Different possibilities for protein crosslinking are examined in this review, with special emphasis on enzymatic crosslinking and its impact on food structure. Among potential enzymes for protein crosslinking are transglutaminase (TG) and various oxidative enzymes. Crosslinking enzymes can be applied in cereal, dairy, meat, and fish processing to improve the texture of the product. Most of the current commercial applications are based on TG. The reaction mechanisms of the crosslinking enzymes differ, which in turn results in different technological properties.

Whey protein isolate (WPI) was chemically modified by vanillic acid in order to enhance its cross-linkability by laccase enzyme. Incorporation of methoxyphenol groups created reactive sites for laccase on the surface of the protein and improved the efficiency of cross-linking. The vanillic acid modified WPI (Van-WPI) was characterized using MALDI-TOF mass spectrometry, and the laccase-catalyzed cross-linking of Van-WPI was studied. Furthermore, the vanillic acid modification was compared with the conventional approach to improve laccase-catalyzed cross-linking by adding free phenolic compounds. A small extent of the vanillic acid modification significantly improved the cross-linkability of the protein and made it possible to avoid color formation in a system that is free of small phenolic compounds. Moreover, the potential application of Van-WPI as emulsifier and the effect of cross-linking on the stability of Van-WPI emulsion were investigated. The post-emulsification cross-linking by laccase was proven to enhance the storage stability of Van-WPI emulsion.

Sodium caseinate was modified by transglutaminase catalyzed cross-linking reaction prior to the emulsification process in order to study the effect of cross-linking on the oxidative stability of protein stabilized emulsions. The extent of the cross-linking catalyzed by different dosages of transglutaminase was investigated by following the ammonia production during the reaction and using SDS-PAGE gel. O/W emulsions prepared with the cross-linked and non-cross-linked sodium caseinates were stored for 30 days under the same conditions. Peroxide value measurement, oxygen consumption measurement, and headspace gas chromatography analysis were used to study the oxidative stability of the emulsions. The emulsion made of the cross-linked sodium caseinate showed an improved oxidative stability with reduced formation of fatty acid hydroperoxides and volatiles and a longer period of low rate oxygen consumption. The improving effect of transglutaminase catalyzed cross-linking could be most likely attributed to the enhanced physical stability of the interfacial protein layer against competitive adsorption by oil oxidation products.

Sodium caseinate was chemically modified in order to alter its isoelectric point (pI). Negatively charged carboxylic groups were introduced to lower the pI, and positively charged amino groups to achieve the opposite. Different chemical amino acid modification approaches were studied and the modified proteins were characterized using free amino group assays, SDS-PAGE, MALDI-TOF mass spectrometry, and zeta potential measurements. Oil-in-water emulsions were prepared using these modified caseinates. The pH stability behavior of the emulsions was monitored, and interestingly, the stability of the emulsion could be modulated through steering the pI of caseinate. Using different modified caseinates, it was possible to create emulsions that were stable in the acid, neutral, and alkaline regions of the pH spectrum. The stability behavior of the emulsions correlated well with the theoretical and experimentally determined pI values of the caseinates. Storage stability of emulsions was also studied at pH values around 7, and emulsions made of modified caseinates showed storage stability similar to that of unmodified caseinate emulsions.

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