Characterization of physicochemical properties of caseinomacropeptide treated with transglutaminase

Caseinomacropeptide (CMP) is released to the whey during cheese production process by rennet enzyme which breaks down κ-casein. Whey protein contains 12-15% g/L protein in its structure and around 20 to 25% of this protein amount is CMP. Caseinomacropeptide is water-soluble hydrophilic and negatively charged peptide even at acidic pH values. CMP is isolated from whey and available for use in foods as a functional bioactive peptide. Transglutaminase is a cross-linking enzyme used for improvement of structural functions of proteins. In this study, effect of transglutaminase treatment on physicochemical properties of a commercial caseinomacropeptide isolate (CMPI) and textural properties of the gel obtained from it was investigated. CMPI samples were prepared at a concentration of 20% (w/w) with deionized and distilled water. Since CMPI contained whey proteins at a concentration of 3.6% (w/w), aqueous WPI solution was prepared at this concentration to determine the possible effect of whey proteins on gelation of CMPI. Solutions were treated with transglutaminase at an enzyme activity in the range of 0-12.5 U/g protein at their pH. Solutions were incubated at 40C for 1 h with gentle stirring for cross-linking with the enzyme, then cooled in an ice-water bath to stop the enzyme reaction. Control samples were prepared and incubated the same way without the enzyme for both CMPI and WPI. The pH of the solutions was adjusted to pH 3 as this pH was found to provide a strong gel of CMP. Physicochemical properties of the samples were determined by measurements of hydrophobicity, zeta potential and gelation properties. Textural properties of the gels obtained from the sample solutions were also determined. Zeta potential (ζ) of the solutions were measured by using a particle size instrument with the capability to measure zeta potential. Caseinomacropeptide solutions were diluted to 0.2% (w/w) concentration and adjusted to pH values in the range of 2.5-6.7. Control and enzyme-treated samples were treated the same way. Isoelectric point of the samples was determined by fitting a polynomial equation to the data and finding the point at which the equation gives 0 zeta potential. To measure hydrophobicity, CMPI samples were diluted to concentrations in the range of 0.01-0.1%. The fluorescent 8-aniline-1-naphthalene sulfonic acid (ANS) probe was used with a concentration of 3 mM in phosphate buffer at pH 7.0. CMPI and ANS solution were mixed and kept under dark at room temperature for 15 min. Fluorescence intensities measured at 480 nm after excitation at 390 nm. Gelation of the samples was measured with a rheometer. Temperature sweep in the range of 25-70C was applied to determine the gelation temperature, then held at 70ºC for 30 min for completion of gelation and after, cooled to 25ºC. Gelation temperature recorded when elastic modulus (G΄) passed over 1 Pa. Same measurements were also carried out with WPI samples to scrutinize the possible role of whey protein on the gel formation of CMPI. Gels were prepared from the samples in water bath at 70ºC for 30 min for determination of textural properties and syneresis of the samples. Textural properties of the gel obtained were measured with a texture analyzer. Penetration test was applied for determination of hardness and work of penetration. Syneresis in the samples were measured by centrifuge method. Zeta potential measurements showed that surface charge changed from positive to negative values with increasing pH. pI of CMPI and enzyme-treated CMPI were at the pH 3.47 and 2.98, respectively. Enzyme-treated CMPI had lower hydrophobicity compared to that of the CMPI itself. In rheological analysis, no significant difference in gelation temperature and G' values during gelation was found between untreated CMPI and transglutaminase-treated CMPI. No gel formation for WPI and enzyme-treated WPI was observed which meant that whey proteins were not affecting the gelation of CMP under the conditions of this study. Hardness and penetration work of the CMPI gels significantly enhanced by treatment with increasing activity of transglutaminase in texture analyses. These significant changes showed that covalent cross-linking of CMPI with transglutaminase strengthened the gel of CMPI. No serum separation was observed in all the samples.