulsification properties of different digests; the samples were screened for the optimal emulsification properties, and then ultrafiltration was performed to obtain the <5 kDa, 5-10 kDa and >10 kDa fractions. After screening the samples with optimal emulsification characteristics, the samples were separated by ultrafiltration to obtain <5 kDa, 5~10 kDa and >10 kDa components, and the relationships between the interfacial properties and emulsion stability of peptides with different molecular weights were investigated by the indexes of interfacial tension, dispersive quartz crystals microbalance, particle size, microstructure, storage stability, and so on.

 

The results showed that the yield of trypsin products was the highest; compared with rice protein, the surface hydrophobicity of all the enzyme products was reduced except for the trypsin product with a hydrolysis degree of 6%; the β-folding of the enzyme was significantly reduced after the enzyme digestion, and the structure of the protein was more stretched; the trypsin 2% had a better emulsifying property; the emulsion prepared with <5 kDa was the most unstable, and the size of the emulsion was increased from 2.59 μm to 7.82 μm after 7 d of storage; while the >10 kDa group had a lower interfacial tension and a thicker interfacial layer, which indicated that the peptide with a larger molecular weight could stabilize the emulsion more effectively. After 7 d of storage, the particle size increased from 2.59 μm to 7.82 μm; while the >10 kDa group had lower interfacial tension, thicker interfacial layer, and better emulsion storage stability, which indicated that peptides with larger molecular weights could stabilize the emulsion more effectively.

 

Synthetic and animal-based emulsifiers are commonly used in the food industry [1]. Plant-based emulsifiers (e.g., plant proteins) are the main trend in the development of food emulsifiers due to their low cost and environmental friendliness, and are in line with the concept of sustainable development of the Food and Agriculture Organization of the United Nations (FAO). As one of the high-quality plant proteins, rice protein has the advantages of high bioefficacy, hypoallergenic, reasonable amino acid ratio and easy digestion and absorption [2], which has attracted much attention and gradually become a hot research topic in the field of food. However, the poor water solubility of rice protein due to cross-linking and high hydrophobicity of its molecular chains[2] limits its application in the field of food emulsifiers. Therefore, rice protein is usually modified to improve its emulsifying properties.

 

Enzymatic modification is widely used in protein modification because of its mild reaction and energy and cost saving [3]. Proteases have specific cleavage sites and different optimal pH, e.g., acidic proteases mainly act on the C-terminal hydrophobic amino acid residues [7], with the optimal pH ranging from 2 to 4; papain acts on the carboxyl terminus of arginine, lysine, and phenylalanine [8] and reacts at pH 7; trypsin acts on the carboxyl terminus of arginine and lysine [8] and reacts at pH 8 or so.

 

According to the theory of "structure determines nature", both the type of protease and the degree of hydrolysis affect the structure of the digested product, which in turn changes the functional properties, such as emulsifying properties. It is worth noting that excessive hydrolysis of proteins can reduce functional properties such as emulsifying properties, so restrictive digestion is essential [9]. Jiang Lianzhou et al.[10] used a variety of proteases to digest soybean proteins, and found that the emulsification properties of alkaline proteases and complex proteases did not change much, while other enzymes increased the emulsification properties of the products to varying degrees. It has been reported that the emulsification activity of soybean isolates treated with papain decreased with increasing degree of hydrolysis [8]. Therefore, it is worthwhile to investigate the effects of enzyme type and degree of hydrolysis on the structural and functional properties of rice protein digests. Enzymatic proteins are mixtures of peptides with different molecular weight segments. It has been shown that the >5 kDa peptide fraction of whey proteins is more stable than the <5 kDa peptide fraction of whey proteins in the formation of emulsions [11]. However, the main components of plant proteins, especially rice proteins, that are better emulsified in enzymatic products are not known.

 

Therefore, in this paper, different types of enzymes were used to treat rice protein, and the structural and functional properties of the enzyme products were investigated, and three peptide fractions with different molecular weights, namely, <5 kDa, 5-10 kDa and >10 kDa, were separated from the enzyme products with better emulsification properties, and the correlation between the interfacial properties and the stability of the emulsion was investigated, which provided theoretical guidance for the development and application of rice protein as a new type of plant-based emulsifier in a commercial scale. The study was conducted to provide theoretical guidance for the commercialization of rice protein as a new plant-based emulsifier.

 

1 Materials and Methods

1.1 Materials and Instruments

Rice protein (purity 90%) Jiangxi Jinnong Bio-technology Co., Ltd; Acid protease (100000 U/g) Angie's yeast Co. Ltd; 8-Anilino-1- naph- thalenesulfonic acid (ANS), Florisil sieve (60~100 mesh), Nile Red Shanghai McLean Biochemical Technology Co. All other reagents were analytically pure.

F2700 Fluorescence Spectrophotometer, Hitachi, Japan; T18 Digital High Speed Disperser, IKA Instruments, Germany; Nicolet islo Fourier Transform Infrared (FTIR) Spectrometer, Thermo Nicolet, U.S.A.; Labscale Ultra Filtration System, Millipore, U.S.A.; JY92- IIDN Ultrasonic Cell Breaker (900 W), Ningbo Xinzhi Biotechnology Co. JY92-IDN Ultrasonic Cell Breaker (900 W), Ningbo Xinzhi Bio-Technology Co., Ltd; DSA100S Optical Contact Angle Measuring Instrument, Krüss, Germany; S3500 Particle Sizing Analyzer, Mackinac Corporation, USA; TCS SP5 Laser Confocal Microscope, Agilent, USA; Q-Sense Explorer Dissipative Quartz Crystal Microbalance, Sweden BioLin Technology Co.

 

1.2 Experimental Methods

The research roadmap of this paper is shown in Figure 1.

 

1.2.1 Preparation of rice protein enzymatic products    

The raw materials used in this paper were obtained from rice flour by direct alkaline extraction and acid precipitation, and the enzymatic hydrolysis of rice protein was carried out according to the method of Xu et al[12]: 30 g of rice protein was dispersed in 500 mL of distilled water for 1 h at room temperature with stirring, and then subjected to enzymatic hydrolysis at the optimal temperature and pH, respectively. The mass ratio of enzyme to substrate was 1:100.

The enzyme conditions were trypsin at 50 ℃, pH 8; papain at 50 ℃, pH 7; and acid protease at 37 ℃, pH 3. During the enzyme digestion, 0.1023 mol/L NaOH or 0.1051 mol/L HCl were added dropwise to keep the pH stable. After the enzyme digestion, the sample was heated at 90 ℃ for 10 min to destroy the enzyme, then quickly cooled to room temperature in an ice bath, and finally adjusted to pH 7. The supernatant was lyophilized by centrifugation (3000 r/min, 15 min) for later use. The degree of hydrolysis (DH) was determined by pH- stat method [13] with the following formula:

Where: B and Nb are the volume of NaOH consumed (mL) and the concentration of NaOH (mol/L), respectively; Mp is the mass of protein (g); α is the degree of dissociation of α-NH3+ (0.885) at pH 8 and 50 °C treatment; and htot is the theoretical number of millimoles of peptide bonds per gram of rice protein (7.40 meq/g) [12].

Trypsin products with a hydrolysis degree of 2% are named "Trypsin 2%", and those with a hydrolysis degree of 6% are called "Trypsin 6%", and the other enzyme products are named similarly.

 

1.2.2 Determination of structural properties

1.2.2.1 Surface hydrophobicity    

Surface hydrophobicity was determined by the ANS fluorescence probe method with a slight modification of the procedure of Chen et al [14]. The proteolytic products were dissolved in phosphate buffer (10 mmol/L, pH 7) at concentrations of 0.01, 0.02, 0.04, 0.08 and 0.1 mg/mL, and the excitation and emission wavelengths were set to 390 and 470 nm, respectively. The excitation wavelength and emission wavelength were set at 390 and 470 nm, respectively. 20 μL of 8 mmol/L ANS was added into the enzyme solution, and then the solution was vortexed for 5 s. The solution was allowed to stand for 10 min, avoiding light, and then the fluorescence intensity was determined after shaking. The fluorescence intensity was plotted against the concentration of enzyme digestion products in different samples, and the surface hydrophobicity index was expressed as the slope of the initial segment.

 

1.2.2.2 Fourier Infrared Spectroscopy (FIR)    

A suitable amount of the enzyme powder was ground with potassium bromide and pressed into a tablet, and the resolution parameter was set at 4 cm-1 and scanned 32 times, and the measurement was carried out at 4000~400 cm-1 .

 

1.2.3 Determination of emulsifying activity and emulsion stability    

The emulsification characteristics were determined by the method of Zheng et al.[6] with a slight modification. Sixteen mL of the enzyme product (1 g/100 mL) and 4 mL of corn oil were dispersed in a high-speed disperser at 12,000 r/min for 1 min. 70 μL of the emulsion was immediately measured accurately at a distance of 0.5 cm from the bottom of the container and vortexed with 7 mL of SDS (0.1 g/100 mL) for 10 s. The absorbance A0 was measured at 500 nm, and A10 was measured again 10 min later to calculate the emulsification index. The absorbance value A0 was measured at 500 nm, and A10 was measured again after 10 min. The emulsifying activity index (EAI) and emulsifying stability index (ESI) were calculated with the following equations:

            × A0 × dilution factor Eq. (2)

                           Equation (3)

where C is the concentration of the digested product (g/mL) and φ is the volume fraction of the oil phase (L/L), which was 0.20 in this experiment.

 

1.2.4 Ultrafiltration separation of enzymatic products   

The product of the enzymatic digestion of pancreatic 2% was separated using a Labscale ultrafiltration (UF) system based on the method of K.K. Song et al. The product was first ultrafiltered using a 10 kDa ultrafiltration membrane. The ultrafiltration was stopped when about 5% of the retentate volume remained, and the retentate was collected as ">10 kDa". The permeate continues to be separated by 5 kDa ultrafiltration membrane, and the retained liquid is collected as "5~10 kDa" and the permeate as "<5 kDa". During the ultrafiltration process, the circulating pressure and inlet pressure were set at 0.2 MPa, and the graded peptide liquid obtained from ultrafiltration was pre-frozen at -80 ℃ and then lyophilized in a vacuum freeze dryer. The yields of <5 kDa, 5-10 kDa and >10 kDa were 40.20%, 13.88% and 45.92%, respectively.

 

1.2.5 Interfacial characterization of peptides of different molecular weights

1.2.5.1 Interface tension   

The variation of interfacial tension ( σ ) of adsorbed proteins at the oil-water interface with adsorption time was determined by the pendant drop method with reference to the method of Jinmei Wang [16]. Because of the small amount of surface-active components in corn oil, it was first purified by Florisil molecular sieve. The following method was applied to 100 g of corn oil with the addition of

3 g of Florisil molecular sieves were stirred for 30 min and centrifuged, and the procedure was repeated three times until the interfacial tension of 10 mmol/L phosphate buffer was at the level of

No significant change occurred within 30 min. The purified corn oil was placed in a glass bath, and the graded peptide solutions at different concentrations (0.01, 0.1 g/100 mL) were passed through a syringe to form 45 μL droplets at the tip of the needle. During the experiments, the entire system was protected from external vibrations that could interfere with the measurements. Based on the droplet shape, the interfacial tension σ was calculated from the Young-Laplace equation (accurate to 0.01 mN/m).

 

1.2.5.2 Determination of peptide adsorption behavior on hydrophobic surfaces   

The adsorption behavior of peptides on hydrophobic surfaces was investigated using a quartz crystal microbalance with dissipation (QCM-D) with reference to the method of Zhang et al [17]. A clean gold chip (QSX 301, 4.95 MHz) was immersed in 1-hexadecanethiol for at least 8 h to form a self-assembled hydrophobic layer to simulate the oil-water interface, and rinsed with ethanol and ultrapure water to remove the unadsorbed 1-hexadecanethiol. A stabilizing baseline was obtained by first passing 10 mmol/L pH 7 phosphate buffer, followed by pumping 1 g/100 mL of sample solution at a flow rate of 0.1 mL/min. Phosphate buffer was reintroduced after 90 minutes to flush out unbound or loosely bound emulsifiers. Finally, the frequency and dissipation were analyzed by Q-ToolsTM software at different octaves, and the thickness of adsorption was analyzed by Sauerbrey model. In this study, the third octave was used to analyze the data because the resonance at this octave is the least mechanically affected [17].

 

1.2.6 Emulsion preparation    

The method was modified from that of Wang Xia et al[18] . 4 g of graded peptide was dispersed in 100 mL phosphate buffer (10 mmol/L, pH 7), stirred for 1 h and hydrated at 4 ℃ overnight. On the second day, 18 g of the graded peptide solution was dispersed with 2 g of corn oil at high speed (10000 r/min, 2 min) to prepare a crude emulsion, which was then sonicated at 450 W for 8 min, during which time an ice-water bath was used to prevent overheating. After emulsion preparation, sodium azide (0.01 g/100 mL) was added to inhibit microbial growth.

 

1.2.7 Determination of storage stability of emulsions

1.2.7.1 Average droplet size of emulsions    

Emulsions were measured at 0 and 7 d surface area mean particle size (d3,2) using a particle size analyzer. The refractive index of corn oil was set at 1.45, the refractive index of the dispersed phase at 1.33, and the stirring speed at 1200 r/min. All samples were diluted with 10 mmol/L phosphate buffer solution (pH 7) to avoid multiple scattering effects.

 

1.2.7.2 Microstructural analysis of emulsions    

Confocal laser scanning microscope (CLSM) was used to observe the microstructure of the emulsions. 1 mL of emulsion was stained with 20 μL of Nile Red solution (1 mg/mL in ethanol), and 6 μL of the sample was taken on a slide and covered with a coverslip. A drop of cedar oil was placed in the center of the coverslip, and the emulsion was observed using a 488 nm laser excitation source and an HCX PLAPO 63× oil microscope.

1.3 Data processing

Each group of experiments was repeated three times and the results were expressed as mean ± standard deviation. SPSS Statistics 20 was used for univariate analysis and Tukey analysis for significant differences between samples (P<0.05), and Origin 2021 was used for data graphing.

 

2 Results and analysis

2.1 Effect of enzyme digestion time on hydrolysis degree of rice protein

Rice protein was subjected to enzymatic hydrolysis under the optimum conditions of the three proteases, and the results are shown in Fig. 2. The degree of hydrolysis increased rapidly in the first 10 min, which indicated that a large number of peptide bonds were broken, and then the degree of hydrolysis increased slowly, which might be due to the reduction of amino acid digestion sites or enzyme inactivation, which is consistent with the report of Hanafi et al[19] . Trypsin showed the highest degree of hydrolysis, followed by acid protease and papain, which may be related to the different enzyme activities of proteases. This may be related to the different enzyme activities of the proteases. In view of the enzyme hydrolysis time and sample yield, 2% and 6% hydrolyzed products were selected for the follow-up study. The enzyme hydrolysis times were 1.29 and 43.03 min for acid protease, 6.57 and 57.92 min for papain, and 1.64 and 25.49 min for trypsin, respectively, and the protein yields of the samples treated with the different enzymes are shown in Table 1, which shows that the protein yields of the samples increased as the hydrolysis degree was increased. The different yields of the proteolytic products of different proteases may be due to the inconsistency of the cleavage sites of the enzymes, which may lead to the difference in the size of the peptides in the supernatant [20].

 

2.2 Effect of enzyme type on the structural properties of enzymatic products

2.2.1 Surface hydrophobicity analysis As shown in Fig. 3, the surface hydrophobicity of the digested products of acid protease and papain was significantly (P<0.05) lower than that of rice protein. This is in agreement with the results of Singh et al[5] . They found that the hydrophobicity of the surface of rice bran protein concentrate was significantly reduced after treatment with papain. This may be due to the release of peptides with hydrophilic sites after enzymatic hydrolysis, which reduces the binding sites of the ANS probe, and the destruction of surface hydrophobic regions or the aggregation of peptides by hydrophobic interactions, which bury the hydrophobic regions within the aggregates[21] . The surface hydrophobicity of tryptic 6% is higher than that of tryptic 2%, probably due to unfolding of the peptide fragments, which prevents peptide aggregation and exposes more hydrophobic groups[22] . The differences in the surface hydrophobicity of the digests of different enzyme types may be due to the inconsistency in the sites where the enzymes act on the proteins, resulting in different hydrophilic and hydrophobic sites of the peptides produced by the digests. This is consistent with the results of Han RJ et al[23] . They used six proteases to restrictively digest whey protein, and found that the hydrophobicity of the digested products was reduced to different degrees, which was mainly due to the different cleavage sites of different enzymes and the inconsistent exposure of hydrophobic amino acid termini.

 

Fig. 3 Surface hydrophobicity of rice proteins and different enzymatic products

Note: Different lowercase letters indicate significant differences (P<0.05).

 

2.2.2 Fourier Infrared Spectral Analysis   

Quantitative analysis of the secondary structure of proteins (Table 2) showed that the content of α-helices and β-horns in the enzymatic products of rice proteins increased significantly, while the content of ordered β-folds decreased significantly (P<0.05). Compared with α-helices and β-horns, β-folds were more stable, and with the decrease of β-folds, the secondary structure of proteins was more relaxed [24]. Proteins with a more flexible structure are more likely to adsorb and stretch at the interface, and can form interfacial membranes with higher viscoelasticity [25]. Therefore, compared to rice proteins, the enzymatic products of rice proteins are more likely to adsorb to the oil-water interface to stabilize the emulsion.

 

2.3 Effect of enzyme type on emulsification activity and emulsion stability of enzymatic products

The amphiphilic nature of proteins allows them to be used as emulsifiers for the preparation of emulsions. The emulsification activity index (EAI) characterizes the ability of proteins to form an oil-water interface, and the emulsion stability (ESI) characterizes the ability of emulsion droplets to resist strain, and is related to the molecular weight of the protein, surface hydrophobicity, and other factors [26]. As shown in Table 2, the EAI of rice protein digests was significantly increased (P<0.05) compared to rice protein, probably due to the fact that enzymatic digestion lowered the molecular weight of the protein to a certain extent, which resulted in the stretching of peptide chains. The EAI of tryptic 6% was lower than that of tryptic 2%, probably due to the fact that the molecular weight of the proteins was further reduced by excessive digestion, smaller peptides might not be able to form a viscoelastic film on the surface of the emulsified droplets, and peptide amphiphilicity was reduced, which inhibited the interaction at the oil-water interface and decreased the stability of the emulsions [27]. The ESI of the digested products was found to be less than that of the original protein. Ghribi et al [28] also found that the ESI of chickpea proteins was lower than that of the original proteins, which may be attributed to the reduced ability of the peptides produced by enzymatic digestion to interact with each other at the interface, thus reducing the viscoelasticity of the interfacial membrane.

Papain and tryptic 2% emulsified rice proteins better than the three proteases, but the yield of papain product was lower than that of tryptic 2%. Taking into account the enzyme digestion time and yield, as well as the energy consumption, trypsin 2% was selected for ultrafiltration separation to obtain graded peptides of different sizes for the study of the interfacial properties and emulsion stability of the enzyme products with high emulsification characteristics.

 

2.4 Characterization of graded peptide interfaces

2.4.1 Analysis of interfacial tension Adsorption kinetics plays an important role in the study of protein emulsion formation and stabilization. Protein adsorption onto the oil-water interface generally involves three main stages: diffusion of protein molecules into the interface, permeation at the interface and structural rearrangement at the interface, which has an important influence on its emulsification properties [29]. As shown in Fig. 4, the interfacial tension decreases with time, which is related to the adsorption of proteins at the oil-water interface. At the initial stage of adsorption, the interfacial tension decreases faster, which may be due to the many free adsorption sites at the oil-water interface. The rate of decrease slows down with the increase of time. This is due to the gradual filling of adsorption sites with peptides, which creates a higher spatial and energy barrier at the interface [30]. The decrease in interfacial tension will reduce the Gibbs free energy of the interface and enhance the stability of the interfacial layer [31]. In this study, the lowest interfacial tension was found in the >10 kDa group, which was suggested to have better emulsification stability.

 

2.4.2 QCM-D analysis    

QCM-D was used to analyze the adsorption-desorption behavior of different peptides at a simulated oil-water interface. As shown in Fig. 5, the frequency decreases rapidly and the dissipation increases sharply during the adsorption process, which is due to the rapid adsorption of the peptide on the hydrophobic surface. As the adsorption time increases, the frequency and dissipation values change slowly, which is due to the slow adsorption of the peptide molecules, as most of the adsorption sites are filled and part of the emulsifier continues to be adsorbed onto the already formed interfacial layer, resulting in multilayer adsorption [32]. After rinsing with phosphate buffer, the frequency increases and the dissipation decreases, but does not return to its original value, indicating that some weakly adsorbed emulsifier molecules are washed away from the interface. The adsorption thicknesses of the samples in Table 3 show that the thickness of the interfacial film increases with the molecular weight of the peptide. The thickness of the interfacial film plays an important role in the stability of the emulsion, and the thicker the interfacial film, the better the stability of the emulsion [33]. In this study, the >10 kDa component formed a thicker interfacial layer, so it was hypothesized to have better emulsion stability.

 

2.5 Analysis of storage stability of graded peptide emulsions

In general, emulsions with smaller particle size are more resistant to droplet aggregation and gravity-induced phase separation. As shown in Table 3, the surface area of the freshly prepared emulsions showed an average particle size of <5 kDa, >10 kDa, and 5~10 kDa in descending order, and the microstructure (Fig. 6) showed that the droplets were more uniformly distributed. After 7 d of storage, the average particle size of the emulsions increased to different degrees, with the <5 kDa emulsion showing the largest change in particle size and flocculation. This may be due to the fact that the <5 kDa component is more enzymatically soluble and smaller in size, which reduces the thickness and strength of the interfacial layer and does not effectively inhibit droplet aggregation, leading to the destabilization of the emulsions [11]. In general, the smaller the droplet size, the stronger the ability to resist droplet aggregation and phase separation, and the more stable the emulsion is. The results show that >10 kDa can form stable emulsions more effectively than <5 kDa.

 

3 Conclusion

The structural and functional properties of three different protease-treated rice protein digests (2% and 6% hydrolysis) showed that the hydrophobicity of the digested proteins varied greatly, and the emulsifying activity increased significantly; the enzymatic digestion increased the α-helix and β-angle content and decreased the β-fold content; and the tryptic 2% had a better emulsifying property and yield compared with the other enzymatic digests. After the separation, we obtained <5 kDa, 5~10 kDa and >10 kDa fractions, and investigated the relationship between interfacial properties and emulsion stability, and found that >10 kDa fraction had the lowest interfacial tension, the largest adsorption thickness, and the best emulsion storage stability. Therefore, >10 kDa can form more stable emulsions, which provides a new idea for the design of highly emulsifiable phytoalexin-based products.

 

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