Rui Pedro
Neto Queirós
a,
Carlos Alberto
Cruz Pinto
b,
José António
Lopes-da-Silva
b and
Jorge Manuel
Alexandre Saraiva
*b
aDepartment of Applications and Food Processing, Hiperbaric S.A., Calle Condado de Treviño, 6, 09001 Burgos, Spain. E-mail: r.queiros@hiperbaric.com
bLAQV-REQUIMTE, Chemistry Department, University of Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, Portugal. E-mail: jals@ua.pt; jorgesaraiva@ua.pt
First published on 29th August 2023
Microbial transglutaminase (MTG) is an enzyme broadly used to improve the technological properties of proteins; however, many globular proteins are poorly susceptible or unsusceptible to its action. High-pressure processing (HPP) can change the conformation of proteins; thus it may be a useful tool to increase the accessibility of MTG to some proteins. Still, HPP conditions and the concentration of MTG need to be carefully studied to achieve the desired effects. The effects of combined MTG (up to 30 U per g of protein) and HPP (200–600 MPa; 5–15 minutes) on the solubility, the content of accessible sulfhydryl groups and surface hydrophobicity of pea (PPI) and soy (SPI) protein isolates were evaluated employing response surface methodology. The regression models obtained presented high coefficients of determination and high F values. Overall, all three parameters were differently affected by pressure. HPP increased solubility in both PPI and SPI (up to ∼200% at 600 MPa); however, it decreased the concentration of accessible sulfhydryl groups in PPI (∼80% at 600/10 min) and increased it in SPI (up to 28% at 200 MPa/10 min). HPP also affected the surface hydrophobicity of both protein isolates differently, decreasing it in PPI (up to ∼25% at 200 MPa/10 min) and increasing it in the SPI (up to ∼30% at 200 MPa/10 min). Non-HPP protein isolates were not affected by MTG, most likely due to the low accessibility of the enzyme to the proteins. However, when combined, HPP and MTG seem to have both synergistic and antagonistic effects, thus broadening the potential to alter the properties of these proteins. These results suggest that simultaneous HPP and MTG treatments can be used to modify the structure of proteins to tailor their techno-functional properties.
The extent of the crosslinking reaction is dependent on environmental conditions (pH, temperature, and the absence of enzyme inhibitors) and the structure and conformation of the target protein(s). Several studies have shown that non-globular proteins are more easily accessible to MTG crosslinking activity than globular proteins.6,7 Also, different vegetable proteins have shown different susceptibilities to the MTG crosslinking activity.4 Therefore, despite the ease of MTG in crosslinking various proteins, many of them, particularly globular proteins, are not affected by MTG in their native state due to the inaccessibility of the reactive residues buried within the protein tertiary structure.
High pressure processing (HPP) is an environmentally friendly non-thermal treatment that requires water (which is largely recycled), compressed air, and electricity (which can be produced from renewable sources) and does not produce any effluents. It may induce structural changes in proteins that could expose the mentioned residues, making them (more) accessible to the MTG's acyl active site.8 With this in mind, research studies have already been performed9–11 regarding MTG stability under high pressure, both in buffer solutions and food products, and how the crosslinking activity of the enzyme is affected by pressure. Overall, MTG is stable under pressure, particularly at pH 6 and 7, retaining more than 40% of its activity even at 600 MPa for 30 min.12
The possible increase in crosslinking obtained by MTG and HPP combined treatments may be a suitable tool for the enhanced modification of proteins, allowing the improvement of functional properties without requiring a pre-treatment or using reducing agents.1,13 Hence, it is possible to infer that the combination of MTG and HPP may offer new perspectives for the modification of proteins and may allow desirable functional and technological changes in the protein matrix to be tailor-made . Despite what is already known regarding HPP effects on protein functionality and MTG effects on protein crosslinking and structure development, knowledge concerning the influence of combined physical and enzymatic treatments on vegetable proteins' techno-functional properties is still needed, as most studies were performed on fish,14 meat15 and dairy16,17 products. Therefore, the objective of this work was to evaluate the combined effects of MTG and HPP on protein solubility, the content of accessible sulfhydryl (SH) groups, and surface hydrophobicity (H0) of pea (PPI) and soy (SPI) protein isolates. Tests were performed based on a factorial experimental design to analyze the effect of HPP conditions, pressure (200, 400, and 600 MPa) and holding time (5, 10, and 15 min) and MTG's concentration (0, 15 and 30 U per g protein). An experimental design approach could be further utilized to identify the optimal conditions for protein modification.
![]() | (1) |
Level (−1) | Level (0) | Level (+1) | |
---|---|---|---|
Pressure, P (MPa) (x1) | 200 | 400 | 600 |
Holding time, t (min) (x2) | 5 | 10 | 15 |
MTG (U per g of protein) (x3) | 0 | 15 | 30 |
Therefore, six models were constructed: two for soluble protein–pea protein isolate (PPISOL) and soy protein isolate (SPISOL); two for accessible sulfhydryl groups – pea protein isolate (PPISH) and soy protein isolate (SPISH); and two for surface hydrophobicity – pea protein isolate (PPIH0) and soy protein isolate (SPIH0).
Pressure(MPa) | Time (min) | MTG (U g−1) | Pea protein isolates | Soy protein isolates | ||||
---|---|---|---|---|---|---|---|---|
Soluble protein (mg mL−1) | SH groups (μmol per g protein) | Surface hydrophobicity | Soluble protein (mg mL−1) | SH groups (μmol per g protein) | Surface hydrophobicity | |||
a Values are presented as a mean ± standard deviation (n = 3). | ||||||||
200 | 5 | 15 | 0.67 ± 0.02 | 2.47 ± 0.26 | 1939 ± 108 | 2.78 ± 0.04 | 3.12 ± 0.09 | 3487 ± 104 |
200 | 10 | 0 | 2.02 ± 0.15 | 2.59 ± 0.19 | 1896 ± 66 | 2.79 ± 0.07 | 3.57 ± 0.16 | 3467 ± 108 |
200 | 10 | 30 | 1.19 ± 0.07 | 1.45 ± 0.17 | 1951 ± 151 | 2.33 ± 0.10 | 2.07 ± 0.22 | 3083 ± 86 |
200 | 15 | 15 | 1.40 ± 0.06 | 0.63 ± 0.09 | 1678 ± 87 | 2.57 ± 0.09 | 2.11 ± 0.20 | 2036 ± 105 |
400 | 5 | 0 | 1.55 ± 0.09 | 1.11 ± 0.14 | 2853 ± 112 | 3.00 ± 0.02 | 2.90 ± 0.13 | 3035 ± 110 |
400 | 5 | 30 | 1.34 ± 0.11 | 1.41 ± 0.21 | 1649 ± 111 | 2.46 ± 0.08 | 2.12 ± 0.09 | 2387 ± 51 |
400 | 10 | 15 | 1.67 ± 0.07 | 0.68 ± 0.22 | 1470 ± 57 | 2.26 ± 0.04 | 2.48 ± 0.23 | 2080 ± 91 |
400 | 10 | 15 | 1.80 ± 0.05 | 0.54 ± 0.10 | 1550 ± 123 | 2.27 ± 0.09 | 2.61 ± 0.09 | 1960 ± 71 |
400 | 10 | 15 | 1.74 ± 0.04 | 0.70 ± 0.21 | 1474 ± 83 | 2.29 ± 0.09 | 2.64 ± 0.16 | 2013 ± 54 |
400 | 15 | 0 | 2.02 ± 0.09 | 0.54 ± 0.20 | 2021 ± 45 | 3.21 ± 0.09 | 2.86 ± 0.03 | 2813 ± 112 |
400 | 15 | 30 | 1.61 ± 0.05 | 0.64 ± 0.09 | 2057 ± 127 | 2.57 ± 0.07 | 2.14 ± 0.20 | 2182 ± 102 |
600 | 5 | 15 | 1.67 ± 0.07 | 0.55 ± 0.20 | 1587 ± 64 | 2.70 ± 0.08 | 2.07 ± 0.06 | 1491 ± 106 |
600 | 10 | 0 | 2.10 ± 0.07 | 0.40 ± 0.18 | 2563 ± 103 | 2.86 ± 0.10 | 2.11 ± 0.22 | 2675 ± 99 |
600 | 10 | 30 | 2.49 ± 0.11 | 2.12 ± 0.24 | 1367 ± 108 | 2.27 ± 0.10 | 2.43 ± 0.16 | 2269 ± 64 |
600 | 15 | 15 | 1.76 ± 0.14 | 1.76 ± 0.20 | 1926 ± 58 | 2.90 ± 0.13 | 2.87 ± 0.23 | 2352 ± 16 |
Estimated coded coefficients | Pea protein isolates | Soy protein isolates | ||||
---|---|---|---|---|---|---|
Soluble protein | SH groups | Surface hydrophobicity | Soluble protein | SH groups | Surface hydrophobicity | |
a Values are presented as a mean ± standard error; x1, x2, and x3 represent dimensionless coded forms of pressure, holding time, and transglutaminase concentration, respectively. All terms are significant (p < 0.001) otherwise marked: *0.001 ≤ p < 0.01; **0.01 ≤ p < 0.05. | ||||||
Constant | 1.72 ± 0.03 | 0.64 ± 0.06 | 1524 ± 35 | 2.27 ± 0.04 | 2.54 ± 0.03 | 2035 ± 32 |
x 1 | 0.34 ± 0.02 | −0.29 ± 0.05 | — | — | −0.17 ± 0.04 | −411 ± 23 |
x 2 | 0.19 ± 0.02 | −0.25 ± 0.05 | — | — | — | −127 ± 23 |
x 3 | −0.13 ± 0.02 | 0.12 ± 0.05** | −289 ± 26 | −0.28 ± 0.02 | −0.34 ± 0.04 | −259 ± 23 |
x 1·x1 | — | 0.71 ± 0.07 | — | 0.11 ± 0.03* | — | — |
x 2·x2 | −0.34 ± 0.03 | — | 239 ± 38 | 0.35 ± 0.03 | — | 294 ± 34 |
x 3·x3 | 0.24 ± 0.03 | 0.28 ± 0.07 | 401 ± 38 | 0.18 ± 0.03 | — | 557 ± 34 |
x 1·x2 | −0.16 ± 0.03 | 0.76 ± 0.07 | 150 ± 36 | 0.11 ± 0.03* | 0.45 ± 0.06 | 578 ± 33 |
x 1·x3 | 0.30 ± 0.03 | 0.71 ± 0.07 | −313 ± 36 | — | 0.45 ± 0.06 | — |
x 2·x3 | — | — | 310 ± 36 | — | — | — |
Overall, the addition of MTG to non-HPP protein isolates (up to 30 U per g protein and to a reaction time of 180 min) resulted in no significant differences (p > 0.05) in the concentration of soluble proteins relative to the control samples – Table 4. The lower solubility of the protein isolates, particularly in the case of globular proteins that have a compact structure, may limit the accessibility of MTG to glutamine and lysine residues, limiting the enzyme effects.26 An exception was verified for PPI, where a MTG's concentration of 30 U per g protein and a reaction time above 120 minutes led to an increase in the concentration of soluble protein of approximately 31%. MTG catalyses the acyl transfer reaction between glutamine and lysine residues; however, in the absence of lysine or other primary amines, water will react as a nucleophile, resulting in deamidation.26 Pea proteins are rich in glutamine and asparagine, which can be converted through MTG into glutamic acid and aspartic acid, respectively. In the absence of conditions that could lead to a pronounced crosslinking and formation of large protein aggregates that would decrease protein solubility, the resulting increased electrostatic repulsion between deamidated proteins may increase their solubility.2,27
MTG (U per g protein) | Time (min) | Pea protein isolates | Soy protein isolates | ||||
---|---|---|---|---|---|---|---|
Soluble protein (mg mL−1) | SH groups (μmol per g protein) | Surface hydrophobicity | Soluble protein (mg mL−1) | SH groups (μmol per g protein) | Surface hydrophobicity | ||
a Values are presented as a mean ± standard deviation (n = 3). Different letters indicate significant differences (p < 0.05). | |||||||
10 | 0 | 0.79 ± 0.08a | 2.48 ± 0.18a | 2517 ± 63a | 2.09 ± 0.20a | 1.98 ± 0.22a | 2595 ± 89a |
60 | 0.81 ± 0.13a | 2.48 ± 0.09a | 2586 ± 78a | 1.93 ± 0.08a | 2.01 ± 0.07a | 2573 ± 31a | |
120 | 0.82 ± 0.13a | 2.46 ± 0.05a | 2535 ± 85a | 1.92 ± 0.07a | 2.04 ± 0.06a | 2555 ± 86a | |
180 | 0.93 ± 0.04a | 2.43 ± 0.18a | 2510 ± 57a | 2.10 ± 0.13a | 1.88 ± 0.09a | 2622 ± 96a | |
20 | 0 | 0.86 ± 0.05a | 2.34 ± 0.15a | 2528 ± 72a | 2.05 ± 0.10a | 1.93 ± 0.06a | 2630 ± 86a |
60 | 0.93 ± 0.07a | 2.42 ± 0.12a | 2509 ± 60a | 1.94 ± 0.12a | 2.11 ± 0.12a | 2643 ± 107a | |
120 | 0.90 ± 0.02a | 2.39 ± 0.11a | 2519 ± 51a | 2.00 ± 0.08a | 1.90 ± 0.05a | 2590 ± 62a | |
180 | 0.91 ± 0.04a | 2.38 ± 0.06a | 2499 ± 86a | 1.99 ± 0.10a | 1.92 ± 0.10a | 2484 ± 103a | |
30 | 0 | 0.80 ± 0.07a | 2.33 ± 0.16a | 2571 ± 74a | 1.99 ± 0.20a | 1.97 ± 0.17a | 2524 ± 76a |
60 | 0.91 ± 0.03a | 2.36 ± 0.09a | 2504 ± 60a | 1.99 ± 0.14a | 1.94 ± 0.14a | 2557 ± 44a | |
120 | 1.09 ± 0.08 b | 2.40 ± 0.14a | 2558 ± 57a | 1.94 ± 0.13a | 1.98 ± 0.16a | 2490 ± 92a | |
180 | 1.05 ± 0.09 b | 2.51 ± 0.13a | 2629 ± 51a | 2.00 ± 0.22a | 2.00 ± 0.08a | 2538 ± 120 a |
For pressurized samples, all tested conditions increased the concentration of soluble proteins for PPI, except for 200 MPa/5 min/15 U g−1 that did not have a significant effect, and also for SPI. Fig. 1 shows the individual effects of each parameter on the concentration of soluble proteins present in the PPI (Fig. 1A), as predicted by the model PPISOL, and in SPI (Fig. 1B), as predicted by the PPISOL model. For PPI, it is evident that raising pressure increases the amount of soluble proteins.
Similarly, increasing holding time up to approximately 13 min also increased proteins' solubility; however, a further increase in time does not further increase the concentration of soluble proteins.
Fig. 2A shows the interaction effects of HPP parameters (pressure and holding time) on the concentration of soluble proteins without the addition of MTG. The smallest increase in the concentration of soluble proteins (an increase of 91%) was verified under the lowest HPP conditions (i.e., 200 MPa/5 min). Overall, increasing pressure and increasing holding time, up to ca. 13 min, led to an increase of up to approximately 200% in the concentration of soluble protein compared to control samples. When a pressure above 400 MPa is considered, a longer holding time does not further increase the proteins' solubility.
In what concerns SPI, every tested condition involving pressure increased proteins' solubility (Table 2). Considering only the individual parameters, although pressure increased the concentration of soluble protein, the pressure level seemed to not have much influence (Fig. 1B).
On the other hand, varying the holding time impacted the proteins' solubility, as intermediate holding times (around 10 min) resulted in a smaller increase than at 5 or 15 min. Although there are some interaction effects of pressure and holding time, these are not very large.
All combinations of pressure and holding time led to an increase in soluble proteins and a synergetic effect was observable at the higher pressures and longer times, peaking at 600 MPa/15 min where the concentration of soluble proteins increased by 77% relative to unprocessed samples (Fig. 3A).
The smaller increase in the concentration of soluble proteins, around 44%, was verified under intermediate HPP conditions (375 MPa/9 min). In general HPP caused an increase in protein solubility, although with a magnitude dependent on the type of protein and HPP conditions, which is in accordance with what is described in the literature, especially for soy proteins.28–31 This increase is most likely due to some unfolding of the proteins and the dissociation of aggregates promoted by pressure.32,33 It is worth mentioning that pressure may enhance interactions between the protein and solvent, thus increasing solubility; however, it may also expose hydrophobic residues increasing intermolecular interactions and the formation of insoluble aggregates, therefore, reducing solubility.34,35 These phenomena may explain why longer holding times did not further increase the pea proteins' solubility or why there was a smaller increase in SPI's solubility at intermediate holding times compared to 5 or 15 min.
MTG had the contrary effect of the HPP parameters, since the presence of the enzyme during the HPP treatments led to a general decrease in protein solubility, for both PPI (Fig. 1A) and SPI (Fig. 1B). However, when interactions were considered, the addition of MTG resulted in different effects depending on the pressure level and type of protein.
For PPI, at low-pressure levels, i.e. <400 MPa, increasing MTG's concentration decreased proteins' solubility, reaching levels near the control samples at 200 MPa, counteracting the effects of pressure (Fig. 2B). On the other hand, at pressure levels above 400 MPa and 10 min holding time, increasing MTG's concentration increased the concentration of soluble proteins up to 2.48 ± 0.05 mg mL−1 (∼250% increase compared to control), showing a synergetic effect between pressure and the MTG concentration. In a general way increasing holding time, at moderate pressure (400 MPa), increased the concentration of soluble proteins; however, this parameter seems to have no interaction effect with the concentration of MTG – Fig. 2C and Table 2. The increase in the concentration of MTG progressively reduced the proteins' solubility present in the SPI – Fig. 1B. Overall, the higher the concentration of MTG the more pronounced the reduction of protein solubility, counteracting, to some extent, the increase promoted by pressure (Fig. 3B).
Overall, there were no synergetic or antagonistic effects between holding time and the MTG concentration – Fig. 3C and Table 2.
Information about the combined effects of HPP and MTG on the solubility of proteins from legumes is scarce. Still, when considering other types of proteins (e.g., β-lactoglobulin, casein, bovine serum albumin, ovalbumin, etc.), one can expect that HPP facilitates the crosslinking of proteins catalysed by MTG.10,13 HPP might induce structural changes in proteins that may expose glutamine and lysine residues, making them accessible to the MTG's acyl active site.8 Studies on the effects of MTG on SPI indicate that β-conglycinin (subunits α, α′and β) and the acidic subunits of glycinin can be crosslinked by MTG, forming high molecular weight biopolymers, whereas the basic subunits of glycinin remain intact. As a result, the solubility of the protein isolates decreased due to MTG activity. In addition, since the glycinin's basic subunits may remain intact, they can form aggregates that add to the reduction of solubility.5,36 A similar reduction in solubility of crosslinked PPI due to the formation of large molecular weight compounds was also reported.37 Therefore, the results suggest that HPP may dissociate proteins' aggregates present in PPI and SPI increasing their solubility. In doing so, and by altering the structure of proteins, pressure can make them more accessible to the action of MTG, which can result in the formation of high molecular weight biopolymers. Still, for PPI, a pressure >400 MPa and an increase in MTG's concentration from 15 to 30 U per g protein led to an increase in proteins' solubility, suggesting that these conditions may have promoted the deamidation of glutamine and asparagine.
When considering the pressure individually, this parameter decreases the concentration of SH groups, particularly at intermediate pressures, within the range analysed. At 400 MPa, the holding time decreases the SH content linearly, but the effect seems to be dependent on the applied pressure (Fig. 5A).
A maximum SH content was predicted at 200 MPa/5 min and represents a 91% increase relative to non-processed samples (Fig. 5A).
However, increasing the severity of HPP conditions led to a progressive reduction of the concentration of accessible SH groups, reaching a predicted minimum at 600 MPa/5 min. Under more severe pressure conditions, i.e., 500 – 600 MPa, longer holding times appear to have a less negative effect on accessible SH than short times.
When MTG was added to PPI, and only considering its main effect, it had a contrary effect to the other processing parameters, as concentrations above 15 U per g protein increased the content of SH (Fig. 4A). The effects of combined pressure and MTG are complex (Fig. 5B), and it seems that these parameters had an antagonistic effect, with the presence of MTG counteracting the reduction of SH caused by HPP. No significant interaction effect was observed between MTG's concentration and the holding time (Table 3).
Moreover, it appears that the PPI with MTG is associated with an increase in the accessible SH content, and this in turn is correlated with an increase in protein solubility.
However, it has been observed that the application of pressure to PPI can lead to a decrease in SH content, yet still result in improved protein solubility. This suggests that the relationship between SH groups and protein solubility is not always linear and that a variety of other factors may come into play.
With respect to SPI, Fig. 4B illustrates the individual main effects predicted by the model SPISH. Although HPP caused a slight increase in the SH concentration, compared to the non-pressurized sample, the increase in pressure also caused a decrease in SH for the soy proteins, whereas the holding time seemed to have no impact (Fig. 4B).
When considering the combined effects of HPP parameters (pressure and time), and without the addition of MTG, the decrease in SH promoted by increasing pressure is clearly dependent on the holding time and somewhat unexpected; the higher the holding time the less pronounced the effect of increasing pressure (Fig. 6A), nevertheless presenting a behaviour similar to that observed for PPI. It is worth mentioning that even so all pressurized samples showed higher SH than the SPI unprocessed samples (Table 2), with the maximum increase predicted to be approximately 110% at 200 MPa/5 min.
The minimum predicted concentration of SH in processed SPI was verified at 600 MPa/5 min, comparable to the value of unprocessed samples. Overall, the results suggest that applying pressure resulted in the dissociation/unfolding of the proteins exposing buried SH, particularly at lower pressure values (<400 MPa).
However, higher pressure values may have promoted hydrophobic interactions that led to S–S exchange and/or formation of new disulphide bonds resulting in a decrease in SH.38,39 The results here described agree with the available literature concerning soy proteins and proteins from other sources, where it is reported that pressure below 300 MPa may preserve or improve the content of SH, whereas higher pressure values or longer holding times seem to decrease it.35,38,40
The individual main effect of MTG was the decrease in the amount of SH for soy proteins, as can be seen in Fig. 4B. In fact, the higher the MTG's concentration, the higher the observed decrease in SH. However, when considering its combined effect with pressure, this reduction seemed to be much more accentuated at lower pressure values (e.g., 200 MPa) than at 600 MPa, where the effect of MTG is barely noticeable (Fig. 6B). Similarly to PPI, an interaction effect between MTG's concentration and the holding time was not verified .
Therefore, most treatments with MTG led to a decrease in SH. This decrease after MTG catalytic action was already reported for SPI41 and a vicilin-rich kidney protein isolate.33 This is most likely due to the crosslinking between proteins, which may have promoted some changes in the conformation of the proteins and consequent formation of new disulphide bonds and/or buried SH into the resulting high molecular weight aggregates making it no longer measurable as free/accessible sulfhydryl groups.33,41 Still, some conditions have led to an increase in SH present in PPI after MTG, namely when pressure >300 MPa was applied. Similar results were reported for sweet potato protein isolate and peanut protein isolate treated with HPP and micro-fluidization, respectively, and subsequent MTG crosslinking.42,43 The mechanism by which this happens is not yet fully understood; still, it is suggested that the conformational changes mentioned above promoted by the crosslinking may expose buried free sulfhydryl groups.43 Furthermore, analysing Fig. 3 and 6, a noticeable trend can be observed in SPI samples treated with MTG. Specifically, a decrease in the content of accessible sulfhydryl groups appeared to correspond with a decrease in protein solubility. This observation may suggest a potential correlation between these two parameters.
![]() | ||
Fig. 7 Main effects of the independent variables (pressure, holding time, and concentration of transglutaminase) on the surface hydrophobicity of (A) pea and (B) soy protein isolates. |
It seems that the effect of pressure on the surface hydrophobicity was not dependent on the pressure level. On the other hand, intermediate holding times seem to have resulted in lower surface hydrophobicity values than low or high holding time values. When considering the combination of the HPP parameters (pressure and time – Fig. 8A) it was observed that the holding time and pressure values had interaction effects.
For instance, the H0 decreased to 38% when the holding time increased from 5 to 15 min at 200 MPa, whereas a reduction of 14% with the same time increase was observed at 600 MPa. An increase in H0 occurred with increasing pressure, more markedly at longer times. For instance, for a holding time of 5 min, increasing pressure from 200 to 600 MPa resulted in a small increase (12%), while the same increase in pressure with a 15 min holding time led to an increase of 56%. Still, most HPP conditions with higher holding times decrease the H0 when compared with untreated samples, particularly at lower 200 MPa and 15 min.
In what concerns SPI, considering the individual main effects of HPP (i.e. pressure and holding time), the increase in both led to lower values of H0; still, an additional increase of pressure from ca. 500 MPa did not further decrease H0 – Fig. 7B. Considering the combined effect of HPP parameters (Fig. 9A) it was possible to observe distinct behaviours at low and high pressure and at shorter and longer holding times.
The predicted higher value of H0 was 4261 ± 60 at 200 MPa/5 min, which represents an increase of 67% relative to the unprocessed samples. From this point, increasing pressure up to 600 MPa resulted in a decrease of 46% of the H0 and increasing holding time to 15 min caused a 33% decrease. In contrast, increasing the holding time, from 5 to 15 min, at 600 MPa increased H0 by 40%. Overall, it seems that pressure variations had more impact on the H0 at shorter than longer holding times, under most pressure conditions resulting in an increase in H0 compared to the untreated SPI.
Applying pressure may result in the unfolding of the proteins, thus exposing the number of hydrophobic groups on their surface. As a result, in general, HPP increases the H0 of proteins due to the resulting conformational changes.35 Still, as described here, the combination of low pressure (i.e. 200 MPa) and high holding time (15 min) has led to a decrease in H0 of PPI, whereas more intensive HPP conditions did not mainly impact this parameter. Pressure may promote interactions between the proteins' hydrophobic regions and solvent or between proteins and change the equilibrium between aggregation and dissociation processes decreasing H0.44
In this particular case, it is possible that these low HPP conditions may have promoted protein–water interactions of PPI, reducing H0 and consequently increasing protein solubility, as the solubility of PPI was higher under these conditions – Fig. 2A. The described results for SPI are in agreement with what is reported in the literature; for instance, applying low pressure to soy's glycinin and β-conglycinin increased their H0.45,46 Still, increasing pressure may lead to a smaller increase in H0 or even decrease it.
This lower H0 under more severe HPP conditions compared to that under mild conditions was already reported for SPI,31,38 which is likely due to an increased aggregation accompanied by conformational changes (dissociation of protein subunits, changes in the tertiary and secondary structures, increasing exposure of hydrophobic groups, etc.).35
The addition of MTG to PPI changed the effect of pressure on H0 – Fig. 8B. While without MTG the increase in pressure from 200 to 600 MPa led to an increase of approximately 30% of H0 when in combination with MTG, the same increase in pressure led to a decrease of also ca. 30% with 30 U·per (g protein). An interaction effect was also observed between holding time and MTG's concentration – Fig. 8C. While, at 400 MPa and no MTG, an increase in holding time from 5 to 15 min led to a 25% reduction of H0, the combination with 30 U·per (g protein) of MTG had a contrary effect, i.e. the same increase in holding time increased H0 by approximately 30%. A lower concentration of MTG (i.e. around 15 U·per (g protein)) seemed to stabilize H0 regarding HPP parameter variations, as H0 did not vary considerably with pressure or holding time changes when this concentration of MTG was used – Fig. 8B and C.
Considering SPI, the addition of MTG at a lower concentration (15 U·per (g protein)) decreased H0; however, it did not seem to have an antagonistic or synergetic effect with any of the other studied parameters, as can be seen in Table 3 and Fig. 9B and C. Still, increasing the concentration of MTG to 30 U·per (g protein) appeared to have a smaller impact on the H0. Broadly, adding MTG during HPP yielded a lower H0; however, in most cases, the reduction was higher with an MTG concentration of 15 than with 30 U·per (g protein). There are reports where MTG both increases47,48 and decreases49,50 the H0 of different proteins. The crosslink promoted by MTG may occlude hydrophobic residues inside the structure of the higher molecular weight polymers formed, decreasing H0.49 Additionally, the deamination of glutamic and aspartic acids may increase the overall negative charge of the proteins, leading to a decrease in H0.26 In contrast, the crosslinking of proteins may also change their structure consequently resulting in unfolding, thus exposing hydrophobic regions and increasing the H0.42 Therefore, the concentration of MTG may lead to different degrees of crosslinking and deamination reaction rates, differently affecting the H0 of proteins. Similar to the observed possible correlation between accessible SH content and protein solubility, there appears to be a potential relationship between the H0 and protein solubility. Specifically, a decrease in the H0 appeared to correspond with a decrease in protein solubility, as observed in the analysed data.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3fb00039g |
This journal is © The Royal Society of Chemistry 2023 |