Prepared foods containing triglyceride-recrystallized non-esterified phytosterols Number:7,144,595 from the United States Patent and Trademark Office (PTO) owispatent A prepared food product for ingestion by mammals is described and methods for preparing such products. The food product includes an oxidation-resistant fat-based composition substantially free of exogenous solubilizing and dispersing agents for phytosterols. The fat-based composition includes between 75% and 98% by weight of at least one triglyceride-based edible oil or fat, and between 2% and 25% by weight of non-esterified phytosterols. Typically, approximately 1.5% by weight of phytosterols remain soluble at room temperature, and between 0.5% and 23.5% by weight are converted to triglyceride-recrystallized phytosterols (TRPs). The fat-based composition which has been partially oxidized in the prepared food by exposure to air (and typically heat), contains a reduced amount of oxidative by-products compared to a similar fat-based composition lacking these non-esterified phytosterols.<H recrystallized, phytosterols, Prepared, foods, , 7144595.html, Patent, pto, 7144595

Title: Prepared foods containing triglyceride-recrystallized non-esterified phytosterols

Abstract: A prepared food product for ingestion by mammals is described and methods for preparing such products. The food product includes an oxidation-resistant fat-based composition substantially free of exogenous solubilizing and dispersing agents for phytosterols. The fat-based composition includes between 75% and 98% by weight of at least one triglyceride-based edible oil or fat, and between 2% and 25% by weight of non-esterified phytosterols. Typically, approximately 1.5% by weight of phytosterols remain soluble at room temperature, and between 0.5% and 23.5% by weight are converted to triglyceride-recrystallized phytosterols (TRPs). The fat-based composition which has been partially oxidized in the prepared food by exposure to air (and typically heat), contains a reduced amount of oxidative by-products compared to a similar fat-based composition lacking these non-esterified phytosterols.

Patent Number: 7,144,595 Issued on 12/05/2006 to Perlman, et al.

Inventors: Perlman; Daniel (Arlington, MA), Hayes; Kenneth (Wellesley, MA), Pronczuk; Andrzej (Boston, MA)
Assignee: Brandeis University (Waltham, MA)

Appl. No.: 10/677,634

Filed: October 1, 2003

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
PCT/US02/36809	Nov., 2002
10295929	Nov., 2002	6638547
60332434	Nov., 2001

Current U.S. Class: 426/611 ; 426/607

Current International Class: A23D 9/00 (20060101)

References Cited [Referenced By]

U.S. Patent Documents


3881005	April 1975	Thakkar et al.
4195084	March 1980	Ong
5244887	September 1993	Straub
5419925	May 1995	Seiden et al.
5502045	March 1996	Miettinen et al.
5932562	August 1999	Ostlund, Jr.
5952393	September 1999	Sorkin et al.
5998396	December 1999	Nakano et al.
6025348	February 2000	Goto et al.
6031118	February 2000	Van Amerongen et al.
6106886	August 2000	Van Amerongen et al.
6117475	September 2000	van Amerongen et al.
6129944	October 2000	Tiainen et al.
6139897	October 2000	Goto et al.
6214534	April 2001	Horowitz et al.
6267963	July 2001	Akashe et al.
6326050	December 2001	Goto et al.
6391370	May 2002	Rogers et al.
6531463	March 2003	Yliruusi et al.
6638547	October 2003	Perlman et al.
2001/0034338	October 2001	Sorkin
2004/0156887	August 2004	Auriou
2004/0260104	December 2004	Sicre et al.

Foreign Patent Documents


1005859	Jun., 2000	EP
WO 99/43218	Sep., 1999	WO
WO/9943218	Sep., 1999	WO
WO/0132029	May., 2001	WO

Other References

Faquhar, et al., "The Effect of Beta Sitosterol on the Serum Lipids of Young Men Arteriosclerotic Heart Disease," Circulation, 14:77-82, (1956). cited by other .
Gertz, et al., "Testing and Comparing Oxidative Stability of Vegetable Oils and Fats at Frying Temperature", Eur. J. Lipid Sci. Technol., 102:543-551, (2000). cited by other .
Jones, et al., "Cholesterol-Lowering Efficacy of a Sitostanol-Containing Phytosterol Mixture with a Prudent Diet in Hyperlipidemic men," Am. J. Clin. Nutr., 69:1144-1150, (1999). cited by other .
Kim, et al., "Direct Determination of the Free Cholesterol and Individual Cholesteryl Esters in Serum by High Pressure Liquid Chromatography," Korean J. Biochem., 16:69-77, (1984). cited by other .
Kucchodkar, et al., "Effects of Plant Sterols on Cholesterol Metabolism in Man," Atherosclerosis, 23:239-248, (1976). cited by other .
Lees, et al., "Plant Sterols as Cholesterol-Lowering Agents: Clinical Trials in Patients with Hypercholesterolemia and Studies of Sterol Balance," Atherosclerosis, 28:325-333, (1977). cited by other .
Ntanios and Jones, "Effects of Variable Dietary Sitostanol Concentrations on Plasma Lipid Profile and Phytosterol Metabolism in Hamsters," Biochem. Biophys. Acta., 1390:237-244, (1998). cited by other .
Ntanios, et al., "Dietary Sitostanol Reduces Plaque Formation But Not Lecithin Cholesterol Acyl Transferase Activity in Rabbits," Atherosclerosis, 138:101-110, (1998). cited by other .
Plat et al., "Therapeutic Potential of Plant Sterols and Stanols," Current Opinion in Lipidology, 11:571-576, (2000). cited by other .
Wang, et al., "Antioxidant Activity of Phytosterols, Oryzanol, and Other Phytosterol Conjugates", JAOCS, 79(12):1201-1206, (2002). cited by other .
Weingand, and Daggy, "Quantification of High-Density-Lipoprotein Cholesterol in Plasma From Hamsters by Differential Precipitation," Clinical Chemistry, 36(3):575 (1990). cited by other .
White, Pamela J. and Armstrong, Lillian S., "Effect of Selected Oat Sterols on the Deterioration of Heated Soybean Oil", JAOCS, 63(4):525-529, (1986). cited by other .
Witztum, et al., "Cholestyramine-Induced Changes in Low Density Lipoprotein Composition and Metabolism," Journal of Lipid Research, 26:92-103, (1985). cited by other .
Yanishlieva, Nedyalka and Schiller, Helmut, "Effect of Sitosterol on Autoxidation Rate and Product Composition in a Model Lipid System", J. Sci. Food Agric., 35:219-224, (1983). cited by other.

Primary Examiner: Paden; Carolyn
Attorney, Agent or Firm: Foley & Lardner LLP

Parent Case Text

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation-in-part of PCT Application PCT/US02/36809 and a continuation-in-part of U.S. application Ser. No. 10/295,929, filed Nov. 14, 2002, now U.S. Pat. No. 6,638,547, which claim the benefit of U.S. Provisional application 60/332,434, filed Nov. 16, 2001, each of which is incorporated herein by reference in its entirety, including all figures and tables.

Claims

What is claimed is:

1. A prepared food product for ingestion by mammals, comprising: an oxidation-resistant fat-based composition substantially free of exogenous solubilizing and dispersing agents for phytosterols, comprising: at least one triglyceride-based edible oil or fat; and 2% to 25% by weight of non-esterified triglyceride-recrystallized phytosterols (TRPs), wherein said TRPs precipitate and recrystallize at room temperature; wherein said TRPs are formed without segregating said phytosterols from said triglyceride-based edible oil or fat; wherein said TRPs have a reduced melting temperature compared to segregated phytosterols; wherein said fat-based composition has been partially oxidized by an interval of exposure to air; wherein said fat-based composition contains a reduced amount of polar and oxidative by-products compared to a similar fat-based composition lacking said non-esterified phytosterols; and wherein said reduced amount of oxidative by-products is reduced by at least 10%.

2. The prepared food product of claim 1, wherein said fat-based composition comprises 2 5% of said TRPs.

3. The prepared food product of claim 1, wherein said fat-based composition comprises 5 10% of said TRPs.

4. The prepared food product of claim 1, wherein said fat-based composition comprises 10 15% of said TRPs.

5. The prepared food product of claim 1, wherein said fat-based composition comprises 15 20% of said TRPs.

6. The prepared food product of claim 1, wherein said fat-based composition comprises 20 25% of said TRPs.

7. The prepared food product of claim 1, wherein said TRPs are formed by heating said fat-based composition combined with one or more other components of said food product to a temperature of greater than 60.degree. C. to dissolve said non-esterified phytosterols, and subsequently cooling said prepared food product to allow said TRPs to be formed.

8. The prepared food product of claim 1, wherein said fat-based composition is substantially free of exogenous solubilizing and dispersing agents for phytosterols.

9. The prepared food product of claim 1, wherein said prepared food product is a margarine.

10. The prepared food product of claim 1, wherein said prepared food product is a frying or baking shortening.

11. The prepared food product of claim 1, wherein said prepared food product is a mayonnaise.

12. The prepared food product of claim 1, wherein said prepared food product is a salad dressing.

13. The prepared food product of claim 1, wherein said prepared food product is a filled dairy product.

14. The prepared food product of claim 1, wherein said prepared food product is a processed cheese.

15. The prepared food product of claim 1, wherein said prepared food product is a nut, seed, or kernel butter.

16. The prepared food product of claim 1, wherein said prepared food product is peanut butter.

17. The prepared food product of claim 1, wherein said prepared food product is a chocolate product.

18. The prepared food product of claim 1, wherein said prepared food product is a pastry.

19. The prepared food product of claim 1, wherein said prepared food product is cake.

20. The prepared food product of claim 1, wherein said prepared food product is a fried snack food.

21. The prepared food product of claim 1, wherein said prepared food product is potato chips.

22. The prepared food product of claim 1, wherein said prepared food product is French fries.

23. The prepared food product of claim 1, wherein said prepared food product is corn chips.

24. The prepared food of claim 1, wherein said prepared food product is tortilla chips.

25. The prepared food of claim 1, wherein said prepared food product is popcorn.

26. The prepared food of claim 1, wherein said prepared food product is a cracker.

27. The prepared food product of claim 1, wherein the oxidation rate of said oxidation-resistant fat-based composition is at least 20% lower than the oxidation rate for the same oil lacking phytosterols.

28. The prepared food product of claim 1, wherein the oxidation rate of said oxidation-resistant fat-based composition is at least 40% lower than the oxidation rate for the same oil lacking phytosterols.

29. The prepared food product of claim 1, wherein said oxidation-resistant fat-based composition is heated to a temperature of greater than 100 degrees C.

30. A method of preparing a TRP-containing fat-based composition comprising: heating a mixture of a triglyceride-based edible fat-containing composition, and non-esterified phytosterols, wherein said mixture comprises not more than 98% by weight of edible fat or oil and 2% to 25% by weight of non-esterified phytosterols for sufficient time and temperature to dissolve said non-esterified phytosterols, and cooling said composition to room temperature; wherein said TRPs precipitate and recrystallize at room temperature; wherein said TRPs are formed without segregating said phytosterols from said triglyceride-based edible fat-containing composition; and wherein said TRPs have a reduced melting temperature compared to segregated phytosterols.

Description

BACKGROUND OF THE INVENTION

The present invention relates to prepared foods, such as fried snack foods, fortified with non-esterified phytosterols delivered in fats or oils that are essentially free of emulsifiers and the like, and to the utility of such phytosterols for stabilizing heated fats and oils against oxidation, as well as to the surprising bioavailability of triglyceride-recrystallized phytosterols in such foods, for decreasing plasma cholesterol levels in mammals.

It has been a widely held belief that to obtain appreciable benefit from phytosterols, i.e., either plant sterols, stanols, or combinations thereof [including beta-sitosterol, beta-sitostanol, campesterol, campestanol, stigmasterol, stigmastanol, brassicasterol, brassicastanol, clionasterol and clionastanol (collectively termed phytosterol or phytosterols)] for lowering plasma cholesterol, the phytosterol should be dissolved in an edible oil or other solvent so that it can enter micelles in the small intestine to inhibit the absorption of cholesterol.

This belief has been supported by early research carried out in the 1950s through the 1970s showing that large doses of phytosterols in their solid form, i.e., coarse powders, were required to achieve meaningful decreases in plasma cholesterol levels. For example, in 1956, Faquhar et al., (Circulation, 14 77 82, 1956) showed that doses of 12 18 g per day of beta sitosterol (provided in divided doses) were required to achieve a 15 20% lowering of serum cholesterol in males with atherosclerosis. In another study, 9 g per day (3 g t.i.d.) of soybean-derived phytosterols were required to lower plasma cholesterol approximately 9% (Kucchodkar et al., Atherosclerosis, 23, 239 248, 1976). In yet another study, 3 9 g per day of tall oil-derived phytosterols were required to lower plasma cholesterol approximately 12% (Lees et al., Atherosclerosis, 28: 325 333, 1977). In a recent study, 1.7 g per day of finely powdered tall oil-derived phytosterols were sufficient to lower total plasma cholesterol by 9% and LDL-cholesterol by about 15% (Jones et al., Am J Clin Nutr, 69: 1144 1150, 1999).

It has been generally appreciated that phytosterols such as alpha and beta sitosterol, stigmosterol, campesterol, and the corresponding saturated (chemically reduced or hydrogenated) "stanol" species, are insoluble in water, and only slightly soluble in edible oils. Accordingly, to promote the solubilization of phytosterols, and their efficacy in lowering plasma cholesterol, U.S. Pat. No. 6,025,348 by Goto et al. describes the incorporation of at least 15% and as much as 70% by weight or more of a polyhydric alcohol/fatty acid ester (including glycerol fatty acid esters containing at least two esterified and at least one unesterified hydroxyl group such as diacylglycerols or diglycerides), into a fat. Between 1.2% and 4.7% by weight of phytosterol is incorporated into the polyhydric alcohol/fatty acid ester containing fat composition.

U.S. Pat. No. 6,139,897 by Goto et al. describes an oil or fat composition containing 80% or more diacylglycerol and up to 20% phytosterol. The high proportion of diacylglycerol assures solubility or dispersal of the phytosterol to provide a cholesterol-lowering fat substitute.

U.S. Pat. No. 5,998,396 by Nakano et al., describes an edible oil containing a phytosterol, vitamin E, and an emulsifier rendering the phytosterol soluble in both the vitamin E and the edible oil.

U.S. Pat. No. 5,419,925 by Seiden et al. describes a reduced calorie fat composition based upon a substantially non-digestible polyol fatty acid polyester plus reduced calorie medium chain triglycerides and other reduced calorie fats or noncaloric fat replacements including plant sterol esters that are soluble in such fat compositions. Free fatty acids, vitamin E and tocotrienol have each been utilized by other inventors to promote the solubilization of phytosterols in fats and oils, with the expectation that the cholesterol lowering properties of various phytosterols would be improved.

U.S. Pat. No. 5,244,887 by Straub describes the preparation of a cholesterol-lowering food additive composition with plant stanols, including: (i) an edible carrier such as an oil, monoglyceride, diglyceride, triglyceride, tocopherol, alcohol or polyol, (ii) an antioxidant and (iii) a dispersant or detergent-like material such as lecithin, or other phospholipids, sodium lauryl sulfate, a fatty acid, salts of fatty acids, or a fatty acid ester. Straub cites research showing that 1.5 grams per day of a stanol mixture derived from soybean sterols lowered blood cholesterol by 15% after 4 weeks of therapy, and believes that these stanols are preferred to sterols based upon less stanol absorption from the G.I. tract and better heat stability in air than sterols.

U.S. Pat. No.5,932,562 by Ostlund, Jr. describes an aqueous micellar mixture of plant sterol and lecithin (in a 1:1 to 1:10 mole ratio) which has been dried to a water soluble powder and which is useful as a food additive for reducing cholesterol absorption.

U.S. Pat. No.4,195,084 by Ong describes a taste-stabilized pharmaceutical suspension of sitosterols to reduce hypercholesterolemia, in which the suspension includes the plant sterol, a chelator such as calcium disodium EDTA, a surfactant and other ingredients to assure suspension and dispersal of the phytosterol.

U.S. Pat. No. 3,881,005 by Thakkar et al. describes a pharmaceutical dispersible powder for oral administration in which sitosterols are combined with any one of a variety of excipients, and any one of a variety of pharmaceutically acceptable surfactants.

U.S. Pat. No. 6,267,963 by Akashe et al. describes a plant sterol/emulsifier complex that has a lower melting temperature than the plant sterol alone. The complex, e.g., a co-crystallized monoglyceride and plant sterol mixture, is said to facilitate incorporation of the sterol into food products without adversely affecting the texture of the food products.

As indicated above, it has been widely believed that increasing the solubility of phytosterols in fat increases their bioavailability and reduces the dose required to achieve a specified degree of cholesterol reduction. Thus, U.S. Pat. No. 5,502,045 by Miettinen et al., describes the preparation and use of the plant stanol, beta sitostanol, in the form of a fatty acid ester which is readily soluble in an edible oil, to reduce the serum cholesterol level in humans. This technology has been utilized in manufacturing the margarine product marketed under the tradename Benecol.RTM..

U.S. Pat. Nos. 6,031,118 and 6,106,886 by van Amerongen et al. describe similar stanol fatty acid esters but provide different and reportedly improved chemical methods for their preparation. Plant sterols(from soybean oil) have also been interesterified with fatty acid esters to produce the margarine marketed under the tradename Take Control.RTM.. Clinical studies suggest that with mildly hypercholesterolemic individuals, dietary intake of between 1.5 and 3 grams per day of the free phytosterol (provided in a fatty acid esterified form) is required to decrease plasma cholesterol approximately 15%.

U.S. Pat. No. 5,932,562 by Ostlund, Jr. points out that cholesterol is absorbed from an intestinal micellar phase containing bile salts and phospholipids which is in equilibrium with an oil phase inside the intestine. Prior to recent experiments, delivery of phytosterol as a solid powder or aqueous suspension was thought to not be preferred because of the limited rate and extent of solubility in intestinal liquid phases. In fact, at least two earlier human studies showed that as much as 9 18 grams of sitosterol per day were required to decrease the plasma cholesterol level by approximately 15% when the sitosterol was provided in a coarse powdered (rather than soluble) form. Yet, esterification of phytosterols, coupled with the use of edible oils to deliver these sterols is not always practical, e.g., in formulating fat-free foods. It is in this context that Ostlund, Jr. provides a water-dispersible mixture of plant sterol and lecithin.

Using a finely milled powdered form of free phytosterols (from tall oil) suspended in a margarine (not fully dissolved or recrystallized in fat), Jones et al. have described cholesterol reduction in hypercholesterolemic humans (Jones et al., Am J Clin Nutr 69: 1144 1150, 1999) and other mammals (Ntanios et al., Atherosclerosis, 138: 101 110, 1998; Ntanios et al., Biochim Biophys Acta, 1390: 237 244, 1998). In these studies, the efficacy based on cholesterol reduction appears to be equal to that of phytosterol and stanol esters reported by others.

Still another method of producing a fine suspension of microparticulate phytosterols in fat and water has been described by Yliruusi et al. in PCT International Publication Number WO 99/43218. The method involves first heating and dissolving beta-sitosterol in a fat or oil, and then precipitating the phytosterol with water to form a homogenous microcrystalline suspension. While this process appears more cost-effective than grinding, emulsification of fat with water causes any fat to become susceptible to oxidation and necessitates refrigeration.

The production of microparticulate phytosterols described in the prior art involves increased cost and inconvenience, e.g., the use of grinding, and can result in a mixed emulsified product that is more susceptible to oxidation and rancidity, particularly when an aqueous fat-phytosterol emulsion is involved. In fact, there are limitations and disadvantages inherent in most of the above prior methods of phytosterol preparation and delivery. These methods have included grinding, formation of fat and water mixed phytosterol emulsions, chemical modification of phytosterols, e.g., esterification, and mixing of phytosterols with substantial amounts of specialized solubilizing and dispersing agents.

A recent review article entitled "Therapeutic potential of plant sterols and stanols" (Plat et al., Current Opinion in Lipidology, 11: 571 576, 2000) has summarized the results of a number of independent clinical studies in which human plasma cholesterol levels were monitored before and after ingestion of food products enriched with plant sterols and sterol esters (approximately 2 2.5 g per day). The authors conclude that LDL cholesterol levels decreased significantly, i.e., an average of 10 14%.

The description above is provided to assist the understanding of the reader, and does not constitute an admission that the cited references are prior art to the present invention.

SUMMARY OF THE INVENTION

The present invention concerns the use of non-esterified phytosterols in fortifying fat-containing prepared foods. Non-esterified phytosterols were found to have the unexpected property of decreasing the oxidation of fats used in prepared foods, particularly when the fats are heated and become particularly susceptible to oxidation. It is believed that soluble phytosterols e.g., the heat-solubilized non-esterified phytosterols described herein, are also able to protect polyunsaturated fatty acid moieties in fats by quenching, i.e., scavenging, oxidative free radicals and/or peroxides and hydroperoxides that are formed during fat oxidation, and that are particularly problematic in heated fats.

Thus, in addition to functioning as a plasma cholesterol-lowering neutraceutical ingredient in prepared foods, phytosterols can actually protect fats against oxidation during cooking and storage. These two different and compatible functionalities each support the novel introduction of phytosterols into fat-based compositions or fat-containing prepared foods, e.g., into frying and baking shortenings that are absorbed (e.g., into potato chips) or otherwise incorporated into such prepared foods.

Heat-solubilizing non-esterified phytosterols in fat or oil, followed by cooling and recrystallization, results in formation of triglyceride-recrystallized non-esterified phytosterols (TRPs). The inventors have found that when ingested, regardless of the crystalline size of these fat-recrystallized phytosterols, they were effective at reducing mammalian plasma cholesterol levels. By using cost-effective non-esterified phytosterols, and rendering them bioavailable by thermal recrystallization in fat (i.e., heating and cooling in the frying fat or in the recipe ingredient fat), the invention provides an effective alternative to using more costly forms of phytosterols for lowering plasma and liver cholesterol levels. Such more costly phytosterols include microparticulate powders (ultrafine micron-sized phytosterol powders), chemically modified fat-soluble phytosterols, e.g., fatty acid-esterified phytosterols, emulsified phytosterols, and the more perishable water-oil microparticulate suspensions of phytosterols. Underlying this new method for utilizing phytosterols is the discovery that although a chemically unmodified phytosterol (such as beta-sitosterol) is insoluble in water and poorly soluble in fat, it need not be converted to a microparticulate powder to be effective at reducing plasma cholesterol levels in vivo.

Accordingly, in a first aspect, this invention provides a prepared food product for ingestion by mammals, e.g., by humans. The food product includes an oxidation-resistant fat-based composition substantially free of exogenous solubilizing and dispersing agents for phytosterols. This fat-based composition includes between 75% and 98% by weight of at least one triglyceride-based edible oil or fat, and between 2% and 25% by weight of non-esterified tryglyceride-recrystallized phytosterols (TRPs). At room temperature a limited amount of phytosterol will solubilize, typically such that a fat will include approximately 1.5% by weight of the phytosterols in solution, with any remaining phytosterols remaining insoluble. Thus, if phytosterols are added to the fat to a level from 2% to 25% by weight at room temperature, the fat composition will contain approximately 1.5% solubilized phytosterol and between 0.5% and 23.5% by weight of the phytosterols will remain insoluble at that temperature. Typically the fat-based composition has been partially oxidized by an interval of exposure to air during the manufacture and storage of the prepared food product, and contains a reduced amount of oxidative by-products compared to a similar fat-based composition lacking these non-esterified phytosterols.

Storage stability of the food product may also be referred to as the shelf-life of the product at ambient temperatures. Depending upon the food packaging materials and inert gases utilized in the packaging process, the shelf life for such products may range from approximately one week to a year or more. This fat-based composition has been shown to be cholesterol-reducing as measured in the plasma of mammals, and the TRPs when ingested, are essentially as effective, i.e., as bioavailable, as fat-soluble esterified phytosterols in lowering plasma cholesterol levels. Preferably the shelf-life of a prepared food product containing TRPs is increased at least 5%, 10%, 20%, 30%, 50%, 100%, or even more compared to an otherwise equivalent food product not containing the TRPs.

In particular embodiments, the fat composition includes phytosterols at a level of 2 5%, 5 10%, 10 15%, 15 20%, or 20 25%. In some cases even higher levels maybe added.

In a related aspect, a prepared food product for ingestion by mammals is provided as above except that the fat-based or fat-containing composition has been partially oxidized by an interval of heating, e.g., frying, baking, cooking and the like, in air, and contains a reduced amount of oxidative by-products compared to a similar fat-based composition lacking said non-esterified phytosterols. An upper limit for the interval of heating in air has not been established. However, it is believed that any duration of heating of a conventional fat (one that is free of phytosterols) that results in an acceptable (not excessive accumulation of oxidative by-products, (such as free fatty acids and conjugated dienes), will be satisfactory for the phytosterol-fortified fat. For example, fats and vegetable oils may be exposed to temperatures of approximately 180.degree. C. during deep fat frying for periods of time ranging from 5 hr to 25 hr while the prepared food cooked in the oil is exposed to such heat for much shorter intervals, e.g., during cooking (typically several minutes rather than several hours). In any event, a prepared food product as described above may be fried, baked or otherwise heated at least for a time period and to a temperature at least sufficient to dissolve a desired amount (preferably all) of the non-esterified phytosterols added to the fat portion of the fat composition. The fat composition is substantially free of exogenous phytosterol-solubilizing and dispersing agents. Phytosterol enrichment of the fat composition decreases the amount of polar and other oxidative by-products accumulated in the fat and in the prepared food during heating and exposure to air. At least a portion of the non-esterified phytosterols in the fat composition are converted by heating, fully dissolving and subsequent cooling, to triglyceride-recrystallized phytosterols, i.e. TRPs, in which the TRPs contained in the fat composition and in the prepared food product are bioavailable when ingested, to reduce mammalian plasma cholesterol levels.

In certain embodiments, the amount of the edible fat composition in the prepared food product is between 10% and 75% by weight of the food product, e.g., 10 20%, 20 30%, 30 40%, 40 50%, 50 60%, or 60 75%. In other embodiments, the amount of the edible fat composition in the prepared food is lower or higher, e.g., 1 5%, 2 5%, 3 5%, or 4 5%.

In preferred embodiments, the TRPs are formed by heating at least the fat-based composition (or heating the prepared food product as it contains the fat-based composition) to a temperature of greater than 60.degree. C., and fully dissolving the non-esterified phytosterols in the fat composition, and subsequently cooling this composition to room temperature to allow the TRPs to crystallize and be formed.

In another related aspect, a prepared food product for ingestion by mammals is provided that includes a plasma cholesterol-reducing oil or fat composition with improved resistance to oxidation. The oil or fat composition is substantially free of exogenous solubilizing and dispersing agents for phytosterols, and includes between 75% and 95% by weight of at least one triglyceride-based edible oil or fat, and at least 5% by weight of non-esterified triglyceride-recrystallized phytosterols. As described above, typically the phytosterols are soluble in the oil or fat composition at room temperature to a level of approximately 1.5% by weight, so that at least 3% by weight of phytosterols are insoluble at room temperature and have been converted by heating, fully dissolving, and cooling to form triglyceride-recrystallized phytosterols, i.e., TRPs. These TRPs, when ingested, are essentially as effective as fat-soluble esterified phytosterols in lowering plasma cholesterol levels in mammals.

In preferred embodiments, the oil or fat composition includes at least 8%, 10%, 12%, 15%, 17%, or 20% by weight of non-esterified phytosterols or is in a range defined by taking any two of those values as endpoints of the range. As described above, typically the phytosterols are soluble in the fat or oil at room temperature to a level of approximately 1.5% by weight, and the remainder (e.g., at least 6.5%, 8.5%, 10.5%, 12.5%, 15.5%, or 17.5% respectively) is insoluble at room temperature, but is dissolved and tryglyceride-recrystallized by heating to dissolve the phytosterols and cooling. These TRPs, when ingested, are essentially as effective as fat-soluble esterified phytosterols in lowering plasma cholesterol levels in mammals.

In preferred embodiments, the TRPs described above are formed by heating at least the above referenced oil or fat composition (or a prepared food product containing the oil or fat composition, or the oil or fat and the phytosterols as ingredients of the prepared food) to a temperature of greater than 60.degree. C., fully dissolving the non-esterified phytosterols in the composition, and subsequently cooling the composition to room temperature to cause the TRPs to be formed.

In certain embodiments, prepared food products are selected from the group consisting of margarine, frying and baking shortenings, mayonnaise, salad dressing, filled dairy products, nut, seed and kernel butters and chocolate (containing cocoa butter). In each of these examples, the phytosterols are dissolved by heating them in the fat portion of these prepared foods, i.e., heating without any aqueous components present. In other embodiments, the prepared food product is a pastry or cake.

In certain embodiments, the prepared food product is fried, baked, or otherwise heat-processed with the oil or fat composition, and/or where the oil or fat composition and phytosterols are added as ingredients in the preparation of the prepared food, wherein such heating allows a portion of non-esterified phytosterols that is insoluble in the oil or fat composition at room temperature to be solubilized and thereby enter and be incorporated into the food product, whereupon during cooling, TRPs are formed in the food product.

In preferred embodiments, the prepared food product is selected from the group consisting of potato chips, French fries, corn chips, tortilla chips, popcorn, and crackers.

Also in preferred embodiments, the food product is cooked, baked, or otherwise heat-processed with the above-described oil or fat composition, allowing a portion of non-esterified phytosterols that is insoluble in the composition at room temperature to be solubilized. During subsequent cooling to room temperature and crystallization of non-esterified phytosterols, a partial or complete solidification of the oil or fat composition can occur. This solidification decreases the oiliness, particularly the surface oiliness, perceived by hand contact with the food product compared to the same food product prepared without non-esterified phytosterols (due to the formation of TRPs in the fat or oil). Solidification or "hardening" of oil can also reduce or prevent oil separation in certain prepared foods, and is particularly useful in such foods as peanut butter, soybean butter, sesame seed butter and other seed, bean and nut kernel butters. "Hardening" of an edible oil may be compared to that resulting from partial hydrogenation of vegetable oils. Both modifications tend to solidify a vegetable oil by increasing the oil's melting temperature. However, from a nutritional perspective, addition of phytosterols to ones diet advantageously decreases the level of plasma LDL cholesterol, while addition of partially hydrogenated oils disadvantageously increases the LDL level.

In preferred embodiments, the food product, and more particularly the oil or fat composition within the food product, when heated in air, is more resistant to oxidation and formation of chemically polar degradation products than the same product lacking the non-esterified phytosterols, e.g., as described in Example 3 below.

In preferred embodiments, the food product incorporating the oil or fat composition has a reduced calorie content compared to a similar food product prepared without non-esterified phytosterols, owing to the presence of the non-esterified phytosterols that are calorie-free, and substitute for a portion of triglyceride-based oil or fat normally absorbed or otherwise incorporated into the food product. This statement is explained and supported by Example 4 below.

In preferred embodiments, the non-esterified phytosterols are selected from the group consisting of tall oil-derived phytosterols (such as those obtained from the manufacture of wood pulp from pine trees) and vegetable oil-derived phytosterols (such as those derived from soybean oil).

In another aspect, the invention provides an oxidation-resistant frying or baking shortening that includes: (a) from 75% to 98% by weight of at least one edible triglyceride-based fat or oil; and (b) from 2.0% to 25% by weight TRPs (produced from at least one non-esterified phytosterol compound being solubilized by heating, and allowed to recrystallize in the fat or oil upon cooling). As explained above, typically from 0.5% to 23.5% by weight of phytosterols are recrystallized in the solid phase, and approximately 1.5% by weight of non-esterified phytosterol remains solubilized in the fat at room temperature.

Highly preferably the shortening is substantially free of exogenous solubilizing and dispersing agents for phytosterols, and the rate of formation of polar oxidation products upon heating the shortening to between 160.degree. C. and 190.degree. C. is reduced, compared to the same shortening lacking the at least one non-esterified phytosterol compound.

Referring to this aspect, the formation of polar oxidation products was determined by measurement of the dielectric constant of the shortening after two hours of heating as described elsewhere herein (see Example 3, second experiment). The term "reduced," referring to the rate of formation of polar oxidation products, indicates that the increase in dielectric constant of the shortening is reduced at least 5%, and preferably 7, 8, or 10% or more for the phytosterol-supplemented shortening, compared to the non-supplemented shortening.

In preferred embodiments, the oxidation-resistant frying or baking shortening includes at least one edible triglyceride-based fat or oil selected from the group consisting of natural vegetable oils or fats, natural animal fats and oils, structurally rearranged or modified vegetable and/or animal fats, and combinations thereof.

In preferred embodiments, the oxidation-resistant frying or baking shortening includes at least one non-esterified phytosterol compound selected from the group consisting of vegetable oil-derived phytosterols, tall oil-derived phytosterols, and combinations thereof.

In preferred embodiments, the oxidation-resistant frying or baking shortening includes at least one non-esterified phytosterol selected from the group consisting of beta-sitosterol, beta-sitostanol, campesterol, campestanol, stigmasterol, stigmastanol, brassicasterol, brassicastanol, clionasterol and clionastanol, and combinations thereof.

In another aspect, the invention features a method for reducing plasma cholesterol levels in mammals. The method includes providing a heat-processed prepared food containing an edible fat-based composition that includes between 75% and 98% by weight of at least one triglyceride-based edible fat, and between 2% and 25% by weight of non-esterified triglyceride-recrystallized phytosterols, for ingestion by the mammal(s). Generally, the phytosterol is soluble to a level of approximately 1.5% by weight, such that the insoluble phytosterols in the fat-based composition at room temperature consititute between 0.5% and 23.5%. The fat-based composition is substantially free of exogenous phytosterol-solubilizing and dispersing agents. The insoluble phytosterols have been heat-solubilized and subsequently cooled to form triglyceride-recrystallized phytosterols i.e., TRPs. The TRPs when ingested are essentially as effective as fat-soluble esterified phytosterols in reducing plasma cholesterol levels.

In preferred embodiments, the proportion of non-esterified phytosterols used in the edible fat-based composition for a prepared food is between 3% and 15% by weight of the composition, and more preferably between 5 and 10% of the composition (or other percentage as described for food products herein). Thus, with the latter range, a serving of food containing 10 grams of a fat-based composition, would contain between 0.5 g and 1.0 g of non-esterified phytosterols. This amount is consistent with current recommendations published by the U.S. Food and Drug Administration.

The edible fat-based composition is heated to a temperature of greater than 60.degree. C., and preferably between 75.degree. C. and 150.degree. C., or higher, to dissolve the non-esterified phytosterols in the composition. At a temperature of 60.degree. C. or below, the rate of dissolution is slower than desirable, and the concentration of dissolved phytosterols in a fat-based medium is lower than generally desired to be commercially useful or practical.

In preferred embodiments, between 0.5 g and 4.0 g of the non-esterified phytosterols contained in the above prepared food are ingested daily by humans.

In preferred embodiments, the TRPs are formed by heating at least the edible fat-based composition to a temperature exceeding 60.degree. C. for a period of time sufficient to dissolve the non-esterified phytosterols in the fat, and subsequently cooling the composition (or the food containing this composition) to room temperature to cause the TRPs to be formed.

In a related aspect, the invention features a method for reducing plasma cholesterol levels in mammals, including providing and regularly ingesting a heat-processed prepared food containing an edible fat-based composition that contains between 75% and 97% by weight of at least one triglyceride-based edible fat, and at least 3% by weight of non-esterified triglyceride-recrystallized phytosterols. Typically the phytosterols are soluble in the fat at a level of approximately 1.5% and the remainder (e.g., 1.5% from a total of 3%) is insoluble at room temperature. The fat-based composition is substantially free of exogenous phytosterol-solubilizing and dispersing agents. The insoluble phytosterols are heat-solubilized and subsequently cooled to form triglyceride-recrystallized phytosterols, i.e., TRPs. The TRPs when ingested are essentially as effective as fat-soluble esterified phytosterols in reducing plasma cholesterol levels.

In certain embodiments, the fat composition contains at least 5%, 7%, 10%, 12%, 15%, 17% or 20% by weight of non-esterified phytosterols (typically the phytosterols are soluble to a level of approximately 1.5% at room temperature and the remainder is insoluble).

In another aspect, a method is provided for preparing a TRP-containing fat-based composition. The method includes (i) providing a triglyceride-based edible fat-containing composition that includes between 2% and 25% by weight of non-esterified phytosterols and not more than 98% by weight of edible fat or oil, in which the composition is substantially free of exogenous phytosterol-solubilizing and dispersing agents, (ii) heating the composition to dissolve (preferably fully dissolve) the non-esterified phytosterols, and, (iii) cooling the composition to room temperature, allowing formation of TRPs. Typically the phytosterols are soluble in the edible fat or oil at room temperature to a level of approximately 1.5%, while the remainder is insoluble at room temperature. In general the fat-containing composition is heated to a temperature of 60 180.degree. C., usually 75 150.degree. C.

Similarly, in a related aspect the invention concerns a method of preparing a TRP-containing fat-based composition by heating a mixture of a triglyceride-based edible fat-containing composition, and non-esterified phytosterols, where the mixture includes not more than 98% by weight of edible fat or oil and 2% to 25% by weight of non-esterified phytosterols for sufficient time and temperature to dissolve said non-esterified phytosterols, and cooling the composition to room temperature.

In yet another aspect, a method is provided for preparing a non-esterified phytosterol-fortified prepared food. The method includes: (i) providing an edible fat-based composition that includes between 2% and 25% by weight of non-esterified phytosterols and between 75% and 98% by weight of at least one edible fat or oil, in which the composition is substantially free of exogenous phytosterol-solubilizing and dispersing agents, and one or more other ingredients for the prepared food if any such additional ingredients are used; (ii) cooking or otherwise heating the prepared food ingredients with the composition to allow the non-esterified phytosterols to dissolve in the oil or fat and enter or become integrated into the food product; and (iii) cooling the food product to room temperature to allow formation of TRPs in the composition within the prepared food.

In certain embodiments, the fat-based composition can be used as an ingredient mixed with other ingredients in the preparation of the prepared food, and/or the prepared food product can be cooked in the fat-based composition.

While in most cases the non-esterified phytosterols are recrystallized in the oil or fat prior to combining with other ingredients, for some prepared foods, the phytosterols can be combined with the oil or fat in preparation of the prepared food. Thus, alternatively, the fat or oil and the phytosterols can be added as separate ingredients in such manner that the phytosterols will dissolve in the fat or oil upon heating of the combined ingredients. In some cases, only a portion of the phytosterols added as ingredients will become solubilized, e.g., where only a portion of the phytosterols are in contact with the fat or oil during heating. In cases where the fat-based composition, or the oil or fat and the phytosterols are added as ingredients in preparing the prepared food, typically a number of different ingredients are blended or mixed such that the various ingredients are relatively uniformly distributed throughout the mixture.

Another aspect concerns a method of increasing the oxidative stability of a heated frying fat composition, where the method involves maintaining a fat composition that contains at least 8% by weight non-esterified phytosterols at a temperature of at least 100 degrees C., wherein said fat composition is used for frying.

The frying fat composition can be held at the elevated temperature for a suitable length of time considering the purpose, e.g., at least 0.5 hr, 1 hr, 2 hrs, 4 hrs, 6 hrs, 8 hrs, 10 hrs, or longer. Of course, as with any frying fat composition, eventually the fat will degrade sufficiently that it will not be used any longer for frying, and may be replaced with fresh fat composition. In particular embodiments, the fat composition oxidizes at a rate that is only 90, 80, 70, 60, 50, 40, 30, 20% or even less of the rate for the same fat composition without phytosterols or other non-fat oxidation rate reducing components.

In the particular embodiments, the fat composition containing non-esterified phytosterols is a composition as described for other aspects herein.

Yet another aspect concerns a dietary supplement that includes at least one triglyceride-based edible fat, and between 3% and 50% by weight of triglyceride recrystallized phytosterols. Such a dietary supplement can also be regarded as a nutraceutical. The supplement can be in numerous different forms, e.g., capsule, pill, wafer. The TRP-fat compostion can be combined with other dietary components, such as protein, vitamins, minerals, and combinations of such components.

In certain embodiments, the phytosterol content, fat content, preparation method for the composition, and other parameters are as described herein for other aspects involving a fat/TRP composition.

The term "prepared" in the context of a "prepared food product" refers to a commercially processed and packaged food product containing multiple combined ingredients, in which the processing includes at least one step in which the assembled food product (or one or more triglyceride-based fat or oil ingredients that are either contacting, or being combined into the food product), are heated together with a suitable quantity of phytosterol ingredient(s), to a temperature sufficient to dissolve the phytosterols in the fat or oil, and often substantially higher than this temperature, and for a period of time sufficient to process, cook, fry or otherwise complete the heat-preparation of the food product. Upon cooling, a portion of the phytosterols recrystallize in a fat or oil component of the processed prepared food product. Examples of such prepared food products include potato chips (containing at least potatoes, frying fat or oil, and phytosterols), French fries, corn chips, tortilla chips, popcorn, crackers, peanut butter, soybean butter, sesame seed butter and other nut kernel butters, mayonnaise, processed cheese, chocolate and the like.

The term "fat" may be used broadly and generally, referring to an edible triglyceride that may be either liquid (also specifically termed oil) or solid at room temperature (also specifically termed fat), and that is derived from a single vegetable source (e.g., soybean, cottonseed, corn) or an animal source (beef tallow, pork lard) or a blended combination of sources. Unless specifically limited to edible triglyceride compositions that are solid at room temperature, use of the term "fat" includes oils. Also unless clearly indicated to the contrary, the term "fat" also includes chemically and enzymatically modified triglyceride-based liquid and solid fats and blends thereof (e.g., hydrogenated, partially hydrogenated, chemically or enzymatically interesterified, or assembled, i.e., "structured" triglycerides and combinations thereof.

The phrase "improved resistance to oxidation" for a fat that contains non-esterified phytosterols refers to a fat exhibiting at least a 10% reduced rate of degradation by oxidation in air, compared to oxidation of the same fat without phytosterols. This differential oxidation rate is particularly evident during heating of the oil, e.g., frying with the oil at a temperature of 160 190.degree. C. Oxidation rate is evidenced by one or more physical measurements such as dielectric constant measurement of polar oxidation products formed in the fat, AOM (accelerated oxidation measurement, OSI (oxidative stability index), or organoleptic quality (tasting f or rancidity). The extent of oxidative protection provided by non-esterified phytosterols dissolved in fat heated to 180.degree. C. is a function of the type of fat and the concentration of phytosterols in the fat. Improved resistance to oxidation is particularly evident in a vegetable oil containing polyunsaturated fatty acids, e.g., soybean, corn and canola oil. When 10% by weight soybean-derived phytosterols is dissolved in such oils, the rate of oxidation, i.e., formation of polar oxidation products, in the heated oils is at least 10% lower than the rate in the same oil lacking phytosterols. Preferably, the rate of oxidation is at least 20% lower, and more preferably, the rate is 30%, 40% or even 50% lower than the rate in the same oil lacking phytosterols.

The term "partially oxidized" refers to a fat-based composition that has been exposed to air either with or without heating, e.g., frying or baking and that has at least begun to accumulate oxidative by-products whose concentrations are measurable either in the oil or in the vapor above the oil by conventional means, e.g., by conductivity, dielectric constant, and free fatty acid content.

It is believed that oxidative protection of fats and oils provided by phytosterols has not been reported previously. Also, phytosterols are not recognized as antioxidants or as scavengers or quenchers of free-radicals or peroxides and hydroperoxides formed during oxidation of polyunsaturated fatty acid moieties. In searching for a rational explanation for this oxidative protection, Applicants have looked to literature describing various properties of cholesterol. Of course "cholesterol fortification" of a food product would be nutritionally undesirable and, indeed, phytosterol fortification is intended to reduce cholesterol uptake. However, the cholesterol molecule is structurally related to the phytosterols, i.e., addition of an ethyl side group to beta-sitosterol generates cholesterol. U.S. Pat. No. 6,214,534 by Horowitz et al. describes several UV light photodynamic quenchers including vitamins, thiols, cholesterol, and several other compounds that react with, and inactivate both free radicals and reactive forms of oxygen. Since free radicals, peroxides and hydroperoxides are produced during the oxidation of polyunsaturated fatty acid groups in triglycerides, phytosterols dissolved in fat may inactivate these reactive compounds, as with cholesterol described in the photodynamic system of Horowitz et al. While the phytosterols may act in this manner, the present invention is not limited by this explanation.

The term "edible" in the context of an oil or fat-based composition means that the composition is suitable for use in mammalian, e.g., human, foods, dietary supplements and pharmaceutical preparations.

The term "exogenous phytosterol-solubilizing and dispersing agents" refers to agents other than triglycerides in the prior art, that have been added to triglyceride-based oils and fats to promote the cholesterol-lowering efficacy of phytosterols (see discussion above in the Background section). A partial list of these agents includes monoglycerides, diglycerides, lecithin, vitamin E, the sorbitans and other surfactants, and fatty acids chemically esterified with phytosterols.

The term "substantially free," referring to any presence of exogenous solubilizing and dispersing agents for phytosterols, means that either zero percent, or in any event, less than 50% (and preferably less than 25%) of the amount of such an agent or agents that would be required in the absence of triglycerides, to achieve solubilization or dispersal of non-esterified phytosterols (at room temperature) that have been added to the referenced composition. Provided that the phytosterols are recrystallized in triglycerides, triglycerides alone are sufficient for phytosterol bioavailability, i.e., effectiveness in plasma cholesterol reduction. Therefore, any addition of such a non-triglyceride solubilizing or dispersing agent to a fat-based composition containing TRPs is considered gratuitous and optional.

The term "phytosterol" refers to any of a group of sterols derived from plants (see examples below in Example 1).

The term "non-esterified phytosterols" refers to forms of phytosterols that are free of ester chemical side chains. Conversely, esterified phytosterols are most commonly fatty acid-esterified phytosterols manufactured to promote phytosterol solubility in fat. Non-esterified phytosterols are defined herein to include both the non-esterified sterol and stanol forms of phytosterols (see Example 1 below). According to the present invention, phytosterols are dissolved in oil or fat before recrystallization, and therefore the particle size, texture, etc. of the material can be coarse for reasons of economy, i.e., chemical dissolution reduces the material to molecular dimensions. Dissolution of more costly forms of phytosterols, e.g., ultrafine micron-sized phytosterol powders, would be economically wasteful, but can also be done.

The composition which includes between 75% and 98% by weight of at least one triglyceride-based edible oil or fat, allows between 2% and 25% by weight of non-esterified phytosterols to be added to the same composition. A 3% to 10% by weight concentration range is a preferred range. Accordingly, at the 3% level, a food that contains 10 g of fat per serving will provide at least 0.3 g of phytosterols per serving. In the case of pharmaceutical preparations, the composition may include as little as 50% by weight of at least one triglyceride-based edible oil or fat, to allow between 3% and 50% by weight of non-esterified phytosterols to be added to the same composition.

The process of treating the non-esterified phytosterols by "heating, fully dissolving, and cooling" refers to a process that: (i) heats the phytosterols together with triglyceride-based fat or oil (and optionally other food ingredients constituting a prepared food product) to a temperature of greater than 60.degree. C. until the phytosterols have dissolved, and then (ii) cooling the heated product and allowing the triglycerides to associate with the recrystallizing phytosterols. Flash-chilling with chilled air or with a chilled water jacket may tend to precipitate and segregate the phytosterols from the triglycerides, preventing optimal recrystallization. Conventional or normal ambient air cooling r