3.1. FTIR
FTIR, as a biochemical fingerprint of protein, provides valuable information about protein secondary structure, composition, and molecular structure in functional groups (Sinthusamran et al. 2015). Studies indicated, to some extent, similar FTIR spectra for different gelatin types. The five distinguished regions in FTIR spectra of gelatin are apparent, including amide I, amide II, amide III, amide A, and amide B (Casanova et al. 2020).
Amide I region observes in the wavenumber range of 1600–1700 cm− 1. The stretching vibrations of C = O along with C-N stretching vibration in peptide bonds, Cα-C-N bending, and in-plane bending vibrations of N-H are related to amide I. An absorption peak at 1633 cm− 1 is the distinctive peak of gelatin helical structure. As shown in Fig. 1, this peak can be seen from 1631 to 1637 cm− 1 depending on the applied treatment. According to Ahmad and Benjakul (2011), when the peak shifts to a higher wavenumber, gelatin has lost molecular order as a result of the disintegration of intermolecular cross-links (Ahmad et al. 2011). Results indicated that three HPH cycles of gelatin with different concentrations increased wavenumber compared with the control, which suggests the greater loss of molecular order in this treatment. However, two and three HPH cycles treatment of gelatin solutions decreased the wavenumber of the amide I band. It seems that protein-protein cross-links are formed due to the unfolding of gelatin. Chen et al. (2017) reported that HPH treatment of myofibrillar proteins could disrupt intra-molecular hydrogen bonds and change the protein structure (Chen et al. 2017). HPH treatment induces forces through cavitation, impact, shear, and turbulence. These forces may modify the stabilizing force of protein structures like hydrogen bonds, ionic interactions, hydrophobic interactions, and disulfide bonds (Schultz et al. 2004). Among them, hydrogen bonds are sensitive to high pressure and could form and break easily (Carullo et al. 2020). It seems that a single HPH cycle causes disruption of molecular interactions, which results in the unfolding of gelatin structure and the highest internal functional groups were exposed on the surface. The application of two and three homogenization cycles increases the collision probability between functional groups. These collisions lead to the formation of intermolecular hydrogen bonds, hydrophobic interactions, and covalent bonds, which produce molecular aggregates. Furthermore, Dai et al. (2020) reported that the increasing hydrogen bond in gelatin molecules expands the space in gelatin molecules (Dai et al. 2020). These changes in the microstructure of gelatin gels are observable in FESEM images (Fig. 5).
Amide II region appeared in the wavenumber range of 1574 to 1480 cm− 1 and is related to the stretching vibration of amino acids NH3+ groups and bending vibration of NH along with out of plane CN stretching vibration. Amide II characteristic peak was observed at 1533 cm− 1 (Fig. 1). Dry collagen showed the amide II band at the wavenumber range of 1530–1540 cm− 1 with a small peak at lower frequencies (Tu et al. 2015). The reduction of amide II band wavenumber indicates higher participation of the NH group in forming hydrogen bonds with adjacent molecules (Ahmad and Benjakul 2011). The two and three HPH cycles of gelatin solution significantly decreased the wavenumber of the amide II band (Fig. 1), suggesting the involvement of the NH group in the hydrogen bond formation. The vigorous mechanical forces, i.e., the combination of high pressure with turbulence, shearing, and cavitation, applied during HPH treatment, can unfold gelatin protein and influence protein structure through dissociation of the hydrogen bond (Chen et al. 2017). It has been found that the hypsochromic shift occurs after gelatin and collagen denaturation. Therefore, three HPH cycles of 9% gelatin solution (10-cm hypsochromic shift) denatured gelatin and unfolded polypeptide chains due to reducing intra-molecular hydrogen bonds. The unfolded protein structure allowed protein alignment very closely in neighboring chains, which provided the opportunity to form very strong intermolecular bonds (Harnkarnsujarit et al. 2015).
The amide III region at 1200–1400 cm− 1 includes C-N stretching vibration and N-H bending vibration that is created due to Amide bonds (Baia et al. 2016). Amide III band of gelatin is seen in 1234 cm− 1 (Sinthusamran et al. 2014). Three homogenization cycles of 9% gelatin solution lead to the exhibition of the amide III at lower wavenumber, suggesting the higher gelatin irregularity associated with higher dissociation (Karnjanapratum et al. 2017).
Amide A represents the stretching vibration of NH groups paired with hydrogen bonding and appears at the wavenumber range of 3400–3440 cm− 1. When the peptide NH group participates in a hydrogen bond, the amide A band indicates a hypsochromic shift to 3300 cm− 1 (Ahmad et al. 2018). Sinthusamran et al. (2014) stated that the bathochromic shift of the amide A band is related to the higher amino group content due to protein degradation (Sinthusamran et al. 2014). It seems that protein unfolding and exposure of NH groups in the side chain of amino acid is probably related to the change of band situation.
Amide B band arises from asymmetric vibration of = CH and –NH3+ groups (Sila et al. 2017). All samples indicated amide B shift to the lower wavenumbers, proposing an increase in free –NH3 groups on the protein chains (Fig. 1).
3.2. Free sulfhydryl
Free sulfhydryl groups and disulfide bonds have a critical role in the structure and function of the protein as well as protein-protein interaction. They are also necessary for the functional properties of proteins and can influence the gelling properties, digestibility, and aggregation of proteins (Briviba et al. 2016). According to Fig. 2, the free sulfhydryl content of gelatin increased upon HPH treatment while it was reduced by increasing the number of homogenization cycles. The alteration of free sulfhydryl content indicates the tertiary and quaternary structure change (Zhang et al. 2017). The exposure of interior free sulfhydryl groups, as a result of protein extension and unfolding, caused the increasing free sulfhydryl content (Zhang et al. 2015b). Shi et al. (2021) also found that three cycles of whey protein isolate HPH up to 100 MPa could increase free sulfhydryl content (Shi et al. 2021).
HPH treatment can induce water penetration into the internal parts of proteins and leads to the stretching and unfolding of the molecules. In this condition, the tertiary and quaternary structure of protein destabilized. The hydrophobic regions, which were placed in internal parts of the protein and restricted by a non-polar environment, were thereupon subjected to an aqueous environment with the unfolded structure.
The free sulfhydryl content decreased substantially with increasing homogenization cycles at all gelatin concentrations (Fig. 2), suggesting the reaction of sulfhydryl groups to form S-S bonds at higher homogenization cycles (Chen et al. 2018). The SH groups can take part in the oxidation reaction and produce protein oligomers and aggregates through disulfide bond formation (Shi et al. 2021). Briviba et al. (2016) believed that in addition to oxidative conditions, the relatively high energy applied during HPH could result in the formation of disulfide bonds. Also, sulfoxide and sulfonyl groups are formed due to further oxidation, which can reduce free sulfhydryl content (Briviba et al. 2016).
3.3. UV absorption spectra
The UV absorbent chromophores in proteins are categorized into two groups: aromatic side chains and peptide bonds. Aromatic side chains absorb UV at 280 nm. The absorbance at this wavelength has been generally applied for determining protein concentration as well as monitoring the structural alteration of proteins. Absorbance at 190 nm by amide bonds indicates conformational changes of proteins (Liu et al. 2009b). If the peak intensity and position change after HPH treatment, it can represent that a change in the micro-environment of aromatic amino acids has happened (Han et al. 2020).
The UV spectra and absorbance of the HPH treated gelatin were evaluated as a function of gelatin concentration and homogenization cycles to track alteration in protein conformation and structural modification. According to Fig. 3, the absorption intensity of the HPH treated gelatin after one pass was higher than that of the untreated one, probably as a result of hydrophobic groups' exposure due to gelatin unfolding. Also, the increasing absorption spectra suggest the protein trend for rearrangement/aggregation in comparison with native protein (Ekezie et al. 2019). After HPH treatment of β-lactoglobulin, Dumay et al. (1994) reported a considerable increase in surface hydrophobicity due to the protein unfolding, which was immediately pursued by the production of soluble aggregates (Dumay et al. 1994). Rosenheck and Doty (1961) stated that conditions like oxidation of disulfide bonds or heat treatment that leads to the protein unfolding are associated with higher absorbance of protein (Rosenheck and Doty 1961). However, with increasing the number HPH cycles, the absorbance intensity at λmax was decreased, suggesting the large aggregates formation or embedding of hydrophobic groups in the protein (Huang et al. 2019). Hu et al. (2021) proposed the reassembly of the partially unfolded protein molecules as a reason for the decrease of UV absorption inrensity (Hu et al. 2021). Han et al. (2020) indicated that the UV/visible absorption intensity of casein after HPH treatment decreased with increasing pressure to 120 MPa. They proposed that HPH treatment can cause masking of the tyrosine and tryptophane chromophores that were subjected to the outside (Han et al. 2020). Against Han et al. (2020), Xu et al. (2018) related the reduction of UV absorption after HPH treatment of soy protein isolate to the exposure of chromophore amino acid residues to the protein surface (Xu et al. 2018b).
The UV absorption spectra of intact protein are principally characterized using aromatic amino acid residues, mainly tryptophane and tyrosine. Phenylalanine residues and disulfide bonds, however, have a minor impact on the UV spectra. Results indicated that the maximum absorbance peak of gelatin was at 191, 196, and 196 nm for 3, 6, and 9% gelatin solution. The red-shift of UV absorption spectra with increasing gelatin concentration is due to the high amount of functional groups C = O, –COOH, and CONH2, which change the λmax at UV range (Akram and Zhang 2020). The maximum UV absorption was altered to 204, 207, and 209 nm after one cycle of homogenization, respectively (Fig. 3). The bathochromic shift of gelatin spectra after homogenization shows the electrostatic interactions between chromophores like glutamic and aspartic acid as well as carboxylic groups with amino groups, which absorb near-UV wavelengths (Derkach et al. 2020a). Hu et al. (2021) demonstrated that the addition of chitosan to potato protein isolate made a bathochromic shift due to the alteration of tryptophane and tyrosine microenvironment, which weakened the hydrophobic interactions (Hu et al. 2021). The results of FTIR also confirmed that one cycle of HPH unfolded the protein structure which was followed by protein aggregation at higher HPH cycles.
3.4. Circular dichroism spectroscopy
Circular dichroism (CD) spectroscopy is applied for estimating the polypeptide and protein secondary structure (Gopal et al. 2012). Based on data obtained from circular dichroism spectroscopy, any activity in the region of 190–230 nm except that related to the presence of aromatic amino acids is basically related to the peptide structure of proteins. The lateral structure of aliphatic amino acids plays no role in the formation of this region in the spectrum (Liu et al. 2017). The peptides with unordered structures indicate a single band around 200 nm, while α-helix structures commonly demonstrate a single positive band near 192 nm as well as two negative bands at 208 and 222 nm. A positive band at 195 nm and a negative band at 217 nm is characteristic of β-sheet structure (Gopal et al. 2012).
As can be seen in Fig. 4, the CD spectra of gelatin indicated a positive peak at 220 nm characteristic of the gelatin triple helix structure and a negative peak at 198 nm, suggesting a random coil structure (Liu et al. 2017). Compared to untreated gelatin, homogenized gelatin indicated a considerable decrease in the negative peak of the dichroic spectrum along with a slight increase or decrease in the intensity of the positive band at 220 nm. It has been found that if gelatin denatures completely, the negative band at 198 nm moves to 230 nm while the positive band disappeared (Wüstneck et al. 1988). The results demonstrated that the integrity of the gelatin structure preserves after homogenization without denaturation.
The negative peak at 198 nm displayed a remarkable increase with the increasing homogenization cycle. Mahmood et al. (2019) stated that the increase in negative ellipticity at 198 nm, suggesting the alteration of gelatin structural packing (Mahmood et al. 2019). Another reason for the deeper negative ellipticity at 198 nm may be related to the increase in the telopeptide region of gelatin. Holmes, Kirk, Tronci, Yang, and Wood (2017) displayed that monomeric atelocollagen has lower intensity at 198 nm compared with monomeric collagen. In atelocollagen, the terminal telopeptides are cleaved, and intact triple-helical are produced (Holmes et al. 2017). Therefore, it seems that the presence of the telopeptide region can increase the intensity of the negative peak at 198 nm. It seems that upon collision of the protein to narrow valve of high-pressure homogenizer, cavitation, turbulence, and high shear stress cause some destruction to the helical structures (Han et al. 2020). Hydrogen bonds are disrupted by HPH treatment, eventuating in a secondary structure transition (Shi et al. 2019) and increasing the telopeptide regions. Therefore, more hydrogen bonds are broken with increasing the number of homogenization cycles; which leads to the enhancement of the telopeptide region and deeper negative ellipticity at 198 nm. Results of FTIR and UV absorption spectra indicated that at two and three homogenization cycles, protein aggregation occurs. Since the α-helix structures decreased with increasing homogenization cycles, it seems that the formation of hydrogen bonds at two and three HPH cycles has led to the creation of unordered structures like random coils. On the other hand, the intensity of this peak increased at higher gelatin concentrations due to the presence of more telopeptide regions.
3.5. Field emission scanning electron microscope
One of the variables considered in this study was gelatin concentration. As can be seen in Figs. 5-A, E, and I, the pocket-shape pores with different sizes, which were partitioned using thin walls and did not interconnect, were formed in untreated gelatin gel. Also, the untreated gelatin had a tendency to non-uniformity.
The porosity and pore size of gelatin gel was influenced by gelatin concentration. It is evident that the pore size and porosity decreased with an increasing gelatin amount. Two hypotheses can be stated for the observations. First, it seems that with increasing gelatin concentration, the nucleation rate is enhanced and results in a more pore number. Nucleation occurs following the liquid phase instability as well as atom diffusion into clusters. The consequence of the increasing gelatin concentration is higher atom mobility and diffusivity into the cluster, increasing the nucleation rate. Second, the protein transfer in the concentrated gel is lower, resulting in smaller pore size.
Figures 5-B, C and D indicate the FE-SEM images of 3% gelatin gels after one, two, and three HPH cycles, respectively. The destruction of massive blocks in the untreated sample was observed, which provided a porous structure with defined shapes. Gelatin gels indicate some difference in pore size with an increasing homogenization cycle. Furthermore, the pore shape changed from a polygon, a well-oriented shape to an unordered structure after three homogenization cycles. Another observable change is the significant reduction in cell wall thickness alongside with increasing homogenization cycles.
Proteins are regularly in a metastable state based on molecular configuration (Bechtel 1995). HPH contributes to macromolecules structural changes through a combination of shear, cavitation, and impact forces. Therefore, the HPH can interrupt the wrinkled structure of protein because of the change in the disulfide content (Fig. 2) and intermolecular hydrogen bonds (Fig. 1). Homogenization of the gelatin with one pass at 100 MPa leads to some extent disruption of the protein structure. The limited unfolding of the protein subject a small amount of intrinsic group on the surface. Thus, the hydrophobic interaction, intermolecular hydrogen, and disulfide bonds enhance, which can promote the formation of a regular network with thick walls (Fig. 5-B). When the gelatin is homogenized with double HPH cycles, the disintegration of folded structure of the protein increases considerably. The intermolecular functional groups are exposed to the surface of the protein molecules. Hence, the possibility of functional group interaction increases alongside their exposure on the surface which leads to the formation of hydrophobic interactions, hydrogen and covalent bonds between protein strands (Fig. 5-C). The three times passe of gelatin at 100 MPa could reach the protein to the highest degree of disintegration. It seems that at this point, the electrostatic repulsion is at the maximum extent. The repulsive force does not allow protein strands to get close to each other. The integral effect of maximum unfolding and electrostatic repulsion promoted the formation of large pores with thin cell walls (Fig. 5-D).
At the gelatin concentrations of 6 and 9%, increasing the homogenization cycles did not have a significant effect on cell wall thickness; however, pore size was decreased to some extent. Also, the polygonal shape of cavities was apparent (Figs. 5-F, G, H and J, K, L). As mentioned before, during HPH treatment of gelatin solution, the cavitation, shear, and impact forces are exerted on the protein simultaneously. Al Mahmud, Hasan, Khan, and Adnan (2020) evaluated the effect of cavitation in gelatin hydrogel. They explained that after 25 ps, the interaction of bubbles with the gel networks starts. Therefore, the bubble spherically index in the gel is reduced compared to the bubble spherically index in water, and the surface area of bubbles in the gel is enhanced in comparison with the bubble in water. Hence, higher energy is needed to grow cavitation bubbles in gel (Al Mahmud et al. 2020). During HPH treatment of gelatin solution, although there was no gel network, the viscosity of the solution increased alongside gelatin concentration. At the concentrations of 6 and 9%, higher energy is required to overcome gelatin solution viscosity. In this condition, the cavity bubbles grow; however, it seems that the bubble collapse does not occur. Consequently, bigger pores are observable in 6 and 9% gelatin solutions compared to 3% protein solution, which bubbles implosive more easily. Also, higher gelatin concentration led to a reduction of bubble spherically index.
A Computational Fluid Dynamic (CFD) model was used by Miller et al. (2002) to explain the influence of fluid viscosity on different fluid dynamics parameters that seem to participate in HPH treatment. They predicted that the viscosity of fluid would affect the impact pressure, extensional stress, and turbulence. When a fluid moves over a surface at high speed, turbulence occurs. Surface roughness leads to the deviation of fluid streamline from the surface shape above a given velocity, which consequence is vortices formation. After this event, fluid particles move disorderly. Based on Reynold's number, turbulence is more predominant in low viscosity fluids than high ones, which participate in cell disruption. Miller et al. (2002) also estimate a significant decrease in impact pressure with increasing viscosity (Miller et al. 2002), justifying the lower influence of HPH treatment on 6 and 9% gelatin gel microstructure than 3% one.
Sha et al. (2018) homogenized the fish gelatin at 80, 120, and 160 MPa. They observed cellular shape pores with a rough and loose structure in gelatin gel (Sha et al. 2018). After processing of whey protein isolate with a microfluidizer, Liu et al. (2011) found that the globular shape of whey proteins was changed to a massive blocky structure. The reduction of blocky whey protein size and loosening of texture was observed with increasing pressure to 80, 120, and 160 MPa (Liu et al. 2011). Scanning electron microscopy images of jelly fig pectin indicated the formation of many pores after high-pressure microfluidization at 40, 80, and 160 MPa, which increased their diameter with microfluidization pressure (Chen et al. 2012). Morphology of Eucommia ulmoides Oliv. seed meal proteins are also altered after dynamic high-pressure microfluidization. The compact structure and smooth surface of the untreated proteins were converted to small irregular fragments after microfluidization treatment (Ge et al. 2021).
3.6. SDS-PAGE
According to Fig. 6, two faint protein bands were detected in untreated gelatin, attributed to α1-, and α2-chains. Also, the proteins with molecular weights higher than α-chains were represented in electrophoretic bands. A similar electrophoretic pattern was observed by Jellouli et al. (2011) for commercial halal bovine gelatin (Jellouli et al. 2011). Proteins with molecular weights lower than α-chain were perceived, which may be related to some collagen hydrolysis during gelatin extraction.
Overall, HPH treatment could not have a significant influence on the protein pattern of gelatin. It seems that the observable difference between the electrophoretic pattern of proteins is related to protein solubility. It is worth mentioning that the lack of new bands with lower molecular weights can demonstrate that HPH treatment does not lead to polypeptide hydrolysis. Therefore, pressure up to 100 MPa and homogenization cycles up to three times were not adequate for the disruption of the protein.
There were different reports about the effect of HPH treatment on the electrophoretic pattern of various proteins. Hu, Zhao, Sun, Zhao, and Ren (2018) studied the effect of microfluidization under high pressure on the functional and structural properties of gelatin obtained from flakes of bighead carp at 40, 80, 120, and 160 MPa. They did not observe any change in the electrophoretic pattern of gelatin after high-pressure microfluidization treatment (Hu et al. 2011). Dynamic high-pressure microfluidization of potato protein isolate up to 120 MPa also did not have any considerable effect on the protein pattern (Hu et al. 2021). Three HPH cycles treatment of oyster protein isolate at 20, 40, 60, 80, and 100 MPa did not change the protein pattern (Yu et al. 2018). However, after HPH treatment of gingko protein at 100 and 200 MPa, Zhou et al. (2016) observed that the protein band near 39 kDa vanished. Pressure increase also decreased the intensity of protein bands when almond proteins are subjected to HPH treatment in the presence of water (Zhou et al. 2016). Microfluidization of whole cowpea flour can change the electrophoretic pattern of two variety cowpea proteins. However, a distinct variation was observed in the polypeptide pattern of Red Bisbee and Rough et Noir varieties after microfluidization (Adjei-Fremah et al. 2019). Therefore, it can be concluded that the effect of HPH treatment is related to the type and structure of the protein.
3.7. Gel strength
Gel strength is considered as a pivotal mechanical indicator of gels texture. As expected, gelatin gels with 3% and 9% concentrations showed the lowest and highest gel strength, respectively (Fig. 7, p < 0.05). Except for 3% gelatin gels, HPH treatment of 6 and 9% gelatin gels leads to the reduction of gel strength.
Gelatin can solve in water at temperatures higher than the melting point. Dissolved gelatin is present in the form of a flexible random coil. Along with the reduction of temperature below the melting point, arranged structure of the protein molecules re-build. Previous studies forcefully recommended that the reformed, regular structures have an identical structure to collagen. Gelatin molecules in semi-diluted solutions penetrate into each other so that the complete reversion of helix-coil structure to the collagen state faces topological obstacles. Therefore, a part of gelatin molecule can reform the triple helical structure of collagen. These parts are connected to each other through the coiled structure peptides along the gelatin contour. At very low gelatin concentrations, the helix-coil transformation is mainly intermolecular, while in the semidiluted solutions, it changes to intramolecular bonds. Therefore, gel forms at concentrations higher than 5% due to the constitution of unlimited molecular lattice in the gelatin solution (Guo et al. 2003). It seems that in the diluted gelatin solution with a 3% concentration, gelatin molecules can not penetrate each other, and the intermolecular helix-coil reversion happens; thereupon, gel strength did not change after HPH treatment.
With increasing gelatin concentration, the probability of two or three protein chains being participated in the gel network formation also increased. However, Joly-Duhamel et al. (2002) indicated that the α-helix amount plays a vital role in final gel strength (Joly-Duhamel et al. 2002). The reduction of α-helix structures upon increasing HPH cycles, which was proved with CD spectra, could be a reason for the reduction of gel strength after HPH treatment. Furthermore, HPH treatment might cause an increase in the network free volume. Increasing free volume resulted in decreased intermolecular interactions, which weakened the hydrogen bonding between the free hydroxyl group of the amino acid chain and water. Such change is obvious in FESEM images (Fig. 5). Montero, Fernández-Dı́az, and Gómez-Guillén (2002) reported that the high hydrostatic-pressure treated megrim gelatin has lower gel strength than heat-treated one. They suggested that the unfolding of megrim gelatin after high-pressure treatment may destabilize the existing triple-helices leading to weaker gel formation (Montero et al. 2002).
During high-pressure microfluidization treatment of gelatin obtained from bighead carp, Sha et al. (2018) found no significant difference in the gel strength of treated and untreated gelatin gels. They proposed that high-pressure microfluidization could not significantly influence gel strength because the molecular weight of fish gelatin did not change considerably (Sha et al. 2018). On the other hand, exposure of the protein to mechanical forces exerted during HPH treatment can influence the protein structures through dissociation of covalent and non-covalent bonds as well as cleavage of protein molecules (Song et al. 2013). Therefore, the unfolded and shorter protein molecules can not participate in the formation of a strong gel. The results of FESEM (Fig. 5) also indicated the increase in porosity of gel structure which results from covalent and non-covalent bonds cleavage and can decrease the gel strength.
3.8. Rheology
Strain sweep
Strain sweep measurement was applied to specification the linear viscoelastic region (LVR) of the gels. In this region, G' (storage modulus) and G" (loss modulus) stay constant with an increase in strain. The gels with higher resistance to the applied deformation indicate a greater LVR extension which is related to the structural strength of a gel (Romanelli Vicente Bertolo et al. 2021).
Figure 8 indicates the storage modulus of gelatin gels as a function of strain. The extracted parameters from the presented curves in Fig. 8 are summed up in Table 1. The γL named critical strain is the largest strain that the gel tolerated before the LVR leaving. The gels with more stability against the applied strain illustrated the higher γL value. Based on the results, the gel prepared with 9% gelatin had the highest γL value. The HPH treatment of gelatin decreased this value so that the lowest critical deformation was obtained after three passes. This trend was observed in all gelatin concentrations, suggesting the reduction of interactions in gelatin gel as a function of homogenization cycles.
Table 1
Effect of HPH treatment at different gelatin concentrations parameters determined during strain sweep measurements at an angular frequency of 10 rad/s and 4oC.
| Gelatin concentration | Number of homogenization cycle | γL (%) | G' LVR (Pa) | G" LVR (Pa) | tan δ |
| 3 | 0 | 21.50 | 490.05 | 12.70 | 0.03 |
| 1 | 21.50 | 419.22 | 11.50 | 0.03 |
| 2 | 21.50 | 348.53 | 10.67 | 0.03 |
| 3 | 14.70 | 226.46 | 9.40 | 0.04 |
| 6 | 0 | 31.60 | 2220.17 | 43.70 | 0.02 |
| 1 | 21.50 | 1825.08 | 40.70 | 0.02 |
| 2 | 21.50 | 1370.52 | 34.50 | 0.03 |
| 3 | 14.70 | 915.15 | 26.70 | 0.03 |
| 9 | 0 | 46.50 | 13770.87 | 115.50 | 0.01 |
| 1 | 31.60 | 10275.69 | 88.00 | 0.01 |
| 2 | 21.50 | 6700.92 | 75.00 | 0.01 |
| 3 | 14.70 | 3945.34 | 53.00 | 0.01 |
γL: Critical strain (), G'LVR: G' at the limit of LVR, G”LVR: G” at the limit of LVR, tanδ: loss tangent value
Other variables are G'LVR and G" LVR, indicating the storage and loss modulus values of gels at the LVR limit, respectively (Table 1). The gels before HPH treatment had the highest G' and G" values. However, every time the solution passes the homogenizer, G'LVR and G" LVR decreases, representing the reduction in the viscoelastic behavior. The changes in G'LVR and G" LVR were more considerable when gelatin with 9% concentration was exposed to HPH treatment. It might be due to the higher exposure of protein strands to turbulence, shear force, and cavitation resulting from HPH treatment.
The alteration of G"/G' as a function of strain can determine the gelatin gel strength. The gels with tan δ lower than 0.1 have a solid-like appearance (Kaewruang et al. 2013). The comparison of tan δ gelatin gels with different concentrations indicates the stronger gel network of gelatin with a 9% concentration (Table 1). Also, tan δ of all gels increased upon strain increased, suggesting a propensity toward weakening gel manner at higher strain (Fig. 8). Meanwhile, tan δ of gels increased as homogenization cycles increased. However, the increase in tan δ was more evident in gelatin gels with 9% concentration, and the reduction of gelatin concentration decreased the effect of homogenization cycles on tan δ.
Figure 8 illustrates that the G" compared with G' values were lower at all gelatin concentrations. Furthermore, G' and G" values decreased along with enhancement of the number of passes, illustrating that the gelatin intermolecular interactions become weaker with exposure to higher homogenization cycles. Zhang et al. (2016) indicated that the telopeptide region of gelatin plays a vital role in the mechanical properties of gelatin gel. The specific residues in the telopeptide region can cross-link with the helical structure of the neighbor gelatin strand to consolidate the gel network (Zhang et al. 2016). As mentioned before, HPH treatment can decrease the telopeptide region of gelatin, leading to the reduction of viscoelastic properties and tan δ of gels.
Another parameter that can influence the rheological properties of gelatin is fibril orientation. Hydrogels are composed of hydrophobic and hydrophilic regions organized in a random network structure in which a large amount of water is trapped. In the aligned fibrils, the junction zones can come near each other so that more intermolecular interactions are formed. However, the inhomogeneous regions in the hydrogel network prevent the appropriate intermolecular interactions (Chen et al. 2011). HPH treatment can create turbulence in the solution, increasing the randomized orientation of gelatin fibrils. Therefore, the higher homogenization cycle increases the randomized fibrils in the gelatin solution, interfering with chain entanglements and physical gelation. As a consequence, the elasticity of the gel network decreases, and the viscosity increases.
Temperature sweep
To determine gel properties and kinetic of gelation, the temperature sweep test was performed at strain and frequency of 0.5% and 1 Hz, respectively. Temperature sweep evaluation is a critical stride in the rheological behavior description of gel-forming materials at variable temperatures. If gelatin is added to foods heated during preparation or before consumption, or exposed to temperatures higher than the predicted value during transport and distribution, knowing its rheological properties upon a broad range of temperatures is essential (Romanelli Vicente Bertolo et al. 2021).
Storage and loss moduli of gelatin gels at the temperature range of 4 to 40 oC were indicated in Fig. 9. The very low storage and loss moduli, which is an indicator of liquid behavior, were seen at 40 oC. As the temperature decreases, gelatin aggregation begins and gel forms, G' being increased significantly. The sol-gel transition of gelatin with the cooling of the solution is related to a phase transition temperature in which the solution is cross-linked and transformed from liquid to solid-like (Fonkwe et al. 2003).
As can be seen in Fig. 9, the G' and G" of all gels diminished with increasing homogenization cycles. The HPH treatment could change the protein structure to prevent the most important hydrogen bonds formed in the spatial network of gelatin, causing the reduction of thermal stability and moduli of the gels. HPH treatment reduced the elastic and loss modulus value of treated gelatin at low temperatures, causing melting and solidification of gelatin gel at a higher temperature. It was affirmed that by reducing the temperature to about 35 oC in mammalian gelatins, the transformation of the random coil to helix structure terminates. Below this temperature, the triple-helices of gelatin have formed a network with thermo-reversible viscoelastic behavior (Derkach et al. 2020b). The reduction in the thermal stability of gelatin gel indicates decreased refolding ability of gelatin into a triple helix (Gómez-Guillén et al. 2002).
A large amount of energy exerted using the HPH process can produce shear force, leading to cavitation and alteration of physicochemical properties of the medium. These events can disrupt some intra- and inter-molecular bonds, causing lower viscoelasticity of gels (Silva and Sato 2019).
After HPH treatment of peanut protein isolate, Gong et al. (2019) proposed that a combination of electrostatic and hydrophobic interactions as well as disulfide and hydrogen bonds participate in the alteration of protein structure. They found that protein disintegration was preferred at pressures below 120 MPa because, in this range of pressure, the repulsive electrostatic force is predominant over intermolecular interactions (Gong et al. 2019), which is confirmed with moduli reduction during the temperature sweep test.
The crossover point of G' and G" during heating of gelatin is considered as melting temperature. This is while the gelation temperature was obtained from the crossover point during the cooling phase (Osorio et al. 2007). Table 2 indicates the gelation and melting point of gelatin after 1, 2, and 3 HPH cycles. The higher melting than gelation point of gelatin gel is related to the thermal hysteresis of the gel (Huang et al. 2017a). Based on Table 2, gelation and melting temperature of gelatin were concentration-dependent. Increasing gelatin concentration directly influenced gelation and melting point. The effect of concentration on these parameters might be due to the higher protein-dissolvent and protein-protein interactions, which require a higher temperature for breaking the higher number of created hydrogen bonds and protein cross-linking (Sarbon et al. 2013; Osorio et al. 2007).
Table 2
Effect of HPH treatment at different gelatin concentrations on Melting and gelation temperature of gelatin gel.
| Gelatin Concentration (%) | Number of homogenization cycle | Melting temperature (oC) | Gelation temperature (oC) |
| 3 | 0 | 30.6 | 28.2 |
| 1 | 30.0 | 26.5 |
| 2 | 29.2 | 26.7 |
| 3 | 28.5 | 25.6 |
| 6 | 0 | 31.5 | 29.2 |
| 1 | 31.0 | 27.7 |
| 2 | 30.2 | 27.5 |
| 3 | 29.7 | 26.6 |
| 9 | 0 | 32.2 | 30.4 |
| 1 | 31.5 | 29.7 |
| 2 | 30.5 | 28.5 |
| 3 | 29.7 | 28.0 |
In addition, the preliminary results showing a mild decrease in the melting and gelation temperature as a function of the homogenization cycle. So that, the sol-gel transition during heating and cooling of gelatin decreased with the increasing number of passes. Sha et al. (2018) observed that the melting temperature of the bighead carp scale decreased as a function of pressure. They explained that the reduction in melting point is not related to the molecular weight; however, the surface hydrophobicity can change it (Sha et al. 2018). Also, cavitation and shear force create mechanical effects, which weaken the gel structure (Huang et al. 2017b).
3.9. Color evaluation
Color parameters of gelatin before and after HPH treatment at different concentrations are illustrated in Table 3. The gelatin gels treated with HPH tended to be lighter and yellowish with higher L* and b* values in comparison with untreated samples. However, with increasing the number of passes to three times, L* value decreased. The increase in L* value upon HPH treatment stems from an increase in light diffraction caused by the change in gel microstructure. Abby et al. (2020) reported that HPH is a parameter influencing L* value of sea buckthorn puree. They explained that particle size reduction creates a larger surface, which enhances light scattering and produces a lighter color. Light scattering in each system depends on molecular uniformity (Madadlou et al. 2005) and microstructures (Rudan et al. 1999). Results obtained through microstructural evaluation (Fig. 5) indicated that gelatin's microstructure has changed under HPH and earned a more ordered conformation. The light scattering increases in such a structure and enhances lightness. However, with increasing the number of homogenization cycles, bigger holes formed in the gel's structure, resulting in a darker color. Also, HPH caused the conformational change in protein with unfolding protein structure and subjecting of hydrophobic groups on the surface. This condition is favored for protein aggregation, intensifying by increasing the number of passes which could reduce the lightness. The L* value of rosehip puree and tomato juice also increased after HPH treatment (Kubo et al. 2013; Saricaoglu et al. 2019).
Table 3
Effect of HPH treatment at different gelatin concentrations on the color of the gel.
| Gelatin concentration | Number of homogenization cycles | L* | a* | b* |
| 3% | 0 | 49.43 Cb±1.20 | 0.49Aa ± 0.30 | 0.43Db ± 0.20 |
| 1 | 89.35Aa ± 0.84 | 0.28Aa ± 0.11 | 5.52Cb ± 0.10 |
| 2 | 87.75Aab ± 0.56 | 0.36Aa ± 0.10 | 7.22Ba ± 0.05 |
| 3 | 85.31Bab ± 0.51 | 0.35Aa ± 0.01 | 8.91Aa ± 0.26 |
| 6% | 0 | 49.08Ba ± 1.83 | -0.74Bb ± 0.07 | 1.24Cb ± 0.86 |
| 1 | 87.65Aa ± 1.54 | -0.09Ba ± 0.01 | 6.04ABab ± 0.15 |
| 2 | 89.98Aa ± 1.69 | -0.57Bb ± 0.08 | 5.58Bb ± 0.38 |
| 3 | 88.57Aa ± 0.72 | 1.20Aa ± 0.22 | 6.75Ab ± 0.82 |
| 9% | 0 | 50.41Ca ± 0.57 | -1.51Bb ± 1.09 | 3.49Ca ± 0.06 |
| 1 | 88.89Aa ± 0.02 | -0.36Ca ± 0.04 | 6.45Aa ± 0.22 |
| 2 | 88.61Aa ± 0.65 | 0.31Aa ± 0.27 | 5.74ABb ± 0.39 |
| 3 | 84.07Bb ± 0.37 | 0.47Aa ± 0.24 | 5.46Bc ± 0.24 |
The different capital letters indicate significant differences between different homogenization cycles at the same gelatin concentration and the different lowercase in the same column indicates significant differences between gelatin concentration at the same homogenization cycle.
Results indicated that a* value decreased after HPH treatment, and then it increased with increasing the number of homogenization cycles. The yellowness also increased with increasing the number of passes at the concentration of 3 and 6%, but at the concentration of 9%, b* value raised after HPH and then decreased with the increasing number of homogenization cycles. These changes in a* and b* values can be attributed to the molecular size and concentration change, resulting in the variation of light absorption (Chantrapornchai et al. 1998). Chantrapornchai et al. (1998) found that a* and L* values of emulsion decreased, and the b* value of hijiki-based edible films increased with increasing the droplet size. Lee and Min (2012) reported that a* and b* values diminished as HPH pressure diminished to 103 MPa and then raised (Lee and Min 2012). Nonetheless, Nagarajan et al. (2012) did not observe any significant effect of homogenization conditions on the color of nanocomposite films prepared from gelatin (Nagarajan et al. 2014).
3.10. Emulsifying properties
Emulsifying capacity and emulsion stability are two important functional properties of proteins. The amphoteric nature of gelatin with the hydrophobic regions on its peptide chain, leading gelatin to act as an emulsifier (Koli et al., 2012). Emulsifying activity and emulsion stability of gelatin are depicted in Table 4. Based on the results, the emulsifying capacity of gelatin samples increased significantly after a single homogenization cycle. It seems that the increase in emulsifying capacity is related to the unfolding of gelatin molecules, exposing buried hydrophobic regions on the protein surface. The hydrophobic amino acids of proteins are naturally hidden inside the protein molecules. The surface hydrophobicity of protein increases by exposing hydrophobic residue on the surface (Zhang et al. 2015a), which in turn will improve emulsifying capacity. Moreover, Gomez, Giménez, López-Caballero, & Montero (2011) found that by exerting force to polypeptides chain, they can participate more in the emulsification process (Gómez-Guillén et al. 2011). Lee et al. (2009), by structural evaluation of whey protein after ultra-high pressure homogenization, concluded that whey protein partially unfolded and exposed hydrophobic group on the surface. The interaction of the protein with other adsorbed whey protein and the lipid droplets would facilitate after this structural change.
Table 4
Effect of HPH treatment at different gelatin concentrations on the emulsifying capacity and emulsion stability.
| Gelatin concentration | Number of passes | Emulsifying capacity (m2/g) | Emulsion stability (%) |
| 3% | 0 | 4.05Cc ± 0.45 | 0.00Bb ± 0.00 |
| 1 | 44.10Ac ± 0.74 | 40.50Ac ± 1.95 |
| 2 | 40.00Bc ± 0.57 | 36.13Ac ± 2.67 |
| 3 | 41.50Bc ± 0.46 | 36.31Ab ± 1.91 |
| 6% | 0 | 8.15Db ± 0.49 | 0.14Dab ± 0.21 |
| 1 | 99.60Aa ± 1.27 | 85.05Aa ± 1.83 |
| 2 | 61.75Bb ± 0.74 | 58.17Bb ± 1.08 |
| 3 | 51.50Cb ± 0.98 | 55.23Ca ± 0.94 |
| 9% | 0 | 11.65Da ± 0.44 | 0.62Da ± 0.23 |
| 1 | 91.25Ab ± 1.12 | 73.50Ab ± 1.26 |
| 2 | 81.50Ba ± 1.34 | 67.17Ba ± 1.14 |
| 3 | 70.75Ca ± 1.76 | 55.29Ca ± 1.27 |
The different capital letters indicate significant differences between different homogenization cycles at the same gelatin concentration and the different lowercase in the same column indicates significant differences between gelatin concentration at the same homogenization cycle.
On the other hand, with increasing the number of homogenization cycles, emulsifying capacity decreased. Investigations have shown that treatment conditions during homogenization, such as high pressure, shear stress, and temperature, can lead to changes in the emulsifying properties of the protein. Currently, it is indicated that the structure of the protein is sensitive to high pressure. In fact, high pressure not only alters the ternary and tertiary structure of the protein but also can change the secondary structure to some extent (Dumay et al. 1994). The unfolding of protein leads to the formation of disulfide bonds and hydrophobic interactions, which promotes protein aggregation. It can consequently decrease emulsifying capacity (Villamonte, Pottier, & de Lamballerie, 2016). Binsi et al. (2009) proposed that the gelatin molecules with higher exposed hydrophilic regions can interact together and cause less gelatin accessibility at the oil-water interface (Binsi et al. 2009). HPH provides the required energy to persuade further changes in proteins structure and their interaction (Lee et al. 2009). Hence, the increase in homogenization cycles, due to the higher kinetic energy, favored protein interaction and the formation of aggregates.
Results of emulsifying capacity indicated that by increasing the gelatin's concentration, emulsifying capacity significantly increased. As the amount of soluble protein plays a vital role in emulsifying properties, increasing the protein concentration would also increase its emulsifying capacity. Since gelatin would be placed at the water and oil interface, surface absorption would be facilitated by increasing its concentration, and consequently, emulsifying capacity would be enhanced. Results obtained by FESEM also indicated that more protein strands are found in the environment upon increasing protein concentration which subsequently facilitates the absorption at the surface oil droplets.
The emulsion stability of gelatin significantly increased after HPH treatment. However, increasing the number of passes had a detrimental effect on emulsion stability. It seems that the unfolding of protein structure is responsible for these changes. HPH treatment can unfold protein and enhance its surface activity. The unfolded protein has better emulsifying properties through higher efficiency for interfacial layer formation (Bouaouina et al. 2006). The same changes in emulsifying properties were reported for hazelnut meal protein (Saricaoglu et al. 2018), lupin protein (Bader et al. 2011), peanut protein (Dong et al. 2011), and soy protein (Floury et al. 2002).
Villamonte, Pottier, and Lamballerie (2016) proposed that after HPH treatment of sarcoplasmic protein, the amount of adsorbed proteins enhances as a consequence of enhanced surface hydrophobicity. The greater hydrophobicity improves the adsorption of protein to the surface of oil droplets. Then, increasing pressure promotes hydrophobic interactions between the reactive groups of unfolded adsorbed proteins, decreasing the protein stability (Villamonte et al. 2016).
Yang, Liu, Zeng, and Chen (2018) reported flocculation tendency as destabilizing mechanisms in emulsion prepared with HPH treated faba bean protein. The major forces that participate in the flocculation of emulsion are osmotic forces, hydrophobic interactions, and van der Waals forces (Yang et al. 2018). For instance, the aggregated micelle of casein in the continuous phase of emulsion promotes the flocculation of emulsion droplets through local osmotic forces (Singh et al. 2003). The development of flocculation is depended on the aggregated protein size. When the size of unadsorbed protein aggregated is approximately one-tenth of oil droplets in emulsion, powerful flocculation happens as a result of osmotic forces (Yang et al. 2018). It seems that the unadsorbed supramolecular aggregates formed after several passes from a high-pressure homogenizer can participate in the destabilization and flocculation of the emulsion.
Results show that by increasing the gelatin concentration, the emulsion stability was increased. In fact, the availability of emulsifying protein molecules increased at higher concentrations of gelatin, producing a network with gel-like particles due to the aggregation of proteins. On the other hand, the environment viscosity increases as the protein concentration enhance, increasing the number of active sites or hydrophilic amino acid residue. In this condition, higher water-binding capacity increases the emulsion viscosity and improves emulsion stability (Ipsen et al. 2000).
3.11. Foaming capacity and foaming stability
Foaming capacity and foaming stability are important attributes in describing the functional properties of a protein. For suitable foam, protein should be completely solved, flexible, and form a film at the air and water interface (Wagner and Gueguen 1999).
The foaming capacity and stability of untreated and HPH treated gelatin are indicated in Table 5. The foaming capacity significantly increased after HPH treatment of 3% gelatin gel; however, the parameter greatly decreased as the number of passes increased. The improvement in the foaming capacity of HPH treated gelatin might be related to the rapid diffusion of the protein at the air/water interface (Yang et al. 2018). It seems that the gelatin structure is unfolded after HPH treatment. The unfolded protein can easily and rapidly diffuse at the air/water interface and cover air bubbles (Han et al. 2020). Also, HPH treatment can increase protein hydrophobicity which decreases the energy barrier at the interface (Yang et al. 2018). The adverse influence of HPH on gelatin unfolding, resulting in a reduction of foaming capacity, was significant at higher homogenization cycles. The decreased foaming capacity with increased number of passes could be related to the aggregation of protein induced by protein unfolding. The aggregated structures have lower flexibility and can not effectively cover the air bubbles, which leads to the reduction of foaming capacity. In several other literatures, it was found that several passes, regardless of exerted pressure, lead to a reduction of foaming capacity (Gong et al. 2019; Maresca et al. 2017). Chao et al. (2018), after HPH treatment of isolated pea protein, reported that pressure could unfold the protein and increase the foaming capacity. Then, foaming capacity decreased with increasing pressure intensity due to protein aggregation (Chao et al. 2018). Evaluating dynamic high-pressure microfluidization of potato protein isolate, Hu, Xiong, Xiong, Chen, and Zhang (2021) found that foaming capacity and stability increased from 0–9 k psi. However, the detrimental effect of 12 k psi pressure on foaming capacity and stability was reported (Hu et al. 2021).
Table 5
Effect of HPH treatment at different gelatin concentrations on the foaming capacity and stability.
| Gelatin concentration | Number of passes | Foaming capacity (%) | Foam stability after 30 min (%) | Foam stability after 60 min (%) |
| 3% | 0 | 316.66Ba ± 10.54 | 93.00Ba ± 3.20 | 91.00Ba ± 8.60 |
| 1 | 512.50Aa ± 12.47 | 100.00Aa ± 2.59 | 100.00Aa ± 3.53 |
| 2 | 287.50Ca ± 17.75 | 100.00Aa ± 1.76 | 98.63Aa ± 4.78 |
| 3 | 237.50Da ± 11.32 | 99.31Aa ± 5.91 | 97.53Aa ± 5.60 |
| 6% | 0 | 37.50Cb ± 2.49 | 84.84Cb ± 4.21 | 78.78Bb ± 7.07 |
| 1 | 191.66Ab ± 10.27 | 100.00Aa ± 8.38 | 97.14Aa ± 2.68 |
| 2 | 187.5Ab ± 9.62 | 98.57Ab ± 7.58 | 97.10Aa ± 3.53 |
| 3 | 40.00Bb ± 3.63 | 97.93Aa ± 4.94 | 97.10Aa ± 6.24 |
| 9% | 0 | 33.33Bb ± 2.44 | 90.62Cb ± 3.53 | 90.62Aa ± 5.97 |
| 1 | 40.00Ac ± 3.59 | 100.00Aa ± 2.62 | 96.87Aa ± 10.60 |
| 2 | 33.33Bc ± 3.82 | 96.77Ac ± 1.41 | 93.54Ab ± 3.53 |
| 3 | 29.10Cc ± 1.89 | 95.23Ab ± 6.72 | 92.26Ab ± 4.94 |
| The different capital letters indicate significant differences between different homogenization cycles at the same gelatin concentration and the different lowercase in the same column indicates significant differences between gelatin concentrations at the same homogenization cycle. |
Increasing protein concentration had unfavorable effects on the foaming capacity so that by increasing the concentration to 6%, a considerable decrease in foaming capacity was observed. Britten and Lavoie (1992) suggested that the reduction of foaming capacity at high protein concentration correspond to the decrease in protein solubility. Carp, Wagner, Bartholomai, and Pilosof (1997) also observed that the foaming capacity attained maximum value at 5% protein concentration for native soy protein and 3% for denatured ones. In fact, protein concentration plays a vital role in foaming properties due to reducing surface tension, modifying mechanical properties of the interfacial layer, and increasing viscosity (Carp et al. 1997). A remarkable point of the protein concentration effect on foaming capacity could be originated from the alteration of continuous phase viscosity. High viscosity can reduce the incorporation of air into protein solution (Vani and Zayas 1995). The increasing viscosity along with protein concentration was evident during the preparation of the gelatin solution.
The foaming stability of gelatins was recorded after 30 and 60 min. The modification of the protein by HPH treatment caused a drastic increase in foaming stability. The possible reason for the production of a more stable foam after single pass was that HPH treatment could increase the hydrophobicity at the protein surface, decrease the interfacial tension and improve the protein flexibility to readily adsorb at the air/water interface (Zhao et al. 2021). Interestingly, there was a direct relationship between foam stability and the solubility of gelatin, demonstrating that desirable solubility could simplify the formation of a stable interfacial layer that was hardly ruptured.