pH-shifting Treatment Research Articles

Interaction between pH-shifted β-conglycinin and flavonoids hesperetin/hesperidin: Characterization of nanocomplexes and binding mechanism

This study characterized the nanoparticles and the binding mechanism between pH-shifted soy β-conglycinin (β-CG) and hesperetin (Ht)/hesperidin (Hd). Different structural characteristics and concentrations of Ht/Hd were found to affect the encapsulation efficiency, loading amount, and particle size of pH-shifted β-CG‒Ht/Hd nanoparticles. This was the result of two glycosyl substituents (7-position of the C-ring) in the presence of Hd containing six hydroxyl groups interacting with the polar groups of proteins to form hydrogen bonds, which facilitated the formation of nanocomplexes. Besides, the exposed hydrophobic groups in the β-CG proteins resulting from pH shift treatment developed hydrophobic interactions with the benzene ring or glucoside in the flavonoids. Moreover, Ht/Hd strongly quenched the fluorescence of pH-shifted β-CG in static mode. After binding with flavonoids, the tertiary and secondary structures of pH-shifted β-CG were significantly changed. The finding presented in this study provided evidence of the binding mechanism of soy proteins and different structural flavonoids and will aid in the design of soy protein/flavonoid enriched-food.

Effect of pH-shifting treatment on the structural and functional properties of soybean protein isolate and its interactions with (–)-epigallocatechin-3-gallate

Polyphenols are readily affected by environmental factors, such as heat and light, which cause them to degrade; however, the functional properties of polyphenols can be protected by complexing them with proteins. In this study, we investigate the effects of acid and alkali treatments on the functional properties of soybean protein isolate (SPI) and interactions between the treated protein and (−)-epigallocatechin-3-gallate (EGCG). The results show that alkali treatment (pH 12–7) affects the structure and functional performance of the protein more significantly than acid treatment (pH 2–7) because the protein structure unfolds, resulting in improved solubility, surface hydrophobicity, and emulsification. In addition, because alkali treatment can change the structure of a protein more than acid treatment, its structure loosens. The interactions between alkali-treated proteins and EGCG were also found to be stronger than those of acid-treated proteins and the control protein, and EGCG binds to a protein through van der Waals forces and hydrophobic interactions; these interactions protect EGCG from degradation, ensuring that its antioxidant properties are maintained and its bioavailability rate is improved.

Synergistic effect of pH shifting and mild heating in improving heat induced gel properties of peanut protein isolate

Peanut protein isolate (PPI) was first subjected to pH shifting treatment at pH 7 or pH 10 with temperatures ranging from 25 °C to 70 °C. Then the structure and heat induced gel properties of PPI were investigated. The breaking force and water holding capacity of PPI10-40 (PPI after pH shifting with pH 10 at 40 °C) gel were 2.2 times and 2.15 times that of PPI7-25. Moreover, they were higher (p = 0.001637, p = 0.000916, p = 0.000021, p = 0.23) than that PPI7-40 or PPI10-25, indicating the synergistic effect of pH shifting and mild heating in improving the gel properties of PPI. These results were probably because the particle size of PPI10-40 decreased was 1.8 times that of PPI7-25 measured from DLS. Moreover, the solubility, free sulfhydryl content and surface hydrophobicity of PPI10-40 were 1.3 times, 1.8 times and 1.6 times that of PPI7-25 respectively, which resulted in enhanced covalent or non-covalent interactions between proteins. Circular dichroism and intrinsic fluorescence spectroscopy suggested that both heating and pH shifting significantly changed the secondary and tertiary structure of PPI. So the combined treatment of pH-shifting and mild heating is an efficient method to improve the gel properties of PPI (PPI10-40) to the maximum extent.

Modification of rapeseed protein by ultrasound-assisted pH shift treatment: Ultrasonic mode and frequency screening, changes in protein solubility and structural characteristics

We investigated the effect of ultrasound-assisted pH shift treatment on the micro-particle, molecular, and spatial structure of rapeseed protein isolates (RPI). Various ultrasonic frequency modes (fixed, and sweep) was used. Protein characterization by the indexes: particle size, zeta potential, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), scanning electron microscopy (SEM), free sulfhydryl (SH), surface hydrophobicity (Ho), Fourier transform infrared Spectrum (FTIR) and fluorescence intensity was studied to elucidate the changes in solubility and structural attributes of RPI. The results showed that ultrasonic frequency and working modes substantially altered the structure, and modified the solubility of RPI. Ultra + pH mode at fixed frequency of 20 kHz had the best effect on the solubility of RPI. Under the condition of ultra + pH mode, 20 kHz at pH 12.5, solubility, compared to control, increased from 8.90% to 66.84%; and the change in molecular structure of RPI was characterized by smaller particles (from 330.90 to 115.77 nm), high zeta potential (from −17.95 to −14.43 mV, p < 0.05), and increased free sulfhydryl (from 11.63 to 24.50 µmol/g) compared to control. Likewise, surface hydrophobicity increased (from 2053.9 to 2649.4, p < 0.05), whilst ɑ-helix and random coil decreased (p < 0.05), compared to control. The fluorescence spectroscopy and FTIR spectroscopy showed that the secondary and tertiary structure of the RPI were altered. These observations revealed that changes in RPI structure was the direct factor affecting solubility. In conclusion, ultrasound assisted pH shift treatment was proven to be an effective method for the modification of protein, with promising application in food industry.

Low pH-shifting treatment would improve functional properties of black turtle bean (Phaseolus vulgaris L.) protein isolate with immunoreactivity reduction.

Low pH-shifting was firstly applied in the black turtle bean (Phaseolus vulgaris L.) protein isolate treatment by acidic (pH 1.0-3.0) buffer incubation for 8h, then was adjusted to pH 7.2 and kept 3h for protein stabilizing. Mild loss of secondary structure was confirmed in the protein isolate after low pH-shifting treatment by CD and FT-IR analyses. Intrinsic fluorescence, UV spectra, surface hydrophobicity, SH content and SDS-PAGE analyses indicated the protein conformation was unfolded with the exposure of much more buried hydrophobic residues, which would result in the enhancement of emulsifying properties, foaming properties and fat holding capacity, and lead to the reduction of solubility and water holding capacity. Furthermore, lower immunoreactivity was observed by the ELISA, and improved digestibility was found in in vitro digestion assay. Our results suggested the low pH-shifting treatments would broaden the application of bean protein isolate with better hydrophobic processing functions and safety.

Combined effects of pH and thermal treatments on IgE-binding capacity and conformational structures of lectin from black kidney bean (Phaseolus vulgaris L.).

Combined effects of pH and thermal treatments on black kidney bean lectin (BKBL) were investigated by response surface methodology (RSM). Low-pH (1.0, 2.0, 3.0) incubation decreased hemagglutination activity (HA) and IgE-binding capacity, but the activities would be restored when the lectin was treated by pH shifting to 7.2. Conformational structure analyses indicated that low-pH induced protein unfolding and pH-shifting treatment resulted in a limited structural rearrangement. Mild heating, such as 60°C for 3min, slightly increased the HA and IgE-binding activities of pH shifted BKBL, but no obvious effects in the pH 1.0 incubated BKBL. High-temperature and long-time treatment might induce the protein aggregation, further decreased HA and IgE-binding capacities. RSM results showed both IgE-binding capacity and HA were the lowest under the combination of pH 1.0 incubation with 80°C heating for 15min or pH shifting from 1.0 to 7.2 with 100°C heating for 10min.

The role of conformational state of pH-shifted β-conglycinin on the oil/water interfacial properties and emulsifying capacities

This study investigated the influence of pH-shift treatment on the conformational state, interfacial properties and emulsifying capacities of β-conglycinin (7S proteins). The conformational state was evaluated using dynamic light scattering, intrinsic tryptophan fluorescence spectroscopy, and far-UV circular dichroism spectroscopy. The interfacial properties, including adsorption kinetics and dilatational viscoelasticity modulus were characterized at the oil/water interface. Emulsifying capacities and emulsion stability against thermal treatment (85 °C, 15min) were characterized by mean droplet size, flocculation index, adsorbed protein content, interfacial protein concentration and Zeta potential values. The results indicated that pH-shift treatment changed the conformational state and interfacial viscoelasticity of 7S proteins. The conformational changes of 7S proteins significantly affected their adsorption at the interface, improving the emulsion stability against thermal treatment.

Effect of pH-shifting treatment on structural and heat induced gel properties of peanut protein isolate

Modifying proteins with new methods to improve their functional properties is essential to extend their application in food related field. In this study, the effect of pH-shifting treatment on structural and heat induced gel properties of peanut protein isolate (PPI) were investigated. PPI was subject to different pH conditions (pH 2, pH 4, pH 10, and pH 12) for 1 h, then adjusted back to pH 7 and they were marked as PPI2, PPI4, PPI10, and PPI12, respectively. It was found that the breaking force (BF) and water holding capacity (WHC) of PPI10 gel were significantly improved due to the decreased particle size, increased solubility, free sulfhydryl group content and surface hydrophobicity of PPI10. While PPI2 or PPI12 lost their gel ability due to the formation of large protein aggregates. These results indicated that pH-shifting treatment might be a convenient and economical method to prepare PPI with different gel and structural properties.

Food functionalities and bioactivities of protein isolates recovered from skipjack tuna roe by isoelectric solubilization and precipitation.

Four roe protein isolates (RPIs) from skipjack tuna were prepared using isoelectric solubilization (pH 11 and 12) and precipitation (pH 4.5 and 5.5) (ISP) at different pH points to evaluate their physicochemical and functional properties and in vitro bioactivities. Moisture (<6.3%) and protein (71%–77%) content were maintained. Sulfur, sodium, phosphorus, and potassium were the major elements, and glutamic acid and leucine were the prevalent amino acids (12.2–12.8 and 9.6–9.8 g/100 g protein, respectively) in RPIs. RPI‐1 showed the highest buffering capacity at pH 7–12. RPIs and casein showed similar water‐holding capacities. At pH 12, RPI‐1(pH 11/4.5) showed the highest solubility, followed by RPI‐3(pH 12/4.5), RPI‐2(pH 11/5.5), and RPI‐4(pH 12/5.5) (p < .05). Oil‐in‐water emulsifying activity indices of RPI‐1 and RPI‐3 significantly differed. At pH 2 and 7–12, pH‐shift treatment improved the food functionality of RPIs, which was superior to positive controls (casein and hemoglobin). RPI‐1 showed ABTS+ radical scavenging (102.7 μg/ml) and angiotensin‐converting enzyme inhibitory activities (44.0%).

Structure Changes in Relation to Digestibility and IgE-Binding of Glycinin Induced by pH-Shifting Combined with Microbial Transglutaminase-Mediated Modification

This study aimed to evaluate the effects of pH-shifting treatment combined with microbial transglutaminase (MTG)-mediated modification on the structure, digestibility, and IgE-binding of glycinin. Glycinin was incubated in acidic (pH 1.0) or alkaline (pH 13.0) solutions to induce protein structure to unfolding followed by refolding for 1 h at pH 7.0. Afterwards, glycinin was incubated with MTG under appropriate conditions. Sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE), circular dichroism, UV absorption spectra, and surface hydrophobicity were considered to measure the changes in the structure of glycinin. The digestibility and IgE-binding of glycinin were determined by Tricine-SDS-PAGE and ELISA, respectively. The results showed that pH 1.0 shifting caused the unfolding of the spatial structure of glycinin and the formation of some polymers via disulfide bond. After glycinin was incubated with MTG, the protein preferentially underwent embedding and folding. The acidic compound-modified glycinin was stable for digestion. Under pH 13.0 shifting treatment, glycinin was partially hydrolyzed, and the MTG-modified alkaline-treated glycinin was slightly affected with a good digestibility. Compound modification could reduce the IgE-binding of glycinin, especially under alkaline conditions. Our findings suggested that alkaline pH shifting combined with MTG cross-linking can be an efficient approach to reduce the IgE-binding of glycinin with a labile digestion.

Effect of pH-shifting treatment on structural and functional properties of whey protein isolate and its interaction with (−)-epigallocatechin-3-gallate

Effect of pH-shifting on structural and functional properties of whey protein isolate and its interaction with (−)-epigallocatechin-3-gallate were investigated. Circular dichroism spectra showed that pH-shifting induced the decrease in α-helix content by 12.18% and β-sheet content by 3.24%, but β-turn and random coil content increased by 4.26% and 5.91%, respectively. Increase of fluorescence intensity and red-shift of maximum emission wavelength indicated the structural unfolding and exposure of tyrosine. The treatment also significantly increased the surface hydrophobicity, disulfide bonds content, solubility, emulsifying activity and emulsion stability of whey protein isolate at P < 0.05 level. Fluorescence quenching analysis revealed that treated whey protein isolate have a stronger binding affinity to (−)-epigallocatechin-3-gallate, resulting a better protection against the degradation of (−)-epigallocatechin-3-gallate and its antioxidant activity. This study confirmed that pH-shifting treatment can improve functional properties of whey protein isolate and its potential as a protective carrier for polyphones.

High pressure homogenization combined with pH shift treatment: A process to produce physically and oxidatively stable hemp milk

Hemp milk, an emerging beverage with high nutritional value and low allergenicity, is an attractive alternative to dairy, soy, and nut milks. To obtain a non-thermally processed, physically and oxidatively stable hemp milk, high pressure homogenization (HPH) combined with pH shift treatment was investigated. For hemp milk (4% protein, 5% fat) without pH shift, increasing the homogenization pressure (up to 60 MPa) resulted in a more uniform distribution of emulsion droplets (2.2–2.7 μm). When pH shift was applied prior to HPH, large clusters and aggregates of oil droplets (3.5–8.2 μm) were formed. Interestingly, hemp milk with such interactive structures was remarkably stable, showing negligible phase separation within 3-day storage at 4 °C. Moreover, hemp milk made by combined pH shift and HPH exhibited delayed hydroperoxides (expressed as peroxide value, PV) and malondialdehyde (expressed as thiobarbituric acid-reactive substances, TBARS) production, suggesting the resistance of such emulsion cluster structures to radicals. On the other hand, a significant reduction of microbial population was observed in hemp milk prepared by pH shift combined with HPH. The results indicate that the pH shift + HPH combination treatment may potentially be employed for the production of non-thermally processed hemp milk.

Effect of PH-Shifting Treatment on the Biochemical and Thermal Properties of Myofibril Protein

The pH-shifting treatment (including acid treatment and alkali treatment) caused partly denaturation and degrading of myofibril. The pH-shifting treatment increased the surface hydrophobicity, surface -SH content and total -SH content, reduced the Ca-ATPase activity. Acid treated myofibril (ACM) did not show any endothermic transition peak in differential scanning calorimetry (DSC); while alkali treated myofibril (AKM) showed reduced T max value for myosin and actin. The SDS-PAGE indicated that acid treatment caused severe degrading of myosin heavy chain. High turbidity of ACM indicated more myofibril aggregated during acid treatment. The final G’ was in the decline order of M (550 Pa), AKM (135 Pa), and ACM (25 Pa). In conclusion, the alkali treatment was milder than the acid treatment.

Role of β‐Conglycinin and Glycinin Subunits in the pH‐Shifting‐Induced Structural and Physicochemical Changes of Soy Protein Isolate

Soy β-conglycinin (7S) and glycinin (11S) were incubated up to 4 h in acidic (pH 1.5 to 3.5) or alkaline (pH 10 to 12) solutions to induce protein structural unfolding followed by refolding 1 h at pH 7.0, a process known as pH-shifting. The pH-shifting markedly increased (P < 0.05) emulsifying activity of 11S and to a lesser extent 7S; the former also produced more uniform oil droplets. The emulsifying activity improvements were accompanied by a significant rise in protein surface hydrophobicity, slight loss of the secondary structure (circular dichroism), and substantial dissociation of disulfide-linked basic and acidic 11S subunits. The findings suggested that 11S globulins of soy protein isolate (SPI) were more responsive to pH-shifting treatments than were 7S globulins, and the resulting emulsifying activity enhancements of 11S, in parallel with that of SPI, were indicative of its determinant role in the overall emulsifying properties of pH-shifting-treated SPI. Extreme alkaline (pH 12) and acidic (pH 1.5) medium treatments can significantly modify the structure and enhance the emulsifying properties of both β-conglycinin and glycinin components of SPI. The functionality improvement by the pH processes is more remarkable for the glycinin protein fraction. Therefore, SPI enriched with glycinin seems to be particularly suitable for extreme acidic or alkaline processes to produce surface-active functional ingredients for food applications.

PH Shifting Alters Solubility Characteristics and Thermal Stability of Soy Protein Isolate and Its Globulin Fractions in Different pH, Salt Concentration, and Temperature Conditions

Soy protein isolate (SPI), beta-conglycinin (7S), and glycinin (11S) were subjected to pH-shifting treatments, that is, unfolding at pH 1.5 or 12.0 followed by refolding at pH 7.0, to induce molten globule structures. Treated samples were analyzed for protein solubility, thermal stability, and aggregation in 0, 0.1, and 0.6 M NaCl solutions at pH 2.0-8.0. The pH(12) shifting resulted in drastic increases (up to 2.5-fold) in SPI solubility in the pH 6.0-7.0 range, especially at 0 M NaCl. The pH(1.5) shifting had a generally lesser effect on solubility. 11S exhibited a solubility pattern similar to that of SPI, but the solubility of 7S was unaffected by pH shifting except at 0.6 M NaCl. The pH shifting, notably at pH 12.0, produced soluble, disulfide-linked polymers from 11S and reduced (P < 0.05) its enthalpy but not its temperature of denaturation. Soy proteins structurally altered by pH shifting had a reduced sensitivity to thermal aggregation.

Structural and Emulsifying Properties of Soy Protein Isolate Subjected to Acid and Alkaline pH-Shifting Processes

Structural unfolding of soy protein isolate (SPI) as induced by holding (0, 0.5, 1, 2, and 4 h) in acidic (pH 1.5-3.5) and alkaline (pH 10.0-12.0) pH solutions, followed by refolding (1 h) at pH 7.0, was analyzed. Changes in emulsifying properties of treated SPI were then examined. The pH-shifting treatments resulted in a substantial increase in protein surface hydrophobicity, intrinsic tryptophan fluorescence intensity, and disulfide-mediated aggregation, along with the exposure of tyrosine. After the pH-shifting processes, soy protein adopted a molten globule-like conformation that largely maintained the original secondary structure and overall compactness but lost some tertiary structure. These structural modifications, consequently, led to markedly improved emulsifying activity of SPI as well as the emulsion stability.

ATP production after pH shift in membrane vesicles from maize coleoptiles.

Microsomal membrane vesicles and purified plasma membranevesicles obtained from coleoptiles of maize (Zea mays L.) weresubjected to pH shifts from pH 7.8 to 4.7. In the presence of ATPaseinhibitors such as vanadate, net accumulation of radiolabelled butyricand indole-3-acetic acid (IAA) remained higher than in controls. When 2min after the pH shift, at 4°C, the microsomal vesicles weredenatured the amount of ATP could be determined using theluciferin/luciferase assay. Significantly increased ATP production overcontrol values – no pH-shift or ionophore treatment – wasfound. Therefore, such vesicles might produce ATP for in vitrotransport processes such as auxin efflux.