HEAT STABILIZED PEPTIDE TABLE SALT SUBSTITUTES

Abstract: A thermally stable table salt substitute which comprises a complex of a heat stable dipeptide containing at least one cycloalkyl amino acid residue in the peptide molecule and a polysaccharide gum stabilizer. Preferred dipeptides are those formed from coupling taurine with cycloalkyl ornithine or cycloalkyl lysine or ornithine coupled with cycloalkyl alanine. Preferred gums are gum tragacanth or gum acacia which are complexed with the peptides. The ratio of the peptide to the gum can vary considerably depending on the end use of the seasoning composition and texture required of the product.

Description: HEAT STABILIZED PEPTIDE TABLE SALT SUBSTITUTES RELATED APPLICATIONS This application is a continuation-in-part applica¬ tion of my pending United States Patent Application Serial Number 688,507, filed January 3, 1985, entitled "HEAT STABILIZED PEPTIDE SALT SUBSTITUTES". BACKGROUND OF THE INVENTION 1. Field of the Invention The general field of the present invention is new heat stable dipeptide salty tasting compounds which are capable of being substituted for sodium and potassium chloride seasoning agents in baking and cooking. A process for salting, i.e., seasoning, comestibles with these new peptide salt substitutes is also described, as well as a novel food composition for making such process operable. Finally, a sodium free and sugar free baking formula is presented for those afflicted by diabetes or high blood pressure symptoms. The particular new salty peptides are uniquely characterized in that they are heat stable and do not decompose when added to edible food compositions which are to be baked or cooked at or above or about 360°F before consumption. 2. Prior Art Found In The Field of The Invention A long felt need has existed for a seasoning sub¬ stance which is capable of imparting a salty character or flavor note to foods which does not at the same time donate sodium to the consumer. To many consumers, especially those suffering from chronic hypertension, ordinary sodium chloride or table salt is not to be ingested, since the intake of sodium in any level has been established to effect an unwanted eleva¬ tion in blood pressure in the person passing the salt into their bodily fluids. In an effort to avoid these problems, and yet to enable the hypertensive consumer some measure of salty flavor for his food, several potassium chloride flavor compositions which mix this other alkali metal salt with monosodium or monopotassium glutamate have been introduced into the market¬ place. However, these sodium salt substitutes have in some cases a salty flavor which is not to the liking of many consumers who have known the true sodium chloride flavor. In December 1984, several Japanese chemists at the University of Hiroshima, Japan, announced that they, by accident, had discovered a dipeptide compound of two known amino acids, ornithine and taurine, which had a salty taste approximately equal to sodium chloride. M. Tada et al. , J. Agri. Food Chem. 1984, Vol. 32, pp. 992-998. Each of these amino acids occur in mammals, the first in bird excretia, the second in bile acids of man. When combined, however, these two amino acids are capable of replacing sodium chloride for those consumers who cannot tolerate the ingestion of sodium- containing compounds. However, this new dipeptide salt substitute compound has a serious drawback, in that it cannot be employed where cooking or baking temperatures are required. It is thermally labile and the peptide linkages between the two amino acid compounds fracture at temperatures of 300°F or higher. In addition to this, the instability of the salty composition leads to a loss of the salty flavor and the formation of such bitter tasting reformation compounds as diketo piperazine (DKP) which is readily formed in all normally structured dipeptides formed by the union of two normally structured amino acids. The urgent need hence exists for a further advance in the art to create a salty dipeptide composition which does not denature, degrade, or rupture the peptide bonds when used in cooking or baking foods. The object of the present invention is to describe and claim such a prouct and to illustrate to those skilled in the art of food chemistry how to employ such a new and unique composition of matter. GENERAL DESCRIPTION OF THE INVENTION Accordingly, the present invention comprises a heat stable peptide salt composition which includes a peptide component composed of a plurality of amino acid residue components, at least one of which has been modified to include a cycloalkyl bridge in the structure and, in some cases, an edible hydrocolloidal gum. The composition in its preferred product form comprises a soluble powder which is readily miscible with other food additives and food formulas. The term "cycloalkyl" as employed in this specifica¬ tion is intended to refer to such cyclic functions as cyclo- propyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like which are by means of their manner of preparation built into the particular amino acid concerned. When these modified amino acids are noted herein, they will be distinguished in some cases by the symbol " ^ « prefixed to the name or abbreviation of the amino acid. This added alkyl function has been found to act as a stabilizer of the peptide link between amino acids, at least one of which must exhibit the unique configuration. The preferred peptide components are dipeptides of the normal or cycloalkyl substituted amino acids taurine .or ornithine coupled with such amino acids as cycloalkyl sub¬ stituted ornithine cycloalkyl substituted lysine or cycloalkyl substituted alanine. These compounds and their mineral acid salts and lower alkyl esters exhibit unusual thermal stability, especially when complexed with a quantity of an edible hydro- colloidal gum such as gum acacia or gum tragacanth. The hydrocolloidal gum is preferably a polysaccharide type hydrocolloidal gum, such as gum acacia, gum tragacanth, pectin gum, gum karaya, guar gum, larch gum, psyllium seed gum, or locust bean gum, for instance. For most, but not all, baking purposes and prepara¬ tion of food compositions, a major amount of the hydrocolloidal gum is complexed with a minor amount of the peptide after moistening the dry mix with a water to form a paste. If it is desired, one may dry by known means such as spray drying, drum drying, etc. the complex to form a powder for use in baking or cooking of foods in place of ordinary table salt, i.e., sodium chloride. BRIEF DESCRIPTION OF THE DRAWING Figure 1 of the attached drawing employed is a schematic representation of two alternate routes for the synthesis of the new peptides of the invention as the synthesis is detailed in example 1 hereinbelow. The amino acid reactants are either known and commercially available materials or are cycloalkylized amino acids prepared by the method of H. T. Bucher and V. A. Lieb, Jour, of Prac. Chem. (1934), Vol. 141, pages 5-10, which is incorporated by reference in its entirety into the present specification, although it is schematically represented in the drawing. A recent article by J. W. Tsang et al., reported in Jour, of Med. Chem. 1984, Vol. 27, pp. 1663 to 1668 also describes the synthesis, and is incorporated by reference herein. DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION Preferred Products In its most preferred product aspect, the new peptide containing table salt substitute comprises a soluble complex of: A. A table salt imitating amount of a dipeptide of one of the structural formulae: (1A) H 2 N- or or and the mineral acid, especially hydrochlorides salts, of these compounds wherein R represents either hydrogen or a lower alkyl group of C- | to C4 carbon atoms or benzyl, n is a positive integer from 1 to 4, but preferably 1, and x can be either 1 or 2, depending on whether the cycloalkyl amino acid coupled to taurine is a cycloalkyl ornithine or a cycloalkyl lysine. The coupling of ornithine to cyclopropyl alanine (3A) is the easiest to do, since both the amino acid starting materials are commercially available. However, this peptide is not as salty tasting as the coupling between cyclopropylorni- thine and taurine or taurine to cyclopropyl ornithine. B. A thermally stabilizing amount of a hydrocol¬ loidal gum such as gum acacia or gum tragacanth. The ratio of the amounts of the peptide and gum components in the complex will vary depending on the end use and the food composition being sweetened and the textural requirements of the end product. Generally, 5 to 10 parts of gum are used for each part by weight of peptide, but as much as 100 parts of the former to 1 part of the latter might be useful in some food applications. In still other uses, a minor amount of gum to the peptide could be considered. In the most preferred applications, a major amount of gum to a minor amount of peptide is needed to promote heat stability. A second preferred product aspect of this invention is the peptide ingredients themselves such as the compounds of (1A), (2A), and (3A) hereinabove. In (1A) and (2A), the R represents either hydrogen or a lower alkyl function' such as methyl, ethyl, propyl, or butyl, for instance, either normal or isomeric in configuration or benzyl, x is either 1 or 2, and n is a positive integer from 1 to 4, and hence represents either cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl functions. A third preferred product aspect of the invention is a food composition which requires a salty material as one of its essential ingredients. A cake mix formula, for instance, where the peptide to gum ratio is critical to gain heat stabil¬ ity and concurrent satisfactory texture and other ingredient compatibility. A new peanut butter formula or a pretzel or potato chip dough or pasta dough which can taste like sodium chloride laden dough but actually be salt free is contemplated. It is now possible to manufacture a soup product which imitates present soups but is sodium free, since it is made without sodium chloride. PREFERRED PROCESS ASPECTS OF THE INVENTION One preferred process aspect of the present invention is the synthesis of a new salty tasting peptide by means of the following reaction: ornithine amino acid + cyclopropyl alanine > salty peptide (1) (2) (3A) In the above amino acid coupling reaction, the known a inό acid ornithine is reacted under well known condensation conditions to form the unique peptide (3A). This is the first time this reaction will have been performed, and-the product obtained will be different than other dipeptides. It will be heat resistant and capable of being complexed with a gum to be heat stable, if desired. A second preferred process aspect of the invention is the formulation of a new and unique cake mix which now becomes feasible for baking in the home. In the conventional recipe, sodium chloride is replaced with the new peptide III or (1A), (2A), or (3A) to obtain when baked a new cake which tastes like known cakes but varies in one material respect. It contains no sodium. A final process aspect of the invention is the pre¬ paration of cooked meals and baked foods in the home without the use of sodium or potassium chloride table salts. In a similar manner, this process aspect applies to commercial preparation of prepared food products such as pastas, baked pies, cookies, pretzels, potato chips and corn chips, etc., of many different shapes and formulas. The hydrocolloidal poly.saccharide gums are known commercially available materials, the details of which are available in the Encyclopedia of Chemical Technology (3rd. Edition 1983) by Kirk-Othmer, Vol. 12, pages 57-67, published by John Wiley and Sons, New York, NY. For instance, one of the preferred gum components, gum tragacanth, is known to be a mixture of acidic polysac- charides containing galacturonic acid, galactose, fucose,' and xylose and arabinose. It is an exudate from the Astralagus tree found in Syria, Iran, and Turkey. Solutions are weakly acidic with a pH of 5.0 to 6.0, and a molecular weight range of 10,000 to about 250,000. Gum acacia, on the other hand, is a dried exudate obtained from the Acacia tree found chiefly in the African Sudan. It has a large molecular weight of a range of 200,000 to about 1,160,000, and is stable at a slightly acid to neutral range. These gums, along with all of the other hydrocolloi¬ dal gums, are quite water soluble. As calcium, magnesium, and potassium salts of a branched polysaccharide which contains galactose, rhamnose, glucuronic acid, and arabinose residues, they will exhibit compexing characteristics. They exhibit a high propensity for their free hydroxyl groups to complex with a cycloalkyl bridged dipeptide of the type described herein. What is unexpected about this is that the combination does not impede or interfere with the saltiness attribute of the peptide and does maintain cake texture and stability of the same against heat degradation. EXAMPLE I Step A. Preparation of Cyclopropyl Ornithine Amino Acid Reactant Used in Preparing New Dipeptide Salt Substitute To a solution of 25 grams (290 mmol) of the 3- pentanone J_ as illustrated in Figure 1 of the drawing, add 28.5 grams (438 mmol) of sodium cyanide 2a_ and 119 grams (1.113 mols) of ammonium carbonate b. Reflux mixture for six hours with constant stirring of the reaction mixture. Dilute w,ith water to cool the same to room temperature and acidify the solution to a pH of 5.6 with concentrated hydrochloric acid. Precipitate the crude 3-pentano spiro-5-hydantoin 3_ overnight after cooling the reaction mixture to 5°C. Purify by recrystallizing the product from water. The alkaline hydrolysis of the hydantoin intermediate 3_ to the cyclopropylornithine amino acid reactant is achieved by suspending 6 grams of the hydantoin _3 from above in 100 mis. of 3 N sodium hydroxide and heating to reflux conditions and maintaining this reflux for 21 hours. At the end of this period, cool the reaction mixture and acidify to pH 6.0 with concentrated hydrochloric acid. The cyclopropyl ornithine amino acid 4_ will precipitate from the solution in good yield. Filter the precipitate out and wash with cold water twice, then with acetone, and dry under high vacuo. Wash three more times with warm anhydrous ethanol to further purify. This product can then be recrystallized from a water/acetone mixture to obtain the cycloalkylamino ornithine amino acid _4 in high yield and excellent purity. If one desires to obtain a cyclopropyl lysine amino acid rather than a cyclopropyl ornithine amino acid 4_, one need only change the known substituted 3-pentanone from one having the structure depicted for j_ in the drawing to a starting substituted pentanone of the structure: the rest of the reaction sequence remaining the same. Step B. Preparation of the Dipeptide of Cyclopropyl¬ ornithme and L-Taurine to Obtain a Salty Tasting Peptide 5 Add to 2.7 mmols of a solution of 1-butyloxycarbonyl L-taurine tertiary butyl ester, the cyclopropylornithme amino acid reaction product of Step A hereinabove in 80 cc of anhydrous tetrafuran and 0.3 is. of n-methyl morpholine. Cool this solution to -15°C and add 7.7 mmol of isobutylchloroformate. After 5 minutes, add a solution of 2.76 mmol benzyl aminoisobutyrate HC1 and N-methylmorpholine (2.70 mmol) in 20 cc tetrahydrofuran. Allow the reaction to proceed at -15°C for one hour, then increase the temperature to 5 β C and continue the reaction for 24 hours. Recover the residue by removal of the solvent under low pressure. Dissolve the residue in ether and water wash once. Wash 3 more times with 5% sodium bicarbonate solution. Wash again with water then 6 more times with a 1% sodium bicarbonate solution. Finally, wash for the last time with water, and dry the solid residue over magnesium sulfate and ether. Remove the ether under reduced pressure to obtain the blocked dipeptide n-tertiary butyloxycarbonyl tertiary butyl L-taurine-1-cyclopropylornithme. This is the intermediate noted as 5_. Step C. Preparation of Final Product 6 Add 50 cc of ice cold trifluoroacetic acid to the intermediate 5_ from step B hereinabove. Allow the reaction to proceed for 90 minutes at room temperature. Evaporate the solvent under reduced pressure. Triturate the residue with isopropyl ether to give a white solid after filtration. This solid is the trifluoroacetic acid salt of the peptide. Dissolve this trifluroacetate salt in a mixture of 30 cc of water and 10 cc of ether. Adjust the pH to 5.0 in this solution by adding a 5% sodium bicarbonate solution in ice water and precipitate the final product, L-cyclopropyl ornithyltaurine 6_ from solution in several crops. Purify the product by recrystallization from water to obtain the new peptide of the invention in high yield. EXAMPLE II Preparation of the Dipeptide of Ornithine With Cyclopropyl Alanine to Obtain a Salty Tasting Dipeptide A. General Procedure This peptide is synthesized by known and conventional methods using, however, a new and unique combination of reactive amino acids to yield a heretofore unknown peptide. In brief, dibenzyloxycarbonyl ornithine and cyclopropyl alanine benzyl ester are condensed to yield a new corresponding dibenzyloxycarbonyl dipeptide benzyl ester by the mixed anhydride method. This compound, when deblocked, is a new peptide. The protecting groups of the dipeptide derivatives are removed by catalytic hydrogenation in acetic acid solution. The product is then treated HC1 -dioxane and obtained as .a hydrochloride salt. The purity of the peptide can be confirmed by melting point, elemental analysis, optical rotation, and thin layer chromatography and NMR and HPLC. B. Specific Procedure Step 1. Preparation of Blocked Peptide as its Benzyl Ester Dissolve the dicyclohexylamine salt of L-ornithine (2.77 grams, 5 mmol) in 30 ml of ethyl acetate. Add 10 is. of 1 molar sulfuric acid to the mixture with constant stirring. Wash the organic layer with distilled water and dry over anhydrous sodium sulfate. Concentrate the solution to dryness in vacuo and dissolve the oily residue in 10 mis of tetra- hydrofuran and 0.55 mis of 5 mmol of N-methylmorpholine. Add 0.5 ml (5 mmol) of ethylchloroformate to this mixture at -5 β C and hold in this condition for 15 minutes. After such time, add 1.96 grams (5 mmol) of the benzyl ester of cyclopropyl alanine to the reaction mixture, along with 0.55 mis (5 mmol) of n-methylmorpholine and 10 cc of N,N-dimethyl- formamide. Store this reaction mixture in an ice bath for 1 hour and then maintain at room temperature overnight. Then evaporate the reaction mixture in vacuo and dilute with ethyl acetate. Wash the solution obtained first with distilled water, then with a 2% HC1 solution, again with distilled water, then with a 4% sodium bicarbonate solution. Finally, wash one more time with distilled water and dry the same over anhydrous sodium sulfate and evaporate the filtrate in vacuo. Crystallize the oily residue from ether and petroleum ether to give the blocked peptide as its benzyl ester, Cbz-L- ornithine (-Cbz)-cyclopropyl alanine 0 Bzl. Step 2. Hydrogenation to Deblock Ester The blocked peptide from step 1 above (0.62 grams or 1.2 mmol) is dissolved in 5 is of acetic acid and hydrogenated in the presence of palladium black at room temperature for 3 hours. Remove the catalyst by filtration and evaporate the filtrate to dryness in vacuo. Step 3. Recovery of Final Peptide as its Hydro- chloride Salt Solidify the residue from step 2 hereinabove by the aid of 0.21 mis (1.2 mmols) of 5.6 M HCl-dioxane and ethanol to obtain the final deblocked peptide as its hydrochloride salt. EXAMPLE II I Preparation of the Salty Complex of Peptide and Gum In a suitable dry mixer of the Banberry type, dry blend 10 parts of the new peptide of Example II with 90 parts of gum acacia tragacanth in one experiment, and mix 10 parts of peptide of Example I with 90 parts of a different type of hydrocolloidal gum, i.e., gum acacia or gum arabic as it is popularly referred to. Both ingredients are water soluble solids, and when moistened slightly with water or other aqueous fluids such as whole milk, the mixture will form a pasty complex which is itself water soluble. This complex is the salty flavor ingredient employed as a replacement for sodium chloride in the following Example, which involves the preparation of a new baking formulation useful for baking of naturally tasting yellow cakes, for example, or other baked products. These food products will contain no sodium, yet will have all the attributes of food products prepared and baked using a formula which contains sodium chloride. The cake mix formula of following Example IV will also contain no sugar but will have this ingredient replaced by the dipeptide sweetener of my pending United States Patent Application Serial Number 680,345, filed December 11, 1984, whose disclosure is incorporated by reference herein to enable those skilled in the art to make the improved food product described in the following Example ' IV. EXAMPLE IV Preparation of a Cake Formula Employing a Complex of Gum Acacia and a Dipeptide of Example I A cake mix recipe for standard yellow cake taken from page 67 of Chapter 4 of the Better Homes and Gardens Cookbook (1972) Ed. printed by Better Homes and Gardens Magazine, New York, NY, can be altered to substitute the new salt substitute of Example III for the normal sodium chloride component. The sugar ingredient is replaced by the sweetener complex of Example II of the cited copending patent application. The new improved cake formula hence is as follows: YELLOW CAKE FORMULA Ingredient Amount in Grams Corn oil margarine 141.7 Sweetener 208S (from U.S.S.N 680, ,345) 340.5 Peptide 30.4 Stabilizer gum 310.1 Eggs 23.0 Whole milk 283.0 Sodium Bicarbonate 1.1 Vanilla extract 0.28 Cake flour 679.2 Salt substitute of Example I 1.0 The above margarine is creamed, and the synthetic sweetener as a wet paste is added slowly over 10 minutes with constant stirring till light. The two eggs are then added. along with the vanilla flavor ingredient.. The mixture is then beaten at a moderate speed after each addition. Beat the entire mixture as a dough briskly for about two minutes. Place the doughy batter into a greased and lightly floured 9" x 2" round cake pan and place into an oven heated to baking temperature of 350 β F. Bake the batter for from 30 to 35 minutes at the 350 β F constant temperature to obtain a browned cake. Remove from the oven and cool for about 10 minutes before removing the cake pan. Cool to room temperature to obtain a tasty sweet yellow cake in all respects similar to one baked with sugar and salt, i.e., sodium chloride, except that neither of these were employed in its preparation. * EXAMPLE V Preparation of a Cake Formula Employing a Complex of Gum Tragacanth and a Peptide of Example I in Replacement of Normal Sodium Chloride Repeat the procedure of Example IV, except to substitute 90 parts by weight of gum tragacanth (a water soluble hydrocolloidal polysaccharide gum) for the gum acacia employed in that example. The complex will retain both its sweet and salty flavor during the bake cycle, and the cake obtained will be quite tasty and not able to be distinguished from cakes baked with addition of both sugar and sodium chloride. The edible heat stable dipeptides of the present invention are particularly useful as salting and sweetening agents for baking pies and cakes, breads and other cooked or baked goods which must be heated to temperatures of the order of 350°F in the course of their preparation. Although the foregoing Examples spell out several concrete illustrations of the practice of the present invention, they are not intended to measure the scope of this entirely new concept in seasoning and salt flavors. Only the several appended claims are capable of performing that function. As an alternative synthesis of the dipeptide of cycloalkyl ornithine and taurine as shown in Figure 1 and Example I hereinabove, one may, for example, follow the route disclosed by Tada et al., cited above on page 994 of their article entitled "Synthesis of Group III Peptides". In this method, the peptides are coupled by means of the active ester method. In this approach, one merely substitutes the cyclo¬ alkyl ornithine for the dibenzyloxycarbonyl succinimide (of compound 19) or the dibenzyloxycarbonyl ornithine (of compound 21) to be coupled with taurine amino acid. This disclosure is incorporated by reference herein to enable those skilled in the art to practice the invention. It is obvious that more than one means of coupling amino acids is available to those wishing to practice the invention and all such are contemplated with the use of a substitution of the new cycloalkyl amino acids.

Claims: I CLAIM: 1. A thermally stable table salt substitute which comprises a soluble complex of a heat stable peptide containing at least one cycloalkyl substituted amino acid residue in the peptide molecule and an edible hydrocolloidal gum. 2. A composition according to claim 1, wherein the heat stable peptide component is a dipeptide. 3. A composition according to claim 1, wherein the hydrocolloidal gum is selected from gum acacia and gum tragacanth. 4. A thermally stable dipeptide of the group consisting of Tau-vOrn, Tau-vLys, Orn-yAla, wherein Orn represents a normal ornithine amino acid residue, Tau represents a taurine amine acid residue, VOrn represents a cycloalkyl derivative of ornithine, wherein the cycloalkyl group contains C3~Cg carbon atoms, VLys represents a cycloalkyl derivative of lysine wherein the cycloalkyl group contains C3-C5 carbon atoms, and VAla represents a cycloalkyl derivative of alanine wherein the cycloalkyl group contains C3~Cg carbon atoms. 5. A method for the preparation of a salty tasting thermally stable edible dipeptide which comprises reacting under amino acid coupling conditions an ornithine reactant of the formula: with a cyclopropyl alanine of the formula: and forming the acid salt thereof and recovering the purified reaction product obtained thereby. 6. A method of preparing a thermally stable table salt substitute complex which comprises admixing and complexing the dipeptide of Claim 4 with a heat stabilizing amount of an edible hydrocolloidal gum. 7. A thermally stable table salt substitute which comprises a soluble complex of: a salty tasting amount of a dipeptide of the formula: NH I 2 (CH), and its acid chloride salts; and a heat stabilizing amount of an edible hydrocolloidal gum. 8. A food composition which comprises a comestible product containing a table salt imitating amount of the product of claim 7. 9. A method of seasoning food products which comprises adding to the food a table salt imitating amount of the product of claim 7. 10. A table salt substitute according to claim 7, wherein the peptide component is a dipeptide of ornithine and a cycloalkyl substituted alanine and the acid salts thereof.

Inventors: JOYCE PATRICK J, JOYCE, PATRICK, J.
Applicants: JOYCE PATRICK J, JOYCE, PATRICK, J.
Application year: 1986
Main classification: A23L1
IPC codes: A23L1, C07K5