
==== Front
Ultrason Sonochem
Ultrason Sonochem
Ultrasonics Sonochemistry
1350-4177
1873-2828
Elsevier

S1350-4177(24)00272-4
10.1016/j.ultsonch.2024.107024
107024
Original Research Article
Ultrasound-assisted glycosylation of ovalbumin and dextran conjugate carrier for anthocyanins and their stability evaluation
Chen Boyu 1
Chen Lei 1
Li Chen
Huang Wanhuan
Zhao Yanan
Ai Chao
Teng Hui tenghui850610@126.com
⁎
College of Food Science and Technology, Guangdong Ocean University, Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety, Guangdong Province Engineering Laboratory for Marine Biological Products, Zhanjiang 524088, China
⁎ Corresponding author. tenghui850610@126.com
1 This author contributed equally to this work.

10 8 2024
10 2024
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3 8 2024
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© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Anthocyanins (AC) are vulnerable to degradation when affected by external factors. The present study employed ultrasound-assisted glycosylation of ovalbumin (OVA) and dextran (Dex) to generate conjugate carrier for AC to improve its stability. The results showed that sonication significantly improved the progression of Maillard reaction to OVA. Compared to traditional glycosylation, ultrasound treatment showed a higher degree of grafting, a lower number of free-SH, and smaller particle size and uniform distribution. The SDS-PAGE results indicated covalent interaction. Intrinsic fluorescence (INF), Fourier transform infrared spectroscopy (FTIR), and Circular dichroism (CD) analysis results suggested that ultrasound-assisted glycosylation altered the OVA structure. The scanning electron microscope (SEM) and X-ray diffractometer (XRD) observed that the ultrasound-assisted complex had a more compact and smoother structure and protein unfolding were better. The protein solubility increased significantly after glycosylation. Thermal gravimetric analysis (TGA) and Differential scanning calorimetry (DSC) indicated that the glycosylated conjugates can significantly improve the thermal stability of AC In addition, the AC showed an improved processing and storage stability when conjugated with glycosylated carrier. The glycosylated protein-anthocyanins complex may help provide new ideas and scientific basis for the development of naturally sourced anthocyanins-relevant products in pharmaceutical and food industry applications.

Keywords

Anthocyanins
Glycosylation
Stability
Ultrasound
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pmc1 Introduction

Anthocyanins (AC) are a class of flavonoid pigments found in many fruits and vegetables [1]. Due to the presence of the sugar component, they are considered as the main water-soluble group of polyphenols, and more than 700 different types of chemical structure have been identified. According to the substitution position on the B ring, six main subclasses, including centaurin, peonidin, petunia, delphinidin, pelargonidin, and malvins are widely found in natural plants. Hexose and pentose are the most common sugar groups, usually in the form of monosaccharide, diosaccharide or triosaccharide attached to the structure of the glycoside [2]. Different hydroxyl positions on the anthocyanin skeleton produce different colors in different environments, such as yellowish-brown cations (red to orange) in acidic environments, and purple to brown quinones in neutral or slightly acidic conditions [2]. In addition, it was found that they could act as anti-inflammatory and antioxidant against various chronic diseases, such as anti-cancer [3], anti-diabetes [4], anti-obesity [5], etc. Although they have a great potential in health care, their development and application for related products are greatly limited due to the instability in processing and storage, inducing color degradation and reducing biological activity [6].

In order to improve the added value and available application range of anthocyanins, a variety of chemical modification or physical embedding methods have been used to modify the structure and improve their stability in the past few years. Among them, the complex formed by combining anthocyanins with other natural macromolecules such as polysaccharides and proteins is an excellent method to improve anthocyanins' stability [7]. For example, Ren et al. 's study proved that whey protein (WP) can improve the chroma and stability of AC [8]. Chung et al.' s study also proved that the complexation of anthocyanins with WPI can reduce the degradation of anthocyanins caused by ascorbic condensation or oxidation and improve the stability of anthocyanins [9].

Ovalbumin (OVA), the main component of egg whites, is an important globular food source of protein. It is a 43 kDa monomeric protein composed of 385 amino acids, and more than half of them are hydrophobic amino acids and one third are charged residues [10]. However, most of the hydrophobic amino acid residues of natural proteins are inside of their molecular structure, so they have the disadvantages of poor thermal stability and solubility [11]. Glycosylation is a way to induce protein coupling with carbohydrates, which results in rapid adsorption of the air/water interface due to exposure of the hydrophobic part and an increased molecular folding [12], thereby improving its functional properties. It is a relatively safe, simple and effective method to improve the functional properties of proteins. Dextran (Dex) is a kind of microbial exopolysaccharide, which has high flexibility and poor aggregation abilities in aqueous solution. Its neutral charge property also inhibits the electrostatic complexation between protein and polysaccharide, which makes Dex an ideal material for glycosylation with protein [13].

The wet heat method is one of the traditional methods for the preparation of protein-polysaccharide conjugates. In this approach, the Maillard reaction (MR) is carried out in an aqueous solution, which is very prone to denaturation and aggregation under heating conditions, thus affecting the efficiency of the glycosylation reaction [14]. Moreover, wet heating has the disadvantages of a long time, which usually takes a couple of hours to several days [15]. In recent years, ultrasound has attracted a great deal of attention in food processing as a green and cost-effective technology. As a non-thermophysical technique, ultrasound has been widely used in food protein modification. The ultrasonic induced acoustic cavitation effect is a periodic process of rapid bubble growth and violent collapse, which can generate strong mechanical forces (such as shear forces, shock waves, and microscopic turbulence) [16], resulting in protein unfolding and peptide bond braking. At the same time, ultrasonic treatment increased the collision probability between reactive groups and accelerated the conjugation process of protein and polysaccharide [17]. According to Chen et al. [18], ultrasonic treatment can greatly shorten the glycosylation reaction time between whey protein isolate (WPI) and Arabica gum (GA), showing lower browning intensity, better solubility and thermal stability. Qu et al. [19] also demonstrated that ultrasound can promote the Maillard reaction between proteins and polysaccharides by exposing more reactive amino groups, thereby improving the functional properties of proteins.

At present, ultrasound assisted Maillard reaction has been increasingly applied to the preparation of glycosylated proteins, but only a few studies have investigated the influence of ultrasound assisted dextran grafting on ovalbumin, and rarely can found any research on the preparation of complex between glycosylated conjugates to carry anthocyanins to improve the stability of anthocyanins. In this study, the conjugate carrier of OVA and Dex was prepared by ultrasound-assisted glycosylation and traditional wet heating, and the degree of Maillard reaction was investigated by means of browning intensity and glycosylated intermediates. And the structure of glycosylated conjugates was characterized and the functional properties of conjugated carriers were analyzed, and the influence of the glycosylated conjugate carriers on the thermal stability as well as processing and storage stability of anthocyanins was inspected. Based on the above studies, the system is expected to provide a relevant research basis for anthocyanins homeostasis, which may help provide new ideas and scientific basis for the development of naturally sourced anthocyanins-relevant products in pharmaceutical and food industry applications.

2 Materials and methods

2.1 Materials and chemical reagents

The OVA with a purity of 80 % was ordered from Yuanye Biotechnology Co., LTD (Shanghai, China). Dextran (Dex, 98 % purity) and blueberry anthocyanins (AC, 25 % purity), were purchased from Aladdin Co., LTD (Shanghai, China). Other reagents were of analytical grade, and were purchased from Beijing Solaibao Technology Co., LTD. (Beijing, China).

2.2 Preparation of ovalbumin-dextran glycosylated conjugates

2.0 g ovalbumin (OVA) was dissolved in 100 mL ultra-pure water to a protein concentration of 2 mg/mL (w/v) and stirred on a magnetic stirrer at room temperature for 1 h. Then the pH value of 2 % OVA solution was adjusted to 7.0 ± 0.1 with 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide, and was hydrated at 4℃ for one night. In addition, 2.0 g dextran (Dex) was dissolved in 100 mL ultra-pure water to obtain 2 % Dex solution, and the pH value of 2 % Dex solution was also adjusted to 7.0 ± 0.1 with 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide, and was hydrated at 4℃ for one night.

Ovalbumin-dextran (OVA-Dex) glycosylated conjugates was prepared as referred to the method reported by Sheng et al with a slight modification [20]. After the overnight hydration, the OVA solution and Dex solution were mixed with an equal ratio (1:1), and 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide were used to adjust the solution to pH=7.0 ± 0.1, and then the OVA-Dex mixture was obtained. Afterwards, ultrasound-assisted glycosylation was proceeded by transferring the mixture of 30 mL into a tube, and putting it in a constant temperature tank (80℃) and then treated by an ultrasonic cell crusher (equipped with a 16 mm diameter probe) with an output power of 20 W/cm3 for 40 min, 120 min, 200 min, 280 min, and 360 min respectively, with a 5 s interval. After the reaction, the sample was placed in an ice bath for 30 min to stop the reaction to get the OVA-Dex conjugates (UOVA-Dex 40, UOVA-Dex 120, UOVA-Dex 200, UOVA-Dex 280 and UOVA-Dex 360), respectively, using the OVA solution as a blank group.

In addition, the traditional water bath glycosylation was inspected as well. Briefly, the OVA-Dex mixture of 30 mL was transferred into a tube and put in a water bath at 80℃ and the glycosylation was completed by stirring by magnetic bars at 600 rpm and lasted for 8 h, 16 h, 24 h, and 32 h, respectively. After the reaction, the samples were placed in an ice bath for 30 min to stop the reaction, and the conjugated samples were recorded as OVA-Dex 8, OVA-Dex 16, OVA-Dex 24, and OVA-Dex 32, respectively.

The reaction mixture was subsequently transferred to a dialysis bag with a molecular weight cut-off of 10 kDa and then dialyzed at 4℃ for 24 h. Finally, all samples were freeze-dried after the preparation and stored in a desiccator until further analysis.

2.3 Browning index and glycosylation intermediates

The determination of the browning index and glycosylation intermediates was completed after an appropriate modification of the method as referred by Ding et al [21]. Ultraviolet–visible spectrophotometers (Evolution, ThermoFisher Scientific, Waltham, America) were used to measure the absorbance of samples at 294 nm and 420 nm, respectively, in order to determine the middle and late stages of the Maillard reaction process.

2.4 Determination of grafting degree

The grafting rate of the conjugate was determined by OPA method according to the reference reported by Aminikhah et al [22]. Phthalaldehyde (OPA) of 40 mg was dissolved in 1 mL methanol, and then mixed with 25 mL of 0.1 mol/L sodium tetrborate, 2.5 mL of 20 % (W/V) sodium dodecyl sulfate (SDS) and 100 uL of β-mercaptoethanol. After that, the OPA reagent was obtained by adjusting the volume to 50 mL with pure water. Then, the sample of 50 μL was blended with 1 mL OPA solution and incubated at 35℃ for 5 min. The absorbance of the sample was measured at 340 nm by a microplate reader(Varioskan, ThermoFisher Scientific, America). The content of free amino acids in the samples was determined by using lysine as the standard curve. The grafting rate was calculated as follows:DG%=A0-A1/A0×100%

where, A0 and A1 were absorbance values of OVA before and after the glyc osylation, respectively.

2.5 Determination of free sulfhydryl group (−SH)

The determination of free sulfhydryl groups was completed after an appropriate modification according to the method of Fu et al [23]. After mixing 0.5 mL sample solution with 4.5 mL standard buffer solution (consisting of 0.5 % sodium lauryl sulfate, 0.086 mol/L Tris, 0.092 mol/L glycine, and 0.004 mol/L EDTA, pH=8.0), the reaction was activated by adding 50 μL Ellman reagent (4 mg/mL 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) dissolved in the standard buffer solution). Subsequently, the absorbance of the reaction solution was measured at the wavelength of 412 nm using the micro-plate reader after the solution was left to stand in darkness for 15 min. The free sulfhydryl group was calculated as follows:SHμmol/g=73.53×A412×D/C

where A412 was the absorbance of the sample at 412 nm, D was the dilution coefficient, and C was the sample concentration (mg/mL).

2.6 Intrinsic fluorescence analysis (INF)

The INF of the sample was appropriately modified and performed with reference to the method of Aminikhah et al [22]. Firstly, the sample was diluted to a final concentration of 0.1 mg/mL, and the intrinsic fluorescence of the sample was analyzed by fluorescence spectrophotometer (RF-5301PC, SHIMADZU, Japan) at the excitation wavelength of 280 nm, the emission wavelength of 300–500 nm, slit width of 5 nm, and voltage of 400 V.

2.7 Particle size and polydispersion index (PDI)

The particle size and polydispersion index (PDI) of OVA, OVA-Dex and UOVA-Dex solutions were analyzed using dynamic light scattering (DLS) by the ZETASIZER NANOZSE (Guangdong Shengze Technology Co., LTD., Dongguan, Guangdong Province). The sample was equilibrated at 25℃ for 2 min and then measured 3 times [15].

2.8 Scanning electron microscopy (SEM)

The SEM of OVA, OVA-Dex, UOVA-Dex solutions were analyzed by fixing the freeze-dried powder sample to a conductive adhesive in the ion sputtering equipment and coating with gold. Samples were then equilibrated at 25℃ for 1 min and measured three times using a scanning electron microscope at an accelerated voltage of 3 kV [15].

2.9 XRD analysis

The crystal structure of the sample was determined by X-ray diffractometer (D/MAX2500, Rigaku, Japan) in a continuous mode at 2θ angle from 5◦ to 90◦ at a scanning rate of 2◦/min [24]. The total area and amorphous regions area of XRD pattern was calculated by Origin, and the The crystallinity calculated as follows:Crystallinity(%)=[(A-B)/A]×100%.

where A was the total area; B was the amorphous regions area.

2.10 Fourier transform infrared spectral (FTIR) analysis

FTIR analysis of OVA, OVA-Dex and UOVA-Dex were determined using a Fourier Transform infrared spectrometer (TENSOR 27, Bruker, Germany) with a maximum resolution of 0.5 cm−1. Scan sample sheets in the infrared were ranged from 400 to 4000 cm−1 [25].

2.11 Circular dichroism (CD) spectroscopy analysis

Each sample was diluted to 0.15 mg/mL and the secondary structure of the sample was analyzed by far ultraviolet CD spectroscopy (Chirascan V100, Applied Photophysics Ltd, UK). At a bandwidth of 1 nm, the wavelength of the samples were scanned ranging from 190 to 260 nm [26].

2.12 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis

The protein composition of the glycosylated sample was analyzed and determined by the SDS-PAGE method as referred by Li et al [15]. Protein electrophoresis gel was prepared by using 10 % separation gel and 5 % concentrated gel which contained 10 % SDS. Protein sample of 120 μL was added with 30 μL 5 × buffer, shaken evenly, and then boiled at 100℃ for 10 min to promote protein denaturation. The denaturised protein sample of 10 μL was successively loaded into the hole and electrophoresis was performed at 100 V voltage. After electrophoresis, the sample was stained with coomassie bright blue fast color solution, and followed by decolorization with ultrapure water. And the band strength at the top of the gel of OVA-Dex 24 conjugate, UOVA-Dex 40/120 /200/280/360 conjugate in the SDS was calculated by Image J.

2.13 Solubility analysis

The solubility analysis of the sample was completed after appropriate modification according to the method of Zheng et al [25]. Briefly, the sample solution was firstly centrifuged at 4℃ and 10,650 × g for 20 min, and then 1 mL of supernatant was fully mixed with 4 mL of biuret reagent and was incubated in darkness for 30 min. The absorbance of the mixture was determined at 540 nm. Standard bovine serum albumin (BAS) was utilized to draw a standard curve, according to which the solubility of the sample was calculated.

2.14 Preparation of the protein-anthocyanin complex

Firstly, 0.4 g UOVA-Dex 360 protein freeze-dried sample was dissolved in ultra-pure water to prepare the protein solution of 4 mg/mL, and then it was hydrated at 4℃ overnight. Anthocyanins (AC) of 0.8 g was weighed and dissolved in 200 mL ultra-pure water to obtain 4 mg/mL AC solution.

The hydrated protein solution was blended with the AC solution at a ratio of 1:1, and the pH of the mixture was adjusted to pH=2 ± 0.1 with 0.1 mol/L hydrochloric acid and 0.1 mol /L sodium hydroxide to get the UOVA-Dex 360-AC complex. Free AC was used as the control.

2.15 Thermal stability analysis

The thermal stability analysis for the protein-anthocyanins complex was completed according to Dong et al [24]. The freeze-dried samples (5–10 mg) were weighed and transferred onto a synchronous thermal analyzer (STA 449F3, Jupiter, Netzsch Scientific Instruments Trading Co., Germany) for the Thermal Gravimetric Analysis (TGA) and differential scanning calorimetry (DSC) analysis. The scan range was 45-350℃, the temperature rise rate was 10℃ min−1, and the flow dry nitrogen was 20 mL /min.

2.16 Processing and storage stability analysis

2.16.1 Light stability

The AC and UOVA-Dex 360-AC samples of 4 mL were added to the test tube, respectively, and the light stability of the sample was evaluated by irradiating them in front of the UV lamp (with a distance of 10 cm) at room temperature for different times (1 h ∼ 8h). The effect of light on the storage stability of UOVA-Dex 360-AC was assessed by observing the absorbance variations and the Folin-Ciocalteu method [27].

2.16.2 High-temperature processing stability

The AC and UOVA-Dex 360-AC solution of 4 mL was added to test tubes respectively, and the samples were heated in a water bath at 80℃ for different times (1 h to 5 h), and then quickly dipped into ice water to cool down to room temperature. The influence of temperature on the storage stability of UOVA-Dex 360-AC was evaluated by checking the absorbance change and the Folin-Ciocalteu method [27].

2.16.3 Stability of ascorbic acid

The ascorbate stability of the complex was determined based on the method mentioned by Zang et al [28]. The AC and UOVA-Dex 360-AC solution of 5 mL were added respectively into two serum bottles, and 0.75 mg vitamin C was added. The absorption variation of UOVA-Dex 360-AC was checked for 7 days during the storage at room temperature, and the AC was used as the control, to inspect the effect of ascorbic acid on the storage stability.

2.16.4 Storage stability

The storage stability of the complex was employed according to the method reported by Li et al [29]. In brief, AC and UOVA-Dex 360-AC of 5 mL were added individually into two serum bottles and stored at room temperature away from light for 7 days. Measurements were proceeded at 0/1st/3rd/5th/7th day, and the changes of free AC and UOVA-Dex 360-AC during the storage were determined by checking absorbance variations.

2.17 Statistical analysis

All experiments were repeated more than three times, and the results were calculated as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was employed and significant differences were determined at the significant level of p < 0.05. Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA, USA), DPS, and Origin (Version 8.0, Origin Lab, USA) were used for further data processing and charting.

3 Results and discussion

3.1 The effect of ultrasound on the glycosylation reaction of OVA-Dex conjugate

3.1.1 Effect of ultrasound on glycosylation intermediates and browning intensity

The degree of the Maillard reaction could be evaluated by the absorption values at 294 nm and 420 nm to assess the content of intermediate and late products, respectively [30]. As shown in Table 1, both the browning intensity of traditional water bath heating (Table 1A) and ultrasound-assisted glycosylation (Table 1B) of OVA was significantly higher than untreated OVA group (p < 0.05), indicating the formation of glycosylation conjugate. In addition, the results showed that the reaction degree of ultrasound assisted glycosylation treatment for 280 min and 360 min was comparable to that of traditional treatment for 24 h and 32 h, respectively. This was consistent with the results of Li et al., which suggested that the ultrasound-assisted glycosylation can greatly shorten the glycosylation time of traditional way [15].Table 1 Browning index and glycosylation intermediates for the OVA, OVA-Dex 8/16/24/32 h conjugates (A), and UOVA-Dex 40/120/200/280/360 min conjugates (B).

A		
Absorption wavelength (nm)	Traditional glycosation time in water bath (h)	
0	8	16	24	32		
294	2.483 ± 0.129c	2.617 ± 0.027 bc	2.651 ± 0.018 bc	2.811 ± 0.133 ab	2.991 ± 0.084 a		
420	0.117 ± 0.020 d	0.173 ± 0.013c	0.204 ± 0.018 bc	0.222 ± 0.006 ab	0.245 ± 0.013 a		
B	
Absorption wavelength (nm)	Ultrasound-assisted glycosation time (min)	
0	40	120	200	280	360	
294	2.483 ± 0.129 E	3.080 ± 0.036 D	3.371 ± 0.074C	3.570 ± 0.179 BCE	3.658 ± 0.197 AB	3.871 ± 0.148 A	
420	0.117 ± 0.020 D	0.160 ± 0.019C	0.186 ± 0.018 BCE	0.214 ± 0.021 AB	0.227 ± 0.011 A	0.247 ± 0.011 A	

In order to better explore the differences between glycosylation conjugates formed by the different methods, glycosylation period for the traditional way was optimized to 24 h in the follow-up experiment.

3.1.2 Effect of ultrasound glycosylation on the grafting degree of OVA-Dex

The degree of grafting is an important index to characterize the degree of glycosylation. Glycosylation products are mainly formed by covalent bond between the free amino group of protein and the carbonyl group of polysaccharide [31]. As illustrated in Fig. 1, the degree of grafting increased with the extension of ultrasound time. This is because during ultrasonic treatment, part of the protein is promoted to fold, the protein structure is gradually unfolded, and more active amino groups are exposed on its surface, resulting in increased grafting between protein and polysaccharide molecules [15]. At the same time, we also found that there was no significant difference between the grafting degree of conventional heating glycosylation for 24 h and that of ultrasound-assisted treatment for 40 min. As can be seen from Table 1, ultrasound-assisted treatment at 40 min may interfere with the formation of late MRPS-like melanoids by inhibiting the polymerization of intermediate products during the Maillard reaction. Therefore, ultrasonic treatment not only shortened the glycosylation time between WPI and GA, but also effectively inhibited the degree of product browning, which was consistent with the experimental conclusions of Chen et al [18].Fig. 1 The grafting degree for the conjugates of UOVA-Dex 40/120/200/280/360 obtained by ultrasound-assisted glycosylation, and OVA-Dex 24 obtained via traditional glycosylation.

3.2 Effect of ultrasound on physical properties of the glycosylated conjugate

3.2.1 Effect of ultrasound-assisted glycosylation on the solubility of OVA-Dex conjugate

Solubility plays a crucial role in determining the various functional properties of proteins, as it reflects the concentration of solids in a stable aqueous phase [32]. As shown in Fig. 2A, the solubility of proteins after the traditional heating glycosylation treatment was lower than that of other treatment groups, which might be due to the gradual formation of high molecular weight protein aggregates during the long-term heating treatment [33]. The solubility of the proteins after ultrasound-assisted glycosylation was significantly higher than that in OVA group, and the solubility gradually increased with the extension of reaction time, reaching the maximum solubility at 120 min, and then the extension of ultrasonic reaction time had no significant effect on the solubility. It may be because ultrasound has the ability to destroy any aggregated proteins, changing their structure and unfolding them, thus, the degree of glycosylation is higher, and the protein hydrophilicity gets enhanced, leading to the increase in the solubility [15].Fig. 2 Comparison for the solubility of OVA, OVA-Dex 24 conjugate, UOVA-Dex 40/120/200/280/360 conjugates (A) and the particle size distribution curve for the samples of OVA, OVA-Dex 24 conjugate, and UOVA-Dex 360 conjugate (B).

3.2.2 Effect of ultrasound-assisted on the particle size and polydispersion index (PDI) of glycosylated conjugate

Table 2 shows the particle size (size) and polydispersion index (PDI) of OVA and glycosylated conjugates. Results showed that OVA in the control group had the highest PDI value (0.96 ± 0.06), indicating that ovalbumin molecules were easy to aggregate to form larger particles [34]. The protein particle size and PDI of the conjugates decreased significantly after the treatment of ultrasound-assisted glycosylation or the traditional approach, which may be due to the protective effect of sugar on protein denaturation during heat treatment, thus reducing the aggregation of protein molecules [35]. In addition, the results also showed that the particle size and PDI of the conjugate after ultrasonic-assisted glycosylation treatment were significantly lower than those by traditional water bath heating treatment, and the minimum particle size (31.17 ± 0.36 nm) and PDI value (0.39 ± 0.01) were obtained after 360 min of ultrasonic treatment. It may be that ultrasound-induced cavitation and microbeam interactions break the chemical bonds between OVA molecules. Moreover, with the extension of ultrasonic action time, the cavitation destruction effect was more significant, and the protein splitted into smaller particles, resulting in the reduction of OVA particle size [36]. Fig. 2B shows the particle size distribution curve of the sample. Ovalbumin showed four-peak particle size distribution, OVA-Dex showed three-peak particle size distribution, and UOVA-Dex showed almost single-peak particle size distribution. Zhao et al. [37]found that PPI-X nanoemulsion with stable conjugations of ultrasonic sugar base exhibited unimodal particle size distribution, while NUPPI, a crude emulsion without ultrasonic homogenization, exhibited trimodal particle size distribution. It was basically consistent with our results, indicating that ultrasonic homogenization could form stable and uniform nanoemulsions.Table 2 The size and PDI value of OVA, OVA-Dex 24 conjugate, UOVA-Dex 40/120 /200/280/360 conjugate.

Samples	Particle size (nm)	PDI value	
OVA	84.47 ± 2.03a	0.96 ± 0.06a	
OVA-Dex 24	53.62 ± 2.94b	0.7 ± 0.02b	
UOVA-Dex 40	37.07 ± 0.89c	0.52 ± 0.02c	
UOVA-Dex 120	33.39 ± 0.7cd	0.45 ± 0.01cd	
UOVA-Dex 200	32.74 ± 0.8d	0.4 ± 0.01d	
UOVA-Dex 280	31.32 ± 0.22d	0.39 ± 0.01d	
UOVA-Dex 360	31.17 ± 0.36d	0.39 ± 0.01d	

3.2.3 Effect of ultrasound on crystal structure of glycosylated conjugate

XRD can provide three-dimensional crystal structures of conjugated compounds to investigate the changes in amorphous and crystalline structures. Fig. 3 shows the XRD patterns of OVA, Dex, OVA-Dex 24, UOVA-Dex 40, and UOVA-Dex 360. It exhibits that Dex has a certain crystal structure, OVA mainly presents amorphous, after the Maillard reaction, the obtained conjugates are amorphous, indicating that OVA and Dex have chemical reactions with each other [38]. In addition, compared with OVA, the glycosylation conjugate was more gentle, among which UOVA-Dex360 showed the highest smoothness, and UOVA-Dex 360 had the lowest crystallinity at this time (Table 3). It indicated that with the aid of ultrasound, the glycosylation reaction between OVA and Dex was more complete, which enhanced the fluidity of the protein chain and destroyed the crystalline phase by disrupting the arrangement of the protein chain [39]. This conclusion was consistent with SEM results. Relative results had also approved that protein conjugation with polysaccharide can reduce the degree of protein crystallization [26], [32], [38]. In addition, the profile on the right of Fig. 3 shows that the crystallization angle of the conjugate after glycosylation assisted by ultrasound gradually red-shifts as compared with traditional glycosylation, leading to a reduced crystallization degree. It indicated that the presence of ultrasound was somehow effective in promoting the glycosylation reaction.Fig. 3 The X-ray diffraction partten of OVA, Dex, OVA-Dex 24 conjugate, and UOVA-Dex 40/360 conjugates.

Table 3 The crystallinity of the OVA-Dex 24 conjugate, and UOVA-Dex 40/360 conjugates.

Sample name	Crystallinity (%)	
OVA-Dex 24	41.39	
UOVA-Dex 40	38.52	
UOVA-Dex 360	33.85	

3.2.4 Influence of ultrasound assistance on the apparent morphology of glycosylated conjugate carriers

The microstructure of OVA and different glycosylated conjugates was analyzed by SEM scanning (Fig. 4). As shown in Fig. 4A-B, the untreated protein had a spherical shape and a very smooth surface. However, after the traditional heating treatment for 24 h, as shown in Fig. 4C-D, the protein expanded from the original sphere into an irregular, loosely textured sheet structure, with a rough surface with many fine pores. Compared with traditional wet heating, ultrasound-assisted glycosylation produced denser and smoother structures, and this phenomenon became more obvious with the extension of reaction time (Fig. 4E-H). It may be because the ultrasound-assisted glycosylation could result in a more dense structure of protein fiber, thus the pore size got smaller, the pore edge was smoother, and the interconnection of the network structure was enhanced, leading to an improved functional properties of the protein. Both glycosylation treatments caused the protein structure to unfold and allowed the polymer molecules to diffuse outwards, facilitating polysaccharide aggregation and glycosylation on the protein surface [40]. Compared with the traditional glycosylation method, ultrasonic treatment could induce cavitation bubbles, which generate shear stress, making protein structure unfold and accelerating molecular movement, finally leading to a promotion of the covalent interaction between protein and polysaccharide [20].Fig. 4 The scanning electron microscopy (SEM) images for observing micro-structure variation of the OVA before and after different glycosylation processing. Micro-structures images for the OVA (A), OVA-Dex 24 (C), UOVA-Dex 40 (E), and UOVA-Dex 360 (G) under magnification of 10 K and the amplified images to 4 K for the corresponding samples of OVA (B), OVA-Dex 24 (D), UOVA-Dex 40 (F), and UOVA- Dex 360 (H).

3.3 Effect of ultrasound on the protein structure of conjugate carrier

3.3.1 Effect of ultrasound on primary structure of the protein in conjugate carrier

Fig. 5A-B shows the SDS-PAGE profiles of the samples obtained by different glycosylation conditions, and the two major bands produced by OVA appeared around 45 and 77 kDa, corresponding to ovalbumin and ovaltransferrin [14]. Fig. 5A exhibits that the bands for glycosylation conjugates of OVA-Dex and UOVA-Dex have obvious characteristics as compared with natural ovalbumin (OVA). The intensity of the bands became lighter and the top bands gradually deepen, which proved that OVA and dextran (Dex) generated covalent compounds with higher molecular weight through the glycosylation reaction. For the UOVA-Dex conjugate, the intensity of the characteristic band gradually became lighter with the extension of ultrasound-assisted glycosylation time, and the color of the regionalized band of 77–200 kDa was gradually deepened, and all bands in the figure show a gradual upward trend from left to right, indicating that the covalent coupling degree of ovalbumin and dextran gradually increased with the extension of reaction time. Chen et al. [18] also confirmed this opinion in their study, with the extension of ultrasound time, the intensity of α-La, β-Lg and BSA bands gradually decreased, while the visibility of bands near the top of the gel increased. At the same time, the results also shows that the band strength of UOVA-Dex 200 at the top of the gel was nearly consistent with the conventional grafting (Table 4), which indicated the ultrasonic treatments can significantly shorten the time of the glycosylation reaction. In addition, as shown in Fig. 5B, the strength of 45 and 77 kDa bands became shallow with the extension of traditional water bath glycosylation heating time, and the bands almost disappeared after 24 h of heating treatment, indicating that ovalbumin and ovaltransferritin are involved in the formation of OVA-Dex glycoconjugation [41]. Sheng et al. [42] also found that protein solution did not polymerize at a gentle heat temperature (60℃), and the OVA degraded and polymerized when heated in a water bath at 80℃. As the heating time increased, the band strength decreased, leading to the dimer, trimer, or remaining oligomers of ovalbumin migrated in the separation gel owing to the formation of intermolecular disulfide bonds.Fig. 5 SDS-PAGE profiles for inspecting the effect of (A) ultrasonic reaction time and (B) traditional glycosylation time on the variation in protein components of conjugated samples.

Table 4 The band strength at the top of the gel of OVA-Dex 24 conjugate, UOVA-Dex 40/120 /200/280/360 conjugate in the SDS.

Label	Band strength	
OVA-DEX 24	116,848	
UOVA-DEX 40	106,950	
UOVA-DEX 120	112,579	
UOVA-DEX 200	114,995	
UOVA-DEX 280	121,082	
UOVA-DEX 360	129,390	

3.3.2 Effect of ultrasound on secondary structure of the protein in conjugate carrier

The CD spectrum is a quite convenient way to inspect the secondary structural forming of the protein (α-helix, β-fold, β-turn, and random curl form) as well as folding and unfolding situations [32]. In the CD graph, the β-sheet has a negative peak near 217 nm, the α-helix has a negative peak near 222 nm and 208 nm, a positive peak near 190 nm, and the negative peak near 198 nm mainly reflects the displacement of the random coil [43]. After glycosylation, the negative far ultraviolet intensity is reduced compared to the original protein, and blue shift occurs simultaneously (Fig. 6A), which may be the result of partial unfolding, dimerization, refolding, and rearrangement of proteins that occur during hydrolysis and conjugation [26].Fig. 6 Protein secondary structural variation (A) and the Free sulfhydryl (R-SH) content (B) of OVA, OVA-Dex 24 conjugate, UOVA-Dex 40/120/200/280/360 conjugates through circular binary spectrumand.

Table 5 lists the prediction results of CDNN software for secondary structure content of various samples. After glycosylation, the α-helix of the conjugate decreased and the random curl increased, indicating that the molecular structure of the protein changed from an ordered state to a disordered state [32]. In the early stage of traditional water bath glycosylation and ultrasound-assisted glycosylation, α-helix significantly decreased, and β-fold and β-angle significantly increased, which may be because the structure of OVA gradually unspiral and fold during the heating process of glycosylation reaction [33]. With the extension of ultrasound time, α-helix gradually increased, β-fold and β-angle gradually decreased (p < 0.05). It was found that the internal thermal effect induced by high-intensity ultrasound for a long time in the glycosylation conjugate caused the protein particles to re-aggregate instead of remaining in the unfolded state, and its structure changed from disordered and flexible to ordered and rigid [44].Table 5 Prediction results for the secondary structure contents of OVA, OVA-Dex 24 conjugate, and UOVA-Dex 40/120/200/280/360 conjugates.

Samples	α-Helix (%)	β-Turn (%)	β-sheet (%)	Random. Coil (%)	
OVA	79.2 ± 1.69 a	2.22 ± 1.23b	2.21 ± 0.72 bc	9.75 ± 0.63 d	
OVA-Dex 24	47.79 ± 1.61 d	5.26 ± 0.10 a	10.62 ± 0.66 a	20.50 ± 1.10 a	
UOVA-Dex 40	50.26 ± 4.51 d	5.90 ± 0.15 a	7.93 ± 1.37 a	19.25 ± 3.71 ab	
UOVA-Dex 120	61.86 ± 0.99c	3.48 ± 0.71 ab	4.65 ± 0.14b	19.07 ± 0.42 ab	
UOVA-Dex 200	74.65 ± 1.06b	1.40 ± 0.28b	1.94 ± 0.42 bc	16.83 ± 0.28 bc	
UOVA-Dex 280	77.81 ± 1.81 a	1.11 ± 0.35b	1.37 ± 0.17c	15.77 ± 0.47c	
UOVA-Dex 360	78.28 ± 2.00 a	1.10 ± 0.31b	1.36 ± 0.27c	15.42 ± 0.61c	

3.3.3 Influence of ultrasound assistance on tertiary structure of the protein in conjugate carrier

In most cases, the sulfhydryl group (SH) is enclosed in a compact protein, and the content of free sulfhydryl group is an important index that can reflect the change of the tertiary structure of the protein [33]. As shown in Fig. 6B, the content of free −SH in the samples either treated by traditional water bath or assisted by ultrasound was significantly lower than that in control group OVA (p < 0.05). Among them, the degree of glycosylation in traditional water bath for 24 h was similar to that of ultrasonic-assisted treatment for 120 min, while the amount of free −SH gradually decreased with the extension of ultrasonic time. It may be because the high-intensity ultrasonic-assisted treatment may generate free radicals through the cavitation effect, resulting in the oxidation of exposed free −SH groups to disulfide bonds [45]. In addition, the free −SH group on the protein surface readily reacts with the Schiff base and Amatori rearrangement products produced by glycosylation, further reducing the free −SH group [17].

3.4 Influence of ultrasound on the interactions between protein and polysaccharide in glycosylated conjugate carriers

3.4.1 FTIR

FTIR is an effective method to analyze protein-polysaccharide interaction and structural changes at molecular level. Fig. 7A shows the FTIR spectra of OVA, Dex, OVA-Dex, and UOVA-Dex. Compared with the OVA, the wavelength of the conjugate was blue-shifted at 2951.03 cm−1 after glycosylation, which was caused by the C-H stretching vibration promoted by the introduction of CH3 and CH2 groups in the polysaccharide chain [46]. The infrared spectrum of the protein consists of three characteristic absorption bands, of which the band at 1600–1700 cm−1 corresponds to the N-H-curved amide I band [19]. It can be seen from the figure that the wavelength of OVA-Dex and UOVA-Dex is blue shifted at amide Ⅰ, because the Maillard reaction consumes some functional groups, such as NH2, especially from lysine, and may produce some new substances such as Schiff base (C=N) and pyrizine (CN) [14]. At wavelength 1064.68 cm−1, the glycosylation conjugate produced a new peak, indicating that Dex was covalently connected to OVA mainly by C-N glycosylation bonds. Compared with Dex, the characteristic peaks of the conjugate all moved to short wave, indicating that the formed C-N covalent bonds experienced strong tensile vibration in the conjugated compounds [19]. In addition, compared with traditional water bath glycosylation, the shift of characteristic peaks of ultrasonic-assisted glycosylation was more obvious, indicating that the absorption of ultrasonic treatment group was stronger than that of wet heating group, and ultrasonic treatment promoted the production of glycosylation products, the change of protein structure and the exposure of more hydrophobic groups [15].Fig. 7 The Fourier transform infrared spectroscopy graft (A) and the intrinsic fluorescence intensities (B) of OVA、OVA-Dex 24 conjugate、UOVA-Dex 40/120/200/280/360 conjugate.

3.4.2 INF

INF of proteins can be used to evaluate the conformational changes of hydrophobic tryptophan, tyrosine and phenylalanine groups in local molecular environments [32]. As shown in Fig. 7B, the fluorescence intensity of untreated ovalbumin was the strongest, followed by that of ultrasound-assisted glycosylation modification, and that of traditional water bath heating was the weakest. This is because the covalent reaction between dextran and protein blocks the fluorescence signal of amino acid residues, resulting in fluorescence quenching of hydrophobic amino acid residues [47]. The fluorescence intensity increased gradually with the extension of ultrasonic time. The increase in fluorescence intensity of the conjugates may be caused by structural changes in the proteins promoted by ultrasound resulting in an increase in the number of chromophores on the surface [32]. In addition, for the unprocessed protein OVA, λ max was about 333 nm, and the maximum wavelength of the protein after traditional treatment and ultrasound assisted treatment showed a red-shift phenomenon, and the red-shift phenomenon became more obvious with the extension of ultrasound time. This indicated that the Maillard reaction reduces the hydrophobicity of the environment in which the tryptophan residues reside, and the polarity in the environment increases [40].

3.5 Homeostasis evaluation for the glycosylated conjugate carrier of AC

3.5.1 Effects of glycosylated UOVA-Dex carrier on the thermal stability of anthocyanins

TGA and DSC measurements were employed to investigate and compare the thermal properties of the UOVA-Dex 360-AC complex and free AC [48]. The results of mass loss (TGA) derivative thermogravimetric (DTG) for AC and UOVA-Dex 360-AC are shown in Fig. 8A and B. At the same temperature, we noted that the smaller the weight loss of the sample, the higher the thermal stability. Initially, only a slight mass loss of 6.1 % was observed for anthocyanins in the temperature range of 50-125℃, which was due to the desorption of bound water and small molecular water components on the sample. However, in the subsequent stage (180-270℃), the weight loss rate increased significantly with a mass loss of 28.2 %, and in the final stage (270-350℃) there was a sharp mass loss with a mass loss of 49.1 %, which could be attributed to two processes. It included the evaporation of physically and chemically bound water and the ring-opening reaction and deglycosylation decomposition of anthocyanins, resulting in the production of phenolic acids and aldehydes at elevated temperatures [29]. Compared with free AC, UOVA-Dex 360-AC mainly presented two stages, the first stage is near 50-125℃, with similar endothermic characteristics, and its weight loss was due to the desorption of bound water and small molecular water components on the sample. Besides, UOVA-Dex 360-AC had a smaller mass loss of only 2.1 %. DTG results also showed that the second weight loss peak of UOVA-Dex 360-AC hardly changed. These changes may be caused by the non-covalent intermolecular interaction between proteins and anthocyanins, which enhanced the binding between the complex and water, while the presence of more hydrophilic groups of anthocyanins could increase the binding ability of water molecules to the binary polymer network. Thereby increasing the content of bound water and reducing the loss of water. In the second stage (180-350℃), the mass loss is 47.9 %, due to the degradation of protein structure and subsequent volatilization, anthocyanins are gradually exposed and also begin to degrade [49]. Compared with free AC, the mass loss rate and maximum weight loss rate of UOVA-Dex 360-AC are greatly reduced, indicating that the composite system has good thermal stability for anthocyanins.Fig. 8 Thermal properties analysis for AC and UOVA-Dex360-AC conjugate (A) thermogravimetric analysis (TGA), (B) Derivative Thermogravimetry (DTG) and (C) differential scanning calorimetric (DSC).

DSC is able to confirm the formation and thermal degradation of complexes because the dissociation and thermal degradation of complexes during heating leads to the occurrence of endothermic peaks [50]. The DSC analysis results of AC and UOVA-Dex360-AC (Fig. 8C) show that both of them have an endothermic peak near 50-125℃. Compared with AC, the endothermic peak of the complex was smaller, which also indicated that the water loss was less. In addition, the peak value of the second endothermic peak of AC was 213℃, and that of UOVA-Dex 360-AC was 226℃, which may be explained by the interaction between anthocyanins and proteins to form a dense structure, resulting in higher endothermic peak value. However, near 300℃, UOVA-Dex 360-AC showed a small exothermic peak, which may be due to the fact that the protein began to melt and REDOX reaction occurred [49].

3.5.2 Effect of glycosylated complex carrier on the processing and storage stability of AC

Anthocyanins are easily affected by the external environment and degrade easily under different processing and storage conditions, among which temperature and light are most significant factors.

As shown in Fig. 9A, the absorbance of AC changed significantly, and anthocyanins gradually deepen from red to brown after heating process, as compared to the UOVA-Dex 360-AC. Under the processing of AC samples at 80℃, thermal degradation involves the formation of a pseudobase of colorless methanol and the subsequent opening of the pyran ring to form chalcones, which are finally converted into brown degradation products [51]. The absorbance of UOVA-Dex 360-AC was almost unchanged, because a dense network structure dominated by hydrogen bonds was formed between AC and the glycosylation complex. It required more energy to destroy this structure, so the color change of UOVA-Dex 360-AC was not obvious [52]. At the same time, the degradation of anthocyanins was also accompanied by the change of polyphenol content, and it can be found from Fig. 9B that the polyphenol content in AC was reduced substantially.Fig. 9 Effect of different time under ultraviolet lamp irradiationon storage stability of UOVA-Dex360 (A) and free AC (B); Effect of different treatment time at 80℃ on storage stability of UOVA-Dex 360 (C) and free AC (D); Effect of different time under ascorbic acid storage stability of UOVA-Dex 360-AC and free AC (E); Effect of different time under storage stability of UOVA-Dex 360-AC and free AC (F).

After light treatment, analogous results were obtained (Fig. 9C-D). Compared with UOva-Dex 360-AC, the absorbance of AC changed significantly. The final product of anthocyanin degradation induced by light irradiation is the same as that by thermal, but the degradation mechanism mainly came from the excitation of flavonoid cations [28], and the degradation of anthocyanin under light would eventually produce colorless or brown substances [53]. Protein and anthocyanins could co-pigmentation reaction, and the interaction could reduce the influence of light on anthocyanins and improve the stability of anthocyanins [43].

In addition, the presence of ascorbic acid is a common means in the food industry to prevent food spoilage and increase flavor, especially in fruit juices [28]. It is inevitable that ascorbic acid will affect the structure of anthocyanins and lead to changes in the functional properties of anthocyanins. Fig. 9E shows that in the presence of ascorbic acid, both of the AC and UOVA-Dex 360-AC samples show different degrees of fading, but the degree of influence of protein-bound anthocyanins is significantly lower than that of free anthocyanins. This change may be due to the combination of ascorbic acid and anthocyanin during storage, which is divided into three steps, first a condensation reaction between corbic acid and anthocyanins, then ascorbic acid produces free radicals to break down the pyridine ring of anthocyanins, and finally ascorbic acid into hydrogen peroxide and dehydroascorbic acid [52]. Ren et al. [54] also found in the experiment that the addition of WP significantly reduced the absorbance loss of anthocyanin (ACN) during storage, which may be because ACN has a stronger binding affinity with WP than AA through hydrophobic interaction and hydrogen binding.

Storage stability is one of the necessary conditions to judge the quality of food, so it is very important to study its influence on anthocyanins and their compounds. In this experiment, glycosylated protein-loaded AC was kept in dark at room temperature and observed for 7 days (Fig. 9F). Compared with free AC, the progressive trend of UOVA-Dex 360-AC was significantly lower than that of free AC. It may be that during long-term storage, the presence of protein can bind with AC to form a dense network structure, protecting them from hydration, oxidation and polymerization [55], [56].

4 Conclusion

This study showed that the time of glycosylation between OVA and Dex could be greatly shortened by ultrasound. Compared with traditional heating, the degree of grafting in the glycosylated complex by ultrasound was higher, the degree of browning was significantly reduced, and the number of free sulfhydryl groups was correspondingly reduced. Besides, the solubility was significantly increased, and the particle size was smaller and uniform. With extension for the reaction time of ultrasound-assisted glycosylation, the structure of the protein changed, resulting in the increase of the number of chromophore on the surface and redshift occurred as well. Compared with the original OVA, the structure of the glycosylated protein opened up and became loose. The SDS-PAGE diagram showed that the reaction of OVA and Dex generated covalent compounds with higher molecular weight. The protein structure of ultrasound-assisted treatment was more dense and smooth. In addition, the glycosylated conjugate improved the stability of AC after the complexing.

In summary, ultrasound-assisted glycosylation can not only significantly shorten the reaction time and improve the reaction degree between protein and polysaccharide, but also improve the functional properties of the modified protein. At the same time, the complex formed with ultrasound assisted glycosylation as the carrier can improve the stability of anthocyanins and enhance the application commercial value of anthocyanins. It has certain reference value in developing nutritional health products or functional foods.

CRediT authorship contribution statement

Boyu Chen: Writing – original draft, Validation. Lei Chen: Writing – original draft, Validation. Chen Li: Methodology, Conceptualization. Wanhuan Huang: Data curation. Yanan Zhao: Writing – review & editing. Chao Ai: Writing – review & editing. Hui Teng: Supervision, 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.

Acknowledgement

This work is supported by the 10.13039/501100001809 National Natural Science Foundation of China (NSFC, Grant No. 32272315 , 32372335 ), the 10.13039/501100003453 Natural Science Foundation of Guangdong Province (2022A1515010694 ), the Key Field Special Project of High Education of Guangdong Province (2023ZDZX2026 )
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