Effect of ultrasound on the functional properties and structural changes of chicken liver insoluble proteins isolated by isoelectric solubilization/precipitation

Graphical abstract

Abstract

The studies investigated the effects of different ultrasonic powers (180, 360 and 540 W) on the functional properties and structural changes of chicken liver insoluble proteins (CLIPs) isolated by isoelectric solubilization/precipitation (ISP) (with alkaline solubilization at pH 11.0 and pH 12.0 respectively, and acid precipitation at pH 5.5). Results indicated that ultrasonic significantly increased the solubility of ISP-isolated CLIPs, and narrowed the particle size distribution of D₃,₂ and D₄,₃ (P < 0.05). The highest solubility was observed at pH 11.0 and 360 W ultrasound treatment, reaching 77.26 %. The ultrasonic with 360 W exhibited higher shear stress and apparent viscosity. Spectroscopy revealed that compared to without ultrasonic treatment, there was an increase in β-sheet and random curling content accompanied by a decrease in β-turn and α-helix structure when ultrasonication. Ultrasound altered the tyrosine hydrophobic residues to be exposed to the surface of the ISP-isolated CLIPs, thus improving the hydrophilicity. Overall, ultrasound combined with ISP treatment effectively improved the functional properties of CLIPs, and it might be a potential, safe and efficient method for improving the processing properties and broadening the application of insoluble animal-derived proteins.

1. Introduction

Chicken products are one of the major sources of meat in the human daily diet, which inevitably contributes to the rise in chicken by-products including chicken livers (CLs). As valuable sources of protein, the CLs contain a high protein content (above 20 %), unique amino acid profile and bioactive peptides [1], [2]. However, the high cholesterol content and heavily fishy odor of CLs make them less receptive for people to eat, thereby limiting their utilization [3]. Generally, the majority of CLs are either simply processed or even discarded, resulting in a serious waste of CL protein resources [4]. Consequently, it is urgently necessary to effectively transform CL protein resources into valuable human food. Insoluble (water-insoluble and salt-insoluble) proteins constitute a significant proportion in CLs, which are essential for determining the texture, stability, and general quality of CL products [5]. Nevertheless, the poor aqueous solubility of insoluble protein leads to poor processing characteristics and restricts their utilization. Therefore, it is imperative to explore suitable protein modification methods to improve the solubility and optimize the functional properties of chicken liver insoluble proteins (CLIPs). This will provide novel, high-quality protein resources for human consumption and maximize the effective utilization of CL protein resources.

During the recent years, the isoelectric solubilization/precipitation (ISP) process has gradually attracted attention as an effective method in the field of biotechnology and biochemistry research. Its application for the recovery of animal by-product proteins is not only characterized by a high extraction efficiency and cost-effectiveness [6]. During ISP processing, proteins in the animal byproduct are first unfolded and dissolved in an extreme pH condition (acidic or alkaline). Subsequently, the pH of protein solution is adjusted to its isoelectric point (PI), leading to protein refolding and precipitation, followed by centrifugation to obtain purified protein [5]. During the unfolding and refolding process of proteins, changes in protein conformation can lead to alterations in their functional properties. Utilizing the ISP process to isolate goose liver protein can enhance its emulsification properties. Moreover, compared to acidic conditions, alkaline conditions can yield more stable emulsions [7], [8]. Zhao et al.[9] reported that myofibrillar proteins extracted from chicken breast meat at alkaline pH exhibited superior gelation properties. Abdollahi et al. [10] found that the ISP process resulted in higher recovery rates and better functional properties for blend fish protein recovered from kilka (Clupeonella cultriventris) and silver carp (Hypophthalmichthys molitrix), with this improvement being more pronounced under alkaline conditions. Evidently, employing the ISP process to isolate insoluble proteins from CL is deemed feasible.

The processing characteristics of proteins will directly impact the quality of protein-based products. Hence, it is crucial to choose economically effective methods for modifying protein to improve their processing characteristics. Ultrasound, as a non-thermal physical processing method for protein modification, has been extensively applied in the food industry. The cavitation effects and substantial shear forces generated by ultrasound can disrupt the intermolecular forces that maintain protein structure, leading to alterations in conformation and promoting the enhancement of protein solubility and functionality [11]. High-intensity ultrasonic (20 kHz, 450 W, 6 min, 30 W/cm²) has been conducted to enhance the emulsifying properties of chicken myofibrillar proteins [12]. Li et al. [13] reported that ultrasonic could unfold the conformation of myofibrillar proteins in frozen shrimp, thereby enhancing gel strength and water-holding capacity. Clearly, using ISP process to extract CLIP and treating them with ultrasound may be a feasible way to effectively alter the functional properties of CLIP. However, in reported studies, ultrasound modification of proteins and meat is not always beneficial; excessively long or high-intensity ultrasound may be counterproductive [14]. Therefore, we believe it is essential to explore the structural changes of proteins under combined ISP and ultrasound treatments. The combined effects of these treatments on proteins may be complex, either synergistic or antagonistic. The impact and mechanisms of ultrasound intervention on ISP-isolated CLIPs need further clarification.

Consequently, in this study, we primarily investigated the effects of different ultrasonic treatments (0, 180, 360 and 540 W) on the solubility, particle size distribution, and rheological properties of ISP-isolated CLIPs. Furthermore, we delved into clarifying the response mechanisms of the solubility and functional properties of ISP-isolated CLIPs to ultrasonic power based on changes in secondary and tertiary structures. This understanding will facilitate further processing and utilization of CLIP resources, promoting the application of animal-derived insoluble proteins in the food industry.

2. Materials and methods

2.1. Materials

This experiment employed fresh chicken livers that were purchased from a local slaughterhouse (Liulaoer Roast Chicken Co., Ltd., Suzhou, China). After pre-processing, which included the removal of bile ducts and blood vessels, the CLs were homogenized into a paste using a meat grinder (MM-12, 8 mm plate, Guangdong, China). Subsequently, the obtained chicken liver paste was subjected to freezing preservation (−18 °C). All chemicals used in the experiment were of analytical grade.

2.2. Chicken liver insoluble protein isolation

The extraction of CLIP was performed following a modified protocol based on the method outlined by Xiong et al.[5]. The prepared chicken liver homogenate (Ultra Turrax T-25 BASIC, IKA Company, Germany) was centrifuged (Allgra 64R, American Beckman coulter company, USA) at 10000g for 10 min at 4 °C, followed by dumping the supernatant to remove the water-soluble proteins. Subsequently, phosphate buffer solution (PBS) of pH 7.0 (0.1 mol/L Na₂HPO₄/NaH₂PO₄) was added to the precipitate stirred for 10 min and centrifuged again for 20 min under the same conditions to remove the salt-soluble proteins. Afterward, collected the precipitate, NaOH (1 mol/L) was added dropwise to adjust the pH to 11.0 and 12.0 respectively (Shanghai Yidian Scientific Instrument Co., Ltd., Shanghai, China), stabilized for 10 min, then centrifuged for an additional 10 min. The obtained supernatant was supplemented with HCl (1 mol/L) dropwise to adjust the pH to 5.5 (PI). Finally, centrifugation (10000g for 10 min at 4 °C) was performed to recover the ISP-isolated CLIP isolations.

2.3. Ultrasound treatment

The obtained ISP-isolated CLIP was reconstituted in phosphate buffer solution (PBS, pH 7.0) to prepare a 5 % w/v protein solution. An ultrasonic pulverizer (SCIENTZ-IID, Ningbo Xinzhi Ultrasonic Technology Co., Ltd., Zhejiang, China) was used under ice bath conditions and the probe was submerged in the prepared ISP-isolated CLIP solution. Ultrasound was applied for 3 min at different powers of 0 W, 180 W, 360 W and 540 W, with continuous stirring during ultrasound to ensure uniform treatment. The processed samples were stored in a refrigerator at 4 °C and used within 48 h.

2.4. Protein solubility

According to the method outlined by Li et al. [15], the ISP-isolated CLIP solution was adjusted to a concentration of 5 mg/mL using deionized water. Centrifuged at 10,000g for 15 min at 4 °C and determined the protein content in the supernatant using the Biuret method, using bovine serum albumin (BSA) as the standard protein The solubility of the protein is expressed as the following Eq. (1), the percentage of protein concentration in the supernatant after centrifugation relative to the protein concentration before centrifugation.

$$\begin{array}{c}Solubility\left(\%\right)=\frac{\mathit{Proteincontentinsupernatant}}{\mathit{Totalproteincontentinsample}}\times 100\end{array}$$ (1)

2.5. Determination of particle size

The particle size distribution of the ISP-isolated CLIP solutions was evaluated using an MS-2000 dynamic light scattering instrument (DLS, Malvern Instruments, UK). Deionized water was served as the dispersant, with the sample and dispersant having refractive indices of 1.47 and 1.33, respectively [16]. The detection results are presented as D₃,₂ (volume surface mean diameter) and D₄,₃ (volume mass mean diameter).

2.6. Static rheological measurement

The static shear behavior of the samples was executed using static rheological measurements (MCR302, Anton Paar, Graz, Austria). The ISP-isolated CLIP solution was placed at the center of the sample stage in the rheometer, with a temperature of 25 °C, and allowed to equilibrate for 30 s. Shear rates ranging from 0.1 s⁻¹ to 100 s⁻¹ were applied, and the initial shear rate of 1 s⁻¹ was used to record the viscosity value. The changes in viscosity were assessed at different shear rates [17].

2.7. Fourier infrared spectrum (FTIR)

The freeze-dried chicken liver insoluble protein was mixed with potassium bromide at a ratio of 1:100 and compressed into tablets. Using a Fourier-transform infrared spectrometer the FT-IR spectra of the samples were acquired (Nicolet IS50, Thermo Scientific Inc., Waltham, MA, USA). The scanned results were subjected to smoothing and deconvolution of the amide I region (1600–1700 cm⁻¹) using Peakfit 4.12 (SeaSolve Software Inc., Framingham, USA), followed by multiple Gaussian curve fitting. Ultimately, peak assignment was conducted, followed by calculating the relative percentage of each sub-peak area in the total peak area to ascertain variations in the content of distinct secondary structures [18].

2.8. Surface hydrophobicity (H₀)

The surface hydrophobicity of ISP-isolated CLIP was assessed following the procedure described by Wang et al.[19]. Prepared a mixture by combining 200 μL of 1 mg/mL bromophenol blue (BPB) solution with 1 mL of the protein solution and thoroughly mixing. After allowing it to stand at room temperature for 10 min, centrifuged at 2000 g for 10 min at 4 °C. Finally, diluted the obtained supernatant 10-fold and measured its absorbance at a wavelength of 595 nm, including a blank control group.

The calculation formula was as follows Eq. (2), where the binding capacity of BPB represented the H₀, Abs_blank was the absorbance value of the blank control group at 595 nm, and ABS_sample was the absorbance value of the sample group at 595 nm.

$$\begin{array}{c}BPBbound\left(\mu g\right)=\frac{200\mu g\times ({\mathit{ABS}}_{\mathit{blank}}-{\mathit{ABS}}_{\mathit{sample}})}{{\mathit{ABS}}_{\mathit{blank}}}\end{array}$$ (2)

2.9. Intrinsic fluorescence spectroscopy

The protein solution was diluted to 0.5 mg/L and scanned using a fluorescence spectrophotometer (LS-55, Perkin Elmer, Detroit, America). A quartz fluorescence cuvette with 1 cm path length was employed, utilizing a laser wavelength of 280 nm, excitation and emission slit widths of 2.5 nm, an emission spectrum range of 300 to 420 nm, and a scanning speed of 600 nm/min [20].

2.10. Ultraviolet (UV) spectra

As described by Li et al. [21], the protein solution was diluted to 0.1 mg/L and subjected to scanning using a UV–Vis spectrophotometer (PerkinElmer Ltd., USA) with a wavelength range of 200–600 nm and a scanning speed of 100 nm/min.

2.11. Statistical analysis

Every experiment was conducted in a minimum of three duplicates, and the results were presented as the means ± standard deviation (SD). Data processing and analysis, including analysis of variance (ANOVA), were performed using SPSS 26 (IBM, New York, USA), with a significance level set at P < 0.05. Graphs and charts were generated using Origin 2021 software (OriginLab Corporation, Northampton, USA), and protein secondary structure calculations were performed using Peakfit 4.12 software.

3. Results and discussion

3.1. Protein solubility

Solubility is a crucial functional property of edible proteins, serving as a prerequisite for their excellent emulsifying properties, and thereby influencing their applicative value in food processing [22]. In the absence of ultrasonic treatment, extreme pH (pH 12.0, 0 W) treatment increased the solubility of CLIPs compare to pH 11.0, 0 W treatment, appearing a trend of increasing solubility with increasing pH Fig. 1 (P < 0.05). This behavior could be explained that extremely alkaline conditions enhanced the reciprocal repulsion of isotropic charges on the protein surface, thereby increasing the stability of the protein solution [23]. This was consistent with the findings of Xiong et al.[5], who observed a gradual increase in the solubility of chicken liver proteins from pH 9.0 to 12.0.. Nevertheless, under corresponding ultrasonic power processing, the solubilities of CLIPs all reduced with the increase of pH. A possible explanation for this could be that sonication broke up CLIP aggregates and revealed more polar residues, thereby increasing the quantity of charged groups in solution and strengthening intermolecular repulsion. This effect could be more severe than charge repulsion brought on by extremely high pH [24].

Fig. 1.

Effects of different ultrasonic powers (0, 180, 360 and 540 W) on the protein solubility of ISP-isolated CLIPs. The different lowercase superscripts (a-c) represent significant differences at P < 0.05 level for the same pH sample under different ultrasonic power treatments; the different capital superscripts (A-B) represent significant differences at P < 0.05 level for different pH samples under the same ultrasonic power treatments.

Furthermore, as shown in Fig. 1, the solubility of CLIPs increased significantly after ultrasonic treatment (P < 0.05). Jiang et al. [25] also discovered that the effect became more pronounced following ultrasound treatment. The solubility of CLIPs isolated at pH 11.0 and pH 12.0 with an ultrasonic power of 360 W increased to a maximum of 77.26 % and 76.23 % respectively, representing an obvious improvement of 54.04 % and 44.54 % compared to the non-ultrasound treatment samples (23.22 % and 31.69 % respectively). This behavior could be explained that the cavitation and shearing effects generated by ultrasonic treatment dissociated the protein into smaller particles and disrupted the hydrogen bonds and hydrophobic interactions within the protein, resulting in protein aggregates with a lower volume-to-surface area ratio. As a result, the hydration forces of the protein were strengthened and its solubility increased [12], [26]. However, excessive ultrasonic power, such as 540 W, led to a slight reduction in protein solubility compared to 360 W. This was because excessive ultrasonic power inducing over-denaturation of the protein, leading to the re-aggregation of protein molecules, a phenomenon in line with the findings of Li et al. [26]. These findings demonstrated the effectiveness of ultrasound treatment in improving the solubility of CLIPs, thereby expanding their applicability. Importantly, the appropriateness of ultrasound conditions must be carefully considered.

3.2. Particle size and distribution

The particle size of proteins directly reflects their structure and degree of aggregation, thereby influencing the functional properties of proteins [27]. The particle size distribution and mean particle sizes (D_{4, 3} and D_{3, 2}) of the CLIPs, both without treatment and after ultrasonic treatment, are shown in Fig. 2 and Table 1, respectively. D_4,3 represents the volume-weighted mean diameter, which is more sensitive to the presence of larger particles (aggregates), whereas D_3,2 denotes the surface-weighted mean diameter, providing a better representation of the size of the majority of particles [6].

Fig. 2.

Effects of different ultrasonic powers (0, 180, 360 and 540 W) on particle size distribution of ISP-isolated CLIPs. pH 11.0–0 W, pH 11.0–180 W, pH 11.0–360 W and pH 11.0–540 W represent pH 11.0 solubilization with 0 W, 180 W, 360 W and 540 W ultrasonic power treatment respectively. pH 12.0–0 W, pH 12.0–180 W, pH 12.0–360 W and pH 12.0–540 W were represented pH 12.0 solubilization with 0 W, 180 W, 360 W and 540 W ultrasonic power treatment respectively.

Table 1.

Effect of different ultrasonic powers on the particle sizes of ISP-isolated CLIPs.

Samples	D4,3(µm)	D3,2(µm)
pH 11.0–0 W	105.29 ± 11.56 A,a	23.69 ± 0.63B,a
pH 11.0–180 W	34.14 ± 5.44B,b	22.26 ± 0.38 A,b
pH 11.0–360 W	24.18 ± 1.34B,b	15.88 ± 0.5 A,d
pH 11.0–540 W	29.9 ± 2.7B,b	19.85 ± 0.55 A,c
pH 12.0–0 W	111.36 ± 4.48 A,a	28.49 ± 1.41 A,a
pH 12.0–180 W	76.98 ± 6.03 A,b	22.35 ± 0.3 A,b
pH 12.0–360 W	30.87 ± 1.83 A,d	14 ± 0.25B,d
pH 12.0–540 W	43.63 ± 4.95 A,c	17.99 ± 0.61B,c

Notes: The different lowercase superscripts (a-c) in the table represent significant differences at P < 0.05 level for the same pH sample under different ultrasonic treatments; the different capital superscripts (A-B) in the table represent significant differences at P < 0.05 level for different pH samples under the same ultrasonic treatments. pH 11.0–0 W, pH 11.0–180 W, pH 11.0–360 W and pH 11.0–540 W represent pH 11.0 solubilization with 0 W, 180 W, 360 W and 540 W ultrasonic power treatment respectively. pH 12.0–0 W, pH 12.0–180 W, pH 12.0–360 W and pH 12.0–540 W were represented pH 12.0 solubilization with 0 W, 180 W, 360 W and 540 W ultrasonic power treatment respectively.

The particle size distribution of untreated CLIPs was broader and had lower peak values, as shown in Fig. 2. This indicated that CLIPs without sonication had a higher proportion of large protein aggregates [28]. The particle size distribution curves showed a relative leftward shift following ultrasonication, with the maximum peak values first increasing and then decreasing. Interestingly, these trends were consistent across various pH levels, all reaching their maxima at 360 W. This demonstrated that in the dispersed phase of CLIPs, ultrasonication improved the volume percentage of small particles. Similarly, D_4,3 and D_3,2 were significantly smaller after sonication than they were before (P < 0.05).

At the same pH values, with increasing ultrasonic power, they exhibited a trend of initially decreasing and then increasing (Table 1). These findings suggested that larger protein aggregates were disrupted by cavitation, turbulence, and shear forces produced by ultrasonication, resulting in the fragmentation and reduction in size of the protein particles [29]. On the other hand, overprocessing could result in a decrease in the overall disruption efficiency. For example, the average protein particle size at 540 W ultrasonic power was larger than that at 360 W (Fig. 2). This phenomenon might arise from excessive ultrasonic intensity, resulting in extensive protein denaturation and subsequent protein aggregation. Zhu et al. [30] also reported that excessive ultrasound treatment of walnut protein dispersions could result in an increase in particle size. Therefore, it is essential to optimize ultrasound conditions to ensure efficient disruption of protein aggregates without inducing excessive protein denaturation.

3.3. Rheological properties

The static rheological behavior of protein solutions is a crucial property that describes their movement speed and resistance to external forces [8], [15]. Fig. 3 illustrates the effect of various ultrasonic power treatments on the static rheological behavior of CLIP solutions. The apparent viscosity of the CLIP solutions gradually decreased and tended to stabilize with increasing shear rate, exhibiting shear-thinning behavior, which was a typical characteristic of non-Newtonian pseudoplastic fluids [17].

Fig. 3.

Effects of different ultrasonic powers (0, 180, 360 and 540 W) on static shear behaviors of ISP-isolated CLIPs. pH 11.0–0 W, pH 11.0–180 W, pH 11.0–360 W and pH 11.0–540 W represent pH 11.0 solubilization with 0 W, 180 W, 360 W and 540 W ultrasonic power treatment respectively. pH 12.0–0 W, pH 12.0–180 W, pH 12.0–360 W and pH 12.0–540 W were represented pH 12.0 solubilization with 0 W, 180 W, 360 W and 540 W ultrasonic power treatment respectively.

At the same shear rate, the apparent viscosity of ultrasonicated CLIPs was significantly higher than that of non-ultrasonicated CLIPs. Additionally, under the same pH conditions, the apparent viscosity of CLIPs subjected to different ultrasonic power treatments exhibited the order of 360 W > 540 W > 180 W. This indicated that the cavitation effects of ultrasound disrupted the structure of CLIPs, resulting in a reduction in protein size, an increase in the surface area of protein particles, and consequently, an elevation in fluid flow resistance [31]. Excessive ultrasonic power, on the contrary, might have accumulated cavitation effects, resulting in the disruption of the steady-state structure within proteins and, as a result, a decrease in fluid flow resistance [32].

Moreover, at the same ultrasonic power, the viscosity was greater under pH 11.0 conditions than under pH 12.0. This was consistent with the findings of Xue et al.[8], who observed that when recovering goose liver protein using alkaline treatment, the viscosity was highest at pH 11.0. As the pH became more extreme, the viscosity of the protein solution deteriorated. A popular explanation is that the more extreme pH led to excessively strong collisions between particles in the system, disrupting the interactions between protein molecules and thus reducing viscosity. Therefore, the combination of appropriate alkaline pH and ultrasonication was conducive to enhancing the viscosity of the protein solution, forming a more stable protein solution system. Alkaline isolation at pH 11.0 combined with 360 W ultrasonication might be an optimum process for enhancing the rheological properties of CLIPs.

3.4. Fourier transform infrared spectrum (FT-IR)

The effects of different ultrasonic powers on the secondary structure of CLIPs in the 4000–500 cm⁻¹ frequency range were investigated using FT-IR spectroscopy. The protein spectra of the water region (3500–3000 cm⁻¹), amide I region (1700–1600 cm⁻¹), and amide II region (1600–1500 cm⁻¹) were included in the CLIP spectrum Zhu et al. [33], which was shown in Fig. 4A. Because of the high sensitivity of the amide I region, it could be used to investigate deviations in protein secondary structure, such as α-helix, β-turn, β-sheet, and random coil. The symmetric stretching vibrations of carbonyl (C = O) functional groups and the characteristic hydrogen bonding for each type are used to make this distinction [34].

Fig. 4.

The secondary structure of ISP-isolated CLIPs at different ultrasonic power treatments (0, 180, 360 and 540 W). pH 11.0–0 W, pH 11.0–180 W, pH 11.0–360 W and pH 11.0–540 W represent pH 11.0 solubilization with 0 W, 180 W, 360 W and 540 W ultrasonic power treatment respectively. pH 12.0–0 W, pH 12.0–180 W, pH 12.0–360 W and pH 12.0–540 W were represented pH 12.0 solubilization with 0 W, 180 W, 360 W and 540 W ultrasonic power treatment respectively.

There were variations in the infrared absorbance of CLIPs in the amide I region when compared to the samples that were not ultrasonicated (Fig. 4A). This demonstrated that the secondary structure of CLIPs was considerably changed by ultrasonication. Fig. 4B shows the proportion of different types of secondary structures in proteins. Following ultrasonication, there was a decrease in α-helix and β-turn in CLIPs, whereas β-sheet and random coil increased (P < 0.05). This result could be explained by the dissolution of disulfide and hydrogen bonds as well as the disruption of molecular interactions among the CLIPs caused by ultrasonic treatment. It indicated that ultrasound led to protein denaturation, causing the loosening of protein molecules and a loss of protein structural stability, transitioning towards disordered structures [35]. Similar conclusions were also reported by Wang et al. [36], who found that ultrasound caused protein residues to be exposed to water molecules, to dissolve in some cases, to lose their structure, to form new hydrogen bonds, and to change from α-helix to β-sheet structures.

Furthermore, at the same pH, there was a slight increase in the percentage of α-helix in CLIPs after an initial decrease with an increase in ultrasonic power. In agreement, β-fold showed a rise at first, then a minor fall, with 360 W showing the lowest percentage of α-helix. This phenomenon might be explained because over sonication (540 W) could lead to partially dissolved protein precipitation and unfolded protein re-aggregation, which would be consistent with the findings of solubility (Fig. 1). Nonetheless, at both alkaline pH levels, the percentage of α-helix was lower under pH 11.0 conditions. Proteins unfolded more readily when the α-helix content decreased, exposing more hydrophobic groups and increasing surface hydrophobicity, this was consistent with the results reported by Wang et al. [37]. The findings presented above indicated that appropriate alkaline conditions and ultrasonic power better encouraged the secondary structure of CLIPs to improve, encouraging CLIPs to isolate.

3.5. Surface hydrophobicity (H₀)

Surface hydrophobicity (H₀) plays a crucial role in the stability, conformation, and functional properties of proteins [38]. BPB had the capacity to interact with hydrophobic sites on proteins, serving as an indicator of protein surface hydrophobicity [19]. The H₀ of CLIPs subjected to different treatments was illustrated in Fig. 5. When sonication was not applied, the H₀ at pH 12.0 was significantly higher than that at pH 11.0. This might be attributed to the greater degree of CLIPs unfolding at extremely alkaline conditions, which exposed more hydrophobic amino acids within [39].

Fig. 5.

Effects of different ultrasonic powers (0, 180, 360 and 540 W) on the surface hydrophobicity of ISP-isolated CLIPs. The different lowercase superscripts (a-c) represent significant differences at P < 0.05 level for the same pH sample under different ultrasonic power treatments; the different capital superscripts (A-B) represent significant differences at P < 0.05 level for different pH samples under the same ultrasonic power treatments.

After ultrasonication, at the same ultrasonic power, the H₀ at pH 11.0 was significantly higher than the extremely alkaline pH 12.0. The possible disruption of hydrogen bonds, electrostatic interactions, and hydration interactions between protein molecules caused by ultrasonication might have been the reason for this phenomenon. These effects might have greater significance than the unfolding of proteins in alkaline conditions [40]. Additionally, under identical pH conditions, there was a trend of first increasing and then decreasing in H₀ of CLIPs with an increase in ultrasonic power (P < 0.05). The H₀ increased by 30.09 % (106.85 μg) at pH 11.0 and 51.31 % (111.09 μg) at pH 12.0 under 360 W ultrasonic power. The results presented suggested that the intense turbulence, shear forces, and cavitation effects produced by ultrasound caused a structural reorganization of the protein, decreasing intermolecular binding and raising the H₀ of CLIPs [41]. Nevertheless, the H₀ at 540 W ultrasonic power was lower than that at 360 W. This could be attributed to the excessively high ultrasonic power causing protein aggregation, protecting the hydrophobic regions of CLIPs. This aligns with the findings of Yao et al. [42] regarding the impact of ultrasound on the functional properties of whey proteins.

3.6. Intrinsic fluorescence

Changes in the local tertiary structure of proteins can be reflected in fluorescence intensity and peak shifts. Protein conformational changes impacted the local molecular environment of phenylalanine, tyrosine, and tryptophan residues, which in turn caused changes in the fluorescence spectrum [33]. Fig. 6A showed that after ultrasonication, the fluorescence intensity of CLIPs increased significantly and there was a slight blue shift in comparison to the untreated samples. This was a consequence of the fact that the application of ultrasonic treatment caused the internal structure of the protein to dissociate, exposing hydrophobic groups within the molecules, rearranging the tertiary structure of the protein, and increasing its hydrophobicity [20]. When the ultrasonic power gradually increased at the same pH, the fluorescence intensity of CLIPs showed an initial increase followed by a decrease. Furthermore, there was a shift in the maximum emission wavelength from nearly 335 nm (0 W) to nearly 331 nm (360 W). These results suggested that an appropriate ultrasonic power facilitated the unfolding of the protein structure, exposing more amino acid residues on the protein surface. However, excessive ultrasonic power, leading to cavitation effects and shear forces, raised the temperature, resulting in protein aggregation and burying of amino acid groups. Similar findings were also confirmed by the results of Wang et al. [43].

Fig. 6.

Effect of different ultrasonic powers (0, 180, 360 and 540 W) on the fluorescence emission spectra (A) and UV (B) of ISP-isolated CLIPs. pH 11.0–0 W, pH 11.0–180 W, pH 11.0–360 W and pH 11.0–540 W represent pH 11.0 solubilization with 0 W, 180 W, 360 W and 540 W ultrasonic power treatment respectively. pH 12.0–0 W, pH 12.0–180 W, pH 12.0–360 W and pH 12.0–540 W were represented pH 12.0 solubilization with 0 W, 180 W, 360 W and 540 W ultrasonic power treatment respectively.

The fluorescence intensity of proteins at pH 11.0 was significantly higher than that at pH 12.0 under the corresponding ultrasonic conditions. This implied that a greater degree of unfolding of proteins isolated at pH 11.0 resulted in an increase in the intensity of protein fluorescence. This was consistent with the finding of Xue et al. [8], who found that there was a trend of pH 11.0 > pH 11.5 > pH 12.0 > acidic conditions in the fluorescence intensity of goose liver proteins extracted with various acid-base treatments.

3.7. UV spectra

Ultraviolet (UV) spectroscopy is closely associated with changes in the microenvironment of tryptophan and tyrosine residues, serving as a crucial indicator to reflect alterations in the tertiary structure of proteins. Most protein molecules have characteristic UV absorption spectra due to the light absorption of aromatic heterocycles in amino acids (such as tryptophan, tyrosine, and phenylalanine) [44]. Generally, it is easy to find these maximum absorption values at 280, 275, and 260 nm, respectively. Consequently, the locations of distinctive absorption peaks can be used to infer changes in protein structure [45]. The UV spectra of CLIPs after various treatments were shown in Fig. 6B, with a characteristic absorption peak at 272 nm, which could be attributed to perturbations caused by tyrosine.

Afterwards ultrasonication, the absorption peaks considerably increased in comparison to the untreated samples. This indicated that ultrasound could unfold proteins, exposing hydrophobic amino acid residues within the molecule, leading to an increase in UV absorption intensity [46]. Furthermore, at the same pH, there was a trend in the peak value of the CLIPs characteristic absorption peak that first increased and then decreased as the ultrasonic power increased. Likewise, at an ultrasound power of 360 W, the peak value of characteristic absorption peak was highest, while at an ultrasound power of 540 W power, it decreased. This could be a result of the originally exposed amino acids being reburied due to the cavitation effects produced by excessive ultrasonic power, which decreased the peak value of the characteristic absorption peak. This was in line with the findings of Yang et al.[47], who found that at higher ultrasonic densities (100 W/L), the UV absorption intensity of germ protein decreased.

The absorbance of the characteristic peak at pH 12.0 without ultrasonication was higher than that at pH 11.0 at the same ultrasonic power. The absorbance of the characteristic peak at pH 11.0 increased instead after ultrasonication. The findings implied that the alkaline environment enhanced the polarity of the tyrosine microenvironment in non-ultrasonic settings, increasing the UV absorbance of the protein. However, this enhancement might not be as significant as the impact of ultrasound on the tertiary structure of protein [29].

4. Conclusion

In this study, varying ultrasonic powers (0, 180, 360 and 540 W) were used to modify the structure and functional properties of ISP-isolated CLIPs. Ultrasonic treatment significantly increased the solubility of ISP-isolated CLIPs from 23.22 % (pH 11.0, 0 W) to 77.26 % (pH 11.0, 360 W), decreased the particle size, disrupted protein aggregation and increased shear stress and apparent viscosity of ISP-isolated CLIPs, companied by the changes in protein secondary structure, which causing a decrease in β-turns and α-helices, an increase in β-sheets and random coils, and altering tertiary structure to enhance the exposure of hydrophobic residues of the protein. It is noteworthy that ultrasonic treatment does not always lead to effective modification of protein structure. Excessively high ultrasonic power (such as 540 W) can cause the modifying impact to diminish. Utilizing 360 W ultrasonic treatment in conjunction with alkaline (at pH 11.0) process was an economical and environmentally friendly approach to improve the functional properties and modify the structural changes of CLIPs, which would increase the processing potential of water and salt-insoluble animal-derived proteins.

CRediT authorship contribution statement

Rongrong Mao: Writing – original draft, Methodology, Investigation, Formal analysis. Guoyuan Xiong: Writing – review & editing, Methodology, Investigation, Funding acquisition, Conceptualization. Haibo Zheng: Resources, Investigation. Jun Qi: Resources, Data curation. Chunhui Zhang: 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.

Appendix A. Supplementary material

The following are the Supplementary data to this article: