Fabrication of high-preformance emulsifier from conjugating maltodextrin onto myofibrillar protein peptide with microwave- ultrasound synergy

Graphical abstract

Highlights

• •Novel emulsifying agents based on MP peptide (MPP) and maltodextrin are created.
• •Synergism of ultrasound and microwave greatly improves the glycation efficiency.
• •Single and combined ultrasound-induced glycation enhance emulsifying ability of MP.
• •MPP modified by UM-assisted conjugation produces the most stable emulsion product.

Abstract

In this study, we systematically investigated the emulsifying capabilities of myofibrillar protein (MP)- and MP peptide (MPP)-based conjugates synthesized through intensification techniques: water bath (WB), microwave, ultrasound, and the combined ultrasound-microwave (UM) methods. Compared with WB, microwave, and ultrasound treatments, the combined UM treatment greatly promoted the glycation reaction because ultrasound and microwave mutually reinforced modification effects. The resultant conjugate structure tended to unfold with more flexible conformation and homogeneous morphology. Moreover, the emulsifying properties of conjugates developed with single and combined ultrasound-assisted glycation displayed substantial improvement, and pre-hydrolysis further enhanced these performances, as observed in the Principal Component Analysis as well. Remarkably, MPP grafted by maltodextrin with the assistance of a combined UM field produced the smallest and most uniform emulsion system, positioning it as the most efficient emulsifier among all the fabricated glycoconjugates. Our study highlighted the potential of synergistically applying ultrasound and microwave techniques to develop a well-performance glycation with an ideal conjugate structure, in which they would be associated into a strong film that provided the robust physical barrier, creaming stability, heat retention, and oxidation resistance. These findings offered a basis for better utilizing complex ultrasonic technology to develop novel and improved MP-based food products.

1. Introduction

In the domain of food technology, oil-in-water (O/W) emulsions constitute a critical component in an array of products such as sauces, ice cream, beverages, and desserts. Nonetheless, the emulsion-based systems exhibit inherent instability during processing, storage, and consumption [1], manifesting in phenomena like flocculation, creaming, coalescence, and Ostwald ripening, which collectively cause damage to the integrity of the dispersed system and culminate in macroscopic phase separation [2]. These instabilities can be amplified by some chemical instability effects concurrently, including oxidation of oil droplets and decomposition of emulsifiers [3]. The related changes in emulsification dramatically reduce the application values of emulsions in modern food industry. Therefore, the incorporation of an appropriate emulsifying agent becomes imperative for the development of homogeneous and stable emulsion products.

Recently, “natural” emulsifiers, such as proteins, polysaccharides, and phospholipids, have increasingly attracted consumer interest [4]. In particular, myofibrillar protein (MP), one of the most important protein sources, is always used as a low-cost material to fabricate emulsion-type meat products [4]. Unfortunately, the complex structure and inaccessible hydrophobic components limit its solubility and emulsifying behavior [5], [6]. Hydrolysis processes facilitate the cleavage of proteins into peptides with shorter peptide chains, thereby enriching the availability of both hydrophilic and hydrophobic amino acids [7], [8], which contributes to better surface activity. Meanwhile, hydrolyzing greatly enhances the capabilities of radical scavenging and metal ion sequestering, and the resulting peptide can be beneficial for delaying lipid oxidation in edible emulsion systems [9].

However, the application of peptides as emulsifiers presents challenges in terms of long-term stability due to weak creaming resistance and poor thermal stability [10]. To address this limitation, emerging research has validated the efficacy of covalent peptide–polysaccharide conjugates, synthesized via glycation techniques, as innovative emulsifying agents capable of sustaining long-term emulsion stability [11]. The polysaccharide moiety grafted onto the peptide amplifies the hydrophilic characteristics of the latter and modulates its molecular amphiphilicity, circumventing the constraints imposed by the simple peptide structure [12]. Notably, it has been proved that covalent protein complexes displayed stronger heat, pH and metals ion stability than that of non-covalent complexes, which is more suitable for the application in complex food system. Furthermore, the incorporated carbohydrate is conducive to constructing a viscoelastic interfacial layer during emulsification, wherein the outwardly oriented polysaccharide chains may expose steric hindrance and increase the electrostatic repulsive effect, mitigating the propensity for droplet aggregation [13]. In addition, glycation reaction can also improve other functional performances at the interface, such as antioxidant and antiaging [14], in which the glycoconjugates of whey protein hydrolysates and linear dextrin could dramatically reduce lipid oxidation of O/W emulsions. The pH and storage stability of zein peptide-based emulsion was greatly increased while conjugating with maltodextrin (MD) [15].

Conventional methodologies for achieving peptide-polysaccharide glycation are time-consuming, necessitating several weeks for dry-heating or tens of hours for wet-heating [16], which poses a significant constraint on the widespread adoption of this technology within the food industry. Microwave irradiation, however, offers a promising avenue for accelerating these reactions [17]. The electromagnetic effects generated by microwave fields have the capability to modulate the spatial orientation of charged groups, subsequently enhancing the reaction kinetics between polypeptides and polysaccharides [18]. By contrast, the microwave process is hard to control, and it is mainly associated with two problems: firstly, localized overheating may occur in substrates with elevated microwave energy absorption capacities; secondly, uncontrolled oxidation reactions accelerate the destabilization of proteins [19]. Hence, there is a pressing need for the development of a methodology that can avoid these adverse effects inherent in standalone microwave-based approaches while concurrently amplifying glycation efficiency.

Recent investigations have verified that glycated proteins display enhanced functional characteristics when ultrasound techniques are incorporated into the glycation process [16]. Ultrasonic irradiation leads to cavitation collapse of bubbles in liquid medium, which is induced by the impact of micro-jetting on the surface of matter and collisions between high-speed particles. Besides, ultrasonic energy can be transmitted to biomacromolecule granules through the cavitation, following the formation, growth and collapse of microbubbles. Those process result in instantaneously high temperatures (5,000 K), pressures (100 MPa), and high-intensity shear field in the cavitation zone [20]. These effects provoke changes in molecular structure through the rearrangement of both intra- and inter-molecular forces, increasing the chances of collision between carbohydrate and active sites in protein [21]. It is noteworthy that while the molecular regulatory mechanisms of microwave and ultrasound treatments are somewhat distinct, their synergistic applications have been observed in multiple contexts [22], [23]. Our previous study has also proved that electromagnetic and mechanical waves can synergistically modulate the structural properties of MP, thereby improving their physicochemical characteristics [24]. As the structural properties are closely correlated with functionalities, it is assumed that combined ultrasound-microwave (UM) treatment was more effective in glycated-type modification than single ultrasound or microwave treatment. However, there is little information regarding the effects and mechanisms of combined UM treatment on the glycation process, and how pre-hydrolysis influences single and combined ultrasound-assisted glycated modification on structural properties and emulsifying behavior remains unknown.

In the present study, we proposed glycated MP with MD as a suitable emulsifying and stabilizing agent for developing stable emulsion systems. MP peptides (MPP) were prepared by controlled hydrolysis of trypsin. The degree of grafting (DG) of various conjugates with the assistance of ultrasound, microwave, and the combined UM treatments were compared. According to the determination of the structural changes, morphology, and aggregation behavior, how they influenced the extent of the glycation was clearly illustrated. Moreover, the emulsifying properties of different glycoconjugates were investigated basing on the partical size evolution of the resulting emulsion as well as their creaming stability. In addition, the influences of environmental stress (temperature and oxidation) on different MP-based emulsions were evaluated. The insights garnered from this investigation have the potential to illuminate novel avenues for the high-value utilization of MP derivatives, endowed with enhanced functional properties.

2. Materials and methods

2.1. Materials

Fresh golden threadfin bream (1–1.2 g) was procured from Haixin Foods Co. Ltd. (Fujian, China). The scales, viscera, skin, and bone were removed, and fat was trimmed away, then the muscle was sliced into 20-mm-thick chops. These samples were stored at −80 °C and utilized within a two-day timeframe. Commercial-grade corn oil was sourced from a local retail outlet. O-phthalaldehyde (OPA), 1-anilino-8-naphtalene-sulfonate (ANS), and lyophilized porcine trypsin with an enzymatic activity of 800 U/mg was acquired from Sigma-Aldrich (Shanghai, China). MD with a dextrose equivalent (DE) of 19 with average molecular weight (M_w) of ∼ 8.8 kDa was provided from Hui Yang Biological Technology Co. (Jinan, China). All other chemical reagents employed in this research were of analytical grade. Deionized water, generated via a Milli-Q water purification system (Millipore Co., USA), served as the solvent for all solution preparations.

2.2. MP extraction

Muscle samples were solubilized using six volumes of Solution A (0.1 M NaCl, 2 mM MgCl₂, 1 mM EDTA, and 0.02 % sodium azide (NaN₃)) and maintained at a pH of 7.0. The samples were subsequently subjected to homogenization utilizing a PRO-12S blender (German Pool Ltd., Hong Kong, China) operated at 11,000 rpm for a duration of 5 min. Post-homogenization, the mixture was centrifuged at a force of 2000 × g and a temperature of 4 °C for 15 min, employing an Avanti J-E centrifuge (Beckman Coulter, Brea, CA, USA). The sediment was isolated and subjected to three sequential washes using Solution A. The resultant pellet was redissolved in 2.5 volumes of Solution B (0.1 M NaCl) and re-homogenized at 8000 rpm for 2 min. A subsequent centrifugation step at 3000 × g for 10 min was employed. This purification cycle was repeated three times to effectively eliminate impurities, yielding pure MP product. Protein concentration was quantified employing the Biuret assay, utilizing bovine serum albumin (BSA) as the calibration standard. The final MP extract was stored at a temperature of 4 °C and was used within a 6-hour timeframe.

2.3. MPP preparation

MP samples were prepared as a 1 % (w/v) dispersion through gentle agitation and subsequently hydrated for an overnight period. Prior to the initiation of hydrolysis, the MP dispersion was thermally equilibrated to 37 °C, and its pH was normalized to 7.0 via the addition of 1 M sodium hydroxide. To obtain MPP, the trypsin was introduced to the dispersion at an enzyme-to-substrate ratio of 20 μg per 100 mL of protein dispersion. The extent of hydrolysis was quantified through the pH-stat methodology, conforming to the protocol delineated by Lee & Hur [25].

The change in degree of hydrolysis (DH) with time was illustrated in Fig. 1. A specified DH could be achieved by modulating the duration of the hydrolysis process. Hydrolysis time corresponding to a DH of 5 % was empirically determined to be 13 min 50 sec. To ensure the precision of the hydrolytic procedure, the experiment was replicated three times, yielding a standard deviation of less than ± 0.2 %. After hydrolysis, the enzyme was deactivated via heat treatment (80 °C, 15 min). The resultant samples were lyophilized over a 48-hour period before utilization.

Fig. 1.

The relation between degree of hydrolysis with hydrolysis time (min).

2.4. Preparation of conjugate samples

2.4.1. Ultrasound-assisted glycation

Dispersions comprising 1 % (w/v) MP and MPP were formulated, and their pH was equilibrated to 7.5 by 1 M HCl and 1 M NaOH. These suspensions were mixed with a 2 % (w/v) MD solution and subsequently subjected to thorough dispersion via magnetic stirring at 4 °C overnight. The blend was then introduced into a four-necked flask and equilibrated in water at 25 °C for 25 min. An XH300B UM reactor (Beijing Xianghu Science and Technology Development Co. Ltd., China), outfitted with a cooling-water circulation system, was employed to carry out the glycation. The process parameters were regulated by a UM station (Model OSR-8, Fiso, Canada). Ultrasound exposure was executed at a frequency of 28 kHz and power of 200 W until the internal medium reached a temperature of 75 °C. This temperature was then sustained for an additional 25-minute period within the ultrasound field. A temperature variance of ± 1.5 °C was permissible during the entire operation. Each sample was subjected to rapid cooling via immersion in an ice bath, and then a dialysis bag with a molecular weight cut-off of 10 kDa was used to remove any unbound MD for 96 h at 4 °C. After that the conjugates were lyophilized and stored for further analysis. The MP and MPP samples obtained under ultrasonic assistance were respectively termed U-MP conjugate and U-MPP conjugate, respectively.

2.4.2. Microwave-assisted glycation

Microwave-assisted glycation was conducted in a manner analogous to that of the ultrasound-assisted samples, except the mixture was microwaved at 2.45 kHz at 100 W until the temperature reached 75 °C, and subsequently maintained at 75 °C for 25 min under a microwave field. The ultrasonic probe was switched off during the microwave-treated procedure.. The MP and MPP samples obtained with microwave assistance were labeled as M-MP conjugate and M-MPP conjugate, respectively.

2.4.3. Um-assisted glycation

Both microwave and ultrasound sources were applied in this process. The frequencies of the ultrasound and microwave were set to 28 kHz and 2.45 kHz, respectively. Combined UM-assisted glycation was performed as described above for ultrasound-assisted and microwave-assisted samples, except the mixture was treated with 100 W microwave power coupled with 200 W ultrasonic power until the temperature was 75 °C, and subsequently maintained at 75 °C for 25 min under the combined UM field. Those samples are referred to as UM-MP and UM-MPP, respectively. The same MP-MD and MPP-MD mixtures were treated by WB alone at 75 °C for 12 h as the control groups, which are referred to as W-MP and W-MPP, respectively.

2.5. Characterization of prepared conjugates

2.5.1. Measurement of DG

The peptide content in the conjugate samples was quantified employing the OPA method, as delineated by Wen et al. [26]. A 2 mL of the conjugate sample with a concentration of 2 mg/mL was mixed with 4 mL of the prepared OPA reagent. Distilled water was employed as the blank control for the procedure. The absorbance of the resulting mixture was acquired at a wavelength of 340 nm utilizing a Lambda 20 spectrophotometer (PerkinElmer, USA). The DG was calculated through the following formula:

$$\text{DG}\left(\%\right)=(A_0-A_t)/A_0\times 100\%$$

where A_t and A₀ were the absorbance values of the conjugate and untreated protein, respectively.

2.5.2. Fourier transform infrared spectroscopy (FTIR) analysis

One milligram of lyophilized conjugate was homogeneously blended with 100 mg of potassium bromide (KBr) powder, and the composite material was subsequently compacted into a pellet utilizing a mortar and pestle. FTIR analysis was performed on the pellet across a spectral range of 4000–400 cm⁻¹ using a Thermo Nicolet 5700 FTIR spectrometer (Thermo Electron, Madison, WI, USA). Alterations in the infrared spectra were analysed using an OMNIC software (Thermo Electric Corporation, Chicago, IL, USA)..

2.5.3. Determination of the secondary structure

The secondary structures of conjugate samples were analysed in accordance with previously established protocols [25]. Diluted samples (50 μg/mL) were transferred into a 0.1-cm path-length quartz cell, and a spectropolarimeter (Chirascan, Applied Photophysics Ltd., Leatherhead, UK) was chosen to measure the molecular ellipticity at wavelengths ranging from 200 to 260 nm with a scan rate of 100 nm/min. The secondary structure composition of MPP was determined using the CDNN software (Applied Photophysics Ltd., UK).

2.5.4. Determination of the surface hydrophobicity

The surface hydrophobicity of the conjugates was evaluated utilizing ANS according to a methodology previously delineated by Chen et al. [27], albeit with certain modifications. A 20-μL aliquot of 15 mM ANS solution at pH 7.0 was combined with 4 mL of sample solution at a concentration (1 mg/mL). Following a 20-minute incubation period at ambient temperature, fluorescence characteristics were quantified using a fluorescence spectrophotometer (F-7000; Hitachi Corp., Japan). The excitation wavelength was preset to 380 nm, and emission spectra were documented within the wavelength range of 410–570 nm. Surface hydrophobicity (H₀) was subsequently computed as the slope of fluorescence intensity as a function of protein concentration.

2.5.5. Determination of the reactive sulfhydryl groups (R–SH)

The R–SH of the conjugate samples was quantitatively evaluated in accordance with the procedure delineated by Wang et al. [28] with minor modifications. Each sample was diluted to a concentration of 1 mg/mL and subsequently mixed with 20 μL of a 10 mM 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB) solution at pH 7.0. Following a 20-minute incubation period, the absorbance of the resultant mixtures was quantified at a wavelength of 412 nm using a Lambda 20 spectrophotometer (PerkinElmer). The associated R–SH content was computed utilizing the molar extinction coefficient (EM = 13,600).

2.5.6. Fluorescence spectrometry analysis

The fluorescence spectra of the samples were acquired in alignment with the methodology delineated by Ma et al. [16] with some modifications. Each protein and conjugate was dissolved to a concentration of 0.2 mg/mL. The fluorescence spectra was recorded using a fluorescence spectrophotometer (Model Cary Eclipse, Varian Inc., Palo Alto, CA, USA). The excitation wavelength was set as 280 nm with an emission wavelength of 300–450 nm.

2.5.7. Determination of physicochemical properties

The solubility of conjugates was quantified utilizing the Biuret assay, in which the samples were standardized to a protein concentration of 0.4 % (w/v), and the pH was adjusted to 7.5. Subsequent to centrifugation at 4000 × g for 15 min at 4 °C, the supernatant was isolated. A 200 μL aliquot of this supernatant was then mixed with 1 mL of Biuret reagent and incubated for one hour. Spectrophotometric measurements were conducted at an absorbance wavelength of 540 nm using a Lambda 20 spectrophotometer (PerkinElmer). The BSA was used to develop standard curve for protein concentration determination.

The conjugate containing solutions were transferred into a 1-cm path-length quartz cuvette. A Zetasizer Nano ZS 90 molecular size analyzer (Malvern Instruments Ltd., Great Malvern, UK), outfitted with a 4 mW He–Ne ion laser (λ = 633 nm), was employed to ascertain both particle size and zeta potential.

2.5.8. Scanning electron microscopy (SEM)

A 5 μL diluted sample (10 μg/mL) was adhered to a conductive paste and fixed with 1 % osmium tetroxide gas. The apparent morphology of the specimen was recorded at 15.0 kV using a scanning electron microscope (JSM-6360LV, JEOL, Tokyo, Japan) after gold spraying under a vacuum. Micrographs (100,000 × ) were obtained for subsequent analysis.

2.6. Preparation of oil-in-water emulsions

A homogenous mixture was prepared by combining the dispersion (containing 0.4 wt% conjugate) with corn oil in a volumetric ratio of 4:1. To inhibit microbial growth, 0.1 % NaN₃ was incorporated into the solution. Subsequent emulsification was carried out for 3 min utilizing an UltraTurrax blender (KAT18, IKA-Werke GmbH & Co. KG, Germany) operating at a speed of 15,000 rpm. Following homogenization, a 5-mL aliquot of the prepared emulsion was transferred into a glass vial, which was sealed using a black plastic cap.

2.7. Emulsification properties evaluation

2.7.1. Measurement of average size of emulsion droplets

A Mastersizer 3500 (Microtrac Instruments, USA) was employed to record the particle size distribution and the average size of the emulsion according to the method described by Hou et al.[29]. Emulsions were transferred to glass bottles and incubated at 4 °C for 0, 5, 10, and 20 days. After storage, the test emulsion droplets were diluted 10 times. The refractive index of droplet particles was set to 1.520 and the continuous phase refractive index was selected as 1.333. The emulsion sample was equilibrated for 2 min inside the static light scattering with a laser obscuration level of 10 %. The changes in volume weighted mean diameter d_{4, 3} was used to reflect the evolution of average droplet size on storage, which was calculated as follows:

$$d_{4,3}=\mathrm{\Sigma }{n}_i{d}_i^4/\mathrm{\Sigma }{n}_i{d}_i^3,$$

where n_i was the number of droplets with diameter d_i.

2.7.2. Determination of creaming stability of emulsion droplets

The creaming stability of emulsions was assessed utilizing a LUMiSizer centrifugal analyzer (L.U.M. GmbH, Berlin, Germany). This device substantially accelerated the rate of phase separation under a high gravitational field. Experimental parameters were configured as follows: centrifugal force was maintained at a rotational speed of 3500 × g, the instrumental temperature was stabilized at 4 °C, and the time intervals for measurements were set at 30-second increments.

2.7.3. Stability against different thermal stresses

All the MP-based emulsions were transferred into glass containers and subjected to incubation at three distinct temperatures: 4 °C for 4 h, 60 °C for 4 h, and 85 °C for 1 h. Subsequent to these thermal treatments, the changes in the average droplet size (d_{4, 3}​) were quantitatively measured utilizing a Mastersizer.

2.7.4. Stability against different oxidative stress

The oxidative stability of emulsions was quantitatively assessed through the measurement of 2-thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides, in accordance with a slightly modified methodology previously delineated by Pan et al.[14].

As for the TBARSs analysis, one milliliter of emulsion sample was blended with 2 mL TBA reagent (15 % (w/v) trichloroacetic acid, 0.375 % thiobarbituric acid, and 0.25 M HCl) and boiled for 25 min. Following this thermal treatment, the sample was rapidly cooled to a temperature of 25 °C using an ice bath. Centrifugation was performed at 500 × g for 15 min to isolate the supernatant. The optical absorbance of the supernatant was quantified spectrophotometrically at a wavelength of 532 nm. 1,1,3,3-Tetraethoxypropane was chosen as a reference product for a standard curve for calculating the TBARS concentration.

In terms of lipid hydroperoxides analysis, a 1.5 mL isooctane/2-propanol mixture (3:1, v:v) was mixed with 0.3 mL of the emulsion. This blend was vortexed and then subjected to centrifugation at 3000 × g for 5 min. A 0.4 mL sample of the resultant upper phase was extracted and mixed with a 14-fold volume of a methanol/1-butanol mixture (2:1, v:v). Afterwards, 100 μL of a ferrous solution (an equimolar blend of 0.144 M FeSO₄, 0.1 M HCl, and 0.132 M BaCl₂) and 100 μL of 3.94 M ammonium thiocyanate were added. The resulting solution was allowed to react for 20 min, after which its optical absorbance was quantified at a wavelength of 510 nm using a Lambda 20 spectrophotometer (PerkinElmer). A standard curve was established using cumene hydroperoxide as the reference compound.

2.8. Statistical analysis

All experimental procedures were executed in triplicate. Statistical discrepancies among various treatments were determined utilizing Duncan's multiple-range tests at a significance level of p < 0.05. Data were subjected to Analysis of Variance (ANOVA) through the employment of SPSS Statistics Version 20.0 software (IBM Corp., Armonk, NY, USA). The findings were expressed as the mean ± standard deviation. Principal Component Analysis (PCA) was carried out using Minitab Statistical software.

3. Results and discussions

3.1. Conjugates fabrication and properties

3.1.1. Degree of grafting

The DG values for both MP and MPP conjugate, developed with varying assistant methods, were presented in Fig. 2. In comparison to the WB-treated conjugate, the application of a microwave field considerably augmented the DG in a shorter time. This observation was consistent with prior research and demonstrated the microwave irradiation was a viable technique for enhancing glycation efficiency [17]. The ultrasound-assisted methodology also promoted glycation, elevating the DG to around 18.56 %. High-intensity ultrasound expanded the excluded volume and the flexibility of protein, which increased the contact area between protein and carbohydrate for more effective collision. And sonocatalysis effect also contributed to speeding up the reaction rate of glycation-driven conjugation. Utilizing the combined UM technique led to a further improvement in DG to about 21.19 %, surpassing the efficiencies of individual microwave and ultrasound methods. It was deemed that the combined UM treatment exerted a stronger modification effect on MP, which was prone to unfold completely into an extended conformation for a more thorough glycation process. Moreover, it could also be observed that MPP conjugates exhibited higher DG values compared with those of MP conjugates. This was ascribed to mild hydrolysis promoting the degradation of polypeptide and increasing the number of available active residues (cysteine, lysine) on molecular surface.

Fig. 2.

The degree of glycation (DG) for various MP and MPP conjugates. Samples designated with different letters indicated significant difference (p < 0.05). MP: myofibrillar protein; MPP: myofibrillar protein peptide; W-, M-, U-, UM- denoted to water bath, microwave, ultrasound and combined ultrasound & microwave, respectively.

3.1.2. FTIR

As shown in Fig. 3, native MP possessed characteristic bands at 1650, 1530, and 1150 cm⁻¹, which was attributed to the C = O stretching vibration of amide I, N–H bending and C–N stretching vibration of amide II, N–H angular vibration and C–N stretching vibration of amide III, respectively. A slight blueshift of these typical peaks was observed after glycation, implying that carbohydrate might be successfully grafted onto the ε-group of polypeptide chains [30]. These changes became more obvious when the extrinsic microwave, ultrasound, and UM techniques were applied. A new absorption peak at 2932 cm⁻¹ was observed, which was attributable to the enhancement of C–H stretching vibrations due to the insertion of glycosidic bonds [31] and it had been confirmed as a unique feature in the glycation reaction [32]. In addition, the absorption band ranging from 3250 cm⁻¹ to 3400 cm⁻¹ displayed a broadening and intensification to varying extents, which signified an increase in free hydroxyl groups and the presence of polysaccharide-induced O–H in-plane [33].

Fig. 3.

FTIR spectra of (a) MP and (b) MPP treated by different glycation methods. Note: MP: myofibrillar protein; MPP: myofibrillar protein peptide; W-, M-, U-, UM- denoted to water bath, microwave, ultrasound and combined ultrasound & microwave, respectively.

3.1.3. Secondary structure

In general, the α-helical structure is stabilized by intramolecular hydrogen bonds, while β-sheet is formed via interchain hydrogen bonding among adjacent polypeptide chains. β-turn corresponds to weakly hydrogen-bonded structures, and random coil pertains to unfolded polypeptide conformations [34]. As presented in Table 1, the glycation with intensification techniques significantly affected the secondary structure of MP and MPP, where the ordered structures (α-helix and β-sheet) were gradually transformed into disordered forms (β-turn and random coil). The insertion of long-chain carbohydrates induced the breakage and subsequent rearrangement of intra- and inter-molecular forces; under these circumstances, the strong protein–protein hydrogen bonds were replaced by weak protein-polysaccharide and protein-water hydrogen bonds, leading to the formation of disordered structures. This phenomenon was partially magnified in U-MP and U-MPP samples, where the disordered pattern became the preferential structure. However, microwave-assisted conjugates exhibited higher β-sheet content, which was mainly explained by the heat-induced reaggregation of MP for strong hydrogen-bonded networks [35]. It was worth noting the more ordered structure was lost while the pre-hydrolysis and/or combined UM treatments were applied. Interestingly, a random coil of MPP samples was slightly reduced relative to that of MP conjugates. The initial structural complexity was likely responsible for this observed behavior [36], in which MP started with more complicated secondary structures (α-helix and β-sheet), that could be readily disrupted to form more random coils under glycation-induced modification. Nevertheless, MPP had fewer of these elements to start with, resulting in a less potential to generate random coils.

Table 1.

Effect of different fabrication methods on secondary and tertiary structures of MP and MPP conjugates.

Samples	Secondary structure				Tertiary structure
	α-helix	β-sheet	β-turn	random coil	H0	R-SH
MP	48.26 ± 2.21a	25.67 ± 1.36a	10.05 ± 0.65 g	16.02 ± 1.88f	518.25 ± 28.95e	5.87 ± 0.52e
W-MP	42.93 ± 1.06c	20.36 ± 0.88bc	12.08 ± 0.92f	24.63 ± 2.3e	595.07 ± 17.4 cd	6.95 ± 0.18c
M-MP	37.54 ± 0.75d	22.18 ± 2.43b	12.84 ± 0.27f	27.44 ± 3.44d	628.61 ± 33.98c	6.36 ± 0.77 cd
U-MP	31.44 ± 1.46f	16.62 ± 1.33e	16.97 ± 2.81e	34.97 ± 1.81b	714.85 ± 12.1a	7.58 ± 0.73b
UM-MP	26.27 ± 0.67 g	15.1 ± 1.02f	19.12 ± 2.5c	39.51 ± 0.77a	671.8 ± 38.61b	8.03 ± 0.65a
MPP	44.74 ± 1.85b	21.54 ± 1.38b	16.79 ± 0.82e	16.93 ± 2.33f	557.33 ± 7.29d	6.47 ± 1.12 cd
W-MPP	38.26 ± 1.53d	18.96 ± 3.92d	18.52 ± 2.34c	24.26 ± 2.89e	588.9 ± 18.65 cd	6.89 ± 0.9c
M-MPP	35.7 ± 2.15e	20.03 ± 0.49bc	17.87 ± 3.7d	26.4 ± 4.2de	616.21 ± 28.45c	6.11 ± 0.83d
U-MPP	27.42 ± 0.97 g	14.9 ± 1.56f	24.84 ± 1.23b	32.84 ± 1.57c	675.2 ± 12.34b	7.07 ± 1.36c
UM-MPP	23.61 ± 0.55 h	13.77 ± 2.75 g	27.91 ± 0.76a	34.71 ± 1.92b	633.3 ± 9.4c	7.69 ± 0.24b

All the data are expressed as mean ± SD. Samples designated with different letters indicated significant difference (p < 0.05). MP: myofibrillar protein; MPP: myofibrillar protein peptide; W-, M-, U-, UM- denoted to water bath, microwave, ultrasound and combined ultrasound & microwave, respectively.

3.1.4. Tertiary structure

The variations in H₀ and R–SH are used to assess the alterations in the tertiary structure of the protein [37]. Glycated MP under WB and ultrasound treatments obviously increased H₀ and R–SH (Table 1), as hydrophobic residues and sulfhydryl groups tended to transfer from hydrophobic regions to the molecular interface during glycation reaction. Therein, U-MP showed higher values of H₀ and R–SH while microwave-treated method seemed to be inconducive to the synthesis of R–SH, which might be due to that some available sulfhydryl groups were oxidized to disulfide bonds through microwave-induced free radicals. UM-MP exhibited greater R–SH than that of M−MP and U-MP, showing that the combined UM-assisted glycation led to the most distinct structural modification as we discussed above. Therefore, it was posited that the electromagnetic waves would effectively target complex regions less accessible to ultrasonic cavitation, thereby synergistically promoting the structural alterations. In contrast, H₀ was decreased in UM-MP samples because the excess of conjugated polysaccharides in turn limited the accessibility of ANS agent to targeted residues during measurement [16], [38].

3.1.5. Fluorescence analysis

The intrinsic fluorescence spectra of MP and MPP conjugates were presented in Fig. 4. The wavelength of maximum emission intensity (λ_max) of the native MP was observed at a wavelength of 329 nm when excited at 280 nm, manifesting that chromophore groups were mainly presented in a “nonpolar” environment. The λ_max of the covalent protein-polysaccharide complex treated by WB, microwave, and ultrasound were red-shifted to 330 nm, 331 nm, and 334 nm, respectively, and this confirmed that some of the fluorescent amino acids (tryptophan, phenylalanine, and tyrosine) were moved to a more hydrophilic microenvironment. Compared with WB and microwave treatment methods, the modificated effect was more profound for ultrasound-assisted glycation reaction. Aggregates of MP or its hydrolysates could be discharged into smaller fragments under both microwave and ultrasound fields [16], [39]. Subsequently, a rapid increase in temperature and free radicals generated by microwave field might cause oxidation and dehydration effects of conjugates, where they tended to recross-link into newly-formed aggregates [19]. On the contrary, the cavitation effect and interfacial waves resulting from constantly collapsed microbubbles further destroyed protein–protein interactions by inhibition of heat-driven refolding and reaggregation of the polypeptide chain [24], leading to an unfolded conformation for the following sonocatalysed glycation. However, UM-MP and UM-MPP showed weaker fluorescence intensity with no change in λ_max compared with that of corresponding ultrasound-assisted samples. This had been ascribed to that increase in conjugated MD enhanced the shielding effect for fluorophore.

Fig. 4.

Intrinsic fluorescence emission spectra of (a) MP and (b) MPP treated by different glycation methods. Note: MP: myofibrillar protein; MPP: myofibrillar protein peptide; W-, M-, U-, UM- denoted to water bath, microwave, ultrasound and combined ultrasound & microwave, respectively.

3.2. Physicochemical properties of fabricated conjugates

3.2.1. Solubility, particle size, and zeta potential

As presented in Table 2, the solubility of native MP was 15.09 % with the average particle size of 224.92 nm, which reflected that the protein subunits were unstable and tended to aggregate into large particles with poor protein-water interactions. The conjugate fabricated under WB and microwave exhibited significantly higher solubilities and smaller particle sizes than that of native MP, and U-MP conjugate had an even greater solubility (76.30 %) and reduced particle size (57.28 nm). However, the protein solubility of UM-MP began to decrease with an increase in particle size, presumably because the increase of covalent polysaccharide augmented the volume of the resulting conjugate, which counteracted the depolymerization effect induced by the conjugation. The zeta potential gradually reduced under microwave-, ultrasound-, and combined UM-assisted glycation, and it could be explained by that charged residues were partly covered by grafted uncharged polysaccharide chains.

Table 2.

Solubility, particle size, and zeta potential of different MP and MPP conjugates.

Samples	Solubility (%)	Particle size (nm)	Zeta potential (mV)
MP	15.09 ± 2.86 g	224.92 ± 21.47a	−16.8 ± 1.06c
W-MP	38.59 ± 1.51f	150.54 ± 8.65b	−23.08 ± 1.23a
M-MP	43.77 ± 5.93ef	121.26 ± 13.71c	−18.95 ± 1.44b
U-MP	76.30 ± 3.26c	57.28 ± 4.32 fg	−16.14 ± 0.81c
UM-MP	74.26 ± 2.74c	60.93 ± 6.48f	−14.81 ± 0.12d
MPP	41.55 ± 3.75f	142.57 ± 7.8b	−12.51 ± 0.93e
W-MPP	48.65 ± 1.69e	103.60 ± 10.33d	−19.82 ± 1.31b
M-MPP	56.44 ± 6.74d	86.97 ± 14.89e	−17.54. ± 1.22bc
U-MPP	86.09 ± 2.11b	50.12 ± 3.05 g	−14.39 ± 0.28d
UM-MPP	95.20 ± 3.24a	33.04 ± 1.62 h	−11.66 ± 0.84e

All the data are expressed as mean ± SD. Samples designated with the different letters indicated significant difference (p < 0.05). MP: myofibrillar protein; MPP: myofibrillar protein peptide; W-, M-, U-, UM- denoted to water bath, microwave, ultrasound and combined ultrasound & microwave, respectively.

Although the changes in solubility and particle size of MPP conjugates followed the same trend as MP conjugates, they showed smaller particle sizes and higher solubility, especially for UM-MPP. Whereas, a lower absolute zeta potential was observed in MPP conjugate samples. The hydrolysis process decreased the protein part in conjugates for less available charged groups. This phenomenon also demonstrated that glycation-induced dissociation and steric hindrance provided by MD were responsible for the improvement of protein stability rather than electrostatic interactions.

3.2.2. SEM

As illustrated in Fig. 5, native MP exhibited a tendency for aggregation into heterogeneous granular structures of substantial size. Mild hydrolysis into MPP notably mitigated the aggregative capability, despite the persistence of obvious clustered structure. The volume of the hydrophobic region was reduced because the interior of the aggregated protein structures was partly destroyed by trypsin. Moreover, glycation could also dissociate and denature the MP, enabling the grafting of polysaccharides onto the heavy and light meromyosin regions [40], which subsequently imparted steric hindrance against the re-folding and/or re-interaction of polypeptide chains. The morphology that some weak cross-linking between protein pellets emerged in M−MP and M−MPP, and this was mainly due to the rapid increase in temperature generated by strong electromagnetic radiation, which would cause quick aggregation of denatured protein before it was completely glycated. In the case of ultrasound-assisted glycation samples, the small structures became dispersed and more uniform with no distinct cross-scale interactions. It was possibly attributed to the following two reasons: (1) the increase in the amounts of covalent polysaccharide decreased aggregation capability of MP and MPP; (2) ultrasound-induced high-frequency shear forces prevented conjugates against inter-molecular interactions during glycation [41].

Fig. 5.

SEM images of MP and MPP conjugate samples treated by different glycation methods (×100 000). Note: MP: myofibrillar protein; MPP: myofibrillar protein peptide; W-, M-, U-, UM- denoted water bath, microwave, ultrasound and combined ultrasound & microwave, respectively.

In comparison to glycation facilitated solely by ultrasound, the utilization of the combined UM approach further reduced the aggregation behavior of conjugates, resulting in a more homogeneous morphological distribution characterized by smaller structural entities. This outcome could be boiled down to microwave loosening the complex aggregate structures of protein, thereby acoustic waves traveled faster and stronger through these denser structures, expediting the kinetics of ultrasound-assisted glycation. Simultaneously, cavitation and localized high shear forces generated by high-pressure shock mitigated the potential for reaggregation induced by electromagnetic fields. Interestingly, it was notable that the particle sizes visualized via SEM were somewhat smaller than those measured using laser particle sizer technology (Table 1). The conjugate samples underwent a drying process that led to the shrinkage and collapse of granular material before SEM observation, and therefore it might somewhat influence on both the particle size and overall microstructure [42]. In fact, considering desirable solubility, molecular morphology, and structural properties of UM-based glycoconjugates, it was proposed their great potential as a type of outstanding emulsifier for development of a stable emulsion system.

3.3. Emulsifying performance of fabricated conjugates

Following the fabrication of conjugates utilizing various intensification techniques, the functional performances of these conjugates as emulsifiers in O/W systems were rigorously evaluated. Comprehensive assessments were conducted to assess the storage stability, creaming stability, thermal stability, and oxidation stability of emulsions stabilized by MP- and MPP-based conjugates, as detailed in the subsequent subsections.

3.3.1. Storage stability

As shown in Table 3, the average droplet sizes of MP- and MPP-stabilized emulsions were approximately 52.18 μm and 47.56 μm, respectively, and they were completely delaminated after 5 days storage. When using W-MP conjugate and W-MPP conjugate as the emulsifier, smaller droplet sizes of 28.64 μm and 21.27 μm were observed respectively, and the droplets remained stable without delamination under 20 days storage. The robust steric repulsion and elevated the bulk viscosity of the system conferred by outward polysaccharide layer accounted for the improved stability [43]. As expected, glycation assisted by an extrinsic ultrasound field further reduced droplet size for 0–20 days storage in both MP and MPP samples. Ultrasound increased the amount of grafted MD with a more open structure, and the resulting conjugates were more tightly adsorbed in the interface and helped to reduce the interfacial energy required to create a new interface. Although covalent carbohydrate was also somewhat increased with the assistance of microwave, there was no marked change in emulsifying stability compared with that of corresponding WB conjugate. Exerting a mild ultrasound field could effectively solve this problem, in which emulsions emulsified by UM-MP conjugate and UM-MPP conjugates exhibited droplet sizes of 43.04 μm and 22.53 μm, respectively, after 20 days of storage, with no discernible phase separation.

Table 3.

Effects of different glycation methods on volume-weighted average particle size (d_4,3) of various MP- and MPP-based emulsions.

Samples	Average emulsion size (d4, 3) (µm)
	0 day	5 days	10 days	20 days
MP	52.18 ± 5.87a	ND	ND	ND
W-MP	28.64 ± 3.12c	46.31 ± 2.17a	77.9 ± 0.83a	ND
M-MP	26.41 ± 3.54c	43.21 ± 0.45a	68.63 ± 4.98b	ND
U-MP	15.33 ± 1.09e	20.07 ± 1.29c	29.84 ± 2.66e	64.25 ± 5.67a
UM-MP	10.47 ± 1.26f	14.81 ± 2.86d	21.2 ± 1.73f	43.04 ± 2.52b
MPP	47.56 ± 3.65b	ND	ND	ND
W-MPP	21.27 ± 1.65d	35.69 ± 4.55b	58.06 ± 2.6c	ND
M-MPP	19.96 ± 1.44d	32.4 ± 0.78b	53.19 ± 4.11d	ND
U-MPP	7.92 ± 0.68 g	10.44 ± 0.54e	16.3 ± 1.33 g	33.57 ± 2.36c
UM-MPP	4.8 ± 0.91 h	7.69 ± 0.29f	11.28 ± 1.18 h	22.53 ± 1.77d

All the data are expressed as mean ± SD. Samples designated with different letters indicated significant difference (p < 0.05). MP: myofibrillar protein; MPP: myofibrillar protein peptide; M-, U-, UM- denoted to microwave, ultrasound and combined ultrasound & microwave, respectively.

Compared with the corresponding MP-based conjugates, it became evident that the MPP-based conjugates manifested a better capacity for emulsion stabilization. The mild hydrolytic conversion of larger protein aggregation to smaller peptide submits improved the molecular amphiphilicity, and thus facilitated accelerated diffusion, absorption, and structural rearrangement into a favorable interfacial conformation [44]. Also, the resulting unfolded structure increased the availability of potential cross-linking sites during interfacial rearrangement, contributing to the formation of a thick, continuous, and viscoelastic film that possessed great resistance to coalescence and creaming.

3.3.2. Creaming stability

The analytical photocentrifuge LUMiSizer is a specialized instrument used to evaluate the stability of dispersions through accelerated separation tests [45]. This technology uses near-infrared light transmitted through the sample to record time-resolved transmitted intensity profiles. As shown in Fig. 6, the evolution of the transmission profile of emulsion stabilized by MP and MPP quickly moved upward whereas the corresponding conjugate-stabilized emulsions exhibited much slower transmissions. The enhanced viscosity of suspension phase associated with grafted polysaccharide accounted for the improved stability. The UM treatment showed the most noteworthy modification efficiency on interfacial properties as the much more subtle transmission change was observed in the UM-MP- and UM-MPP-stabilized emulsion.

Fig. 6.

Evolution of transmission profiles of emulsions stabilized by (a) MP-based conjugates and (b) MPP-based conjugates. Note: MP: myofibrillar protein; MPP: myofibrillar protein peptide; W-, M-, U-, UM- denoted to water bath, microwave, ultrasound and combined ultrasound & microwave, respectively.

Heavily aggregated structures of native MP indicates its poor amphipathy as most of hydrophobic residues reside in the interior of aggregates with low molecular freedom, limiting the subsequent structural changes for emulsification [6]. Hydrolysis and glycation are two feasible approaches to reducing the aggregate degree of protein in the food industry [46]. Generally, peptides induced by hydrolysis improve surface hydrophobicity at the expense of the loss of molecular flexibility and interfacial stability [44]. In this case, UM-assisted glycation greatly reinforced the film-forming ability and emulsifying stability of protein; however, high-molecular-weight conjugates was hard to be precisely and preferentially absorbed into the interfacial phase, and the unabsorbed parts might act as flocculation bridges among the droplets, speeding up aggregation and coalescence. Therefore, hydrolysis and combined UM-assisted glycation could complement each other for protein structural modification as high-efficiency glycation provided an extra physical barrier for polypeptides against phase separation while well-controlled hydrolysis process contributed to maintaining a suitable molecular size and the balance of hydrophilic/hydrophobic portions for desirable interfacial behavior.

3.3.3. Thermal stability

Since the importance of heating treatment in food processing, the application values of emulsion product is heavily dependent on its thermal stability. The droplet sizes of MP, MPP, W-MP, W-MPP, M−MP, and M−MPP were greatly increased after thermal processing (Fig. 7). Under these circumstances, protein or conjugate components were separated and decomposed from O/W interface because interfacial polypeptide part was easily denatured under heating, and subsequently, the droplet interface became unsaturated coverage for reduced interfacial stability. In contrast, the thermal treatment showed a slighter effect on emulsion stabilized by ultrasound- and UM-treated conjugates with smaller particle sizes. This underscored the advantage of ultrasound over microwave in achieving superior thermal stability of resulting conjugates. Such an advantage derived from the elevated DG, where more covalent carbohydrate provided stronger protection effect to heat-sensitive polypeptide part. As a result, the integrity of dense interfacial film developed by glycoconjugates remained relatively stable, therefore effectively retarding heat transfer and precluding thermally-induced aggregation of the dispersed system.

Fig. 7.

Effect of temperature (4 °C, 60 °C, and 85 °C) on the average size of MP-based emulsions stabilized by various MP and MPP conjugates. MP: myofibrillar protein; MPP: myofibrillar protein peptide; W-, M-, U-, UM- denoted to water bath, microwave, ultrasound and combined ultrasound & microwave, respectively. Samples designated with different letters indicated significant difference (p < 0.05).

Upon the application of an elevated temperature of 85 °C, complete creaming appeared in emulsions stabilized by native MP/MPP and conjugates prepared via WB and microwave methods. And the droplet size of U-MP and UM-MP was markedly increased. The phenomenon would be attributed to the promoted thermal conditions compromising the steric hindrance between droplets [14]. Notably, this effect was substantially blocked in the presence of UM-MPP conjugates with a droplet size of only 20 μm, suggesting the strong shielded layers associated by the ideal conjugate structure seemed to drastically enhance a heat-resistant effect, where high energy water molecules and heat were slowed and buffered from interface.

3.3.4. Oxidation stability

The oxidation stability of emulsion stabilized by MP- and MPP-based conjugates was determined by investigating the changes of TBARSs and lipid hydroperoxides under accelerated oxidation environment. A higher value of TBARS and hydroperoxides is indicative of diminished oxidative stability. As delineated in Fig. 8, the concentrations of TBARS in emulsions stabilized by native MP and MPP were 5.51 μM/L and 4.06 μM/L, respectively. These concentrations were markedly reduced in emulsion system stabilized by conjugates. The resistant effect conferred by the grafted polysaccharides helped to inhibit the transfer of oxidative radicals to the droplets in different degrees. At the same time, glycated protein products could be regarded as edible antioxidants for scavenging reactive oxygen [46]. The corresponding roles showed more distinct in ultrasound- and combined UM-treated samples. The trend observed was corroborated by the fact that emulsions stabilized with the combined UM-treated conjugates demonstrated consistently lower lipid hydroperoxide levels compared to those of individual microwave- and ultrasound-treated samples. This aligned with the observed creaming stability and TBARS formation, where the combined UM technology was further proven as the most proficient at safeguarding method to fabricate protein emulsifiers with great oxidative resistance.

Fig. 8.

Formation of the TBARS(A) and lipid hydroperoxides (B) of MP-based emulsions stabilized by various MP and MPP conjugates. MP: myofibrillar protein; MPP: myofibrillar protein peptide; W-, M-, U-, UM- denoted to water bath, microwave, ultrasound and combined ultrasound & microwave, respectively. Samples designated with different letters indicated significant difference (p < 0.05).

Prior research had observed that both mild hydrolysis and ultrasound treatment augmented glycation, and following intensified fuctional properties and antioxidant activities [14], [21]. Analogous to findings on storage, creaming, and thermal stability, the interfacial films constituted by UM-MPP conjugates demonstrated greater shielding ability against the diffusion of oxygen radicals, manifesting the most robust oxidation resistance among all the conjugates examined. A tailored conjugate conformation, achieved through the moderate hydrolysis of MP into MPP, coupled with UM treatment, served as an efficient route for functional conjugate fabrication. This strategic formulation not only really overcame the interfacial limitations of MP, but also conferred protective barriers against thermal conduction and oxidative radicals. Such insights offered a theoretical foundation for the synthesis of conjugates as efficient emulsifiers, endowing emulsion-based foods with excellent characteristics.

3.4. Principal component analysis

As the emulsifying properties of MP and its conjugates were likely to be affected by many complex factors, the utilization of a score plot derived from Principal Component Analysis (PCA) was necessary for analysing the performance of MP- and MPP-based conjugates due to its capacity for high-dimensional data reduction and interpretation. Abundant variables were assessed in this study, including DG, solubility, particle size, and zeta potential, secondary and tertiary structure change, storage, creaming, thermal, and oxidation stability of emulsions. A score plot of first and second principal components visualized the samples in this reduced-dimensional space, facilitating the identification of clusters that might not be apparent in the higher-dimensional original space. Hence, it could condense the multi-dimensional performance metrics into fewer dimensions and following highlighted the key differences and similarities between MP-based and MPP-based conjugates.

As visible from Fig. 9, U-MP, U-MPP, UM-MP, and UM-MPP were distinctly separated from other conjugates along the axis of the first principal component, which suggested that their physicochemical characteristics and emulsifying capabilities substantially differed from those of the other conjugates. It was consistent with the findings described in the preceding sections, whereby conjugates synthesized with the aid of ultrasound or UM demonstrated more favorable properties and emulsifying performances. Additionally, MP and MPP were readily separated from W-MP, W-MPP, M-MP, and M-MPP along the axis of the second principal component. This implied that as WB and microwave methodologies might not be as effective as ultrasound and UM in conjugate fabrication, they also contributed to glycation, resulting in conjugates that serve as significantly more efficient emulsifiers than native MP and MPP. This corroborated the assessments articulated in the preceding sections as well.

Fig. 9.

The score plot from Principal Component Analysis (PCA) of various conjugates’ performances. MP: myofibrillar protein; MPP: myofibrillar protein peptide; W-, M-, U-, UM- denoted to water bath, microwave, ultrasound and combined ultrasound & microwave, respectively.

Noteworthy, although the synergistic effect between ultrasound and microwave treatments on glycation has been confirmed and the benefit of the pre-hydrolysis was also revealed, how the molecular weight of MD and hydrolysis degree of protein influenced these complementary behavior has not been detailly considered. Therefore, more precise analysis of complex relationship among combined UM fields, mutil-scales behaviors of polypeptides and carbohydrates, and interfacial preformances of resulting conjugates, as well as underlying molecular mechanisms should be explored in future discussions.

4. Conclusions

The current study focused on the structural changes and emulsifying properties of MP and MPP conjugated with the assistance of microwave, ultrasound, and combined UM treatment methods. Hydrolyzing MP into MPP greatly promoted the covalent combination with MD as evidenced by the DG and FTIR results. In this case, the heavily-aggregated MP particle was broken into smaller subunits for following glycation. The ordered structures of MP and MPP were transformed into disordered forms after the covalent conjugation, and meanwhile, more hydrophobic residues were exposed. These phenomena became more pronounced when treating with a combined UM field. Electromagnetic radiation could enter the core of aggregated regions that were unavailable for single mechanical effect while continuous cavitation contributed to dissipation excess heats under combined microwave treatment. This complementary function facilitated a more open and flexible conformation for strong grafting ability. Moreover, the glycoconjugates fabricated via the UM technique exhibited the highest efficiency as an emulsifier, which appeared smaller average particle sizes and more uniform morphology than those of WB, microwave, and ultrasound samples. Furthermore, UM-MPP could be considered as an optimal emulsifying agent, capable of stabilizing emulsions against various forms of thermal, storage, creaming, and oxidative stresses. The PCA result implied that although WB and microwave methodologies were not be as effective as ultrasound and UM in conjugate fabrication, they also contributed to glycation, resulting in conjugates that serve as significantly more efficient emulsifiers than native MP and MPP. Overall, our study proved that mild pre-hydrolysis and the synergistic application of ultrasound and microwave technologies contributed to a well-performed glycation process for a great improvement of a variety of emulsifying performances, and it would be beneficial for the development of different types of desirable MP-based products in the food industry.

CRediT authorship contribution statement

Zhiyu Li: Writing – original draft, Funding acquisition, Data curation, Conceptualization. Xiaomei Zhong: Visualization, Data curation. Cuirong Luan: Visualization, Software, Conceptualization. Nanhua Wen: Software, Conceptualization. Chuanyang Shi: Software, Conceptualization. Xiaoyu Lin: Software, Conceptualization. Chao Zhao: Visualization, Conceptualization. Yang Zhang: Writing – review & editing, Methodology. Lianyu Luo: Visualization, Conceptualization. Liang Zhang: Visualization, Conceptualization. Yijing Wu: Writing – review & editing, Funding acquisition, Conceptualization. Jie Yang: Writing – review & editing, Supervision, Funding acquisition.

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.