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ABSTRACT: Two peptides, [f135-158] and [f135-162]-SH, were used to study the binding of the peptides to native β-lactolobulin, as well as the subsequent effects on aggregation and gelation of β-lactoglobulin. The binding of the peptide [f135-158] to β-lactoglobulin at room temperature was confirmed by SELDI-TOF-MS. It was further illustrated by increased turbidity of mixed solutions of peptide and protein (at pH 7), indicating association of proteins and peptides in larger complexes. At pH below the iso-electric point of the protein the presence of peptides did not lead to an increased turbidity, showing the absence of complexation. The protein-peptide complexes formed at pH 7 were found to dissociate directly upon heating. After prolonged heating, extensive aggregation was observed, while no aggregation was seen for the pure protein or pure peptide solutions. The presence of the free sulfhydryl group in [f135-162]-SH resulted in a 10 times increase in the amount of aggregation of β-lactoglobulin upon heating, illustrating the additional effect of the free sulfhydryl group. Subsequent studies on the gel strength of heat-induced gels also showed a clear difference between these two peptides. The replacement of additional β-lactoglobulin by [f135-158] resulted in a decrease in gel strength, while replacement by peptide [f135-162]-SH increased the gel strength.
Journal of Agricultural and Food Chemistry 04/2013; · 2.82 Impact Factor
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ABSTRACT: α-Lactalbumin was glycated via the Maillard reaction in the dry state using various mono- and oligosaccharides. The reaction resulted not only in coupling of the saccharides to α-lactalbumin but also in cross-linked proteins. The glycation rate and the extent of cross-link formation were highly dependent on the saccharide used. Glycation by arabinose and xylose led to a very fast protein cross-link formation, whereas glucose showed a relatively low protein cross-linking ability. The stability of foams, created using the various glycated protein samples, depended on the type of saccharide used, the extent of glycation, and possibly the amount of cross-linked protein. Compared to nonmodified α-lactalbumin, glycation with rhamnose and fucose improved foam stability, whereas application of glucose, galacturonic acid, and their oligosaccharides did not exert a clear effect. Mass spectrometric analysis revealed that dehydration of the Amadori products is an indicator of the formation of protein cross-links.
Journal of Agricultural and Food Chemistry 12/2011; 59(23):12460-6. · 2.82 Impact Factor
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ABSTRACT: A bovine β-lactoglobulin hydrolysate, obtained by the hydrolysis by the Glu specific enzyme Bacillus licheniformis protease (BLP), was fractionated at pH 7.0 into a soluble and an insoluble fraction and characterized by LC-MS. From the 26 peptides identified in the soluble fraction, five peptides (A[f97-112] = [f115-128], AB[f1-45], AB[f135-157], AB[f135-158], and AB[f138-162]) bound to β-lactoglobulin at room temperature. After heating of β-lactoglobulin in the presence of peptides, eight peptides were identified in the pellet formed, three of them belonging to the previously mentioned peptides. Principle component analysis revealed that the binding at room temperature (to β-lactoglobulin) was related to the total hydrophobicity and the total charge of the peptides. The binding to the unfolded protein could not be attributed to distinct properties of the peptides. The presence of the peptides caused a 50% decrease in denaturation enthalpy (from 148 ± 3 kJ/mol for the protein alone to 74 ± 2 kJ/mol in the presence of peptides), while no change in secondary structure or denaturation temperature was observed. At temperatures <85 °C, the addition of peptides resulted in a 30-40% increase of precipitated β-lactoglobulin. At pH < 6, no differences in the amount of aggregated β-lactoglobulin were observed, which indicates the lack of binding of peptides to β-lactoglobulin at those pH values as was also observed by SELDI-TOF-MS. Although only a few peptides were found to participate in aggregation, suggesting specificity, principal component analysis was unable to identify specific properties responsible for this.
Biomacromolecules 06/2011; 12(6):2159-70. · 5.48 Impact Factor
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ABSTRACT: Surface activity and molecular changes of cuttlefish skin gelatin modified with oxidized linoleic acid (OLA) prepared at 60, 70 and 80 °C at different times were investigated. Modification of gelatin with OLA could improve surface activity of resulting gelatin as evidenced by the decreased surface tension and the increased foaming and emulsifying properties. Interaction between OLA and gelatin led to the generation of carbonyl groups, loss of free amino content and the increase in particle size of resulting gelatin. Emulsion stabilized by modified gelatin had the smaller mean particle diameter with higher stability, compared with that stabilized by gelatin without modification.
International journal of biological macromolecules 02/2011; 48(4):650-60. · 2.37 Impact Factor
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ABSTRACT: The peroxidase-mediated oxidation of calcium-depleted bovine α-lactalbumin generates a mixture of covalently bound protein oligomers with interesting foaming properties. Here, we isolated the initially formed covalent α-lactalbumin dimer and studied its mode of cross-linking. Liquid chromatography-Fourier transform mass spectrometry (LC-FTMS) of proteolytic digests revealed the unambiguous identification of a peroxidase-catalyzed covalent link between Tyr18 and Tyr50. This shows that, although the radical reaction is often regarded as a random reaction, the initial product formation is specific. Protein structural modeling indicates that the conjugation reaction between these tyrosines is sterically favored and involves initial noncovalent protein complex formation through charge compensation, facilitating intermolecular cross-linking. The identification of the Tyr18-Tyr50 cross-link supports the view that the peroxidase-mediated oxidation of apo α-lactalbumin is a sequential process, involving the formation of linear trimers and higher order oligomers.
Journal of Agricultural and Food Chemistry 01/2011; 59(1):444-9. · 2.82 Impact Factor
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ABSTRACT: The thermal unfolding and rheological properties of patatin gels were compared with those of commonly used proteins (β-lactoglobulin, ovalbumin, glycinin).
A significant difference between these proteins was observed in both the denaturation temperature (59 °C for patatin; about 20 °C lower than the other proteins) and the onset temperature of gel formation (50-60 °C, compared to 70-85 °C for the other proteins). At low ionic strength the minimal concentration was only 6% (w/v) for patatin, compared to 8-11% for the other proteins. This effect was attributed to the relatively high exposed hydrophobicity of patatin as determined by hydrophobic interaction chromatography. For gels compared at 'iso-strength', the frequency dependence was found to be close to identical, while small differences were observed in the strain at fracture.
Patatin was found to form gels with comparable small-deformational rheological properties as typical food proteins. In addition, at concentrations where the elastic modulus was similar for all proteins, the frequency and strain dependence were also comparable. From this it is concluded that patatin is a promising protein to be used in food applications as a gelling agent.
Journal of the Science of Food and Agriculture 10/2010; 91(2):253-61. · 1.44 Impact Factor
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ABSTRACT: Enzymatic protein cross-linking is a powerful tool to change protein functionality. For optimal functionality in gel formation, the size of the cross-linked proteins needs to be controlled, prior to heating. In the current study, we addressed the optimization of the horseradish peroxidase-mediated cross-linking of calcium-depleted bovine alpha-lactalbumin. To characterize the formed products, the molecular weight distribution of the cross-linked protein was determined by size exclusion chromatography. At low ionic strength, more dimers of alpha-lactalbumin are formed than at high ionic strength, while the same conversion of monomers is observed. Similarly, at pH 5.9 more higher oligomers are formed than at pH 6.8. This is proposed to be caused by local changes in apo alpha-lactalbumin conformation as indicated by circular dichroism spectroscopy. A gradual supply of hydrogen peroxide improves the yield of cross-linked products and increases the proportion of higher oligomers. In conclusion, this study shows that the size distribution of peroxidase-mediated cross-linked alpha-lactalbumin can be directed toward the protein oligomers desired.
Journal of Agricultural and Food Chemistry 03/2010; 58(9):5692-7. · 2.82 Impact Factor
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ABSTRACT: Many of the macroscopic properties of foams and emulsions are controlled by the mesoscopic properties of the thin films separating the bubbles or droplets. The properties of these films depend on contributions (1) from the adsorbed surface layers and (2) from the liquid that separates these adsorbed layers. To separate in the experimental studies the effects of these two contributions, we developed a new modified version of the capillary cell for foam film studies (originally developed by Scheludko and Exerowa (Scheludko, A.; Exerowa, D. Kolloid Z. 1959, 165, 148-151), which allows exchange of the film-forming liquid between the air-water surfaces. This modified cell allows one to distinguish between the role of the adsorbed species (e.g., proteins, particles, or long-chain synthetic polymers) and the species present in the film interior (e.g., particles, electrolytes, or surfactants). The film properties that can be studied in this way include film stability, rate of film thinning, and surface forces stabilizing the film. These properties are of significant interest in understanding and controlling the stability of dispersed systems. The experimental procedure and the capabilities of the modified cell are demonstrated in several examples.
Langmuir 05/2009; 25(11):6035-9. · 4.19 Impact Factor
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ABSTRACT: We have synthesized a new amphiphilic block oligomer by the enzymatic linking of a fatty acid (lauric acid) to a fructan oligomer (inulin) and tested the functionality of this carbohydrate derivative in foam stabilization. The structure of the modified oligosaccharide was found to be (Fruc)n(Glc)1CO-C11H23, which implies that on average one lauric acid molecule was linked to one inulin molecule. The new component produces foams with exceptional stability. Our results show that enzymatic acylation can produce an entirely new class of amphiphilic materials, with functionality comparable to that of synthetic block copolymers.
Langmuir 02/2008; 24(2):359-61. · 4.19 Impact Factor
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ABSTRACT: Unfolding of proteins has often been mentioned as an important factor during the adsorption process at air-water interfaces and in the increase of surface pressure at later stages of the adsorption process. This work focuses on the question whether the folding state of the adsorbed protein depends on the rate of adsorption to the interface, which can be controlled by bulk concentration. Therefore, the adsorption of proteins with varying structural stabilities at several protein concentrations was studied using ellipsometry and surface tensiometry. For beta-lactoglobulin the adsorbed amount (Gamma) needed to reach a certain surface pressure (Pi) decreased with decreasing bulk concentration. Ovalbumin showed no such dependence. To verify whether this difference in behavior is caused by the difference in structural stability, similar experiments were performed with cytochrome c and a destabilized variant of this protein. Both proteins showed identical Pi-Gamma, and no dependence on bulk concentration. From this work it was concluded that unfolding will only take place if the kinetics of adsorption is similar or slower than the kinetics of unfolding. The latter depends on the activation energy of unfolding (which is in the order of 100-300 kJ/mol), rather than the free energy of unfolding (typically 10-50 kJ/mol).
Journal of Colloid and Interface Science 08/2006; 299(2):850-7. · 3.07 Impact Factor
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ABSTRACT: There is an ongoing debate about whether a protein surface film at an air-water interface can be regarded as a gelled layer. There is literature reporting that such films show macroscopic fracture behavior and a rheology comparable to three-dimensional protein bulk-networks. If this is the case, a complete description of the formation of adsorbed layers should include a transition from single, freely moving proteins to a gelled layer. This report presents studies using spectroscopic techniques, such as infrared, fluorescence and neutron spectroscopy, or ellipsometry, to derive molecular insight in situ to substantiate the intermolecular networking in surface films of chicken egg ovalbumin. It is concluded that protein films, generated by equilibrium adsorption from the bulk, behave as a densely packed colloidal repulsive particle system, where the proteins still have a significant rotational mobility, have a predominantly retained globular fold, and show distinct (lateral) diffusion. Applied stresses on the surface film (by compressions of the interface) may result in protein denaturation and aggregation. This process renders a surface film from a colloidal particle into that of a gelled system.
Biopolymers 08/2006; 82(4):384-9. · 2.87 Impact Factor
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ABSTRACT: The stability of adsorbed protein layers against deformation has in literature been attributed to the formation of a continuous gel-like network. This hypothesis is mostly based on measurements of the increase of the surface shear elasticity with time. For several proteins this increase has been attributed to the formation of intermolecular disulfide bridges between adsorbed proteins. However, according to an alternative model the shear elasticity results from the low mobility of the densely packed proteins. To contribute to this discussion, the actual role of disulfide bridges in interfacial layers is studied. Ovalbumin was thiolated with S-acetylmercaptosuccinic anhydride (S-AMSA), followed by removal of the acetylblock on the sulphur atom, resulting in respectively blocked (SX) and deblocked (SH) ovalbumin variants. This allows comparison of proteins with identical amino acid sequence and similar globular packing and charge distribution, but different chemical reactivity. The presence and reactivity of the introduced, deblocked sulfhydryl groups were confirmed using the sulfhydryl-disulfide exchange index (SEI). Despite the reactivity of the introduced sulfhydryl groups measured in solution, no increase in the surface shear elasticity could be detected with increasing reactivity. This indicates that physical rather than chemical interactions determine the surface shear behaviour. Further experiments were performed in bulk solution to study the conditions needed to induce covalent aggregate formation. From these studies it was found that mere concentration of proteins (to 200 mg/mL, equivalent to a surface concentration of around 2 mg/m(2)) is not sufficient to induce significant aggregation to form a continuous network. In view of these results, it was concluded that the adsorbed layer should not be considered a gelled network of aggregated material (in analogy with three-dimensional gels formed from heating protein solutions). Rather, it would appear that the adsorbed proteins form a highly packed system of proteins with net-repulsive interactions.
Advances in Colloid and Interface Science 03/2006; 119(2-3):131-9. · 8.12 Impact Factor
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ABSTRACT: In this study a set of chemically engineered variants of ovalbumin was produced to study the effects of electrostatic charge on the adsorption kinetics and resulting surface pressure at the air-water interface. The modification itself was based on the coupling of succinic anhydride to lysine residues on the protein surface. After purification of the modified proteins, five homogeneous batches were obtained with increasing degrees of modification and zeta-potentials ranging from -19 to -26 mV (-17 mV for native ovalbumin). These batches showed no changes in secondary, tertiary, or quaternary structure compared to the native protein. However, the rate of adsorption as measured with ellipsometry was found to decrease with increasing net charge, even at the initial stages of adsorption. This indicates an energy barrier to adsorption. With the use of a model based on the random sequential adsorption model, the energy barrier for adsorption was calculated and found to increase from 4.7 kT to 6.1 kT when the protein net charge was increased from -12 to -26. A second effect was that the increased electrostatic repulsion resulted in a larger apparent size of the adsorbed proteins, which went from 19 to 31 nm2 (native and highest modification, respectively), corresponding to similar interaction energies at saturation. The interaction energy was found to determine not only the saturation surface load but also the surface pressure as a function of the surface load. This work shows that, in order to describe the functionality of proteins at interfaces, they can be described as hard colloidal particles. Further, it is shown that the build-up of protein surface layers can be described by the coulombic interactions, exposed protein hydrophobicity, and size.
The Journal of Physical Chemistry B 10/2005; 109(35):16946-52. · 3.70 Impact Factor
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ABSTRACT: Using a variety of spectroscopic techniques, a number of molecular functionalities have been studied in relation to the adsorption process of proteins to air-water interfaces. While ellipsometry and drop tensiometry are used to derive information on adsorbed amount and exerted surface pressure, external reflection circular dichroism, infrared, and fluorescence spectroscopy provide, next to insight in layer thickness and surface layer concentration, molecular details like structural (un)folding, local mobility, and degree of protonation of carboxylates. It is shown that the exposed hydrophobicity of the protein or chemical reactivity of solvent-exposed groups may accelerate adsorption, while increased electrostatic repulsion slows down the process. Also aggregate formation enhances the fast development of a surface pressure. A more bulky appearance of proteins lowers the collision intensity in the surface layer, and thereby the surface pressure, while it is shown to be difficult to affect protein interactions within the surface layer on basis of electrostatic interactions. This work illustrates that the adsorption properties of a protein are a combination of molecular details, rather than determined by a single one.
Biopolymers 74(1-2):131-5. · 2.87 Impact Factor
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ABSTRACT: Horseradish peroxidase can induce the oxidative cross-linking of proteins through the radicalization of tyrosine residues and subsequent formation of dityrosine bonds. The dityrosine bond absorbs light at 318 nm which can be used to monitor in-line the peroxidase-catalyzed cross-linking of proteins in a microfluidic system. In this study calcium-depleted α-lactalbumin is used as model protein. To quantify the progress of the reaction, the absorbance increase at 318 nm was monitored in-line and compared with the amount of reacted monomeric α-lactalbumin as determined with size-exclusion chromatography (SEC) at various residence times. The increase in absorbance at 318 nm shows a logarithmic relation with the extent of reacted monomer. The logarithmic relation can be explained using a reaction model describing minimum and maximum formation of dityrosine cross-links to reacted monomer. Since the size distribution of reaction products was found to be reproducible, the absorbance increase at 318 nm can be used as a fast in-line screening method for the peroxidase-mediated cross-linking of proteins.
Chemical Engineering Journal.
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ABSTRACT: In food industry, protein isolates are often used to help in the formation and stabilisation of food foams. Subsequently there is great interest in (1) understanding the effect of processing parameters on the functional properties of the isolate, and (2) methods and techniques that can help to predict the foam properties. This article describes the foaming properties of proteins that were modified in the Maillard reaction. From these relatively simple experiments results were obtained that indicate that for certain protein solutions the foam properties can vary significantly, while the interfacial properties are constant.Commercial protein isolates originate from only a few sources, mainly egg white and whey, and sometimes plant proteins (e.g. soy). Despite these limited sources a large variety of isolates with a wide range of properties is produced. One source of variation is the isolation procedure, but at least equally important are the conditions used before, during and after drying the protein solution to form the dry powder.From the literature it was found that one of the major changes to the protein during processing of the isolates is the covalent coupling of sugars via the Maillard reaction. To study the effects of these reactions, a model system was produced that consists of proteins that were glycated to different degrees using Maillard reaction. For each sample, interfacial properties (e.g. surface pressure, dilatational modulus) were determined, and foam experiments were performed. The results show that at constant concentration of both the protein (0.5 g/L) and sugar (0.7 g/L), the foam-ability and stability could be significantly improved (e.g. non-modified lysozyme does not foam, the highest modification is easily foamed and the foam has a half-life time of 200 s).Interestingly, the improved foam properties could not be related to any change in interfacial properties. While foam stability improved with increasing modification, the measured interfacial properties were not significantly affected. These observations seem to go against the general view that changes in foam behaviour should be reflected in changes in the interfacial properties. Additional experiments on thin liquid films were performed, where the disjoining isotherm was measured. These isotherms did not show significant differences in the interactions between the adsorbed layers. This indicates that the electrostatic and steric interactions between the adsorbed layers do not depend on the degree of modification. Only the thin film stability against rupture was found to increase with increasing modification. The thin film experiments lead to the hypothesis that aggregates (or oligomeric proteins) formed during modification might become trapped in the film. The presence of these oligomeric proteins could result in an increase of the apparent viscosity in these films, or in gelling or jamming of the liquid phase between the two interfaces. In other words, the observed behaviour is the result of the confined geometry of the thin films.The results confirm other observations that Maillard reactions improve foaming properties. Moreover, strong indications were found that to predict foam stability we need more than the traditional parameters (i.e. (dynamic) surface pressure, interfacial reology, and disjoining pressure).
Colloids and Surfaces A: Physicochemical and Engineering Aspects.
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ABSTRACT: The stability of adsorbed protein layers against deformation has in literature been attributed to the formation of a continuous gel-like network. This hypothesis is mostly based on measurements of the increase of the surface shear elasticity with time. For several proteins this increase has been attributed to the formation of intermolecular disulfide bridges between adsorbed proteins. However, according to an alternative model the shear elasticity results from the low mobility of the densely packed proteins. To contribute to this discussion, the actual role of disulfide bridges in interfacial layers is studied. Ovalbumin was thiolated with S-acetylmercaptosuccinic anhydride (S-AMSA), followed by removal of the acetylblock on the sulphur atom, resulting in respectively blocked (SX) and deblocked (SH) ovalbumin variants. This allows comparison of proteins with identical amino acid sequence and similar globular packing and charge distribution, but different chemical reactivity. The presence and reactivity of the introduced, deblocked sulfhydryl groups were confirmed using the sulfhydryl–disulfide exchange index (SEI). Despite the reactivity of the introduced sulfhydryl groups measured in solution, no increase in the surface shear elasticity could be detected with increasing reactivity. This indicates that physical rather than chemical interactions determine the surface shear behaviour. Further experiments were performed in bulk solution to study the conditions needed to induce covalent aggregate formation. From these studies it was found that mere concentration of proteins (to 200 mg/mL, equivalent to a surface concentration of around 2 mg/m2) is not sufficient to induce significant aggregation to form a continuous network. In view of these results, it was concluded that the adsorbed layer should not be considered a gelled network of aggregated material (in analogy with three-dimensional gels formed from heating protein solutions). Rather, it would appear that the adsorbed proteins form a highly packed system of proteins with net-repulsive interactions.
Advances in Colloid and Interface Science.
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ABSTRACT: Antioxidative activity and emulsifying properties of cuttlefish skin gelatin modified by different oxidised phenolic compounds including caffeic acid, ferulic acid and tannic acid at different concentrations were investigated. Oxidised phenolic compounds were covalently attached to gelatin as indicated by the decrease in amino groups. Fourier transform infrared spectroscopic studies indicated the presence of an aromatic ring and a hydroxyl group in gelatin after modification. Modified gelatin had the increased antioxidative activity but the decreased surface hydrophobicity. Gelatin modified with 5% oxidised tannic acid had no change in emulsifying properties. Emulsion stability and oxidative stability of menhaden oil-in-water emulsion stabilised by 0.5% and 1.0% gelatin without and with modification by 5% oxidised tannic acid were studied. Both gelatins at a higher concentration (1.0%) yielded an emulsion with a smaller particle size. Modified gelatin inhibited the formation of TBARS in the emulsion more effectively than the control gelatin throughout the 12 days of storage.
Food Chemistry.
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ABSTRACT: Conformation and foaming properties of cuttlefish skin gelatin modified by N-hydroxysuccinimide esters of different saturated fatty acids including capric acid (C10:0), lauric acid (C12:0) and myristic acid (C14:0) at different molar ratios (0.25, 0.50, 1.00 and 2.00) were investigated. Covalent attachment of fatty acids into gelatin was observed as evidenced by the decrease in amino groups. Fourier transform infrared spectroscopic study indicated the presence of alkyl group of modified gelatin. The higher increase in surface activity with coincidental increase in surface hydrophobicity was observed in gelatin modified with fatty acid ester having a longer chain, especially at the higher molar ratio. The increase in foam expansion was related with the improved surface activity mediated by the modification by N-hydroxysuccinimide esters of fatty acid.Graphical abstract
Food Hydrocolloids 25(5):1277-1284. · 3.47 Impact Factor
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ABSTRACT: The non-covalent interaction between cuttlefish skin gelatin and tannic acid was observed in gelatin modified with unoxidized tannic acid at pH 7, whereas covalent interaction was found in gelatin modified with oxidized tannic acid at pH 9. Degree of tannic acid incorporation into gelatin via non-covalent interaction was more pronounced than that found via covalent interaction as evidenced by lowered free amino group content and increased total phenolic content and hydroxyl group and aromatic ring determined by FTIR. Gelatin modified with oxidized tannic acid had the slight decrease in surface hydrophobicity, with no changes in particle size distribution of the emulsions. Modification of gelatin with tannic acid, especially via non-covalent interaction, increased in vitro antioxidative activity, compared with the control gelatin. Gelatin modified with tannic acid via covalent interaction rendered the emulsion with high stability and could inhibit lipid oxidation of menhaden oil-in-water emulsion effectively throughout the storage of 12 days.Industrial relevanceCuttlefish skin gelatin modified with tannic acid possessing both emulsifying activity and the improved antioxidative activity can be used as a natural and safe additive in food industry. Therefore, cuttlefish skin, a by-product from seafood processing industry, can be produced as the high value added product with wider applications.
Innovative Food Science & Emerging Technologies 11(4):712-720. · 3.03 Impact Factor
