A nutritional supplement, prepared food product, or direct food additive for ingestion by mammals, and methods for preparing such products, are provided. Products of the invention comprise an oxidation-resistant fat-based composition substantially free of exogenous solubilizing and dispersing agents for phytosterols. The fat-based composition includes greater than 25% and less than 75% by weight of one or more triglyceride-based edible oil or fat, and greater than 25% and less than 75% by weight of one or more non-esterified phytosterols. The fat-based composition, when exposed to air, contains a reduced amount of oxidative by-products compared to a similar fat-based composition lacking non-esterified phytosterols. Also provided are methods for reducing plasma cholesterol in mammals, and methods for protecting plasma lipoproteins and cholesterol from oxidation by ingestion of products of the invention.

Inventors: Perlman; Daniel; (Arlington, MA) ; Hayes; Kenneth; (Wellesley, MA) ; Pronczuk; Andrzej; (Boston, MA) 
Correspondence Name and Address:      FOLEY & LARDNER LLP
    P.O. BOX 80278
    SAN DIEGO
    CA
    92138-0278
    US

 
Serial No.:  222512
Series Code:  11 
Filed:  September 7, 2005

U.S. Current Class: 426/601 
U.S. Class at Publication: 426/601 
Intern'l Class:  A23D 9/00 20060101 A23D009/00

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Claims

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1. A product comprising: a fat-based composition substantially free of exogenous solubilizing and dispersing agents for phytosterols, wherein said fat-based composition comprises: greater than 25% and less than 75% by weight of one or more triglyceride-based edible oil or fat; and greater than 25% and less than 75% by weight of one or more non-esterified phytosterols that have been converted to triglyceride-recrystallized phytosterols.

2. The product of claim 1, wherein said product is edible; wherein said product is useful as a component of a member of the group consisting of prepared foods, dietary supplements, and direct food additives.

3. The product of claim 1, wherein said fat-based composition is exposed to air and contains a reduced amount of oxidative by-products compared to an otherwise similar fat-based composition lacking said non-esterified phytosterols.

4. The product of claim 1, wherein said one or more triglyceride-based edible oil or fat is selected from the group consisting of vegetable oils, vegetable fats, animal oils, animal fats, and mixtures thereof.

5. The product of claim 1, wherein said one or more triglyceride-based edible oil or fat is further selected from the group consisting of safflower oil, sunflower oil, corn oil, cottonseed oil, soybean oil, canola oil, peanut oil, coconut oil, cocoa butter, palm oil, palm olein, palm super-olein, palm kernel oil, algae oil, flaxseed oil, and combinations thereof.

6. The product of claim 1, wherein said one or more triglyceride-based edible oil or fat is selected from the group consisting of butter, anhydrous milk fat, tallow, lard, mutton fat, poultry fat, fish oil, and combinations thereof.

7. The product of claim 6, wherein said one or more triglyceride-based edible oil or fat is cholesterol-free or cholesterol-reduced.

8. The product of claim 1 wherein said one or more triglyceride-based edible oil or fat is selected from the group consisting of natural vegetable and animal fats, structurally rearranged or otherwise modified vegetable and animal fats, and combinations thereof.

9. The product of claim 1 wherein one or more of said one or more non-esterified phytosterols is selected from the group consisting of vegetable oil-derived phytosterols, tall oil-derived phytosterols, and combinations thereof.

10. The product of claim 1 wherein one or more of said one or more non-esterified phytosterols is selected from the group consisting of beta-sitosterol, beta-sitostanol, campesterol, campestanol, stigmasterol, stigmastanol, brassicasterol, brassicastanol, clionasterol, clionastanol, and combinations thereof.

11. The product of claim 1, wherein said triglyceride-recrystallized phytosterols are formed by heating said fat-based composition either alone or combined in said product to a temperature and for a time sufficient to dissolve said one or more non-esterified phytosterols, and subsequently cooling said fat-based composition to allow said triglyceride-recrystallized phytosterols to be formed.

12. The product of claim 1, wherein said product comprises a member selected from the group consisting of margarines, spreads, butter, frying shortenings, baking shortenings, mayonnaises, salad dressings, dairy products, nut butters, seed butters, kernel butters, peanut butter, chocolate, fried snack foods, leavened bakery products comprising a flour, potato chips, French fries, corn chips, tortilla chips, popcorn, crackers, condiments, and sauces.

13. The product of claim 12, wherein said leavened bakery product is selected from the group consisting of cakes, muffins, donuts, pastries, breads and rolls.

14. The product of claim 12, wherein said condiment is selected from the group consisting of tomato catsup, mustard, barbecue sauce, steak sauce, Worchestershire sauce, cocktail sauce, tartar sauce, and pickle relish.

15. The product of claim 12, wherein said sauce is selected from the group consisting of tomato-based pasta sauce, pizza sauce, prepared chili, and dessert sauce.

16. The product of claim 1, wherein the oxidation rate of said fat-based composition is at least 20% lower than the oxidation rate for the same one or more triglyceride-based edible oil or fat lacking phytosterols.

17. The product of claim 2 useful as a dietary supplement wherein said one or more triglyceride-based edible oil or fat comprises at least 10% by weight omega-3 fatty acids.

18. The product of claim 17 wherein said omega-3 fatty acids are selected from the group consisting of DHA, EPA, alpha-linolenic acid, and combinations thereof.

19. The product of claim 17 wherein said one or more triglyceride-based edible oil or fat is selected from the group consisting of fish oil, cholesterol-reduced fish oil, cholesterol-free fish oil, algae oil, flaxseed oil, and combinations thereof.

20. The product of claim 17 wherein said fat-based composition is packaged in edible gelatin capsules.

21. A method for reducing plasma cholesterol levels in mammals, comprising: regularly ingesting a product according to claim 2.

22. The method of claim 21 wherein between 0.4 g and 4.0 g of said non-esterified phytosterols are ingested daily by humans.

23. The method of claim 21 wherein said triglyceride-recrystallized phytosterols are formed by heating said one or more triglyceride-based edible oil or fat and said one or more non-esterified phytosterols to a temperature of at least 60.degree. C. for a period of time sufficient to dissolve said non-esterified phytosterols, and subsequently cooling said composition to room temperature to cause said triglyceride-recrystallized phytosterols to be formed.

24. The method of claim 21 wherein the plasma concentration of carotenoids comprising alpha- and beta-carotene is maintained essentially constant while said plasma cholesterol levels are reduced.

25. The method of claim 21 wherein said one or more triglyceride-based edible oil or fat comprises at least 10% by weight omega-3 fatty acids comprising DHA or DHA in combination with EPA, wherein the levels of plasma triglycerides and cholesterol are simultaneously reduced.

26. A method for protecting plasma lipoproteins and cholesterol from oxidation in mammals, comprising: regularly ingesting a product according to claim 2.

27. The method of claim 26 wherein between 0.4 g and 4.0 g of said non-esterified phytosterols are ingested daily by humans.

28. The method of claim 26 wherein said triglyceride-recrystallized phytosterols are formed by heating said one or more triglyceride-based edible oil or fat and said one or more non-esterified phytosterols to a temperature of at least 60.degree. C. for a period of time sufficient to dissolve said non-esterified phytosterols, and subsequently cooling said composition to room temperature to cause said triglyceride-recrystallized phytosterols to be formed.

29. A method of preparing a triglyceride-recrystallized phytosterol-containing fat-based composition comprising: heating a mixture comprising: less than 75% by weight of one or more triglyceride-based edible fat or oil, and greater than 25% and less than 75% by weight of one or more non-esterified phytosterols for sufficient time and temperature to dissolve said non-esterified phytosterols; and cooling said triglyceride-recrystallized phytosterol-containing fat-based composition to room temperature.

30. A method of preparing a non-esterified phytosterol-fortified prepared food product, comprising: providing edible fat-based composition substantially free of exogenous phytosterol-solubilizing and dispersing agents, said edible fat-based composition comprising greater than 25% and less than 75% by weight of one or more triglyceride-based edible fat or oil and greater than 25% and less than 75% by weight of one or more non-esterified phytosterols, and other ingredients required for said prepared food product; heating said other ingredients with said edible oil or fat-containing composition to allow said non-esterified phytosterols to dissolve in said oil or fat and disperse in said food product; and allowing said food product to cool to room temperature to allow formation of triglyceride-recrystalized phytosterols in said edible oil or fat-containing composition within said prepared food.

31. A method for producing a fried snack food having reduced surface oiliness, comprising: frying said snack food in a fat-based composition comprising one or more triglyceride-based edible oil or fat, and greater than 25% and less than 75% by weight of one or more non-esterified phytosterols.

32. A prepared food or beverage used in a weight loss regimen for overweight and obese patients, wherein said food or beverage comprises non-esterified phytosterols in the form of triglyceride-recrystallized phytosterols; wherein said patients can benefit from a reduction in the level of plasma LDL cholesterol.

33. A prepared food product comprising endogenous cholesterol, and further comprising triglyceride-recrystallized phytosterols that have been added to said prepared food product during or subsequent to its manufacture; wherein the weight ratio of non-esterified phytosterols contained in said triglyceride-recrystallized phytosterols to said endogenous cholesterol is between 2:1 and 10:1.
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Description

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CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application is related to the following: U.S. application Ser. No. 10/677,634, filed Oct. 1, 2003, and published as U.S. Pat. App. Pub. 2005-0042355, Feb. 24, 2005; PCT Application PCT/US02/36809, filed Nov. 14, 2002, and published as WO 2003/043433, May 30, 2003; U.S. application Ser. No. 10/295,929, filed Nov. 14, 2002, which published as U.S. Pat. App. Pub. 2003-0096035, May 22, 2003, and which issued as U.S. Pat. No. 6,638,547, Oct. 28, 2003; and U.S. Provisional Application No. 60/332,434, filed Nov. 16, 2001, now abandoned, each of which is incorporated herein by reference in its entirety, including all figures and tables and for all purposes.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to dietary supplements and prepared foods fortified with non-esterified phytosterols that have been recrystallized with fats or oils that are essentially free of emulsifiers and the like, and to the utility of such Triglyceride-Recrystallized Phytosterols (TRPs) for reducing rancidity development in fat-containing foods, stabilizing perishable triglycerides such as those found in fish oil, and stabilizing heated fats and oils against oxidation. The present invention also relates to the surprising bioavailability of TRPs provided in the mammalian diet, resulting in a substantial decrease in plasma LDL cholesterol levels.

[0003] It has been a widely held belief that to obtain appreciable benefit from phytosterols, i.e., by definition herein, including 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.

[0004] 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 particles, 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).

[0005] It has been generally appreciated that phytosterols such as alpha- and beta-sitosterol, stigmosterol, campesterol and others, including 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.

[0006] Perlman et al. in U.S. application Ser. No. 10/677,634, filed Oct. 1, 2003, and published as U.S. Pat. Appl. Pub. 2005-0042355, Feb. 24, 2005, describes a prepared food product which comprises an oxidation-resistant fat-based composition substantially free of exogenous solubilizing and dispersing agents for phytosterols, wherein 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 in the form of triglyceride-recrystallized phytosterols (TRPs). Further discussed is a method for producing a fried snack food having reduced surface oiliness, comprising frying said snack food in a fat-based composition comprising at least one triglyceride-based edible oil or fat, and 2% to 25% by weight of non-esterified phytosterols. Further discussed are methods of preparing a TRP-containing fat-based composition comprising not more than 98% by weight of edible fat or oil and 2% to 25% by weight of non-esterified phytosterols in the form of TRPs, wherein the method comprises heating the fat-based composition for sufficient time and temperature to dissolve said non-esterified phytosterols, and cooling said composition to room temperature. Further discussed is a dietary supplement comprising at least one triglyceride-based edible fat; and between 3% and 50% by weight of triglyceride recrystallized phytosterols.

[0007] Perlman et al. in U.S. Pat. No. 6,638,547, issued Oct. 28, 2003, disclose and claim 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 between 75% and 98% by weight of at least one triglyceride-based edible oil or fat, between 2% and 25% by weight TRPs, wherein said fat-based composition has been partially oxidized by an interval of exposure to air, or by an interval of heating in air, and contains a reduced amount of oxidative by-products compared to a similar fat-based composition lacking said non-esterified phytosterols. Also disclosed are a reduced calorie prepared food product comprising between 75% and 98% by weight of at least one triglyceride-based edible oil or fat, between 2% and 25% by weight TRPs, wherein said non-esterified phytosterols are calorie-free and substitute for a portion of triglyceride-based edible oil or fat normally absorbed or otherwise incorporated into a prepared food product. Also disclosed are an oxidation-resistant frying or baking shortening comprising from 75% to 98% by weight of at least one edible triglyceride-based fat or oil; and from 2.0% to 25.0% by weight of TRPs. Also provided is a method for reducing plasma cholesterol levels in mammals, comprising regularly providing and ingesting a heat-processed food containing a fat-based composition comprising between 75% and 97% by weight of at least one triglyceride-based edible fat or oil, and at least 3% by weight of non-esterified TRPs, wherein the fat-based composition is substantially free of exogenous phytosterol-solubilizing and dispersing agents, and wherein said TRPs when ingested are essentially as effective as fat-soluble esterified phytosterols in reducing said plasma cholesterol levels. Also provides are methods of preparing TRP-containing fat-based compositions comprising providing a triglyceride-based edible fat-containing composition comprising between 2% and 25% by weight of non-esterified phytosterols and not more than 98% by weight of edible fat or oil, wherein said composition is substantially free of exogenous phytosterol-solubilizing and dispersing agents, heating said composition to dissolve said non-esterified phytosterols, and cooling said composition to room temperature. Also provided are methods of preparing non-esterified phytosterol-fortified prepared foods comprising providing an edible fat-based composition comprising 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, wherein said composition is substantially free of exogenous phytosterol-solubilizing and dispersing agents, and other ingredients, if any, for said prepared food; cooking or otherwise heating said ingredients with said composition to allow said non-esterified phytosterols to dissolve in said oil or fat and enter or become integrated into said food product, and cooling said food product to room temperature to allow formation of triglyceride-recrystalized phytosterols (TRPs) in said composition within said prepared food.

[0008] Perlman et al. in PCT/US2002/036809, filed Nov. 14, 2002, and published as WO 2003/043433, May 30, 2003, provide 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 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 TRPs, wherein said fat-based-composition has been partially oxidized by an interval of exposure to air, or by an interval of heating in air, and contains a reduced amount of oxidative by-products compared to a similar fat-based composition lacking said non-esterified phytosterols. Further provided is a reduced calorie prepared food product comprising between 75% and 98% by weight of at least one triglyceride-based edible oil or fat, and between 2% and 25% by weight TRPs, wherein said non-esterified phytosterols are calorie-free and substitute for a portion of triglyceride-based edible oil or fat normally absorbed or otherwise incorporated into a prepared food product. Further provided is an oxidation-resistant flying or baking shortening comprising from 75% to 98% by weight of at least one edible triglyceride-based fat or oil; and from 2.0% to 25.0% by weight of TRPs. Further provided is a method for reducing plasma cholesterol levels in mammals, comprising regularly providing and ingesting a heat-processed food containing an edible fat-based composition comprising between 75% and 97% by weight of at least one triglyceride-based edible fat, and at least 3% by weight of non-esterified TRPs, wherein the fat-based composition is substantially free of exogenous phytosterol-solubilizing and dispersing agents, and wherein said TRPs when ingested are essentially as effective as fat-soluble esterified phytosterols in reducing said plasma cholesterol levels. Further provided is a method of preparing a TRP-containing fat-based composition comprising the following: providing a triglyceride-based edible fat-containing composition which in turn comprises between 2% and 25% by weight of non-esterified phytosterols and not more than 98% by weight of edible fat or oil, and wherein said composition is substantially free of exogenous phytosterol-solubilizing and dispersing agents; heating said composition to dissolve said non-esterified phytosterols; and cooling said composition to room temperature. Also provided is a method of preparing non-esterified phytosterol-fortified prepared foods comprising the following: providing an edible fat-based composition comprising 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, wherein said composition is substantially free of exogenous phytosterol-solubilizing and dispersing agents, and other ingredients, if any, for said prepared food; cooking or otherwise heating said ingredients with said composition to allow said non-esterified phytosterols to dissolve in said oil or fat and enter or become integrated into said food product, and cooling said food product to room temperature to allow formation of TRPs in said composition within said prepared food.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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..

[0018] 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%.

[0019] 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.

[0020] 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.

[0021] Still another method of producing a fine suspension of microparticulate phytosterols in fat and water has been described by Yliruusi, et al. in U.S. Pat. No. 6,531,463. The method involves first heating and dissolving beta-sitosterol in a fat or oil, and then precipitating the phytosterol with water to form a microcrystalline suspension of phytosterol particles in a mixture of fat and water. 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.

[0022] The production of microparticulate phytosterols described in the relevant literature 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 methods of phytosterol preparation and delivery described above. 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.

[0023] 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%, under this regimen.

[0024] 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

[0025] The present invention concerns the use of non-esterified phytosterols in formulating fat-containing dietary supplements and direct food additives, and in fortifying prepared foods. Non-esterified phytosterols were found to have the unexpected property of decreasing the oxidation of fats used in these supplements and prepared foods, particularly the oxidation of triglycerides containing polyunsaturated fatty acids including linoleic and alpha-linolenic acid and also the more perishable fatty acids found in fish oil, e.g. DHA and EPA, as well as decreasing the rate of triglyceride oxidation caused by heating, e.g., during frying and baking. It is believed that non-esterified phytosterols described herein are 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 exposure of triglycerides to air, and that are particularly problematic in heated fats. Thus, in addition to their ability to function as a plasma cholesterol-lowering neutraceutical ingredient in dietary supplements and prepared foods, phytosterols can actually protect fats against oxidation during cooking and shelf storage.

[0026] An unanticipated finding emerged recently from analyses of human plasma samples that Applicants obtained during a clinical study in which subjects consumed non-esterified phytosterols (TRPs) incorporated into tortilla chips over a 4 week period (see Example 7B below). In addition to the cholesterol-lowering results from that study reported previously (Hayes et al., J. Nutr. 134: 1395-1399; 2004), it has been discovered that the plasma samples from all of the subjects in the study had a beneficially reduced content (averaging 30% decrease) in thiobarbituric reactive substances (TBARS, see Example 11 below) after the subjects had consumed TRP-fortified tortilla chips for four weeks (compared to "control" chips lacking the phyotsterols). These different and compatible functionalities (i.e., beneficial lowering of LDL cholesterol and TBARS levels, and oxidative stabilization of fats) support the novel combination of phytosterols and fish oil in dietary supplements, and the introduction of phytosterols in the form of TRPs into fat-containing prepared foods, e.g., into frying and baking shortenings that are absorbed into or combined with such prepared foods.

[0027] Heat-solubilizing non-esterified phytosterols in fat or oil, followed by cooling and recrystallization, results in formation of triglyceride-recrystallized non-esterified phytosterols (herein termed TRPs). Applicants have found that when ingested, regardless of the crystalline size of these fat-recrystallized phytosterols, TRPs were effective at reducing mammalian plasma cholesterol and peroxide levels. By using cost-effective non-esterified phytosterols, and rendering them bioavailable by thermal recrystallization in fat (i.e., heating and cooling in flying fat, baking shortening, receipe ingredient fat, or any other edible fat or oil), 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.

[0028] In the present invention, Applicants describe the combining of higher concentrations of non-esterified phytosterols (greater than 25% to less than or equal to 75% by weight) with vegetable oil as well as other edible oils and fats such as fish oil and flax oil to form TRP complexes. In contrast to the soft and/or fluid TRPs containing between 2% and 25% by weight phytosterols previously described in U.S. Pat. No. 6,638,547, TRPs with higher concentrations of phytosterols and less fat are semi-solid or solid upon cooling to room temperature. It was anticipated that these solid materials would be difficult or impossible for the mammalian GI tract to digest into bioavailable constituents due to the seemingly resistant physical state of the TRPs, given that phytosterols themselves are essentially undigestable and are largely insoluble in vivo. In other words, based on theoretical considerations it was considered unlikely that the phytosterols in these TRP complexes would be capable of reducing plasma cholesterol levels. Surprisingly however, the more concentrated phytosterols in these semi-solid and solid TRPs were nearly as effective (on the basis of weight content of phytosterols) at reducing plasma cholesterol as phytosterols found in liquid TRPs (the latter containing a substantially lower concentration of phytosterols, e.g., typically 10-15% by weight) combined with a larger proportion of fat (typically 85-90% by weight). The same method was used to form these more concentrated TRPs, except that higher temperatures were required to initially dissolve the higher proportions of phytosterols (typically 100-135 degrees C. rather than 60-90 degrees C.). Subsequent cooling of these heated solutions allowed the mixed crystalline TRPs to be formed. Physical characterization, and the testing of these TRPs in the mammalian system are described below. A number of uses for these TRPs, including their use in dietary supplements as well as in additional processed foods are described herein.

[0029] In U.S. Pat. No. 6,638,547, Perlman, et al. describe the improved oxidative stability of fats "stored" as TRP complexes in fat-containing foods, as well as a beneficial reduction in the level of plasma LDL cholesterol. As mentioned above, an additional and unanticipated health benefit was discovered in further analyzing the human plasma samples that had been stored frozen from the same clinical study (see Example 7B below). Applicants tested these plasma samples for the presence of undesirable oxidative molecular species measured by thiobarbituric acid-reactive substances (TBARS). It is generally understood that these oxidative species include oxidized cholesterol, plasma lipids, lipoproteins, proteins and the like. It is further understood that elevated levels of these oxidative species in human plasma may accelerate the onset and progression of a number of diseases including but not limited to atherosclerosis and insulin-independent diabetes. Therefore, it was remarkable and useful to discover that the TBARS levels present in seven out of seven subjects tested who had regularly consumed phytosterol-fortified tortilla chips averaged 30% lower (i.e., better) than the TBARS levels measured in the same subjects both at the beginning of the trial and after these subjects-had consumed "control chips" i.e., similar tortilla chips lacking phytosterols, for the same time duration (i.e., 4 weeks). Without wishing to be bound by theory, since it is known that negligible amounts of non-esterified phytosterols are absorbed into the bloodstream, their effect in decreasing the plasma TBARS levels is probably indirect. Accordingly, it is possible that dietary phytosterols in the GI tract reduce the amount of oxidized cholesterol that is absorbed into the bloodstream, and thereby improve the plasma TBARS status.

[0030] Accordingly, in a first aspect, this invention provides a product. In certain embodiments, the product is useful for ingestion by mammals, e.g., by humans. The products of the invention have utility in a variety of uses, including without limitation as a component of a prepared food or a dietary supplement, or as a direct food additive. In certain embodiments, the product includes an oxidation-resistant fat-based composition substantially free of exogenous solubilizing and dispersing agents for phytosterols. The fat-based composition of the invention includes between greater than 25% and less than 75% by weight of one or more triglyceride-based edible oil or fat, and greater than 25% and less than 75% by weight of one or more non-esterified triglyceride-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 triglycercide-based edible oil or fat to a level from greater than 25% to 75% by weight at room temperature, the fat-based composition will contain approximately 1.5% solubilized phytosterol and between greater than 23.5% and less than 73.5% by weight of the insoluble phytosterols. Advantageously, because 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, in certain embodiments the product contains a reduced amount of oxidative by-products compared to an otherwise similar fat-based composition lacking the non-esterified phytosterols of the invention. In this context, an "otherwise similar fat-based composition" comprises the same percentage by weight of one or more triglyceride-based edible oil or fat but lack the non-esterified phytosterols of the invention.

[0031] In particular embodiments, the invention provides product, the fat-based composition of which comprises phytosterols at a level of greater than 25% to less than or equal to 30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, and 70-75%.

[0032] In particular embodiments, the invention provides a product wherein the triglyceride-based edible oil or fat contained therein is vegetable oil, vegetable fat, animal oil, animal fat, or mixtures thereof. In some embodiments, the triglyceride-based edible oil or fat is safflower oil, sunflower oil, corn oil, cottonseed oil, soybean oil, canola oil, peanut oil, coconut oil, cocoa butter, palm oil, palm olein, palm super-olein, palm kernel oil, algae oil, flaxseed oil, or combinations thereof.

[0033] In further embodiments, the triglyceride-based edible oil or fat is butter, anhydrous milk fat, tallow, lard, mutton fat, poultry fat, fish oil, and combinations thereof. In yet further embodiments, the triglyceride-based edible oil or fat is cholesterol-free or cholester-reduced.

[0034] In yet further embodiments, the triglyceride-based edible oil or fat is selected from the group consisting of natural vegetable and animal fats, structurally rearranged or otherwise modified vegetable and animals fats, and combinations thereof.

[0035] In yet further embodiments, the non-esterified phytosterol provided by the invention is selected from the group consisting of vegetable oil-derived phytosterols, tall oil-derived phytosterols, and combinations thereof.

[0036] In yet further embodiments, the non-esterified phytosterol of the product provided by the invention is selected from the group consisting of beta-sitosterol, beta-sitostanol, campesterol, campestanol, stagmasterol, stigmastanol, brassicasterol, brassicastanol, clionasterol, clionastonal, and combinations thereof.

[0037] Storage stability may also be referred to as the shelf-life of product at ambient temperatures. Depending upon the food packaging materials and inert gases utilized in the packaging process, the shelf life for products of the invention may range from about one week to about one year or more. 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 TRPs.

[0038] In particular embodiments, the fat-based composition of the invention is heated to a temperature at or above 60.degree. C., for example 60.degree. C. to 80.degree. C., 80.degree. C. to 100.degree. C., 100.degree. C. to 120.degree. C., 120.degree. C. to 150.degree. C., 150.degree. C. to 200.degree. C., or even greater than 200.degree. C.

[0039] In a related aspect, a product useful as a prepared food product for ingestion by mammals is provided as above except that the fat-based 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-based composition. The fat-based composition is substantially free of exogenous phytosterol-solubilizing and dispersing agents. Phytosterol enrichment of the fat-based 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-based 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-based composition and in the prepared food product are bioavailable when ingested, to reduce mammalian plasma cholesterol levels and thiobarbituric reactive substances (TBARS).

[0040] In certain embodiments, the amount of the fat-based 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%, 60-75% or even higher.

[0041] In another aspect, the invention provides a frying fat-based composition which can be held at 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. As with any frying fat-based composition, eventually the fat will degrade sufficiently that it will not be used any longer for frying, and may be replaced with fresh fat-based composition. In particular embodiments, the frying fat-based 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-based composition without phytosterols or other non-fat oxidation rate reducing components.

[0042] In preferred embodiments, the invention provides products containing TRPs which are formed by heating at least the fat-based composition (or heating the product as it contains the fat-based composition) to a temperature and for a time sufficient to dissolve the non-esterified phytosterols in the fat-based composition, and subsequently cooling the fat-based composition to room temperature to allow the TRPs to crystallize and be formed. The temperature of the dissolution process may be for example 60.degree. C. to 80.degree. C., 80.degree. C. to 100.degree. C., 100.degree. C. to 150.degree. C., or even greater than 150.degree. C., and the time necessary to achieve dissolution may be for example zero to 1 minute, 1-10 minutes, 10-20 minutes, 20-30 minutes, 30-45 minutes, 45-60 minutes, 1-2 hr, 2-3 hr, 3-4 hr, or longer than 4 hr. As the weight proportion of phytosterols to fat or oil in a mixture is increased, the temperature required to fully dissolve the non-esterified phytosterols, as well as the melting point of the TRP complex produced from that heat-solubilized mixture, increases (see Example 12).

[0043] 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 plasma cholesterol-reducing oil or fat composition is substantially free of exogenous solubilizing and dispersing agents for phytosterols, and includes greater than 25% and less than 75% by weight of one or more triglyceride-based edible oil or fat, and at least greater than 25% and less than 75% by weight of one or more non-esterified triglyceride-recrystallized phytosterols. As described above, typically the phytosterols are soluble in the plasma cholesterol-reducing oil or fat composition at room temperature to a level of approximately 1.5% by weight, so that at least greater than 23.5% 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.

[0044] In preferred embodiments, the fat-based composition includes at least slightly greater than 25% to less than 75%, for example, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or less than 75%, by weight of non-esterified phytosterols, or the fat-based composition 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 24.5%, 25.5%, 26.5%, 27.5%, 28.5%, 33.5%, 37.5%, 43.5%, 48.5%, 53.5%, 58.5%, or 73.5% respectively) is insoluble at room temperature, but is dissolved and triglyceride-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.

[0045] In another aspect, the TRPs of the invention are formed by heating at least the above referenced fat-based composition (or a product containing the fat-based composition, or the triglyceride-based edible oil or fat and the non-esterified phytosterols as ingredients of the product) to a temperature and for a time sufficient to fully dissolve the non-esterified phytosterols in the fat-based composition, and subsequently cooling the fat-based composition to room temperature to cause the TRPs to be formed. In further preferred embodiments, the temperature to achieve dissolution is 60.degree. C. to 80.degree. C., 80.degree. C. to 100.degree. C., 100.degree. C. to 150.degree. C., or even greater than 150.degree. C., and the time necessary to achieve dissolution may be for example zero to 1 minute, 1-10 minutes, 10-20 minutes, 20-30 minutes, 30-45 minutes, 45-60 minutes, 1-2 hr, 2-3 hr, 3-4 hr, or longer than 4 hr. 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 composition is lower than generally desired to be commercially useful or practical as known to one skilled in the art.

[0046] In another preferred embodiment, the invention provides a method for preparing a TRP-containing fat-based composition, which method comprises: i) heating a mixture comprising one or more triglyceride-based edible oil or fat and non-esterified phytosterols, wherein the mixture comprises less than 75% by weight of triglyceride-based edible fat or oil, and greater than 25% and less than 75% by weight of non-esterified phytosterols for sufficient time and temperature to dissolve said non-esterified phytosterols; and ii) cooling said triglyceride-recrystallized phytosterol-containing fat-based composition to room temperature. The heating step i) above employs a temperature in the range of 40.degree. C. to 150.degree. C., for example, 40.degree. C., 50.degree. C., 60.degree. C., 70.degree. C., 80.degree. C., 90.degree. C., 100.degree. C., 110.degree. C., 120.degree. C., or even 150.degree. C., and the time necessary to achieve dissolution may be for example zero to 1 minute, 1-10 minutes, 10-20 minutes, 20-30 minutes, 30-45 minutes, 45-60 minutes, 1-2 hr, 2-3 hr, 3-4 hr, or longer than 4 hr.

[0047] In certain embodiments, the invention provides products useful as prepared foods or useful in the manufacture of prepared foods including margarines, spreads, butter, frying and baking shortenings, mayonnaise, salad dressings, dairy products, nut, seed and kernel butters, or chocolate. In each of these examples, the phytosterols are dissolved by heating them in the fat portion of these prepared foods, i.e., heating without a substantial amount, e.g., less than 10% by weight of the food, of aqueous components present.

[0048] In preferred embodiments, the product of the invention is a leavened bakery product comprising a flour, potato chips, French fries, corn chips, tortilla chips, popcorn, crackers, condiments, and sauces. The term leavened denotes being made light by aerating, as with yeast or baking powder.

[0049] In yet further embodiments, the invention provides a leavened bakery product comprising a flour which is a cake, muffin, donut, pastry, bread or roll.

[0050] In further embodiments, the product of the invention is a condiment which is tomato catsup, mustard, barbecue sauce, steak sauce, Worchestershire sauce, cocktail sauce, tartar sauce, and pickle relish.

[0051] In further embodiments, the product of the invention is a sauce which is a tomato-based pasta sauce, pizza sauce, prepared chili, or a dessert sauce.

[0052] In certain embodiments, the invention provides a product which is a prepared food product which is fried, baked, or otherwise heat-processed with the fat-based phytosterol-containing composition, and/or where the triglyceride-based edible oil or fat and non-esterified 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 triglyercide-based edible oil or fat at room temperature to be solubilized and thereby enter and be incorporated into the prepared food product, whereupon during cooling, TRPs are formed in the prepared food product.

[0053] In further preferred embodiments, the product of the invention is a food product which is cooked, baked, or otherwise heat-processed with the above-described oil or fat plus phytosterol-containing 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 and TBARS, while addition of partially hydrogenated oils disadvantageously increases the LDL level.

[0054] In a further embodiment, the invention provides a method for producing a fried snack food having reduced surface oiliness, said method comprising frying the snack food in a fat-based composition which comprises one or more triglyceride-based edible oil or fat, and greater than 25% and less than 75% by weight of one or more non-esterified phytosterols.

[0055] In certain aspects, the invention provides a product useful as a dietary supplement comprising a fat-based composition wherein one or more triglyceride-based edible oil or fat comprises omega-3 fatty acids at a concentration of at least 10%, for example, 10%-15%, 15%-20%, 20%-30%, and greater than 30%. In certain embodiments of this aspect, the omega-3 fatty acid is DHA, EPA, alpha-linolenic acid, or combinations thereof. In further embodiments of this aspect, the triglyceride-based edible oil or fat is a marine oil. In further embodiments of this aspect, the triglyceride-based edible oil or fat is fish oil, cholesterol-reduced fish oil, cholesterol-free fish oil, algae oil, flaxseed oil, and combinations thereof. In further embodiments of this aspect, the product consists essentially of the triglyceride-based edible oil or fat in combination with phytosterol present as TRPs. In further embodiments of this aspect, the product is packaged in edible capsules, for example edible gelatin capsules.

[0056] In preferred embodiments, the food product, and more particularly the fat-based 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 oxidation rate of the fat-based composition of the invention is at least 20% lower than the corresponding rate for the triglycerisde-based oil or fat of the fat-based composition lacking the non-esterified phytosterol component. In more preferred embodiments, the reduction in oxidation rate of the fat-based composition relative to corresponding composition without non-esterified phytosterols is greater than 20%, for example, 25%, 30%, 35%, 40%, and even 50%.

[0057] In further preferred embodiments, the invention provides a method of increasing the oxidative stability of a heated frying fat composition useful for frying, wherein the method comprises maintaining a heated frying fat-based composition which is greater than 25% by weight non-esterified phytosterols at a temperature of at least 100.degree. C., for example, 100.degree. C., 110.degree. C., 120.degree. C., 130.degree. C., 140.degree. C., 150.degree. C., 180.degree. C., and even 200.degree. C.

[0058] In preferred embodiments, the product of the invention 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 edible oil or fat normally absorbed or otherwise incorporated into the food product. This statement is explained and supported by Example 4 below.

[0059] 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).

[0060] In another aspect, the invention provides an oxidation-resistant frying or baking shortening comprising: i) from greater than 25% to less than 75% by weight of one or more edible triglyceride-based fat or oil; and ii) from greater than 25% to less than 75% by weight TRPs produced from one or more non-esterified phytosterol compound being solubilized by heating and allowed to recrystallize in the fat or oil upon cooling.

[0061] 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 a working temperature, for example 160.degree. C. to 200.degree. C., is reduced, compared to the same shortening lacking the at least one non-esterified phytosterol compound. In this context, a working temperature is a temperature at which frying or baking is routinely conducted by one of skill in the cooking or baking arts.

[0062] 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.

[0063] In preferred embodiments, the oxidation-resistant frying or baking shortening includes one or more 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 (including but not limited to hydrogenated oils), and combinations thereof.

[0064] In preferred embodiments, the oxidation-resistant frying or baking shortening includes one or more non-esterified phytosterol compound selected from the group consisting of vegetable oil-derived phytosterols, tall oil-derived phytosterols, and combinations thereof.

[0065] In preferred embodiments, the oxidation-resistant frying or baking shortening includes one or more 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.

[0066] In another aspect, the invention provides a method for reducing plasma cholesterol levels in mammals. The method comprises providing and ingesting a product of the invention, which product comprises a fat-based composition that comprises greater than 25% and less than 75% by weight of one or more triglyceride-based edible fat or oil, and greater than 25% and less than 75% by weight of one or more non-esterified triglyceride-recrystallized phytosterols. 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 TRPs. The TRPs when ingested are essentially as effective as fat-soluble esterified phytosterols in reducing plasma cholesterol levels.

[0067] In certain embodiments of this aspect of the invention, the proportion of non-esterified phytosterols used in the fat-based composition for a prepared food is greater than 25% and less than 75% by weight of the composition, and more preferably between 26% and 40% of the composition (or other percentage as described for food products herein). Thus, with the latter range, a serving of food containing 10 g of a fat-based composition, would contain between 2.6 g and 4.0 g of non-esterified phytosterols. This amount is consistent with current recommendations published by the U.S. Food and Drug Administration. Further, a serving of food containing 1.5 g of a fat-based composition would contain between 0.4 and 0.6 g of non-esterified phytosterols.

[0068] In preferred embodiments, between 0.4 g and 4.0 g of the non-esterified phytosterols contained in the above prepared food are ingested daily by humans.

[0069] In preferred embodiments of this aspect of the invention providing for the reduction in plasma cholesterol levels in mammals, the TRPs are formed by heating at least the fat-based composition to a temperature of at least 60.degree. C., for example, 60.degree. C., 70-C, 80.degree. C., 90.degree. C., 100.degree. C., 110.degree. C., 120.degree. C., or even 150.degree. C., for a period of time sufficient to dissolve the non-esterified phytosterols in the fat, and subsequently cooling the fat-based composition (or the food containing this composition) to room temperature to cause the TRPs to be formed. The time necessary to achieve dissolution may be for example zero to 1 minute, 1-10 minutes, 10-20 minutes, 20-30 minutes, 30-45 minutes, 45-60 minutes, 1-2 hr, 2-3 hr, 3-4 hr, or longer than 4 hr.

[0070] In certain embodiments, the fat-based composition contains at least slightly greater than 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or less than 75% by weight of non-esterified phytosterols.

[0071] In preferred embodiments, the plasma concentration of carotenoids comprising alpha- and beta-carotene is maintained essentially constant while plasma cholesterol levels are reduced.

[0072] In further preferred embodiments, the triglyceride-based edible oil or fat of the method comprises at least 10%, for example 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, or even 40% by weight omega-3 fatty acids. In further preferred embodiments, the omega-3 fatty acids comprise DHA or DHA plus EPA wherein the levels of plasma triglycerides and cholesterol are simultaneously reduced.

[0073] In another aspect, a method is provided for preparing a non-esterified phytosterol-fortified prepared food. The method comprises: (i) providing an edible fat-based composition that comprises greater than 25% and less than 75% by weight of one or more non-esterified phytosterols and greater than 25% and less than 75% by weight of one or more triglyceride-based edible fat or oil, wherein 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 as known by those of skill in the art; (ii) cooking or otherwise heating the prepared food ingredients with the edible fat-based composition to allow the non-esterified phytosterols to dissolve in the triglyceride-based edible 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.

[0074] 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.

[0075] 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.

[0076] In the particular embodiments, the fat-based composition containing non-esterified phytosterols is a composition as described for other aspects herein.

[0077] In yet another aspect, the invention provides an product useful as a dietary supplement that includes one or more triglyceride-based edible fat or oil, and greater than 25% and less than or equal to 50% by weight of one or more 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 composition can be combined with other dietary components, such as protein, vitamins, minerals, and combinations of such components.

[0078] In certain embodiments, the phytosterol content, fat content, preparation method for the fat-based composition, and other parameters are as described herein for other aspects involving a fat/TRP composition.

[0079] In a certain aspect, the invention provides a method for protecting plasma lipoproteins and cholesterol from oxidation in mammals. The method comprises providing and ingesting a product of the invention, which product comprises a fat-based composition that comprises greater than 25% and less than 75% by weight of one or more triglyceride-based edible fat or oil, and greater than 25% and less than 75% by weight of one or more non-esterified triglyceride-recrystallized phytosterols. The fat-based composition is substantially free of exogenous phytosterol-solubilizing and dispersing agents. In preferred embodiments, between 0.4 g and 4.0 g of the non-esterified phytosterols are ingested daily by a mammal, e.g., a human.

[0080] In a further embodiment of this aspect of the invention providing protection of plasma lipoproteins and cholesterol from oxidation in mammals, the TRPs are formed by heating at least the fat-based composition to a temperature of at least 60.degree. C., for example, 60.degree. C., 70.degree. C., 80.degree. C., 90.degree. C., 100.degree. C., 110.degree. C., 120.degree. C., or even 150.degree. C., for a period of time sufficient to dissolve the non-esterified phytosterols in the fat, and subsequently cooling the fat-based composition (or the food containing this composition) to room temperature to cause the TRPs to be formed. The time necessary to achieve dissolution may be for example zero to 1 minute, 1-10 minutes, 10-20 minutes, 20-30 minutes, 30-45 minutes, 45-60 minutes, 1-2 hr, 2-3 hr, 3-4 hr, or longer than 4 hr.

[0081] In another aspect, the invention provides a food or beverage used in a weight loss regimen for overweight and obese patients wherein the food or beverage comprises non-esterified phytosterols in the form of TRPs wherein the patient can benefit from a reduction in the level of plasma LDL cholesterol. An verweight subject, e.g., a human, can benefit from regular consumption of prepared foods and beverages that include between approximately 400 mg and 800 mg per serving, for example 400 mg, 500 mg, 600 mg, 700 mg, or 800 mg, of non-esterified phytosterols in the form of TRPs that can reduce the level of plasma LDL cholesterol.

[0082] In another aspect, the invention provides a prepared food product comprising endogenous cholesterol, and further comprising TRPs that have been added to the prepared food product during or subsequent to its manufacture, wherein the weight ratio of non-esterified phytosterols contained with the TRPs to the endogenous cholesterol is between 2:1 and 10:1, for example, between 3:1 and 7:1, or between 4:1 and 6:1. In preferred embodiments of this aspect of the invention, the prepared food product is a butter-containing spread comprising at least 50% by weight milkfat. In an additional preferred embodiment, the prepared food product is ice cream. In further preferred embodiments, the prepared food product is ground meat or fish-containing product.

[0083] The term "prepared" in the context of a "prepared food product" or "prepared food" refers to a commercially processed and packaged food or beverage 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 edible 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 triglyceride-based edible 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 that can retain the TRPs include potato chips, tortilla chips, corn chips and French fries (all fried in oils fortified with phytosterols), popcorn, crackers, pastry, cakes, breads, rolls, muffins, peanut butter, soybean butter, sesame seed butter and other nut kernel butters, margarine, frying and baking shortenings, mayonnaise, salad dressing, chocolate and chocolate-containing products, condiments such as tomato catsup, mustard, barbecue sauce, steak sauce, Worcestershire sauce, cocktail sauce, tartar sauce, and pickle relish, and food sauces such as tomato-based pasta and pizza sauces, prepared chili (meat or meatless) dessert sauces, prepared dairy products such as processed cheeses, yogurt, filled milks, butter, cream and the like.

[0084] 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) that is derived from a vegetable source (e.g., soybean, cottonseed, corn, palm, algae), an animal source (beef tallow, pork lard, sheep or mutton fat, poultry fat, e.g., chicken and turkey, and fish oil), or a blended combination of sources. Unless specifically limited to fat-based compositions that are solid at room temperature, use of the term "fat" includes oils. Furthermore, unless clearly indicated to the contrary, the term "fat" also includes synthetic fats and oils, 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. For example, structured triglycerides that are known in the art as short chain and medium chain triglycerides, owing to a decreased number of carbon atoms forming the fatty acid chains in the triglyceride molecules, are also included under the broad term "fats." Saturated, monounsaturated and polyunsaturated fatty acids, that in ester linkage with the glycerol molecule make up triglyceride molecules, are well known in the art. Of particular relevance herein, polyunsaturated fatty acids (containing at least two carbon-carbon double bonds) are "essential" dietary nutrients because the human body does not synthesize them. Polyunsaturated fatty acids include omega-6 and omega-3 fatty acids.

[0085] The "omega-3 fatty acids" as described above and herein, contain three or more carbon-carbon double bonds with a first carbon-carbon double bond between the 3rd and 4th carbon atoms counting from the omega end, i.e., the free end, of the molecule. Without being bound by theory, the omega-3 fatty acids may help to reduce the risk of heart attack by preventing blood from clotting and sticking to the artery walls and reducing the incidence of cardiac arrthymia leading to sudden death. Flax oil with alpha-linoleic acid, algae oil with DHA, and fish oils containing both DHA and EPA, are useful sources for omega-3 fatty acids, and these edible oils are used in dietary supplements described herein.

[0086] The term "cholesterol-free" as defined herein refers to a processed food product or a dietary supplement that contains less than 2 mg of cholesterol per standard serving of food, or per recommended dosage of supplement. "Cholesterol-reduced" (or reduced cholesterol) is meant to indicate that the food product or dietary supplement product contains 50 percent (or less) of the cholesterol found in the product containing the same amount of fat that is not treated or altered to remove cholesterol. Low in cholesterol means that the cholesterol per serving (or per 100 grams of food) is 20 milligrams or less. While the terms "cholesterol-free" and cholesterol-reduced" as defined by the FDA also typically require that the saturated fat content per serving is 2 grams or less, this limitation does not apply herein.

[0087] 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 at the same temperature 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 for rancidity). The extent of oxidative protection provided by non-esterified phytosterols dissolved in fat heated to, for example, 110.degree. C. during stability measurements or 180.degree. C. during frying, 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. Typically, the rate of oil oxidation decreases as the concentration of phytosterols combined with a fat is increased.

[0088] 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.

[0089] It is believed that oxidative protection of fats and oils provided by phytosterols has not been reported previously (before the priority date of U.S. Provisional application 60/332,434). Similarly, phytosterols were 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.

[0090] The term "edible" in the context of a fat-based composition means that said composition is suitable for use in mammalian, e.g., human, foods, dietary supplements and pharmaceutical preparations.

[0091] 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 edible 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.

[0092] 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.

[0093] The term "phytosterol" refers to any of a group of sterols and stanols found naturally in plants, or partially or fully hydrogenated (converting sterols to stanols) after removal from plants. The commercially available phytosterols employed in the present invention are typically referred to as "free." As defined herein, "free" means that greater than 75% by weight of the phytosterol material employed exists in chemically non-esterified form. These free phytosterols are derived from soft plants, e.g., soybeans, or alternatively from so-called "tall oil" extracted from woody plants, e.g., pine trees. Preparations of plant sterols may include combinations of these different sources, and typically include mixtures of both phytosterols and phytostanols. The definition of phytosterols is intended to include any and all combinations of phytosterols and phytostanols such as beta-sitosterol and beta-sitostanol, campesterol and campestanol, stigmasterol and stigmastanol, brassicasterol and brassicastanol, and clionasterol and clionastanol. The term also includes non-esterified phytosterols that have been partially or fully converted to non-esterified stanols, typically by chemical hydrogenation.

[0094] 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.

[0095] 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 edible 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 rates of prepared foods containing heated triglycerides and phytosterols is preferable to flash cooling. For example, in many cases cooling of a fat-based composition or prepared food to room temperature will occur over a period of 5 minutes to 2 hrs, although longer or shorter times can be used.

[0096] The term "triglyceride-recrystallized phytosterols" or TRPs and the process of heating and cooling these ingredients is described elsewhere herein. The term "recrystallized" is distinguished from the term "solubilized" (in which the phytosterols are dissolved to form a clear solution). Recrystallized is meant to indicate that the phytosterols after initially being dissolved in one or more triglyceride-based edible fats or oils, are allowed to cool and recrystallize in the oil or fat. By physical analyses (light microscopy of lipid stained crystals, and melting temperature determinations described elsewhere herein), Applicants have determined that such recrystallization results in fats and/or oils, i.e., triglycerides, becoming intimately associated with crystallizing phytosterols. The resulting products are mixed and/or interrupted crystal structures having melting temperatures reduced below that of the phytosterols alone. It is believed that these physically destabilized, triglyceride-containing crystals are more easily emulsified and/or dissolved in the mammalian gut, resulting in improved phytosterol bioavailability and therefore more effective plasma cholesterol reduction in vivo. As noted above, a proportion of the phytosterols is soluble in the fat at room temperature (typically at a concentration of about 1.5%). Therefore, when a combination of phytosterols and fat is heated to dissolve the solid (e.g., crystalline) phystosterols, and the solution is then cooled, the phytosterols that cannot remain in solution at room temperature solidify or recrystallize, and a portion remains dissolved in the fat. Unless clearly indicated to the contrary, reference herein to "triglyceride-recrystallized phytosterols" or "TRPs" and the content thereof, includes all the phytosterol content in a composition but not its fat content, i.e., both the dissolved phytosterols as well as the re-solidified or recrystallized phytosterols. Thus, the weight proportion of TRPs in a composition produced only from fat and phytosterols is simply calculated based upon the weight of phytosterols relative to the weight of fat plus phytosterols. As an example, if 25 g of non-esterified phytosterols (including any non-esterified phytostanols) is dissolved and recrystallized with 75 g fat, the composition is said to contain 25% by weight TRPs.

[0097] The term "effective" refers to the extent to which plasma cholesterol levels in mammals are reduced by regular, e.g., daily, twice daily, or thrice daily ingestion of the recommended 1-2 gram dose (or the appropriate divided dose) of phytosterols. In a random population of human adults, a 5% to 15% or greater lowering of total cholesterol in the plasma caused by ingestion of phytosterols is considered-effective.

[0098] The term "esterified phytosterols" refers to phytosterols (plant sterols and stanols) that have been joined through an ester linkage to fatty acids using a chemical, enzymatic, combination, or other process. The commercial margarines Benecol.RTM. and Take Control.RTM. discussed above, incorporate such esterified phytosterols. Therefore, "non-esterified phytosterols" refers to phytosterols that have not been esterified to fatty acids as described.

[0099] The term "reduced surface oiliness" means that upon routine handling of the prepared food, less oil is transferred from the food to ones hands (or to an absorbant surface) than would otherwise occur if the food were prepared with the oil or fat alone (see Example 5 below).

[0100] As used herein, the term "dietary supplement" has a specific legal meaning in the U.S. established by the Dietary Supplement Health and Education Act (DSHEA). It refers to a product that supplements the diet and whose label clearly states that it is a dietary supplement (sometimes commonly referred to a nutritional supplement). A dietary supplement is distinct from a drug which is represented as a substance that is intended to diagnose, cure, mitigate, treat, or prevent diseases and which must undergo extensive testing and be preapproved by the FDA before being sold. There are specific cases where dietary supplements may also be labeled as impacting disease. Dietary supplements typically contain the following kinds of ingredients: vitamins, minerals, herbs, botanicals, other plant-derived substances, amino acids (the individual building blocks of protein) and concentrates, metabolites, constituents, and extracts of these substances. They are intended for ingestion typically in pill, capsule, tablet, or liquid form and are not represented for use as a conventional food or as the sole item of a meal or diet. Dietary supplements are typically adapted to supplement, i.e., add to, an individual's dietary intake of one or more dietary components, for example, fish oil supplements providing omega-3 fatty acids. Non-esterified phytosterols refined from vegetable sources can be combined with edible vegetable or animal oils or fats to form TRPs. These may be packaged in gelatin capsules to provide a convenient dietary supplement. In general, gelatin capsule formulations comprise raw gelatin, plasticizer, solvent and optional ingredients such as flavors and colorants, as known by those skilled in the art. If the phytosterols are combined with edible fish oil also forming TRPs, this combination can provide a supplement with the health benefits of both omega-3 fatty acids as well as phytosterols. Both the fish oil and the phytosterol in the form of TRPs are biochemically accessible, i.e., bioavailable, in the mammalian gastrointestinal tract (see Examples 15 and 16). A "neutraceutical" refers to a product isolated or purified from raw or unprocessed food materials, and generally sold in medicinal forms not usually associated with food. By definition, neutraceuticals provide a physiological benefit or provide protection against chronic disease. In the present invention, phytosterols provide a hypocholesterolemic benefit and are a nutraceutical. Phytosterols can be purified from raw vegetable oils such as soybean oil and incorporated into dietary supplements and processed food products.

[0101] The term "condiment" as used herein means a savory, pungent, piquant, spicy, acidic or salty accompaniment to food to enhance flavor or give added flavor, such as a relish, sauce, mixture of spices and so on. Catsup and mustard are two of the most popular condiments in the U.S.

[0102] The term "sauce" as used herein means a fluid, semifluid or sometimes semisolid accompaniment of solid food, such as a meat stock, fish stock, milk or cream that has been thickened and flavored with spices, condiments, and the like (e.g., bechamel, brown sauce, hollandaise, veloute). Some sauces are sweetened mixtures served as toppings on desserts. Other sauces include stewed or canned fruit eaten as an accompaniment with other food. Many condiments are considered sauces, and vice-versa, e.g., steak sauce, hoisin sauce, Worcestershire sauce, hot pepper sauce, and the like.

[0103] For the definition of any fat and oil-related terms that have not been expressly defined herein, the reader is referred to the reference book, Bailey's Industrial Oil and Fat Products, Fourth Edition, Daniel Swem, editor, John Wiley & Sons, N.Y., 1979.

[0104] By "comprising" is meant including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0105] Additional aspects and embodiments will be apparent from the following Detailed Description and from the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0106] Recently, a number of investigators have described a variety of methods for producing very small particles or microcrystals of phytosterols. It is believed that such small particles have greater efficacy in being dispersed in the GI tract and controlling plasma cholesterol levels. U.S. Pat. No. 6,129,944 by Tiainen et al. describes the production of a microcrystalline phytosterol product useful as a cholesterol-lowering agent, formed by pulverizing, i.e., dry or wet grinding, a crystalline phytosterol to produce microparticles having a preferred mean particle size of approximately 5-10 microns. The microcrystalline phytosterol product can be mixed with a sweetening agent and water or alternatively, mixed with another carrier such as fat to form a microparticulate emulsion. There is no suggestion by Tiainen et al. or any other investigator of which the inventors are aware that microcrystalline phytosterols after being formed, should be heated or dissolved in such a fat or oil. Such heating in oil, as described for the present invention, would be expected to destroy the sized microparticles described by Tiainen et al.

[0107] As described herein, phytosterols are recrystallized with triglycerides (e.g., vegetable oil, shortening, or the like). The first step involves heating the triglyceride(s) and phytosterol(s) until the phytosterols are dissolved. This phytosterol-triglyceride solution is used to contact, or be combined with the food product being fried, cooked or otherwise heated. (Alternatively, the fats and the phytosterols are added as separate ingredients in the preparation of a prepared food.) Subsequently, the prepared food product is cooled (preferably by contacting the heated food product with ambient air). Under the light microscope (600.times. magnification), it is seen that phytosterols that have been recrystallized in vegetable oil, e.g., soybean oil, tend to form a diversity of macrocrystalline structures spanning tens or hundreds of microns. This material when tasted, has a surprisingly soft and agreeable mouth feel, and includes elongated hexagonal crystals, radially extending branched crystalline needle structures (appearing as wispy ball-shaped structures), and large extended flat plate crystals. On the other hand, phytosterols that are recrystallized by quick-chilling to room temperature (e.g., by ice chilling to room temperature in a few seconds rather than by ambient air contact), tend to form harder, smaller, more homogeneous needle-like micro-crystals having diameters of only a few microns, i.e., 1-4 microns.

[0108] The temperature required to re-dissolve the above crystals in the surrounding vegetable oil differs significantly depending upon the rapidity of recrystallization. For example, 10% by weight soybean-derived phytosterols that were recrystallized at room temperature in soybean oil, redissolved in the oil at a temperature of 65.degree. C. On the other hand, the more rapidly ice-recrystallized phytosterols described above required a higher temperature (72.degree. C.) to be redissolved. By comparison, the same amount of phytosterol (as a dry powder) initially placed in soybean oil, required a temperature of nearly 85.degree. C. to be dissolved. The observations on recrystallization (coupled with the microscopic analysis of crystalline sizes and shapes) suggested that slower recrystallization allows formation of mixed composition triglyceride-containing (larger) phytosterol crystals. These crystals would be expected to redissolve more easily, i.e., at a lower temperature, than the rapidly formed crystals.

[0109] To determine whether the larger crystals contained any triglycerides, these crystals were washed and centrifuged twice in ethanol. Next, the crystals were stained with a saturated Sudan Black solution (60% by weight ethanol in water) to visualize any lipids. Light microscopy confirmed that the lower melting point larger crystals (but not the higher melting point small needle-shaped crystals) contained multiple internal layers and occlusions of lipid. It is reasonable to conclude that the intimate association of triglycerides and phytosterols that results from fully dissolving and then recrystallizing phytosterols in fats, yields crystals having a reduced melting temperature. These crystals appear to provide dietary phytosterols in a highly bioavailable form useful for reducing plasma cholesterol levels.

[0110] While it has been recently reported that a crystalline complex can be formed by combining phytosterols and monoglyceride emulsifiers (see above, U.S. Pat. No. 6,267,963), the existence and utility of triglyceride-recrystallized phytosterols have not been previously described. In fact, Applicants have not found any prior reference to formation of a mixed crystalline complex or association between triglycerides and phytosterols that enhances phytosterol bioavailability.

[0111] Non-esterified phytosterols are known to have a very limited solubility (to a concentration of approximately 1.5% by weight) in an edible oil or fat at room temperature. Nevertheless, between 1.5% and 75% by weight of non-esterified phytosterols (e.g., semi-pure or purified phytosterols from soybeans or pine tree tall oils), can be readily and conveniently dissolved in edible oil or fat by heating to a temperature of 60.degree. C. or greater, and preferably to 75.degree. C., 100.degree. C., 125.degree. C. or above (the required temperature depending upon the concentration of phytosterols to be dissolved). Subsequently, as the heated composition is cooled to room temperature, a substantial portion of the solubilized phytosterol precipitates, i.e., is recrystallized, in the triglyceride-based edible oil or fat in the form of a Triglyceride-Recrystallized Phytosterol composition or complex (abbreviated "TRP", "TRP composition or TRP complex").

[0112] Remarkably, the TRP composition formed in this manner has been found to be as potent in the mammalian diet at reducing the levels of plasma and liver cholesterol as fatty acid-esterified phytosterols that are fully soluble at room temperature. In the first direct comparison between non-esterified phytosterols and equivalent amounts of phytosterols as sterol esters in the same experiment, it was found that non-esterified phytosterols fully dissolved in oil by heating (>60.degree. C., preferably >80.degree. C., and more preferably to >100.degree. C.), and then subsequently cooled, provided equivalent (or even greater) reductions in plasma and liver cholesterol as compared to equivalent amounts of esterified sterols. In the context of cholesterol reduction, the term "greater" means that the cholesterol reductions measured and reported herein and in Hayes, et al. (J. Nutr. (2004) 134:1395-1399) are greater than those reported by Ntanios and Jones (Biochim. Biophys. Acta (1998) 1390:237-244) for the same levels of sterols, in which the sterols were incompletely dissolved in fat. While TRPs may have been accidentally produced in the past in the course of heating and cooling non-esterified phytosterols and fats, their utility for plasma cholesterol reduction would not have been recognized due to their poor room temperature solubility.

[0113] The presently described TRP composition is more convenient and cost-effective than esterified phytosterols or phytosterol-containing compositions that have been supplemented with solubilizers, emulsifiers, antioxidants and other additives for inclusion in foods. The TRP composition also has a significant advantage over the finely milled and microcrystalline powdered forms of phytosterols described by Tiainen et al. and Jones et al., in light of the considerable cost associated with producing these micron-sized powders. The present composition is particularly useful in preparing fat-based foods such as shortening, margarine, mayonnaise, salad dressing, peanut butter and the like, and processed food products including fried and baked snack foods.

[0114] Surprisingly, as illustrated below, the presence of dissolved phytosterols in a heated oil or fat, improves the triglyceride's oxidative stability, and at ambient temperature, decreases the surface oiliness of foods fried in the triglyceride-based composition. At the same time, the caloric fat content of a food prepared in or with the TRP-containing composition is reduced. While other investigators have found that finely milled or microcrystalline preparations of non-esterified phytosterols that have not been initially heat-solubilized in an oil or fat, can also function efficiently to reduce mammalian plasma cholesterol levels, the additional benefits described above are obtained only after heat-solubilization. For example, heat-solubilization in a triglyceride-based edible oil allows non-esterified phytosterols to freely enter a food product as it is being fried in the oil, whereas particles of phytosterols would be excluded. Likewise, suspended particles would not be expected to improve the oxidative stability of the oil.

[0115] For the purpose of this invention, the fat or oil used as a vehicle or carrier for the phytosterol herein, is a conventional triglyceride-based cooking fat or oil that is substantially free of phytosterol solubilizing agents, dispersants and/or detergents (collectively termed "oil emulsifiers or additives"). Examples of such fats and oils include natural vegetable oils, interesterified fats and oils, and partially hydrogenated vegetable oils, animal fats and combinations thereof.

[0116] Unlike recently described compositions for oils and fats containing phytosterols described above in the Background, the presently described triglyceride-based composition contains substantial amounts of insoluble phytosterol (recrystallized in fat) rather than solubilized phytosterol, and is substantially free of the above-described oil additives for dispersing or solubilizing phytosterols. The composition is particularly useful in preparing fat-containing foods that do not require oil transparency at ambient temperatures. This is true of margarines, shortenings, mayonnaise, cheese and other dairy fat-containing products, some salad dressings, condiments such as tomato catsup, mustard, barbecue sauce, steak sauce, Worchestershire sauce, cocktail sauce, tartar sauce, and pickle relish, and food sauces such as tomato-based pasta and pizza sauce that may contain olive and other vegetable oils, prepared chili and many other foods including processed foods that are fried, baked or otherwise prepared by cooking or heating in, or in combination with fat or oil. Examples of such foods include the snack food category, e.g., potato chips, crackers, and the bakery category, e.g., donuts, pies, cakes, breads, rolls, muffins, cocoa butter-containing chocolate products and the like.

[0117] The present invention describes compositions and methods for introducing substantially fat-insoluble non-esterified phytosterols into dietary supplements such as omega-3 fish oil-containing supplements, and processed food products, including snack foods, by means of the standard fat or oil that is used in the receipes for, or in the frying or baking of such foods. It was the inventors' intention to compare the efficacy of using non-esterified phytosterol preparations recrystallized in edible fat and used in foods, e.g., fried foods, with that of more costly diglyceride-solubilized or fatty acid esterified phytosterols in limiting cholesterol absorption in the gut, and lowering plasma cholesterol levels. Surprisingly, the phytosterols recrystallized in fat that has been incorporated into such foods are very effective, i.e., bioavailable, in reducing plasma and liver cholesterol levels. It is believed that this cholesterol-lowering efficacy compares favorably with that of fully solubilized phytosterol preparations (e.g., phytosterols esterified with fatty acids to assure solubility in fat-containing products such as Benecol.RTM. and Take Control.RTM. margarines).

[0118] As an unanticipated benefit and utility in the present invention, the presence of 5-10% or more by weight of phytosterol that has been recrystallized with triglycerides in the oil portion of fried snack food (e.g., potato chips) has been found to decrease the surface oiliness of fried food when compared to food fried in oil lacking the phytosterol. Applicants have also found that the presence of either soybean oil-derived phytosterols or tall oil-derived phytosterols in vegetable oil during flying, helps in chemically stabilizing the oil against oxidation by reducing the rate of appearance and the amount of polar breakdown products in the oil. To the extent that the phytosterols replace a portion of the oil in such a blend, the phytosterols also serve to reduce the caloric fat content of a food cooked in the blend. Thus, the present invention also provides methods for decreasing the surface oiliness of fried foods, and the resulting fried foods, and methods for providing reduced calorie food, utilizing TRPs as described herein.

[0119] Except for micron-sized finely milled powders of non-esterified phytosterols described by Tiainen et al. and Jones et al. (see above), as well as previously described emulsified preparations, the non-esterified phytosterols have been thought to lack "bioavailability" relative to esterified sterols and stanols, as emphasized in the introductory references. In this instance, bioavailability for a given quantity of phytosterol means the potency of that particular physical and/or chemical form of phytosterol in lowering the plasma level of total and LDL cholesterol. Despite the limited solubility of non-esterified phytosterols in fats and oils at room temperature, it has been discovered that concentrations of between 1.5% and 75% by weight non-esterified phytosterols (e.g., soybean oil-derived mixed prilled sterols or stanols or tall oil-derived sterols and stanols) can be conveniently and rapidly dissolved by mixing or other agitation in diverse oils, fats and fat-containing foods, e.g., cooking or salad oil, shortening, peanut butter and dairy cream, heated to a temperature of greater than 60.degree. C., and preferably between 75.degree. C. and 150.degree. C., or above. At higher temperatures such as 180.degree. C., a heated oil or fat, e.g., corn, canola, cottonseed, soybean oil, or palm oil that contains heat-solubilized phytosterols is useful in the preparation (e.g., frying and baking) of potato chips and other snack foods. When such heat-solubilized phytosterols are cooled and recrystallized in such fats or fat-containing foods, their ability to lower plasma cholesterol levels is excellent (see nutritional studies below).

[0120] The fat compositions and food products of the present invention can be prepared by conventional methods, with the addition of phytosterols (e.g., as described herein). Persons familiar with pre