Cell Microencapsulation: Dripping Methods.

Microencapsulation processes may be divided into three steps, namely: incorporation of the bioactive substance in the matrix, dispersion of the matrix in droplets, and conversion in microcapsules. This contribution is focused on the second step and more specifically using the dripping approach to form droplets by extrusion of liquid through a nozzle. Different technologies of dripping are described, using as an example the production of alginate beads.

Therapeutic cell encapsulation techniques and applications in diabetes.

The encapsulation of therapeutic cells permits the implantation of allogeneic and xenogeneic cells for the regulation of certain physiological processes damaged by the death or senescence of host tissues. The encapsulation of pancreatic cells for the treatment of diabetes is emphasized; however, many of the techniques are applicable to a wide array of mammalian cell applications. The summary of both established and novel encapsulation techniques, clinical trials, and commercial product developments highlights the metered but steady pace of therapeutic cell encapsulation towards implementation.

Development of formulations to improve the controlled-release of linalool to be applied as an insecticide.

In recent studies, insecticide activity of a monoterpene, linalool, has been demonstrated, finding, however, limitations in application because of its rapid volatilization. Potential effectiveness of microcapsules and effects of various types of matrices on its stability as controlled-release systems for the slow volatilization of linalool to be applied as insecticide were evaluated. To study controlled-release, linalool was entrapped into microcapsules, inclusion complexes, and beads, obtained by different methods, inverse gelation (IG1, IG2, IG3, IG4, and IG5), oil-emulsion-entrapment (OEE), interfacial coacervation (INCO), and chemical precipitation (Cyc5 and Cyc10). The encapsulation yield turned out to be different for each formulation, reaching the maximum retention for IG1 and OEE. In controlled-release, OEE followed by INCO presented a long time necessary for releasing as a result of the presence of glycerol or chitosan. These results pointed out remarkable differences in the release behavior of linalool depending on matrix composition and the method of encapsulation.

Encapsulation of a lipid precursor, the eicosapentaenoic acid, to study the development of the Crassostrea gigas oyster flavours.

The present study is part of a larger project whose aim is to understand how the oyster Crassostrea gigas develops its aromas from a lipid precursor, the eicosapentaenoic acid (EPA), in glyceride form. The objective of this study is, therefore, to prepare an encapsulation process that will enable the bivalve to be supplied with this lipid precursor. The complex coacervation method was chosen as it gave the best compatible microcapsules with respect to the nutritional aspects of oyster (i.e. digestibility) and the environmental constraints (i.e. behaviour and stability in seawater). The aim of this study is to manufacture and optimize a process of complex coacervation, to obtain capsules made of gelatin and acacia gum with a size under 100 microm in diameter and containing very small drops of cod liver oil (rich in EPA). The preservation of these microcapsules in seawater has been confirmed.

Characterization of microcapsules: recommended methods based on round-robin testing.

Alginate beads, as well as microcapsules based on alginate, cellulose sulphate and polymethylene-co-guanidine, were produced at diameters of 0.4, 1.0 and 1.5 mm. These standard materials were tested, by independent laboratories, in regards to water activity, bead or capsule size, mechanical resistance and transport behaviour. The water activity and mechanical resistance were observed to increase with bead and capsule size. Transport properties (ingress) were assessed using a variety of low molar mass and macromolecular probes. It was observed that the penetration of Vitamin B12 increased with bead diameter, as did dextran penetration. However, for the membrane-containing microcapsules, larger membrane thickness, observed for the larger capsules, retarded ingress. The authors, who are part of a European working group, recommend that permeability be assessed either using a large range of probes or a broad molar mass standard, with measurements at one or two molar masses insufficient to simulate the behaviour in application. Mechanical compression is seen as a good means to estimate elasticity and rupture of beads and capsules, with the sensitivity of the force transducer, which can vary from microN to tens of N, required to be tuned to the anticipated bead or capsule strength. Overall, with the exception of the mechanical properties, the precision in the inter-laboratory testing was good. Furthermore, the various methods of assessing transport properties agreed, in ranking, for the beads and capsules characterized, with gels having smaller radii being less permeable. For microcapsules, the permeation across the membrane dominates the ingress, and thicker membranes have lower permeability.

Production of alginate beads by emulsification/internal gelation.

Alginate microspheres were produced by emulsification/internal gelation of an alginate sol dispersed within vegetable oil, followed by a reduction in pH to release calcium from an insoluble salt. Microspheres with mean diameters ranging from 50 to 1,000 microm were obtained with standard deviations ranging from 35 to 45% of their mean value. Smooth, spherical beads were obtained with the narrowest size dispersion when using low guluronic and low viscosity alginate and a carbonate complex as calcium vector. The calcium salt must also be included within the alginate sol as a very fine powder to promote homogeneous gelation. Internal gelation was also tested with the dropping method. Observation of the beads produced revealed that the structure of the beads is more homogeneous than observed with external gelation. Shrinking is more important, although the diffusion of large molecules is faster with internal versus external gelation.

Improved performances and control of beer fermentation using encapsulated alpha-acetolactate decarboxylase and modeling.

The use of the enzyme alpha-acetolactate decarboxylase allows the acceleration of beer fermentation/maturation because it shunts diacetyl formation, whose elimination is the rate-limiting step of the process. To obtain a cost reduction by using this exogenous enzyme, we propose a new process involving recoverable encapsulated alpha-acetolactate decarboxylase. The performance of traditional and new processes was investigated by a modeling approach. A simple model, focused on alpha-acetolactate and diacetyl profiles during beer fermentation, was set up. The simulated profiles are consistent with literature data. This study shows also that encapsulated alpha-acetolactate decarboxylase allows the acceleration of beer fermentation as efficiently as free alpha-acetolactate decarboxylase. The advantage of immobilized alpha-acetolactate decarboxylase versus free enzyme is that it is recoverable and reusable, which means a process cost reduction.

High-resolution, human-bovine comparative mapping based on a closed YAC contig spanning the bovine mh locus.

A closed YAC contig spanning the mh locus was assembled by STS content mapping with seven microsatellite markers, eight genes or EST, and nine STS corresponding to YAC ends. The contig comprises 27 YACs, has an average depth of 4.3 YACs, and spans an estimated 1.2 Mb. A linkage map was constructed based on five of the microsatellite markers anchored to the contig and shown to span 7 cM, yielding a ratio of 160 kb/1 cM for the corresponding chromosome region. Comparative mapping data indicate that the constructed contig spans an evolutionary breakpoint connecting two chromosome segments that are syntenic but not adjacent in the human. Consolidation of human gene order by means of whole genome radiation hybrids and its comparison with the bovine order as inferred from the contig confirm conservation of gene order within segments.

Functional analysis of ZNF85 KRAB zinc finger protein, a member of the highly homologous ZNF91 family.

We previously identified the ZNF85 (HPF4) KRAB zinc finger gene, a member of the human ZNF91 family. Here, we show that the ZNF85 gene is highly expressed in normal adult testis, in seminomas, and in the NT2/D1 teratocarcinoma cell line. Immunocytochemical localization of a panel of beta-Gal/ZNF85 fusion proteins revealed that ZNF85 contains at least one nuclear localization signal located in the spacer region connecting the KRAB domain with the zinc finger repeats. Bacterially expressed ZNF85 zinc finger domain bound strongly and exclusively to DNA in vitro in a zinc-dependent manner. The KRAB(A) domain of the ZNF85 protein and of several other members of the ZNF91 family exhibited repressing activity when tested in Gal4 fusion protein assays. The repression was significantly enhanced by the addition of the KRAB (B) domain, whereas further addition of other conserved regions had no effect. The ZNF85 KRAB(A) and (B) domains in vitro bound several nuclear proteins that might constitute critical cofactors for repression.

Kzf1 - a novel KRAB zinc finger protein encoding gene expressed during rat spermatogenesis.

Two novel KRAB (Krüppel associated box) type zinc finger protein encoding cDNAs, named Kzf1 and Kzf2 (Kzf for KRAB zinc finger), were identified by screening of a rat embryonic brain cDNA library with a human ZNF91 KRAB probe. Kzf1 and Kzf2 encode proteins with an amino-terminal KRAB domain and a carboxy-terminal zinc finger cluster containing 9 and 13 zinc finger units, respectively. While Kzf2 appears to be ubiquitously expressed, Kzf1 is preferentially expressed in the testis. Within the testis, Kzf1 mRNA is restricted to germ cells. The Kzf1 protein exhibits DNA binding activity and its KRAB domain can function as a repressor module in transcription. Using somatic cell hybrid analysis, the Kzf1 gene was mapped to chromosome 6.

Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle.

We have determined the entire myostatin coding sequence for 32 double-muscled cattle sampled from ten European cattle breeds. Seven DNA sequence polymorphisms were identified, of which five would be predicted to disrupt the function of the protein, one is a conservative amino acid substitution, and one a silent DNA sequence variant. Four additional DNA sequence polymorphisms were identified in myostatin intronic sequences. In all but two breeds, all double-muscled animals were either homozygous or compound heterozygotes for one of the five loss-of-function mutations. The absence of obvious loss-of-function mutations in the coding sequence of the two remaining breeds points either towards additional mutations in unexplored segments of the gene, or towards locus heterogeneity of double-muscling.

External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation.

Alginate gels produced by an external or internal gelation technique were studied so as to determine the optimal bead matrix within which DNA can be immobilized for in vivo application. Alginates were characterized for guluronic/mannuronic acid (G/M) content and average molecular weight using 1H-NMR and LALLS analysis, respectively. Nonhomogeneous calcium, alginate, and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more uniform gels. Sodium was determined to exchange for calcium ions at a ratio of 2:1 and the levels of calcium complexation with alginate appears related to bead strength and integrity. The encapsulation yield of double-stranded DNA was over 97% and 80%, respectively, for beads formed using external and internal calcium gelation methods, regardless of the composition of alginate. Homogeneous gels formed by internal gelation absorbed half as much DNAse as compared with heterogeneous gels formed by external gelation. Testing of bead weight changes during formation, storage, and simulated gastrointestinal (GI) conditions (pH 1.2 and 7.0) showed that high alginate concentration, high G content, and homogeneous gels (internal gelation) result in the lowest bead shrinkage and alginate leakage. These characteristics appear best suited for stabilizing DNA during GI transit.

Enhanced expression in seminoma of human zinc finger genes located on chromosome 19.

Six Krüppel-type zinc finger (ZF) genes were cloned from a seminoma cDNA library. One, ZFS-1, showed high sequence homology to the ZNF91 KRAB (Krüppel-associated box) ZF gene family and also the same chromosomal assignment. Interestingly, Northern blot analyses using ZFS-1 and ZNF91 revealed that multiple ZF genes on chromosome 19 were predominantly expressed in seminomas. In addition, the testis and the seminoma showed specific expression of 2.3 kb transcript. Our results suggest that ZF genes on chromosome 19 may be implicated in the development and/or growth of seminomas.

A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle.

An exceptional muscle development commonly referred to as 'double-muscled' (Fig. 1) has been seen in several cattle breeds and has attracted considerable attention from beef producers. Double-muscled animals are characterized by an increase in muscle mass of about 20%, due to general skeletal-muscle hyperplasia-that is, an increase in the number of muscle fibers rather than in their individual diameter. Although the hereditary nature of the double-muscled condition was recognized early on, the precise mode of inheritance has remained controversial; monogenic (domainant and recessive), oligogenic and polygenic models have been proposed. In the Belgian Blue cattle breed (BBCB), segregation analysis performed both in experimental crosses and in the outbred population suggested an autosomal recessive inheritance. This was confirmed when the muscular hypertrophy (mh) locus was mapped 3.1 cM from microsatellite TGLA44 on the centromeric end of bovine chromosome 2 (ref. 5). We used a positional candidate approach to demonstrate that a mutation in bovine MSTN, which encodes myostatin, a member of the TGF beta superfamily, is responsible for the double-muscled phenotype. We report an 11-bp deletion in the coding sequence for the bioactive carboxy-terminal domain of the protein causing the muscular hypertrophy observed in Belgian Blue cattle.

Microencapsulation of lobster carotenoids within poly(vinyl alcohol) and poly(D,L-lactic acid) membranes.

The use of natural pigments such as lobster carotenoids in fish feed formulations offers advantages over the use of the synthetic alternatives. Microencapsulation of the pigments, with or without the addition of antioxidants to the formulation, may be of benefit in terms of stabilizing pigment colour. In the present study, lobster carotenoids were extracted from lobster shell into petroleum ether and microencapsulated by phase separation and salt coacervation within (poly vinyl alcohol) and poly(vinyl alcohol)/poly(D,L-lactic acid) membranes. Spherical microcapsules, with smooth, thin and resilient membranes were obtained with mean diameters ranging from 50 to 150 microns, depending on the membrane material, and source of pigment. The microcapsules were pink-orange in colour, and colour stability was followed spectrophotometrically. Enhanced stability was observed in both membrane materials, in comparison to the non-encapsulated control. Rates of discoloration were determined under a variety of storage conditions, including the absence of light, reduced temperatures and under nitrogen atmosphere. The best stability of lobster carotenoids was observed under a nitrogen atmosphere within PVA/PLA membranes, representing an 11-fold enhancement of pigment stability in comparison to the controls. Under ambient conditions, the enhancement in pigment stability was approximately 6-fold. The optimum concentration of PVA during microencapsulation was 3-4%, and the microencapsulated pigments appeared most stable under acidic conditions. The rate of discoloration appeared independent of pigment concentration.

Microencapsulation of DNA within alginate microspheres and crosslinked chitosan membranes for in vivo application.

Calf thymus DNA was microencapsulated within crosslinked chitosan membranes, or immobilized within chitosan-coated alginate microspheres. Microcapsules were prepared by interfacial polymerization of chitosan, and alginate microspheres formed by emulsification/internal gelation. Diameters ranged from 20 to 500 microns, depending on the formulation conditions. Encapsulated DNA was quantified in situ by direct spectrophotometry (260 nm) and ethidium bromide fluorimetry, and compared to DNA measurements on the fractions following disruption and dissolution of the microspheres. Approximately 84% of the DNA was released upon core dissolution and membrane disruption, with 12% membrane bound. The yield of encapsulation was 96%. Leakage of DNA from intact microspheres/capsules was not observed. DNA microcapsules and microspheres were recovered intact from rat feces following gavage and gastrointestinal transit. Higher recoveries (60%) and reduced shrinkage during transit were obtained with the alginate microspheres. DNA was recovered and purified from the microcapsules and microspheres by chromatography and differential precipitation with ethanol. This is the first report of microcapsules or microspheres containing biologically active material (DNA) being passed through the gastrointestinal tract, with the potential for substantial recovery.

Microencapsulation of Lactococcus lactis subsp. cremoris.

Lactococcus lactis subsp. cremoris was microencapsulated within alginate/poly-L-lysine (alg/PLL), nylon or crosslinked polyethyleneimine (PEI) membranes. Toxic effects were observed with solvents and reagents used in nylon and PEI membrane formation. Alg/PLL encapsulation resulted in viable and active cell preparations which acidified milk at a rate proportional to the cell concentration, but at rates less than that of free cell preparations. At 4 x 10(8) colony-forming units (cfu/ml milk), encapsulated cells took 17 per cent longer than free lactococci to reduce the pH of milk to 5.5. Similar activities of free and micro-encapsulated cells may be attained at higher cell concentrations (10(9) cfu/ml milk). The rate of lactic acid production was approximately 2 mmol/h at an encapsulated cell concentration of 4 x 10(8) cfu/ml.

Immobilization of cells for application in the food industry.

Immobilization of cells offers advantages to the food process industries, including enhanced fermentation productivity and cell stability and reduced downstream processing costs due to facilitated cell recovery and recycle. This article summarizes the varied immobilization methodologies, including adsorption, entrapment, covalent binding, and microencapsulation. Examples of interest to the food industry are provided, together with a review of the physiological effects of immobilization. Topics in process engineering include immobilized cell bioreactor configurations and the scale-up potential of the various immobilization techniques.

Microencapsulation within crosslinked polyethyleneimine membranes.

A microencapsulation technique is proposed involving the formation of a polyethyleneimine (PEI) membrane crosslinked by an acid dichloride. The membranes were formed at pH 8 in a non-polar solvent, conditions which are better suited for the encapsulation of biocatalysts or fragile biochemicals than those using polyamide membranes. The mean diameter and size distribution of the PEI microcapsules were similar to that observed with nylon membranes. The resultant microcapsules were spherical, free-flowing with a strong membrane. The mass of membrane was seen to be independent of the reaction time (1-4 min), insensitive to the PEI concentration and proportional to the concentration of crosslinking agent.