The actomyosin complex samples’ surface hydrophobicity was assessed using Chelh’s (35) methodology with a few changes. With 0.6 M KCl phosphate buffer (pH 7.5), the samples were diluted to 0.00125, 0.0025, 0.005, and 0.02 mg/ml, respectively. A total of 20 μL of 8 mM 8-anilino-1-naphthalenesulfonic acid (ANS) solution was then added to 4 ml of the sample solution. A SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA) was used to detect the reaction solution at excitation wavelengths of 390 nm and 470 nm with a slit width of 3.0 nm. The slope of the relative fluorescence vs. the percent protein content (w/v) of the samples was used to determine their H₀.

By utilizing the technique described by Deng et al. (36), the Fourier Transform Infrared Spectroscopy (FT-IR) module (Tensor II, Bruker Co., United States) determined the infrared spectroscopy of MPs. Protein was produced by pressing protein isolates in a vacuum hydraulic press after mixing them with moisture-free potassium bromide at a ratio of 1:100 (w/w). FTIR spectra of protein isolate pellets was recorded in the range of 4000–400 cm⁻¹.

Fluorescence spectra were examined in order to learn more about the modification of protein tertiary structure. The experiment was carried out with a fluorescence spectrophotometer (F-7000, Hitachi Co., Tokyo, Japan) using a modified version of Xu et al.’s (37) approach. The freeze-dried sample was dissolved in 0.01 mol/L of phosphate buffer to create the sample solution (0.2 mg/ml) (pH 7.0). With a constant slit of 5 nm for both excitation and emission, the solution was excited at 290 nm, and emission spectra were obtained from 300 to 460 nm.

The solubility of MPs, as defined by Agyare et al. (38), was calculated with minor adjustment. One gram of sarcoplasmic protein was removed from the muscle, inserting 20 ml of ice-cold 0.1 M phosphate buffer (pH 7.2, 1.1 M potassium iodide), shaking 20 s, 10,000 rpm for three times, and then left on a shaker at 4°C for 12 h. Samples were centrifuged at 1,500 g for 20 min, and the concentration of protein in the supernatants was measured by the Biuret process.

The turbidity was measured according to the method of Benjakul et al. (39), and 5 mL of protein solution which was adjusted to 1 mg/ml was added into a 10-ml centrifuge tube, heated in the water bath at 80°C for 30 min, and then cooled to room temperature. Protein aggregation was monitored continuously by measuring the absorbance at 660 nm.

The emulsifying properties were measured according to Pearce and Kinsella (40) with slight changes. A total of 10 ml of corn oil (filter pressed corn oil, Yihai Kerry Arawana Co., Ltd., China) and 30 ml of 1 mg/ml of SMP solution in 0.1 M phosphate buffer (pH 7.0) were shaken together and then homogenized at 10,000 rpm for 1 min at room temperature. A total of 200 μL of emulsions were taken from the bottom of the container after being placed undisturbed for 10 min and dispersed into 10 ml of 0.1% SDS solution. The absorbance was recorded at 500 nm with 0.1% SDS as a blank control. Emulsifying activity index (EAI) and emulsion stability (ES) were defined as:

where A₀ and A₁₀ represent the absorption value of the emulsion at 0 and 10 min, respectively.

Foaming properties were carried out by using the method of Agyare et al. (41). 50 mL of SMP solution (1 mg/mL, pH 7.0) foaming solution was add into a 100 mL graduated cylinder, and whipped with a rotating anchor at 15000 rpm for 2 min at room temperature, and the height V₀ of foam was recorded after the end of whipping and V₃₀ after 30 min. The foaming capacity (FC) and foaming stability (FS) were calculated according to the following formulas, respectively:

The MPs were suspended at a concentration of 40 mg/ml in phosphate buffer (20 mM, pH 7.0) containing 0.6 M NaCl. At a rate of 1°C min^–1, the solutions were heated from 25 to 75°C. After that, the heat treatment was kept at 75°C for 20 min. The resultant samples were kept at 4°C for 12 h after being chilled to 4°C in an ice bath. The preparation of each gel sample was done in three copies.

The method reported by Xia et al. (42) was somewhat modified to assess gel strength using a TA-XT texture analysis meter fitted with a cylindrical probe (P0.5 diameter). In a nutshell, each sample (15 mm × 15 mm × 15 mm) was subjected to two-cycle compression under the following circumstances: pretest speed of 5 mm/s^–1, test and posttest speed of 1 mm/s^–1, trigger force of 5 g, and ultimate distance of 30%. The breaking force (g) and distance (mm) were multiplied to determine the gel strength (g × mm).

The water-holding capacity (WHC) of the MP gels was calculated using a slightly modified version of the Xia et al. (42) approach. In a nutshell, the supernatant fluid was drained after centrifuging 3 g of the gel samples at 10,000 g for 15 min at 4°C. The weight of the gel after centrifugation to the weight of the gel before centrifugation was used to calculate WHC.

The primary mechanism preserving the protein’s tertiary structure is the interaction of hydrophobic groups. As a result, the distribution of hydrophobic groups on the protein surface can accurately represent the stability of the protein’s structure and its functional characteristics (43). As shown in Figure 2, surface hydrophobicity of all feeding treatments showed a significant increase with storage time (P < 0.05) and reached a maximum at 72 h among different feeding groups, which was consistent with the finding of Mignino et al. (44) on MP of the mantle from frozen-stored squid that surface hydrophobicity increased with aging time prolonged. More hydrophobic groups become exposed during storage, weakening the hydrophobic connection between the protein molecules and increasing the hydrophobicity of the protein’s surface (45). However, MP surface hydrophobicity at the same aging time was basically showing a downward trend with the supplement of selenium yeast and jujube powder. The resemblances among different post-mortem periods were that MP surface hydrophobicity of treatments fed with selenium yeast or jujube powder was significantly lower than that of the CK group, and the surface hydrophobicity of the treatments with jujube powder was lower than that of the treatments without jujube powder. The above results confirmed the significant interaction effect of selenium yeast and jujube powder (P < 0.05) and the significant main effect of selenium yeast or jujube powder (P < 0.01). Selenium is a component of a selenoprotein methionine sulfoxide reductase B, which prevents protein oxidation, a key factor in the deterioration of meat quality during storage (46). Because hydrophobic amino acids are buried within the protein, it is generally accepted that a decreased surface hydrophobicity suggests the folding state of the protein structure which depends on its oxidation degree. In addition, it is generally accepted that a decreased surface hydrophobicity suggests the folding state of the protein structure and that the hydrophobic amino acids are buried within the protein (47). Also, the non-covalent interactions that occur between polyphenols of jujube powder and proteins led to lots of hydroxyl groups linked at the aromatic ring being introduced to the surface of the protein molecule, thus decreasing the surface hydrophobicity (48, 49), which was consistent with the founding reported by Meng and Li (50) that the addition of rye polyphenols to the ration significantly reduced the surface hydrophobicity of loach.

The α-helix and β-sheet characterize the regularity of protein molecules, which are stabilized by intra- and inter-hydrogen bonds between peptide chains (51), whereas random coils usually reflect a looser structure. It can be seen in Figure 2 that all treatments showed a similar changing trend, i.e., the content of α-helix gradually decreased, while that of β-sheet increased as maturation progressed. Similar to the study by Andersen et al. (52), extensive proteolysis of large structural proteins occurred post-mortem. Exposure of hydrophobic groups could lead to a reduction in intramolecular hydrogen bonds; therefore, the increase in β-sheet structure appeared to be due to the decrease in the α-helix and turn structure (53). Following the addition of selenium yeast and jujube powder to the diet, significant effects on the content of α-helix and β-sheet (P < 0.01) were observed, and there was a very significant interaction between these two additives. In the selenium yeast and jujube powder groups, there was an increase in α-helical content and a decrease in β-sheet content in chicken MP compared to that in the CK group. However, a significant difference was observed only between the high-dose selenium yeast and compound group, and no significant difference in α-helix and β-sheet content between the CK+J group and 0.3S group (P > 0.05) was found. There were no significant differences in the irregular curl content between the feeding groups. The highest α-helix content in chicken MP was in the 0.6 mg/kg selenium yeast and jujube powder group at 0 h post-mortem, which was 37.48%; meanwhile, the highest β-sheet content was found after 72 h of aging in the CK group, which was 38.39%, close to the level of highest α-helix content, which illustrated the switch between these two structures when the degree of protein oxidation was changed. Although there is little information regarding the effect of feed additives on protein secondary structure, existing research may provide evidence on how muscle antioxidant capacity is promoted by different dietary supplements. As an integral part of selenoproteins, dietary Se is a crucial factor regulating GSH-Px activity and the efficiency of the antioxidant system (54). By reducing the oxidation levels, the structural integrity of MP is maintained, leading to the prevention of damage to the hydrogen bonds within the α-helix and then turning into an unstable β-sheet (51).

An essential metric for observing changes in the protein matrix is the intrinsic fluorescence spectrum. The primary fluorescent groups are aromatic residues, namely, tryptophan (Trp) and tyrosine (Tyr). The number of aromatic residues exposed inside the protein is represented by the relative intensity of the maximal fluorescence emission wavelength (λ_max). The changes in the tertiary structure of MPs caused by feeding selenium yeast and jujube powder were evaluated by monitoring the changes in the inherent fluorescence intensity of Trp residues. The fluorescence emission spectra of MP modified by feeding selenium yeast and jujube powder compounds are shown in Figure 2. Considering the post-mortem effect, MP fluorescence decreased with prolonged aging within the same treatment group. At the same aging time, the MP of the CK group exhibited the lowest fluorescence intensity compared to that of all treatments. Trp residues are highly sensitive to the polarity of their microenvironment, suggesting that the protein fluorophore may have been altered as a result (55, 56). Trp residues are frequently found in the hydrophobic core of the folded proteins, where they display high quantum yield and high fluorescence intensity (57). Trp residues are exposed to a solvent (hydrophilic environment) when proteins become partially or entirely unfolded, which causes a decrease in fluorescence intensity. Therefore, the more complete the MP structure, the higher the observed fluorescence intensity. The 0.6 S + J group showed the highest fluorescence intensity, which is also shown in Figure 2 (left side). Selenium, an active component of GSH-Px, which is an endogenous antioxidant enzyme widely present in the animal body, participates in the defense system against animal peroxidation and catalyzes the oxidation of glutathione (GSH) to generate oxidized glutathione (GSSG), thus protecting the structure and function of cells (58). Generally, the λ_max of the intrinsic fluorescence of each treatment at the indicated post-mortem time points increases from 334 to 335 nm (Table 4). In contrast, the λ_max of intrinsic fluorescence at the same post-mortem time point decreased from 335 nm in CK to 334 nm in 0.6S+J. In other words, a red shift occurred during the post-mortem period, but a blue shift occurred with the addition of selenium yeast and jujube powder. This might be related to the stability of the protein structure, corresponding to an increase in the helix content in the secondary structure. Dietary selenium yeast and jujube powder may leave most of the secondary structural elements of MP intact by reducing its oxidation; therefore, as a sensitive index of conformational changes in the tertiary structure, Trp residues will fluoresce in a manner that is dependent on the folding of the protein, and the λ_max shift to shorter wavelengths (blue shift) indicating a decrease in the polarity of the Trp residue microenvironment. This might be attributed to the fact that antioxidant supplementation protects the internal hydrophobic interactions of protein molecules and induces molecular folding (36).

Solubility is a practical index to measure protein denaturation and aggregation, and it can affect many other functional properties of the protein. The solubility of MP decreased with the aging period (Table 5). Similar to the research of Xiong and Brekke (59) on protein denaturation in broiler breast meat, the extensive aggregation involving both disulfide bonds and non-disulfide interactions between protein subunits resulted in the solubility reduction which can be proved in the increase in surface hydrophobicity and a decline in α-helix. The addition of selenium yeast and jujube powder in the diet increased the solubility of chicken MP. At 0 h of the aging period, MP solubility of the highest-dose compound group was 34.84%, which was an increase of 33.79% compared with the CK group. However, except for the CK group, there was no significant difference in MP solubility in the other feeding groups; at the 24 and 72 h, the solubility of chicken MP also increased with the supplement of selenium yeast and jujube powder, but the differences were only significant between CK, CK+J, 0.3S, and other three treatments (P < 0.05). Studies have shown that adding selenium yeast to the diet can increase the solubility of chicken MP, but the difference is not significant (P > 0.05); the addition of jujube powder can significantly increase the solubility of chicken myofibril protein after slaughter (P > 0.05). Although the effect of the combination of the two is not significant (P > 0.05), the 0.6S+J feeding group has the best effect on increasing protein solubility. Xu et al. (7) ingested that high dietary valine decreased sarcoplasmic protein solubility, while it did not alter the solubility of MP and total protein relative to the ingestion of lower dietary valine (P > 0.05). Interestingly, Xu et al. (60) found that L-Val/H-Ile treatment led to the highest MP solubility and total protein solubility of pork. This demonstrated that dietary antioxidation could modify the solubility of animal protein, but depend on the kinds of muscle protein and the level of additions. Relkin and Shukat (61) found that the solubility of potato protein decreased due to polyphenols demonstrating that the precipitation was induced by protein–polyphenol conjugates rather than solubilization in an aqueous neutral media.

An essential metric for assessing protein binding and aggregation is turbidity. The degree of protein aggregation increases with increasing turbidity values (61). From Table 5, it can be seen that as storage time increased, the turbidity values increased for the control and addition supplementary groups (P < 0.05). This could be attributed to the increased degree of protein denaturation and aggregation, which affects light scattering and hinders light propagation, thus increasing the absorbance (62). With the increase of selenium yeast content in the diet, the turbidity is extremely reduced (P < 0.01); compared with the treatment group without jujube powder at the same level, the turbidity of the group treated with compound jujube powder was significantly lower (P < 0.01), and there was a significant interaction between selenium yeast and jujube powder (P < 0.05). Compared with the CK group, the MP turbidity in the group was reduced by 53–70%. Compared with the CK group, the addition of selenium yeast and jujube powder or the combination in the diet can significantly reduce the turbidity of chicken myofibril protein (P < 0.05). Studies have shown that both selenium yeast and jujube powder can reduce the turbidity level. Selenium yeast and jujube powder interact significantly (P < 0.05), and the combination of the two has the best effect. The 0.6S+J group chicken myofibril protein turbidity was the lowest among all treatment which indicated that the protein exhibits a higher degree of aggregation. Utrera et al. (63) studied the changes in beef quality during frozen storage and reported similar results that protein solubility represented contrast law to turbidity, the physical factors of antioxidants additives might protect the hydrogen bonds and hydrophobic interactions which were responsible for the intermolecular association of protein aggregates. For this reason, selenium yeast and jujube powder diet affected the oxidation degree of protein, hydrogen bonds inside the secondary structure were protected, and thus the hydrophobic groups were prevented from exposure, hence the solubility of MP increased and turbidity decreased.

The emulsifying activity index (EAI) is determined by protein–protein and protein–lipid interactions. Emulsifying stability (ES) referred to the ability of an emulsion to resist creaming, flocculation, and coalescence (64). As shown in Table 5, the EAI and ES of MP reduced in all feeding groups with the post-mortem time prolonged. Protein structural alterations brought on by oxidation, as shown by surface hydrophobicity and FT-IR studies, affected the protein–protein interactions. When adsorbed on the surface of the oil droplets, the cross-linked MP was too big to maintain their flexibility (65). The supplement of selenium yeast or jujube powder to the diet had a very significant effect on the EAI and ES of chicken MP (P < 0.01), but the two only have significant interactions on EAI (P < 0.05). Compared with the CK group, the EAI and ES of the 0.6S+J histone increased significantly (P < 0.05), which were 1.15 m²/g and 39.76%. Among the J group, 0.3S group, 0.6S group, and 0.3S+J group, though the EAI and ES gradually increased, there was no significant difference (P > 0.05). Zhao et al. (17) have studied that the high-dose GML feed supplementation improved the egg white protein emulsion stability during long-term storage. The higher surface hydrophobicity was indicative of increased protein flexibility, which led to an increase in repulsion between droplets in the emulsion by hydrophobic interactions. Therefore, the addition of selenium yeast or jujube powder could protect the protein from oxidative degradation resulting in the increase of Trp natural fluorescence, leading to the protein–lipid interaction changes. However, different outcomes were found by Zhao et al. (17) that there were no significant differences in EA or ES of fresh egg yolk from laying hens fed docosahexaenoic acid-rich microalgae (P > 0.05), which indicates that DHA enrichment has not changed the emulsification properties of egg yolk.

The foamability depends on the relationship between the final volume of the solution and the volume of the formed foam, while foam stability is based on the ability of the protein to stabilize the foam against gravitational stress (66). Changes in the foaming capacity (FC) and foaming stability (FS) of chicken MP are shown in Table 5. The FC and FS of post-mortem chicken MP of all treatments were reduced with aging time prolonged. As mentioned earlier, the surface hydrophobicity of MP was increased during storage, as Dong et al. (67) indicated that FS is primarily influenced by protein concentration, hydration, and interactions between hydrophobic residues. The addition of selenium yeast and jujube powder in the diet makes the foamability and foam stability of chicken MP, thus showing an upward trend after slaughter. With the increase in selenium yeast addition level, the foaming property and foam stability increased significantly (P < 0.05); and compared with the treatment group without jujube powder at the same level, the foaming property and foam stability of the group treated with compound jujube powder were higher. Compared with the CK group, the addition of selenium yeast and jujube powder to the diet can significantly increase the foaming of chicken MP (P < 0.05). Studies have shown that the addition of selenium yeast and jujube powder to the diet can increase the foaming and foam stability of chicken myofibril protein, and the combination of the two has the best effect. The 0.6S+J group chicken myofibril protein foams had the highest performance and foam stability. Foaming properties of EWP which were got from dietary GML supplementation have been studied, and the same results were found (17). The increase in FC induced by dietary GML supplementation is associated with the effects of GML on the protein composition and concentration of EWP, which lead to the exposure of more hydrophobic groups and improved protein–air interactions. Consistent with our results, dietary selenium yeast and jujube powder supplementation induced partial unfolding of the protein structure, leading to rapid adsorption at the freshly formed air–water interface and forming viscoelastic films, thus leading to greater stability (66).