Plastic and nonmetallic article shaping or treating: processes – Encapsulating normally liquid material – Liquid encapsulation utilizing an emulsion or dispersion to...
Reexamination Certificate
1999-12-17
2001-12-04
Mullis, Jeffrey (Department: 1711)
Plastic and nonmetallic article shaping or treating: processes
Encapsulating normally liquid material
Liquid encapsulation utilizing an emulsion or dispersion to...
C264S004330
Reexamination Certificate
active
06325951
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to protein-encapsulating oil particles by complex coacervation, and more particularly to enzymatic cross-linking of the protein-encapsulating shell.
BACKGROUND OF THE INVENTION
Coacervation is the process by which an aqueous solution of a macromolecular colloid is separated into two liquid phases. One liquid phase, called the coacervate, is composed of many tiny colloid-rich droplets that are bound together. The other liquid phase, called the equilibrium liquid, is an aqueous solution of the coacervating agent.
When two or more oppositely-charged macromolecular colloids are used to form the coacervate, the process is termed complex coacervation. Colloids that bear a positive charge include gelatin and agar; colloids that bear a negative charge include carboxymethylcellulose and gum arabic. Depending upon each colloid's isoelectric point, dilution with water and/or adjustment of pH may be necessary for the particular colloids to be oppositely charged. These reactions must occur at a temperature above the gelling temperature for either colloid, otherwise the colloids will not be in a liquid phase and coacervation will not occur. When coacervation occurs in an environment that contains oil particles, the oil particles act as nucleating agents and the protein colloids deposit as a shell-like structure around each oil particle.
Encapsulating oil particles in the process of complex coacervation is well known in the prior art. U.S. Pat. No. 2,800,457 discloses oil-containing microscopic capsules and method of making them by complex coacervation. The '457 patent teaches dispersing a colloid in water, introducing an oil, forming an emulsion, dispersing a second colloid in water and mixing with the emulsion, and adjusting the pH and/or diluting with water to form a complex coacervate, all at a temperature above a gel point of the colloids, then cooling to cause a gel to form, followed by optional steps of hardening and cross-linking with formaldehyde or an equivalent. In one embodiment, gum arabic and gelatin are used to form a shell-like film of colloid material around an oil nucleus. Once the coacervate is formed, the mixture is allowed to stand for an hour at not over 25° C., after which time the formation of capsules is complete. The capsules may then be used as desired or may undergo the optional hardening step. U.S. Pat. No. 2,800,458 similarly discloses a method for making oil-containing microcapsules. The '458 patent discloses the use of a salt solution to form the coacervate, while the '457 patent discloses either altering pH or diluting with water to form the coacervate.
Cross-linking of the protein shell of the complex coacervate renders the protein-encapsulated oil thermostable, since a protein containing cross-links is a stable structure. The use of known chemical cross-linking agents, such as formaldehyde or glutaraldehyde, to irreversibly cross-link the oil-containing capsules is disclosed in the prior art. Other cross linking agents such as tannic acid (tannin) or potassium aluminum sulfate (alum) are similarly known. The optional hardening step disclosed in both the '457 patent and the '458 patent consists of adjusting a suspension of capsular material to pH 9 to 11, cooling to 0° C. to 5° C., and adding formaldehyde.
Formaldehyde and glutaraldehyde, while effective chemical cross-linking agents, are toxic. Thus, oil capsules that have been cross-linked using such chemicals cannot be used for oils that may be applied to or ingested within a mammalian body. This severely limits the applications for such products.
Certain naturally-occurring enzymes are also good cross-linking agents. Such enzymes work by catalyzing the formation of bonds between certain amino acid side chains in proteins. In addition, because the enzymes are naturally occurring, encapsulated oils that are enzymatically cross-linked do not suffer from the problems inherent with formaldehyde and glutaraldehyde cross-linking, and hence may be ingested or applied without concern about the toxicity of the cross-linking agent. Because cross-linking is a enzyme catalyzed reaction, however, the proper environmental conditions must exist for optimum enzyme activity.
An enzyme that catalyzes protein cross-linking is transglutaminase (amine y-glutamyl transferase, EC 2.3.2.13). Transglutaminase catalyzes an acyl transfer reaction between y-carboxamide groups of glutamine residues in a peptide and various primary amines, frequently e-amino groups of peptide-bound lysine residues. The result is a bond or cross-linkage between a glutamine residue in one protein molecule and a lysine residue in another protein molecule. For optimal activity, transglutaminase requires a divalent metal ion, usually calcium or magnesium, as a cofactor and a pH of around 7.
Japanese patent publication 5-292899 to Ajinomoto Inc. discloses the use of transglutaminase as a cross-linking agent in preparing microcapsules. The structure taught in that publication, however, is not believed to be a complex coacervate as defined by those skilled in the art. It is, rather, an enzyme-modified gelatin emulsion. Additionally, the 5-292899 publication discloses cross-linking at elevated temperatures. Molecular and/or particulate structures maintained at elevated temperatures are more fluid and less stable, resulting in cross-linking a molecule or particles of undefined structure. The Ajinomoto publication “Ajinomoto Co.'s Transglutaminase (TG)” discloses optimum cross-linking conditions for transglutaminase at pH 6-7 and elevated temperatures of 50° C.
SUMMARY OF THE INVENTION
This invention relates to a method of enzymatically protein-encapsulating oil particles by complex coacervation. According to this method, a complex coacervate is first formed and then stabilized by gelling a protein shell around discrete particles of oil. The protein shell of the stabilized protein-encapsulated particles is then cross-linked with an enzyme to provide thermostable microparticles.
The method also achieves a number of advantages over the prior techniques. The method produces microcapsules having defined structures and sizes which have diverse properties for different end uses. For example, flavor oils that are in protein-encapsulated particles ranging from approximately 100 to approximately 300 microns are sized to both provide a significant flavor burst upon chewing and to enable processing in food applications. While particle sizes greater than 300 microns may be formed, such larger particles are not as amenable to the spraying, extruding, and other mechanical shearing forces required in many food applications. Additionally, protein-encapsulated flavor oil particles are thermostable and can withstand baking, frying, and microwaving.
In one preferred method of this invention, a coarse emulsion is first formed between the oil and the colloid dispersion of two oppositely charged colloids. A complex coacervate is then formed with a protein shell around discrete oil particles. The discrete particles are cooled to gel the surrounding protein shell. The protein shell surrounding the discrete particles is then enzymatically cross-linked at low temperatures to form microcapsules of oil. It has been found that at low temperatures of about 20° C. to about 27° C., especially at 5° C. to 10° C., enzymatic cross-linking can be achieved for protein shells of fish and beef gelatins to provide the microcapsules of flavor oils. Furthermore, the cross-linking reaction at such low temperatures is not pH dependent. Thus, a wide pH range of about 2 to about 10 or more may be utilized, which broadens the number and types of enzymes which may be employed.
In a preferred form of the invention, transglutaminase is employed to enzymatically cross-link the protein shell at a pH of about 7 over a temperature range of about 5° C. to about 10° C. Processing times and quantities of microencapsulated oils may be economically achieved for commercial purposes according to the preferred modes of operation.
The objectiv
Soper Jon C.
Thomas M. Teresa
Givaudan Roure Flavors Corporation
Mullis Jeffrey
Wood Herron & Evans L.L.P.
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