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Fluorescent probe and raman spectroscopic investigation of the effects of pH, heating, and [kappa]-carrageenan on whey protein structure Alizadeh-Pasdar, Nooshin


Surface hydrophobicity (S₀) is an important structural parameter describing food proteins due to its correlation with functionality. A new method to measure S₀ was established based on the neutral fluorescent probe, 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN). S₀ was determined for whey protein isolate (WPI), bovine serum albumin (BSA)and β-lactoglobulin (BLG) at various pH (3.0, 5.0, 7.0 and 9.0) under heated (80°C for 30 min) or unheated conditions. S₀ values measured by PRODAN and cis-parinaric acid (CPA) were lower at pH 3.0 than at other pH, while values measured by 1-anilinonaphthalene-8-sulfonic acid (ANS) were highest at pH 3.0. The anionic probes ANS and CPA yielded opposing results on the effects of pH and heating on proteinhydrophobicity. Heating, pH and addition of K-carrageenan (KCG) at low (1.0:62.5), medium(1.0:6.0) or high (1.0:1.2) ratios of KCG:protein, as well as the interactions between these factors, significantly (P<0.05) affected S₀ measured by PRODAN. Generally, proteins had higher S₀ and were more sensitive to heating and KCG addition at pH 9.0, than at other pH. Heating increased S₀ of high ratio KCG:protein mixtures at pH 3.0, but generally decreased SQ of mixtures at higher pH, especially pH 9.0.Raman spectroscopy was used to analyze structural changes at higher protein concentration (15% w/v) than those (0.002-0.01%) used in spectrofluorometry. Decreased SS and Trp band intensities, and lower helical and higher (3-sheet content, were generally observed after heating at pH 9.0 or KCG addition at pH 5.0. Decreased helical and increased (3-sheet contents obtained by heating BLG at pH 7.0 or 9.0 were not observed after heating KCG:BLG mixtures at these pH. Addition of KCG to BSA at pH7.0 or 9.0 resulted in increased helical content. Heating KCG:WPI mixtures at pH 5.0 and9.0 resulted in large increases in SS and Trp band intensities and helical content, and decreased ratio of the tyrosine doublet, indicating a more buried or hydrophobic environment around aromatic residues. Raman and fluorescent probe spectroscopy provided information on protein structural properties as a function of pH, heating and interactions with other macromolecules, which may be important in applications of these proteins in food systems.

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