Influence of Ultrasonication Prior to Glycation on the Physicochemical Properties of Bovine Serum Albumin–galactose Conjugates

Abstract

The effects of different ultrasonic powers, times and temperatures on the physicochemical properties of bovine serum albumin (BSA)–galactose conjugates were investigated. SDS-PAGE and size exclusion chromatography analysis indicated that BSA–galactose conjugates gave a higher molecular weight than native BSA. The BSA–galactose conjugates prepared through ultrasonic pretreatment had higher degree of graft (DG) value, surface hydrophobicity (H₀), emulsifying, foaming property and denaturation temperatures (T_d) as compared to native BSA and untreated BSA-galactose conjugates. The highest DG, H₀, and emulsifying ability index were obtained in ultrasonic time, power and temperature of 10 min, 150 W/cm², and 45°C, respectively. The maximum foaming property and T_d were detected in the sample ultrasonicated at 150 W/cm² for 10 min at 35°C. From this, we conclude that increased DG and H₀ in BSA–galactose conjugates were correlated with the improved emulsifying property and foaming capacity. Overall, ultrasonic pretreatment combined with glycation has great positive effects on the physiochemical properties of BSA.

Introduction

Bovine serum albumin (BSA) is a globular protein and often served as a molecular model for investigating the physicochemical properties and biological functions of protein interactions among proteins in small organic molecules, and metabolism of related small molecule in vivo (Ojha et al., 2010, Ni et al., 2010, Zhang et al., 2016). Numerous methods, such as chemical and heat treatment, enzymatic hydrolysis, high-pressure homogenization and non-enzymatic glycosylation (glycation), are often used to improve the functional properties of proteins, particularly their emulsion and foaming ability (Chen et al., 2011, Peñas et al., 2004). However, the chemical modification in food proteins potentially results in health hazards, and most of chemical treatments cannot be widely applied on food processing. Thus, an effective and safe method must be developed to improve the physicochemical properties of proteins. Glycation is caused by the typical covalent bond between a protein or lipid molecule and a sugar molecule (Serban et al., 2016). Glycation is superior to other types of chemical modification because it proceeds under mild and safe conditions and requires no extraneous chemicals. Thus, glycation can be potentially applied for protein modification in the food industry. The state-of-the-art about the physicochemical properties and structure of glycoconjugates of proteins processed by various methods were summarized by Liu et al. (Liu et al., 2012a). Moreover, some previous studies have been reported about the effect of protein glycation by BSA reaction with polysaccharides (Xia et al., 2015), lactoses (Ledesmaosuna et al., 2009), and monosaccharides (Jian et al., 2016) on the physicochemical properties of proteins. However, studies on the physicochemical properties of proteins that were pretreated by ultrasonication and subjected to glycation reactions are rare.

Recently ultrasonication presents a great deal of successful improvements on the extent of protein glycation (Zhang et al., 2014a). Ultrasonication is the interaction of many kinds of forces that induced heating effects, acoustic cavitation, acoustic streaming, and fluid particles oscillations etc.(Legay et al., 2011). Ultrasonication can improve protein glycation (Zhang et al., 2014b). The cavitation effect induced by ultrasonication can generate transient higher temperature and pressure, thereby increasing the glycation rate (Suslick et al., 1999). Ultrasonic treatment accelerates the glycation of β-lactoglobulin in aqueous model systems, BSA and glucose under neutral conditions (Stanic-Vucinic et al., 2013, Shi et al., 2010). Our previous study demonstrated that ultrasonication alters the BSA structure, accelerates glycation, increases the number of glycation sites in BSA, and increases the galactose reaction was increased from 12 (11 lysines and 1 arginine) to 42 (39 lysines and 3 arginines) after ultrasonic treatment (Zhang et al., 2014a). However, the physicochemical properties of BSA–galactose conjugates are not researched. Thus, the effect of galactose glycation combined with ultrasonication on the physicochemical properties of BSA must be explored.

This study aimed to provide a supplement for previous theoretical study, BSA was pretreated with ultrasonic at different conditions, followed by glycation with galactose. The different ultrasonic pretreatment effect on the physicochemical properties of the BSA-galactose conjugates was evaluated by degree of graft value, surface hydrophobicity, emulsifying property, foaming property and denaturation temperatures. The results clearly revealed the critical role of ultrasonic combined with glycation in improving the physicochemical properties of BSA.

Materials and Methods

Materials    Bovine serum albumin (BSA), 1, 8-anilinonaphthalenesulfonate (ANS) reagent was purchased from Sigma-Aldrich (St Louis, MO, USA). D (+)-Galactose, O-Phthaldialdehyde (OPA), blue prestained low molecular weight protein marker (14.4 ∼ 97.4 kDa), sodium dodecyl sulphate (SDS) and other reagents were obtained from Beijing Solarbio Science & Technology Co., LTd (Beijing, China). Soybean oil was purchased from local supermarket (Nanchang Rainbow Department Store Co. LTd.). All other chemicals were of analytical grade.

Sample preparation    Native BSA (N-BSA) 2 g was dissolved in 200 mL of 50 mM phosphate buffer saline (PBS) at pH 7.4. Fifteen milliliters of BSA were split into 25 mL flat bottom conical flasks, and immersed in ice bath. The solution was treated by probe sonicator (Misonix Qsonica Q700 Sonicator, USA, 20 kHz) equipped with a microtip probe (1/8 in. = 3 mm) with a 5s on and 5s off pulsation at an actual ultrasonic intensity of 0, 60, 90, 120 and 150 W/cm², respectively, for 10 min, and ultrasonic time of 0, 1, 5, 10 and 20 min, respectively, to ensure the temperature of the sample solution is not elevated (lower than 15°C). Otherwise, 15 mL of BSA splited into 25 mL flat bottom conical flasks were immersed in a thermostat water bath (DC-2010, Ningbo Xinzhi Instruments, Inc., Ningbo, China). The solution was ultrasonicated with 5s on and 5s off pulsation at 150 W/cm² for 10 min with the temperature kept at 0, 15, 25, 35 and 45°C, respectively. BSA solution without galactose was used as control.

In case of glycation, 10 mg of galactose was added to 1 mL of the native BSA and ultrasonicated BSA solution, separately. After lyophilization, the powders of N-BSA, native and ultrasonicated BSA-galactose complex were incubated at 65% relative humidity (saturated potassium iodide solution) and 55°C for 1 h. The reaction was stopped by transferring the sample tubes into an ice bath. The free galactose and salts were filtered by a Centricon centrifugal filter unit (10000 Da, Millipore, Bedford, MA, USA). The concentration of native BSA- galactose conjugates (N-BSA-G) and ultrasonicated BSA- galactose conjugates were adjusted to 10 mg/mL, respectively with PBS at pH 7.4 and stored at 4°C for further use.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)    The protein content of all samples was 1 mg/mL. SDS-PAGE was carried out on a Bio-Rad Mini-Protein Tetra Electrophoresis System (Bio-Rad Laboratories, Inc., Hercules, USA) with a 5% stacking gel and 12% separating gel. After electrophoresis, gels were stained with Coomassie R250 dye.

Size exclusion chromatography (SEC)    Sample solution (50 uL, 2.0 mg/mL) were purified by SEC on a TSK Gel 3000 SWXL column (TOSOH Bioscience, King of Prussia, PA) using an Agilent 1200 (Agilent Technologies, Palo Alto, CA) HPLC system. The separation was performed with 0.1 M ammonium acetate (pH 6.8) at a flow rate of 0.5 mL/min and was monitored with 280 nm UV detection.

Degree of graft (DG)    Free amino groups were determined by the o-phthaldialdehyde assay. According the method of Li et al. (Li et al., 2014), the absorbance of sample at 340 nm was measured by a Hitachi U-2910 spectrophotometer (Hitachi, LTd, Tokyo, Japan). Lysine was used as standard. DG was then calculated:   

Where A₀ is the levels of free amino groups in mixtures of BSA-galactose; At is the levels of free amino groups in BSA-galactose conjugates and A is the level of free amino groups in BSA.

Intrinsic fluorescence emission spectroscopy    Samples (1 mg/mL) were prepared with 50 mM, pH 7.4 PBS. The intrinsic fluorescence emission spectra were obtained by a Hitachi F-7000 fluorescence spectrophotometer (Hitachi, LTd, Tokyo, Japan). The emission spectra were recorded from 300 nm to 400 nm (both at a constant slit of 5 nm) with excitation set at 290 nm.

Surface hydrophobicity (H₀)    The H₀ values of BSA were determined by using ANS as fluorescence probe. Fluorescence intensity (FI) of sample were measured excitation wavelength at 390 nm and emission wavelength at 470 nm (both at a constant slit of 2.5 nm) using a Hitachi F-7000 fluorescence spectrophotometer (Hitachi, LTd, Tokyo, Japan). The initial slope of FI versus protein concentration plot was used as the index of H₀.

Emulsifying properties    The emulsifying ability index (EAI) and emulsifying stability index (ESI) were determined by the method of Zhang. (Zhang et al., 2014c). Soybean oil (10 mL) and 30 mL of 1% protein solution (pH 7.4) were mixed. The mixture was homogenized using a homogenizer (Ultra-Turrax T25, IKA, Germany) at a speed of 12,000 rpm for 2 min. An aliquot of the emulsion (50 µL) was pipetted from the bottom of the container at 0 and 10 min after homogenization and mixed with 5 mL of 0.1% SDS solution. The absorbance of the sample solution was measured at 500 nm using a Hitachi U-2910 spectrophotometer (Hitachi, LTd, Tokyo, Japan). EAI and ESI were then calculated:   

  

Where, m is the protein weight (g), A₀ and A₁₀ are the absorbance of the emulsion at 0 min and 10 min, respectively.

Foaming properties    Foaming capacity (FC) and foaming stability (FS) of BSA-galactose conjugates were measured according to the method of Zhang. (Zhang et al., 2014c). BSA or glycated BSA solution (25 mL, 0.5% w/v) in PBS (50 mM, pH 7.4) was homogenized using a homogenizer (Ultra-Turrax T25, IKA, Germany) at a speed of 20,000 rpm for 1 min. After homogenization, the volume of the solution was measured immediately 0 and 30 min later respectively. FC and FS were then calculated:   

  

Where, V₁ (mL) and V₂ (mL) is the volume of the solution measured after 0 and 30 min of homogenization respectively.

Thermal denaturation    Thermal behavior measurements were performed on a Differential Scanning Calorimetry (NETZSCH DSC 200 F3, Germany) using aluminum pans. Approximately 6–10 mg of all samples were weighed into the aluminum pans. The pans were hermetically sealed and then a temperature ramp from 30 to 120°C was applied, at a heating rate of 10 K/min. Denaturation temperature (Td) was calculated using Proteus analysis software (NETZSCH, Germany).

Statistical analysis    All experiments were performed in triplicate and the data obtained were analyzed by one way analysis of variance (One-Way ANOVA) using SPSS for windows version 20 (SPSS Inc., Chicago, IL). Values were expressed as means ± SD. Significant differences (p < 0.05) between means were identified by Tukey's-b test.

Results and Discussion

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis    The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of BSA and BSA–galactose conjugates are shown in Fig. 1. It is not obvious that BSA-galactose conjugates pretreated by ultrasonic migrated broader than native BSA, but they were all above 66.2 kDa. Zhang et al reported that the molecular weights of the native and ultrasonicated BSA–galactose conjugates shift to 66,949.93 and 70,216.84 Da, respectively, which were much higher than that of the native BSA (66,431.36 Da) (Zhang et al., 2014a). This result suggested that ultrasonic pretreatment can increase further the exposure of lysine and arginine residues from BSA, thus promoting the glycation reaction between BSA and galactose (Zhang et al., 2014a). Meanwhile, BSA–galactose conjugates contain an arrangement of BSA molecules with different numbers of coupled galactose residues. However, no significant difference was found between the BSA–galactose conjugates pretreated by different ultrasonic powers levels (Fig. 1A) and pretreated by different ultrasonic times and temperatures (Fig. 1B and 1C). The SDS-PAGE pattern cannot reveal their changes exactly and further work must be performed through size exclusion chromatography and mass spectrometry.

Fig. 1.

The molecular weight of BSA-galactose conjugates pretreated by different power (A), time (B) and temperature (C).

Size exclusion chromatography    Size exclusion chromatography (SEC) separates biomolecules according to their molecular size (Fekete et al., 2013). Fig. 2 displays SEC diagrams for native BSA and ultrasonicated BSA–galactose conjugates. Tetramers, dimers, and monomers of N-BSA were calculated by SEC. The same results were observed in the BSA-galactose conjugates pretreated by different ultrasonic powers, times and temperatures (Fig. 2A ∼ C). The elution time (monomer) of ultrasonicated BSA–galactose conjugates shifted to 16.09 min, which is much shorter than the elution time of NBSA at 17.50 min and untreated conjugates at 16.50 min. This result indicated the formation of proteinaceous high molecular weight components after ultrasonication combined with glycation.

Fig. 2.

Size exclusion chromatography for analyses BSA-galactose conjugates pretreated by different power (A), time (B) and temperature (C).

Degree of graft (DG)    The levels of free amino groups of all the samples were investigated. The results are listed in Table 1. The free amino groups in the BSA–galactose conjugates pretreated by different ultrasonic conditions were significantly lower than that of BSA and untreated conjugates (p < 0.05). Glycation reaction can decrease the amuont of free amino groups in the proteins. Therefore, the result indicated that glycation occurred in the treated samples, and ultrasonic pretreatment can accelerate the glycation reaction of BSA and galactose. The free amino groups of ultrasonicated BSA–galactose conjugates were decreased, whereas the degree of graft (DG) was enhanced with the increase of ultrasonic power and temperature (Table 1A, C). However, free amino groups and the DG of BSA–galactose conjugates had no significant (p > 0.05) change at different ultrasonic times (Table 1B). This finding can be attributed to the structure of BSA, which loosened when pretreated by certain ultrasonic temperature and power. The loosened the structure of BSA facilitated the glycation reaction and then decreased the free amino groups of proteins. The highest DG of 32.9 ± 0.7% was obtained in BSA–galactose conjugates pretreated at 150 W/cm² for 10 min at 45°C; this result indicated that ultrasonic heat-treatment had more effect on glycation reaction, and glycation combined with ultrasonic pretreatment developed much higher DG value than untreated conjugates. This finding may be that the ultrasonic heat-treatment can reduce the unfolding of BSA chains, leading to the exposure of the reactive groups (Kardos et al., 2001) and start of the glycation reaction between BSA and galactose.

Table 1. Levels of free amino groups and degree of graft (DG) and denaturation temperatures (T_d) of BSA-galactose conjugates pretreated by different power (A), time (B) and temperature (C). Different letters (a–d) indicated significant (p < 0.05) difference between the values.

A
Sample	Free amino groups (mg/mL)	DG (%)	Td (°C)
N-BSA	0.045 ± 0.002a		88.9 ± 1.8d
N-BSA-G	0.041 ± 0.002b		94.5 ± 0.5c
60W/cm2	0.035 ± 0.006c	12.8 ± 3.0c	95.9 ± 0.1bc
90W/cm2	0.033 ± 0.005d	17.4 ± 0.4b	96.5 ± 0.5bc
120W/cm2	0.032 ± 0.003d	19.9 ± 0.1b	97.7 ± 1.1b
150W/cm2	0.030 ± 0.005e	25.3 ± 0.4a	103.2 ± 1.5a
B
Sample	Free amino groups (mg/mL)	DG (%)	Td (°C)
N-BSA	0.045 ± 0.002a		88.9 ± 1.8c
N-BSA-G	0.041 ± 0.002b		94.5 ± 0.5b
1min	0.028 ± 0.007d	28.2 ± 1.0a	103.6 ± 1.1a
5min	0.027 ± 0.004d	29.2 ± 2.0a	104.0 ± 0.6a
10min	0.030 ± 0.005c	25.3 ± 0.4b	103.2 ± 1.5a
20min	0.029 ± 0.003d	27.7 ± 2.0a	102.6 ± 0.2a
C
Sample	Free amino groups (mg/mL)	DG (%)	Td (°C)
N-BSA	0.045 ± 0.002a		88.9 ± 1.8e
N-BSA-G	0.041 ± 0.002b		94.5 ± 0.5d
15°C	0.028 ± 0.006c	29.2 ± 0.8b	98.0 ± 0.5c
25°C	0.027 ± 0.004c	30.3 ± 0.3b	102.4 ± 0.8b
35°C	0.026 ± 0.003d	32.6 ± 1.0a	104.7 ± 0.4a
45°C	0.026 ± 0.005d	32.9 ± 0.7a	100.6 ± 0.6b

Intrinsic fluorescence emission spectroscopy    The conformational changes around Trp residues were evaluated by the intrinsic fluorescence spectra of all the samples. The Trp residues of the BSA–galactose conjugates were decreased compared with that of native BSA (Fig. 3). This result indicated that the galactose chain might have shielded the area around the Trp residues (Hattori et al., 1997), and the conformation changes were occurred around the Trp residues due to the glycation reaction between BSA and galactose (Jiménez-Castaño et al., 2005). The maximum fluorescence emission (λ_max) of BSA and untreated conjugates were at 343.8 nm and 343.6 nm, respectively and the λ_max of BSA-galactose conjugates pretreated by different power levels (Fig. 3A) were not significantly different from that pretreated by different temperatures (Fig. 3C). However, with the increase of ultrasonic time, the fluorescence intensity of the conjugates was gradually decreased (Fig. 3B). Thus, the Trp residues in BSA–galactose conjugates were more surrounded to the hydrophobic environment than that in native BSA; this finding leads to the conformational change of BSA during glycation (Kobayashi et al., 2001). The change in λ_max value suggested that the tertiary conformation of BSA became less compact after glycation (Liu et al., 2012b), and BSA–galactose conjugates pretreated by ultrasonication had much less influence on the tertiary conformation of BSA than that untreated.

Fig. 3.

The intrinsic fluorescence spectra of BSA-galactose conjugates pretreated by different power (A), time (B) and temperature (C).

Surface hydrophobicity (H₀)    The surface hydrophobicity (H₀) values of the BSA and BSA-galactose conjugates are shown in Fig. 4. Ultrasonic pretreatment combined with glycation can significantly (p < 0.05) increase the H₀ value of BSA–galactose conjugates. This result was probably due to the increased amounts of galactoses grafted with BSA and rapid increase in surface hydrophobicity (Mu et al., 2010). The aggregate dissociation or protein unfolding, and the exposure of hydrophobic groups initially buries in the interior of protein molecules (Li et al., 2014). Furthermore, with the increase of ultrasonic power and temperature, the H₀ values of BSA–galactose conjugates also increased, and the maximum H₀ was achieved at 150 W/cm² for 10 min at 45°C. These effects can be attributed to the high power and temperature, which expose the hydrophobic groups. The BSA–galactose conjugates ultrasonicated at 45°C had higher H₀ than others, suggesting that temperature plays an important role in the H₀. The H₀ values of BSA–galactose conjugates by different ultrasonic times initially increased and then decreased as shown in Fig. 4B. The maximum H₀ value was detected in the sample ultrasonicated at 150 W/cm² for 5 min. When the sample was ultrasonicated at 150 W/cm² for 10 min and 20 min, the H₀ values were decreased. When ultrasonic time was over 10 min, the hydrophobic groups of BSA were encased in the aqueous environment. In a summary, the ultrasonication combined with glycation can increase the H₀ of protein.

Fig. 4.

The H₀ values of BSA-galactose conjugates pretreated by different power (A), time (B) and temperature (C). Different letters (a–f) indicated significant (p < 0.05) difference between the values.

Emulsifying property    The emulsifying ability index (EAI) and emulsifying stability index (ESI) of BSA and BSA–galactose conjugates are described in Fig. 5. Contrary to the negative effect on emulsifying activity presented by BSA–glucose and BSA–mannose conjugates (Jian et al., 2016), the glycation between BSA and galactose combined with ultrasonic pretreatment exhibited substantial positive effects on emulsifying activity. These effects may be attributed to the prolonged glycation reaction time and different ultrasonic conditions applied in this research. As expected, glycation combined with ultrasonic pretreatment were more effective in improving the emulsification property of BSA than that using dry heating alone. The EAI and ESI of BSA–galactose conjugates by different ultrasonic time were not significantly different from the increasing of ultrasonic times (p > 0.05; Fig. 5B), but the EAI was against it with the improvement of ultrasonic power and temperature (Fig. 5A, C). This finding may be due to the increased grafting accessibility of the major subunits in BSA after ultrasonic pretreatment at different power levels and temperatures, indicating that looser BSA can be grafted readily with galactose and kept soluble around its pI (Lin et al., 2016). The conjugates ultrasonicated at 150 W/cm² for 10 min at 45°C and 35°C generated the maximum EAI and ESI, respectively, owing to the effect of temperature on emulsifying properties by changing the particle size, secondary structure, and surface hydrophobicity of proteins (Zhang et al., 2014a). Previous results revealed that BSA–galactose conjugates pretreated by ultrasonication had high DG values (Table 1), and ultrasonic pretreatment can induce protein unfolding, expose hydrophobic group, and increase the H₀ of BSA-galactose conjugates (Fig. 1–4); these may be responsible for the improved emulsifying properties (Ricky et al., 2013).

Fig. 5.

The EAI and ESI of BSA-galactose conjugates pretreated by different power (A), time (B) and temperature (C). Different letters (a–e) indicated significant (p < 0.05) difference between the values.

Foaming property    Foaming capacity (FC) and forming stability (FS) were used to evaluate the foaming activity of protein. In Figure 6, significant (p < 0.05) difference was noted in the level of FC of BSA-galactose conjugates pretreated at different ultrasonic conditions. The FS levels of BSA and untreated conjugates showed no significant effect. The ultrasonic treatment promoted the glycation reaction between BSA and galactose and improved the conjugation ratio (Zhang et al., 2014a). It also improved the crosslinking between BSA and BSA (Ter Haar et al., 2011), thereby enhancing the foaming properties of BSA. The FS of the BSA–galactose conjugates subjected to ultrasonic treatment had no significant (p > 0.05) improvement at increased ultrasonic power, time, and temperature (Fig. 6A, B, C). The FC of the same conjugates pretreated by ultrasonic power exhibited a similar alternation, as shown in Fig. 6A. However, the FC was initially increased and then decrease with the increase of ultrasonic temperature (Fig. 6C). The maximum FC and FS at 169.4 ± 17.4% and 56.7 ± 13.9%, respectively, were detected in the sample ultrasonicated at 150 W/cm² for 10 min at 25°C. As the temperatures increased, the H₀ of the glycated BSA was affected, leading to the change in the foaming activity. H₀ is a primary factor determining FC, and a higher H₀ means higher affinity for the air/water interface (Townsend et al., 1983). By contrast, the interfacial properties of protein can affect foaming property (Jian et al., 2016). Therefore, ultrasonic pretreatment combined with glycation can improve the foaming property of BSA and BSA–galactose conjugates.

Fig. 6.

The FC and FS of BSA-galactose conjugates pretreated by different power (A), time (B) and temperature (C). Different letters (a–f) indicated significant (p < 0.05) difference between the values.

Denaturation temperatures (T_d)    Differential scanning calorimetry (DSC) was established to investigate the thermal properties of the BSA–galactose conjugates. Generally, a higher denaturation temperature (T_d) value is associated with high thermal stability for a globular protein (Liu et al., 2012a). Table 1 shows the DSC characteristic changes in the BSA and BSA-galactose conjugates pretreated and un-pretreated by ultrasonication. The T_d of BSA and untreated conjugates were 88.9 ± 1.8°C and 94.5 ± 0.5°C, respectively, which were significantly (p < 0.05) lower than those of the pretreated conjugates. Maillard reaction between BSA and galactose can cause covalent binding between BSA and galactose, thereby inhibiting the interaction among proteins and improving the thermal stability of BSA (Broersen et al., 2004). Ultrasonic pretreatment combined with glycation can increase the T_d of BSA, although the difference was nonsignificant (p > 0.05). As shown in Table 1A, the T_d of BSA–galactose conjugates increased with the increase of ultrasonic power. However, the T_d value of BSA- galactose conjugates firstly increased and then reduced with the increase of ultrasonic temperature (Table 1C). The maximum T_d value of 104.7 ± 0.4°C was detected in samples ultrasonicated at 150 W/cm² for 10 min at 35°C. Ultrasonication caused the conformation change of BSA, loosened the BSA structure, and destroyed part of the interaction force (Zhang et al., 2014d). Consequently, the glycation was accelerated, and the amount of the glycation products increased. The T_d values of BSA–galactose conjugates pretreated by ultrasonication were improved. Furthermore, the introduction of galactose molecules, increase in net negative charge of glycation products, and the enlargement of electrostatic repulsion and steric hindrance may hinder the aggregation of glycation products during heat (Ahmed et al., 2016, Tang et al., 2011, He et al., 2014), accordingly the thermal stability of conjugates were improved. Thus, ultrasonic pretreatment combined with glycation can improve the thermal stability of BSA–galactose conjugates.

Conclusion

In this study, the physiochemical properties of BSA and BSA–galactose conjugates pretreated with ultrasonication were fully investigated. Different ultrasonic power, time and temperature pretreatment can accelerate the glycation between BSA and galactose, and produce BSA–galactose conjugates with higher degree of graft, surface hydrophobicity, and emulsifying and foaming properties compared with those of native BSA and untreated sample. Higher surface hydrophobicity and degree of graft may explain the improved physiochemical properties of the BSA–galactose conjugates, particularly their emulsifying and foaming properties. These results indicated that ultrasonic treatment is an efficient method for forming glycated BSA, and ultrasonic pretreatment combined with glycation had a significantly positive effect on the physiochemical properties of BSA. By contrast, further work needs to be conducted to elucidate the molecular basis and directional regulation mechanism of Maillard reaction under ultrasonic treatment.

Acknowledgements    This work was supported by funds from Chinese National Natural Science Foundation (No. 31460395), National Natural Science Foundation of China (No. 31360374), China Agriculture Research System (CARS-45), and National High Technology Research and Development Program of China (863 Program, No. 2013AA102205).

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