Protein ingredient for carrying lipophilic nutrients

Details Protein ingredient for carrying lipophilic nutrients Protein ingredient for carrying lipophilic nutrients

1998-01-20

2001-09-18

Criares, Theodore J. (Department: 1617)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Cosmetic, antiperspirant, dentifrice

C424S439000, C424S440000, C424S442000

Reexamination Certificate

active

06290974

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to complexes of proteins and lipophilic nutrients and method of making the same. In particular, the present invention concerns the use of complexes formed between protein carriers and particular lipophilic nutrients as a food additive.
BACKGROUND OF THE INVENTION
Beta-lactoglobulin (&bgr;-Lg) is the most abundant whey protein in bovine milk. &bgr;-Lg represents up to 50% of whey proteins in the milks of ruminants and some nonruminants such as horses, pigs, dogs, dolphins, kangaroos, cats, and whales, but is absent from the milks from humans, rodents and lagomorphs. Perez and Calvo, (1995)
J. Dairy Sci.
78, 978. &bgr;-Lg is a 162 amino acid polypeptide with five cysteine residues, four of which participate in intramolecular disulfide bonds. There are two major &bgr;-Lg variants, designated &bgr;-Lg A and &bgr;-Lg B, which differ at two amino acid positions. Swaisgood, (1982)
Developments in Dairy Chemistry
1, 1-59 (Fox, P. F., ed.), Applied Science Publishers, London.
In the ruminant, &bgr;-Lg primarily exists as a dimer, but is present as an 18,000 Da monomer in the milk of pigs, horses and donkeys. Perez and Calvo, (1995)
J. Dairy Sci.
78, 978. Self-association to form dimers is affected by pH. Chen et al., (1993)
J. Protein Chem.
12, 613. Alterations in the pH have also been reported to promote tetramerization of &bgr;-Lg to form an octomer. Kumosinski and Timasheff, (1966)
J. Am. Chem. Soc.
88, 5635.
Analysis of the crystal structure of &bgr;-Lg indicates that the core of the molecule contains eight strands of &bgr;-sheets, which form an anti-parallel &bgr;-barrel (i.e., a calyx), with an &agr;-helix and another &bgr;-strand on the surface of the protein. Monaco et al., (1987)
J. Mol. Biol.
197, 695. The interior of the calyx is hydrophobic. The three-dimensional structure of &bgr;-Lg is very similar to that of retinol-binding protein (RBP), the primary transporter of retinol in serum, although there is only a 25-30% homology in amino acid sequence between these two proteins. Godovac-Zimmermann, (1988)
TBS
13, 64.
SUMMARY OF THE INVENTION
The low solubility of lipophilic nutrients (such as fat-soluble vitamins) poses a problem for the food industry. It is often difficult to maintain lipophilic nutrients in low-fat foods because they come out of solution and/or absorb to packaging materials. There is a need in the art for methods of providing lipophilic nutrients as food additives.
Accordingly, it is an object of the present invention to provide complexes of &bgr;-Lg and lipophilic nutrients for use as food additives, particularly in low-fat foods. Another object of the invention is to provide infant formulas, dietary supplements (e.g. nutrient and vitamin supplements), and topical compositions containing the disclosed &bgr;-Lg
utrient complexes. &bgr;-Lactoglobulin is highly soluble in aqueous solutions and acts as an effective carrier protein for lipophilic nutrients. Furthermore, bioselective absorption is employed to isolate &bgr;-Lg for use as a carrier protein. &bgr;-Lg prepared by bioselective absorption exhibits superior properties as a lipophilic carrier protein.
As a first aspect, the present invention provides a composition comprising a complex comprising &bgr;-lactoglobulin and a lipophilic nutrient selected from the group consisting of vitamin E and vitamin K
1
.
As a second aspect, the present invention provides a food composition comprising a complex comprising &bgr;-lactoglobulin and a lipophilic nutrient. In preferred embodiments, the lipophilic nutrient is vitamin A, vitamin D, vitamin E, vitamin K
1
, cholesterol, or conjugated linoleic acid.
As a third aspect, the present invention provides a dietary supplement comprising a complex comprising &bgr;-lactoglobulin and a lipophilic nutrient. In preferred embodiments, the lipophilic nutrient is vitamin A, vitamin D, vitamin E, vitamin K,, cholesterol, or conjugated linoleic acid.
As a fourth aspect, the present invention provides a method of manufacturing a food composition comprising a complex comprising &bgr;-lactoglobulin and a lipophilic nutrient, comprising the steps of: (a) purifying &bgr;-lactoglobulin by affinity purification; (b) forming a complex between the purified &bgr;-lactoglobulin of step a and a lipophilic nutrient; and (c) manufacturing a food composition comprising the complex formed between the isolated &bgr;-lactoglobulin and the lipophilic nutrient of step b.
As a fifth aspect, the present invention provides a topical composition comprising a complex comprising &bgr;-lactoglobulin and a lipophilic nutrient in a topical carrier. Preferably, the lipophilic nutrient is vitamin A or vitamin E.
These and other aspects of the present invention are disclosed in more detail in the description of the invention below.


REFERENCES:
Allen and Swaisgood, Applications for Purified Whey Protein Fractions Derived from Bioselective Adsorption Affinity Chromotography: Continuation,SDFRC 1997 Semi-Annual Reporty,pp. 36, 38-39 (1997).
Allen et al.; Binding of Retinoids to &bgr;-Lactoglobulin Isolated With Bioselective Adsorption,The Faseb Journal,10(3):A751-A752, Abstract 4350, Mar. 1996.
Allen et al.; Applications for Purified Whey Protein Fractions Derived from Bioselective Adsorption Affinity Chromatography, Southeast Dairy Foods Research Center, SDFRC Annual Report 95-96:19-21 (Jul. 1996).
Wang et al; &bgr;-Lactoglobulin Binds Vitamin D and Cholesterol, American Chemical Society, Annual Meeting Aug. 1996, Abstract 057.
Chen et al.; Purification and Characterization of &bgr;-Structural Domains of &bgr;-Lactoglobulin Liberated by Limited Proteolysis,Journal of Protein Chemistry,12(5):613-625 (1993).
Jang et al.; Analysis of Ligand Binding and &bgr;-Lactoglobulin Denaturation by Chromatography on Immobilized Trans-Retinal,J. Dairy Sci.,73(8):2067-2074 (1990).
Wang et al.; Characteristics of &bgr;-Lactoglobulin Binding to the All-Trans-Retinal Moiety Covalently Immobilized on Celite,J. Dairy Sci.,76(7):1895-1901 (1993).
Wang et al.; Binding of Retinoids to &bgr;-Lactoglobulin Isolated by Bioselective Adsorption,J. Dairy Sci.,80(6):1047-1053 (1997).
Wang et al., Binding of Vitamin D and Cholesterol to &bgr;-Lactoglobulin,J. Dairy Sci.,80(6):1054-1059 (1997).
Dufour et al.; Binding of retinoids and &bgr;-carotene to &bgr;-lactoglobulin. Influence of protein modifications,Biochimica et Biophsica Acta,1079:316-320 (1991).
Dufour et al.; &bgr;-Lactoglobulin binds retinol and protoporphyrin IX at two different binding sites,Febs Letters,277(1,2):223-226 (Dec. 1990).
Flower et al.; Structure and sequence relationships in the lipocalins and related proteins,Protein Science,2:753-761 (1993).
Frapin et al.; Probing the Fatty Acid Binding Site of &bgr;-Lactoglobulins,J. of Protein Chemistry,12(4):443-449 (1993).
Jasminka Godovac-Zimmermann; The structural motif of &bgr;-lactoglobulin and retinol-binding protein: a basic framework for binding and transport of small hydorphobic molecules?,TIBS13:64-66 (Feb. 1988).
Clement Ip et al., Retention of conjugated linoleic acid in the mammary gland is associated with tumor inhibition during the post-initiation phase of carcinogenesis,Carcinogenesis,18(4):755-759 (1997).
Clement Ip et al.; Mammary Cancer Prevention by Conjugated Dienoic Derivative of Linoleic Acid,Cancer Research,51:6118-6124 (1991).
Monaco et al.; Crystal Structure of the Trigonal Form of Bovine Beta-lactoglobulin and its Complex with Retinol at 2.5 ÅResolution,J. Mol. Biol.,197:695-706 (1987).
M.Z. Papiz et al.; The structure of &bgr;-lactoglobulin and its similarity to plasma retinol-binding protein,Nature vol.,324:383-385 (Nov. 1986).
Pérez et al.; Interaction of &bgr;-Lactoglobulin with Retinol and Fatty Acids and Its Role as a Possible Biological Function for This Protein: A Review,J. Dairy Sci.,78(5):978-988 (1995).
Puyol et al.; Interaction of Bovine &bgr;-Lactoglobulin and Other Bovine and Human Whey Proteins with Retinol and Fatty Acids,Agric. Biol. Chem.,55(10):2515-2520 (1991).
Spector et al.; Binding of Long Chain Fatt

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