The efficiency of different sugars, polysaccharides and salts for the preservation of frozen enzymes

SANTAGAPITA, PATRICIO R.; GARCÍA CRUZ, ARIEL; MAZZOBRE, M. FLORENCIA; PENICHE, CARLOS; VILLALONGA SANTANA, REYNALDO; BUERA, M. PILAR

Viña del Mar, Chile

Congreso; International Congress of Engeneering and Food (ICEF 10); 2008

Ministerio de Agricultura (Chile), Pontificia Universidad Católica de Chile, Universidad Técnica Federico Santa María.

The conservation of labile biomolecules is generally performed in dehydrated or frozen media. Undesired or uncontrolled crystallization processes have become a main issue in the food industry, since crystallization of the solid phase may significantly affect the mechanisms by which sugars manifest protective effects on biomolecules, and, consequently, the shelf life of a product. 1. Objective The purpose of this work was to test the efficiency of different sugars, natural and modified  polysaccharides and salts to improve the stabilization of enzymes in frozen formulations, in comparison to trehalose. 2. Methods 20% (w/v) solutions containing biopolymers (polyethilenglycol (PEG), chitosan and/ or their covalent modifications with cyclodextrins or by acylation) or trehalose and trehalose-salt mixtures (chlorides, acetates and citrates) were prepared with the corresponding enzyme. The solutions were stored at -26°C and enzyme activity (catalase, amylase, urease and aspartate amino transferase). Thermal transitions (glass transition, water crystallization/melting) were analyzed in all systems after storage. Spin-spin relaxation times and atomic force microscopy of the biopolymer films (obtained by dip coating) were also employed for the systems characterization. 3. Results In frozen systems, the amount of ice formed in the diluted enzymatic extracts was higher than in more concentrated ones, negatively influencing enzyme stability. The amount of water associated to the amorphous frozen matrix increased when salts were present. Thus, enzyme stability impaired due to the increased molecular mobility, and to the high concentration of salt in the unfrozen matrix. Succinylated chitosan, cyclodextrin-modified PEG, and their mixtures with trehalose have shown to be appropriate additives for frozen enzyme formulations. 4. Significance to the food engineering field. The effect of excipients could be related to the delay of water crystallization, in one side, but molecular interactions played a significant role. The adequate modification of biopolymers in order to protect enzyme native conformations may lead to the rational preparation of cryoprotective media, as alternative for trehalose.