The efficiency of a novel modified b-cyclodextrin to improve invertase conservation, in comparison to trehalose and other biopolymers.

SANTAGAPITA, PATRICIO R.; GÓMEZ BRIZUELA, L.; MAZZOBRE, MARÍA F.; VILLALONGA SANTANA, R.; CORTI, H.R.; BUERA, MARÍA DEL P.

Moeller Centre, Churchill College, Cambridge, UK

Congreso; Amorph 2006 - Molecular basis of stability in pharmaceutical and food glasses.; 2006

BioUpdate Foundation

Amorphous and crystalline states of certain sugars are relevant in biological, pharmaceutical and food sciences. 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 amorphous sugars manifest protective effects on biomolecules, and, consequently, the shelf life of a product. Several authors have indicated that the presence of a polymer promoted a delay in the crystallization process of sugars. The purpose of this work was to test the efficiency of different biopolymers to improve the stabilization of the enzyme b-fructofuranosidase (invertase) from Saccharomyces cerevisiae in freeze-dried formulations, in comparison to trehalose. Particularly, a novel b-cyclodextrin modified with alginate (b-CD-etilendiamine-alginate, b-CD-A) was employed as excipient. Combined matrices with trehalose and  controls were also tested for comparative purposes. Enzyme activity and thermal transitions of the systems (Tg and melting) were analyzed. The freeze dried systems consisted of solutions containing 118 enzymatic units/mL and 10% of trehalose or polymers (b-CD-A, alginate or b-CD) or their blends with trehalose. After freeze-drying the systems were transferred to vacuum desiccators and exposed to water activities (aw) of 0.22 and 0.43 at 25°C for one week. An accelerated stability test was performed at 87°C.During freeze-drying, the polymers or their mixtures did not conferred protection to invertase compared to trehalose. The curves describing the decrease of remaining enzymic activity during thermal treatment had an initial phase of fast decay, and reached a plateau value. When maintained in amorphous state (at aw 0.22), trehalose was an effective protectant, but this effect was lost upon sugar crystallization (at aw 0.43). All polymers had an inhibitory effect on trehalose crystallization. b-CD-A (with or without trehalose) and b-CD + trehalose were the best protective agents while b-CD and alginate showed a negative effect on invertase activity preservation at both aw values. The effect of excipients could be related to the delay of trehalose crystallization, in one side, but molecular interactions played a significant role. b-CD was not efficient as protectant. Through the addition of trehalose or by its covalent modification with alginate it becomes a suitable matrix to stabilize invertase. The presence of trehalose and the covalent modification with alginate seems to favor the interaction of b-CD with hydrophobic groups of the enzyme thus preventing its denaturation. b-CD-A have shown to be an appropriate additive for dehydrated formulations of invertase, allowing to protect the enzyme in conditions at which trehalose crystallizes.