Research papers
Protein loss by the microencapsulation of an enzyme (lactase) in alginate beads

https://doi.org/10.1016/S0378-5173(97)00172-5Get rights and content

Abstract

Alginate gel beads are used in many applications as matrices for release or immobilisation. The problems of enzyme stability and low entrapment efficiency by this microencapsulation procedure are well known, especially with water-soluble substances like peptides. Microencapsulation of the enzyme lactase into alginate beads resulted in a protein loss of about 36%. During the crosslinking step, the bead weight decreased due to the `syneresis' phenomenon: the carboxylate groups of the guluronate monomers complex the calcium cations. The water leakage was 44% when 1% sodium alginate was used. The contraction phenomenon by the alginate crosslinking and subsequent water leakage is the reason for the loss of water soluble peptides. A reduction of water leakage was achieved decreasing the pH of the alginate solution from 6.5 to 4.3. To hold back the water in the beads, 1% of bentonite was added and the water leakage was reduced to 28%. The protein adsorption onto alginate beads was shown to be affected by pH. Namely, the maximum of protein adsorption was found at a pH slightly below the isoelectric point of lactase (4.61). On charging the protein positively, a binding to anionic alginate occurred. On decreasing the pH of the alginate solution and adding bentonite, the protein loss was reduced from 36 to 3% without lowering of the enzyme activity.

Introduction

The immobilisation of enzymes within microcapsules has several advantages. The microcapsules can easily be separated from the reaction products by filtration, microencapsulated enzymes can be used for selective cleavage of low molecular weight substrates in the presence of high molecular weight substrates and immobilized enzymes can be used many times. One of the most widespread methods to immobilize enzymes or microorganisms is their inclusion into semipermeable microcapsules (Lim and Sun, 1980Poncelet et al., 1992Monshipouri and Price, 1995). When an aqueous solution of sodium alginate is added dropwise to a solution containing divalent metal ions, spherical alginate beads are produced. An insoluble alginate matrix is formed by cation exchange. The l-guluronan blocks of alginate react with Ca2+ ions according to a cooperative mechanism, giving rise to the formation of junction zones referred to as the eggbox model. Alginate beads have the advantage of being nontoxic as biological entities.

The problems of enzyme stability and low entrapment efficiency by this microencapsulation procedure is well known, especially with water-soluble substances like peptides. Arginase microencapsulation was reported by Kondo (1976)and he concluded that enzyme inactivation during microencapsulation was caused by the contact of arginase with an organic solvent and incorporation of the enzyme into the membrane. Wood and Whateley (1982)investigated the loss of enzyme activity during an interfacial polymerization microencapsulation process. The low yields of activity (about 40%) found in polyamide microcapsules for chymotrypsin are typical (Aisina, 1992).

Significant protein loss was observed by the microencapsulation in alginate beads. Pommersheim et al. (1994)reported that during the formation of initial gel beads, 34.4% of the enzyme was lost by diffusion into solution without denaturation. The main loss (30.2%) happened in the first bath when the droplets are hardened to gel beads. Thus, the enzyme is well protected against diffusion by the gel beads even without an additional membrane (Pommersheim et al., 1994).

Protein adsorption onto alginate beads was shown to be affected by pH. The maximum of adsorption was found at a pH slightly below the isoelectric point of the protein (Velings and Mestdagh, 1994).

The enzyme lactase (β-galactosidase) belongs to the group of sugar converting enzymes in the family of hydrolases as well as other hydrolytic enzymes, for instance, lipases, esterases, peptidases etc.

Lactases have been isolated from different organisms, eukaryotes as well as procaryotes. Thermostable lactases were found in bacterial strains of the genus Thermus (Berger et al., 1995) and Bacillus (Choi et al., 1995). Lactases from Kluyveromyces fragilis (Carrara and Rubiolo, 1996), K. lactis (Cavaille and Combes, 1995), Bifidobacterium bifidum (Passerat and Desmaison, 1995) and E. coli are examples for other important bacterial enzymes.

The β-galactosidase from Xanthomonas manihotis exhibits strong homology to several eucaryotic β-galactosidases from plants, animals and fungi. For this enzyme the strongest similarity was found with lactases of human and mouse lysosomes with 42 and 41% identity respectively (Taron et al., 1995). Fungal β-galactosidase from Aspergillus species is prepared for technical applications (Iwasaki et al., 1996).

Lactases are important tools for the treatment of milk intolerances in adult humans, which are caused by the disaccharide, lactose. The enzymes are used to pre-hydrolyze milk. Lactase preparations are commercially available to be added to milk (Passerat and Desmaison, 1995, Suarez et al., 1995).

The objective of this study was to investigate the reasons which lead to protein loss during enzyme microencapsulation into alginate beads and to find measures to overcome this loss.

Section snippets

Materials

Sodium alginate (Kelco International Ltd. Tadworth, UK), lactase (Amano Pharmaceutical Co. Ltd., Japan), isolated from Aspergillus oryzae, bentonite (Sigma, St. Louis, MO, USA) were used.

All other chemicals were of analytical grade and used without any further purification.

Formation of microcapsules

For the alginate bead preparation the method of Lim and Sun (1980)was applied. For drop formation the DropJet System MJ-K-120 (Microdrop GmbH, Norderstedt, Germany) was used.

Water leakage

Water leakage was determined as follows: 300 ml of

Results and discussion

When drops of alginate solution fall into a CaCl2 solution, beads are immediately formed. For a short time, the beads stay on the top of the solution. After a while they sink due to an increase in the density. This period is known as the maturation step (Velings and Mestdagh, 1995). The bead's weight decreases due to the `syneresis' phenomenon: the carboxylate groups of the guluronate monomers complex with the calcium cations. This network formation reduces the space occupied by the alginate

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