Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-05-26
2001-09-04
Lee, Howard C. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S127000, C536S123120, C514S054000
Reexamination Certificate
active
06284886
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates generally to beta glucans. More particularly, the invention relates to methods and compositions for producing liquid beta glucan preparations from cereal sources.
2. Background of the Invention
Gums are either hydrophobic or hydrophilic high molecular weight substances that in an appropriate solvent produce gels or highly viscous suspensions or solutions at low dry substance content. Gums commonly used in food, medicine, and industrial products include starches, cellulose derivatives, guar gum, locust bean gum, pectin, algin, carrageenan, xanthan, beta glucan, and gum arabic, see Whistler, R. L. (1993)
Industrial Gums: Polysaccharides and Their Derivatives
Eds. Whistler R. L. and BeMiller J. N.(Academic Press) p. 2.
Most gums in a solid state consist of polysaccharide chains grouped in a disorganized manner. The random nature of this structure only partially satisfies the intermolecular interaction potential, for example hydrogen bonding potential is not saturated. The numerous unsatisfied hydrogen bonds are capable of rapid hydration, binding water molecules at hydrogen bonding positions not otherwise involved in intra- and intermolecular bonding of the polysaccharide molecules. When a polysaccharide is placed in water, the water molecules quickly penetrate amorphous regions and bind to available polymer sites, competing for and eventually reducing other interpolysaccharide bonds to negligible numbers. Segments of a polysaccharide chain become fully solvated and move away by kinetic action, tearing apart more interpolysaccharide bonds, which are immediately solvated. This intermediate stage in the dissolution of a polymer molecule represents a transient gel state and portrays a universal stage in the dissolution of all polysaccharides. For carbohydrates not fully soluble in cold water, dissolution is completed by heating with rapid mixing and results in a monodispersed hydrogel solution. Unless mechanisms are adopted to prevent reannealing, the hydrogel will form a gel on cooling.
When linear gum molecules in solution collide they form an association over several chain units. At the junctions of the chains, molecular bonding e.g. hydrogen bonds form and as the interactions spread throughout the solution a great three dimensional network is formed and the liquid becomes a gel. Once formed, a gel may undergo lengthening of junction zones as the molecules slide over each other or by moving together. This causes an overall tightening of network structure and a decrease in solvent filled spaces between molecules. Hence, the solvent is exuded from the gel to produce syneresis or weeping. Cooling or freezing a colloidal solution or gel accelerates this effect.
Industrial gums are sold as powders because of problems with solution stability. In order produce or enhance solubility, and stabilize gel solutions, gums may be chemically modified. For example addition of methyl, ethyl, carboxymethyl, hydroxyethyl, hydroxypropyl, phosphate, sulfate and similar groups enhances solubility and produces stable solutions of high viscosity. Beta glucan is classified as a viscous gum, see Wood, P. J. (1993)
Oat Bran
Ed. P. J. Wood (American Association of Cereal Chemists, Inc., St. Paul, Minn). Glucans are structural polysaccharides present in the cell wall of yeasts, bacteria, fungi, and cereals. For example, (1→3), (1→4), and mixed (1→3), (1→4) beta D glucans are found in the endosperm cell wall of such cereals as barley and oat, among others. Beta glucans affect the viscosity and hence the effectiveness of products derived from these sources. For example, beta glucans appear to influence digestion, assist in glucoregulation, and lower serum cholesterol. Cereal beta glucans are useful nutritional agents and have also been used as bulking agents in place of sucrose. Beta glucans have also been described as potent immune system stimulants and promote the healing of wounds. Yun et al.,
Int. J. Parasitol
. (1997) 27:329337; Estrada et al.,
Microbiol. Immunol
. (1997) 41:991998; Williams et al., (1997) U.S. Pat. No. 5,676,967. Beta glucan gels have also been used in suspending biocompatible particles for tissue injection, Lawin et al. (1995) U.S. Pat. No. 5,451,406. The cosmetics industry favors the use of beta glucan for its viscosity, shear strength and moisture enhancing properties.
The solubility properties of beta glucans differ according to their source. For example cereal beta glucans are normally soluble in aqueous solvents, whereas yeast (
Saccharomyces cerevisiae
) beta glucan are insoluble in aqueous solvents. Soluble glucans are desirable. Yeast beta glucan has been solubilized by the addition of phosphate groups, see Williams et al.,
Immununopharmacol
. 22:139-156 (1991). Jamas et al., U.S. Pat. No. 5,622,939 describes methods to extract soluble beta glucan from
Saccharomyces cerevisiae
. The method described is complex involving acid hydrolysis, base hydrolysis and the extensive use of centrifugation and ultrafiltration. No details are provided as to the stability of the solubilized yeast beta glucan.
Biological buffers are characterized by their zwitterionic properties. Good's buffers (see: Good, N. E. et al., Biochemistry 5:467 (1966); Good N. E. and Izawa S.
Meth. Enzymol
. 24:Part B 53 (1972); Ferguson W. J. and Good N. E.,
Anal Biochem
. 104:300 (1980)) were designed to have a pKa between 6.0 and 8.0, to have minimal salt effects due to the ionic composition of the solution, and to have minimal interactions with cations. The important feature of these buffers in relation to the stabilization of gum solutions is the temperature coefficient (pKa/C) that ensures the variation of pH with temperature. This property may be used to create both a destabilizing effect during formulation in the temperature range of 20° C. to 96° C., specifically the reversal of hydrogen bonding polarities and the transition through a zero polar, neutral state, as well as a stabilizing effect as a solution nears the critical freezing point.
DISCLOSURE OF THE INVENTION
The present invention provides for a simple and efficient method of formulating and producing stable solutions of beta glucan. The invention utilizes a biological, zwitterionic buffer during the purification process, to retard gelation and/or precipitation of the beta glucan upon cooling. The invention thus allows for greater yields of beta glucan, as well as the use of cruder cereal preparations as starting materials for purification of beta glucan.
Accordingly, in one embodiment, the invention is directed to a method for retarding gelation of a beta glucan solution. The method comprises combining a beta glucan-containing preparation with a biological, zwitterionic buffer solution to result in a beta glucan solution, under conditions sufficient to retard gelation of the beta glucan solution when the beta glucan solution is cooled to a temperature of about 10° C. or lower.
In another embodiment, the invention is directed to a method for retarding gelation of an oat beta glucan solution when the beta glucan solution is cooled to a temperature of about 10° C. or lower. The method comprises combining an oat beta glucan preparation with a HEPES buffer solution. The HEPES buffer solution comprises a preservative and HEPES buffer at a concentration of about 2.5 mM to about 5 mM and has a pH of about 7.2.
In yet another embodiment, the invention is directed to a method for purifying a beta glucan from a beta glucan-containing cereal preparation. The method comprises:
(a) combining the preparation with a biological, zwitterionic buffer solution under conditions sufficient to disperse the beta glucan, to form a beta glucan solution; and
(b) heating the beta glucan solution to produce a dispersed hydrocolloidal solution.
In preferred embodiments, the method further comprises filtering the dispersed hydrocolloidal solution to provide a clarified beta glucan solution.
In still a further embodiment, the invention is directed to a method for purifyi
Ceapro INC
Erratt Judy A.
Lee Howard C.
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