EP0204826A4

EP0204826A4 - Heat stabilized peptide sweeteners.

Info

Publication number: EP0204826A4
Application number: EP19860900456
Authority: EP
Inventors: Patrick Joseph Joyce
Original assignee: Individual
Current assignee: University of Georgia Research Foundation Inc UGARF
Priority date: 1984-12-11
Filing date: 1985-12-11
Publication date: 1989-02-09
Also published as: EP0204826A1; SE8603380D0; JPS63500211A; WO1986003378A1

Abstract

A low calorie dipeptide sweetener composition comprising a complex of: (A) a minor amount of a dipetide of this structural formula (I), and, (B) a major amount of a hydrocolloidal polysaccharide gum, such as gum acacia or gum tragacanth. The sweetener is heat stable at baking conditions of 360<o> F for 30 minutes.

Description

HEAT STABILIZED PEPTIDE SWEETENERS RELATED APPLICATION

This application is a continuation in Part Application of Serial No. 680,345, filed December 11, 1984, entitled "HEAT STABILIZED PEPTIDE SWEETENERS" BACKGROUND OF THE INVENTION

1. Field of the Invention

The general Field of the present invention is peptide sweeteners for foods and beverages presently classified in Class 426 and peptide compositions in Class 260. A process for sweetening comestibles with low calorie non-toxic edible synthetic sweeteners is also described as well as a novel composition for making such process operable.

The particular peptide sweeteners illustrated 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 360°F before consumption.

2. Prior Art Found In the Invention A great deal of research effort has been directed during the past years to attain a low calorie sweetener to replace sucrose in baking and cooking. The successful candidate must meet several principal criteria to be considered satisfactory as a sweetener substitute for sugar in baking and cooking. The criteria are:

(a) the sweetener must be at least as sweet as presently known dipeptide sweeteners which are 100-300 times as sweet as an equivalent amount of sucrose. (b) the sweetener must be free of aftertaste and capable of admixture with extenders which maintain mouth feel and texture in baked goods to which it has been added.

(c) the sweetener must be heat stable in cake batters at baking temperatures of the order of 360° F. for 30-45 minutes to maintain sweetness of the baked cake. It cannot degrade or substantially decompose under thermal stress.

(d) the sweetener must be non-toxic, edible, and substantially free of side reactions to form other products under the influence of heat. Although a number of natural extracts and synthetic compositions have been proposed, tested and evaluated for use as artificial sweeteners over the past 15 years, none have successfully complied with all of the above enumerated requirements. Either the particular low calorie sugar substitute suffers the disadvantage of a bitter aftertaste or it exhibits side effects due to chemical modification in vitro, forming upon heating by-products which are no longer sweet and which yield a food product of unsatisfactory taste or texture. A chronology of prior artificial sweetener development will serve to frame the relevance of the present discovery.

In 1969, it was reported by Schlatter in JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol. 91, pages 2684-2691, that lower alkyl esters, particularly the methyl ester of L-aspartyl-L-phenylalanine, has a potent sweetening capacity being 160-200 times as sweet as an equivalent amount of sucrose. It is low calorie and does not exhibit an aftertaste. It performs well at low pH levels of pH 3.0 to 4.0 and is thus widely used in carbonated beverages. However, this sweetener has poor stability at pH 7.0 and baking temperature of 360°F, and can lose as much as 80% of its sweetening capacity by peptide clevage and heat induced formation of diketopiperazines. This causes the baked cake to require impractical amounts of the sweetener to attain sweet taste comparable to sugar.

In 1976, in an effort to overcome these difficulties, Chibata et al reported the synthesis of certain novel ester derivatives of N-aspartyl-aminoalkanols in their issued United States Patent 3,971,822. These compounds were said to be equal in sweetness to the methyl esters of L-aspartyl-L-phenylalanine earlier reported by Schlatter except that the Chibata compounds are stated to be stable under baking conditions. These products have never been commercially accepted as impractical to manufacture.

In 1979, Lipinski et al disclosed in his U.S. Patent No. 4,158,068, a non peptide sweetener which he maintains is capable of use in baking and cooking foods. The product is known popularly as "ACETO SULFAME-K". The compound has been approved in the United Kingdom, but has not yet been approved for use in the United States. It has been reported to have aftertaste problems in some applications, and, hence may be delayed in commercial applications. In 1983, still another synthetic sweetener was described by Dubois in United States Patent 4,381,402. This product was a glycoside of the stevioside variety which is essentially an extract from the steviocide plant and its leaf, which grows in abundance in Paraguay, and is quite intensely sweet. These products are also reported to have aftertaste problems and production cost difficulties.

In August, 1983, a United States Patent No. 4,399,163, was issued to Brennan et al, and discloses thousands of peptide compounds which are said to be sufficiently stable to be able to be used for baking and cooking at elevated temperatures of the order of 360°F. Almost all of these compounds are new and unique organic structures and, as yet, not tested as to their chemical and physical properties. Finally, in March, 1984, a United States Patent was granted to Tsau et al, bearing Patent No. 4,439,460. This patent describes peptide sweeteners which have been transformed into the sulfate and suifonate salts of the dipeptides disclosed therein. These salts are claimed to be heat stable dipeptides of Aspartic Acid coupled to phenylalanine. The materials are specifically alleged to be heat stable up to temperatures of 170°C.

In 1982, a doctoral thesis was published in the name of Stephen King and Lodged at the University of Georgia library, which thesis describes the preparation of a dipeptide sweetener which is said to be stable against enzyme hydrolysis. This dipeptide is a coupling of L-aspartic acid and cyclopropyl phenylalanine methyl ester. In December, 1984, a paper appeared in Journal of Medicinal Chemistry Vol. 27, pages 1663-1668, by M. Goodman et al, which describes the preparation of the methyl esters of a dipeptide of L-aspartic acid and cyclopropyl alanine as new sweeteners.

However, upon review of the prior art on artificial sweeteners, applicant is convinced that prior to the time of the present invention, none of the artificial sweeteners proposed has fulfilled the total requirements of a satisfactory low calorie sweetener replacement for sugar. III. GENERAL DESCRIPTION OF THE INVENTION The principal object of the present invention is to provide a heat stable low caloric dipeptide sweetening product which can be employed to sweeten cakes and pies and cooked foods and retain its sweetness. The composition of the product comprises in admixture a soluble complex of: A. A minor amount of weight of a dipeptide of the structure :

wherein n is a positive integer from 1 to 3 , and R is a lower alkyl group of C₃ to C₇ carbon atoms, or a cycloalkyl group of C₃-C₅ carbons or a lower alkyl substituted cycloalkyl group or a lower alkyl substituted aryl group. A preferred R group is one such as n-propyl, isopropyl, n-butyl or isobutyl for high level sweetness, alone or in combination with,

B. A major amount of a hydrocolloidal polysaccharide gum. The hydrocolloidal gum is preferably a polysaccharide type hydrocolloidal gum, such as gum tragacanth, gum acacia, pectin, gum karaya, psyIlium seed gum, gum gatti, guar gum, larch gum, and locust bean gum.

The two components of the complex can be admixed in a number of ratios as long as the gum components exceeds the peptides component. When one is admixing the peptide with the two preferred gums, which are gum tragacanth and gum acacia, it has been established that an excess of the gum over the peptide is required to preserve thermal stability of the sweetener complex. The preferred range of proportions of the mixture is a ratio of 1 part by weight of peptide to 5 to 10 parts of hydrocolloidal gum.

When the ratio of 1 part gum to 1 part peptide is tested, the rate of decomposition and peptide degradation accelerates under 360°F. thermal stress to an unacceptable degree. On the other hand, when a ratio of 100 parts gum to 1 part peptide is attempted, the texture of the baked product suffers.

The peptide component itself is also novel, and even without the gum complex, has an unexpected resistance to thermal degradation. At a baking temperature of 360°F. for 40 minutes, only 18% of the dipeptide degrades into D.K.P. (diketopiperazine) and amino acid fragments, while a commercial dipeptide sweetener degrades a a level of about 80% loss of sweetness property after baking under the same conditions.

IV. BRIEF DESCRIPTION OF THE DRAWING

Figure 1 of the attached drawing is a schematic representation of synthesis of the new and novel dipeptide as detailed in Example 1. The starting amino acids A and B are known compounds. The coupling is also well known and is not claimed as part of this invention.

V. DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTOR

While one can conceive of several product aspects and several process aspects of the present invention, a single preferred embodiment exists to illustrate the best mode of practice of the discovery.

A. In a most preferred product aspector embodiment, the preferred complex comprises: a. 10 parts, by weight, of a hydrocolloidal polysaccharide gum selected from gum tragacanth and gum acacia: and b. 1 part, by weight, of a dipeptide of the structural formula:

wherein R represents a normal propyl group or a normal butyl or isobutyl functional group. B. Another preferred product embodiment, or aspect as it is sometimes referred is a novel dipeptide itself of the general formula: wherein R represents a normal propyl group C₃ H₇ or normal butyl or isobutyl groups which makes the ester group larger than a methyl ester earlier shown in the art.

C. A third preferred embodiment is a cake mix formula which substitutes the above peptide-gum complex for sucrose in a food composition.

D. A fourth preferred embodiment of the invention is a cake mix formula which substitutes the peptide B in concert with other hydrocolloidal polysaccharide gums, such as pectin, for sucrose in the food composition.

The cycloalkyl alanine component of the new peptide composition, specifically the compound cyclopropylalanine, is a naturally occurring amino acid which is found in small amounts in fruits such as apples. It is also commercially available from Cal-Biochem Corporation, Los Angeles,

California, and Sigma Chemical Co., St. Louis, Missouri. The aspartic acid component is also commercially available from the same sources.

The hydrocolloidal polysaccharide gums are also 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 to 67, published by John Wiley and Sons, New York.

For instance, the preferred gum component, gum tragacanth, is known to be mixture of acidic polysaccharides containing galacturonic acid, galactose, fucose and xylose and arabinose. It is an exudiate from the Astralagus tree found in Iran, Syria, 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 pH to neutral range.

These gums, along with all of the other hydrocolloidal 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 complexing characteristics.

They exhibit a high propensity for their free hydroxyl groups to complex with a cycloalkyl bridged dipeptide of the aspartic acid cyclopropyl alanine propyl ester type described herein. What is unexpected about this is that the combination does not impede or interfere with the sweetness attribute of the peptide and does maintain cake texture and stability of the same against heat degradation.

One unexpected aspect of the present discovery is that the propyl, normal butyl or isobutyl ester of the dipeptide aspartic acid cyclopropylalanine is both sweeter than the commercial peptide sweetner aspartic acid phenylalanine, methyl ester, and greatly sweeter than the methyl ester of aspartic acid cyclopropylalanine dipeptide. In addition to this, the cyclopropylalanine, n-butyl, isobutyl and propyl esters are stable to heat degradation and other hydrolysis, while the cyclopropyl phenylalanine methyl and cyclopropyl phenylalanine propyl esters are not sweet or stable. To further specifically illustrate the preferred embodiments of the invention as enumerated above the following examples are set further herein below.

VI. EXAMPLES OF THE INVENTION WHICH ILLUSTRATE THE

PREFERRED EMBODIMENTS EXAMPLES 1-A

PREPARATION OF THE DIPEPTIDE COMPONENT OF THE COMPLEX

A-INTERMEDIATE 1

N-Propyl α - Amino cyclopropane Carboxylate Hydrochloride

(2)

Dissolve 22 mmol of commercially available cyclopropyl alanine amino acid (1) in 35 mis of a solution of n-propanol chilled to -10°. Under anhydrous conditions dry

HCl gas is bubbled into the solution a 0°C and the solution refluxed for 4 hours.

Evaporate off the solvent under reduced pressure and dry the residue thouroughly under high vacuum. Dissolve the residue in ice cold saturated Na₂ CO₃ solution and extract successively four times with ether.

Combine the ether extracts and wash with dilute Na₂ CO, solution, then with brine and dry over anhydrous magnesium sulfate.

The amino acid propyl ester is precipitated from the ether solution as the hydrochloride salt (2) upon the addition of anhydrous HCl at 0°C. The crystals are collected and recrystallized from ether to provide the product propylα- aminocyclopropane carboxylate hydrochloride (2) in purified form.

B. INTERMEDIATE 2

N-t-Butyloxycarbonyl -B-t-Butyl-L-Aspartyl-l-aminocyclopropane carboxylic acid propyl ester (3)

Add to 2.7 mmol of the product of Part A a solution of t-butyloxy carbonyl-L-aspartic acid-B-t-butyl ester in 80CC. Anhydrous tetrahydrofuran, and 0.3 mis N-methγl morpholine. Cool this solution to -15°C. and add 7.7 mmol of isobutyl chloroformate. After 5 minutes add a solution of 2.76 mmol. benzyl-aminoisobutyrate HCl and N-methylmorpholine (2.76) mmol. in 20cc. tetrahydrofuran. Allow the reaction to proceed at-15°C for 1 hour, increase the temperature to 5°C and continue the reaction for 24 hours. Recover the residue by removal of the solvent under pressure.

Dissolve the residue in ether and water. Wash once, then with 5% sodium bicarbonate solution three times; again with water, then 6 times with a 1% sodium bicarbonate solution and finally with water. Then dry over magnesium sulfate. Remove the ether under reduced pressure to obtain the crude second intermediate (3) as white flakes after recrystallization m.p. 71-72°C.

C. FINAL PRODUCT

L-Aspartyl-l-Aminocyclopropane Carboxylic Acid N- Propyl Ester (4)

Add 50 mls. of ice cold trifluoro acetic acid to the intermediate from step B above in the amount of 11.1 mmol. Allow the reaction to proceed for 90 minutes at room temperature. Evaporate the solvent under reduced pressure. The residue is triturated with isopropyl ether to give, a white solid after filtration. Dissolve this trifluoroacetate salt in a mixture of 30 mls. of water and 10 mls. ether. Adjust the pH of the solution to 5.0 with 5% sodium bicarbonate solution in ice water and precipitate the final product from solution.

Collect the final peptide in several crops from the mother liquor and recrystallize the same from water to obtain purified final product (4) (72% yield) as white needles m.p. 168-170°C (dec.)

EXAMPLE 1-B

PREPARATION OF THE N-BUTYL ESTER OF THE DIPEPTIDE

COUPLING OF L-ASPARTIC ACID AND 1-AMINO 1-CYCLOPROPANE

CARBOXYLIC ACID

Step 1

Preparation of N-Butylα-Amino cyclopropane Carboxylate

Ester

To a solution of n-propanol (690 mis.) and SOCL2

(34.5ml.), chilled to -10°C, add (36 grams 0.11 mol.) of 1-aminocyclopropane-l-carboxylic acid and reflux the solution for 7 hours. Evaporate the solvent to obtain the ester as an oil, comprising a yield of 90.1% product, rf

(IV) 0.60, H-NMR (CD₃OD) : 0.97 (t,J=6Hz,3H, methyl,

1.35-1.74 (m,6H, Cyclopropyl H, CH₂ CH₂ CH₂ CH₃) 4.15 (t J=8Hz, 2H, OCH₂) .

Step 2

Coupling of the Ester from the preceding step with blocked

Aspartic Acid to form the Dipeptide

A. To a solution of N-Boc-Aspartic Acid-B-t-butyl ester (0.11mol.) in 250CC of Tetrahydrofuran, add 12 mis. (0.11 mol.). of N-methyl morpholine and cool the solution to -15°C. Add 0.11 mol. of isobutyl formate and stir the reaction mixture for 10 minutes.

B. A solution of the n-butyl α aminocyclopropane carboxylate ester (18 grams 0.1 mol.) and 0.10 mol. of N-methyl morpholine in another 250cc of tetrahydrofuran was then added slowly to the ester product from step 1. The reaction mixture was allowed to slowly warm up to room temperature and constantly stirred for 3 hours. Evaporate off the solvent in vacuo and dissolve the solid residue in ethyl acetate. Wash this solution three or four times with 0.5m citric acid, each time using about 50cc of acid. Then twice with brine, and three timed with 5% sodium bicarbonate solution and finally, two or three times again with brine.

Dry the above liquid over anhydrous magnesium sulfate and remove the solvent in vacuo. The crude peptide product is purified by further recrystallization from hexane to obtain the pure blocked dipeptide ester in 81.5% yield as white flakes, 71-72°C.

Step 3

Deblocking of Dipeptide Ester to obtain sweet tasting final product

A solution of 24 grams of N-BOC-t-Butyl-L-Aspartyl-α -aminocyclopropane carboxylic acid n-Butyl ester from preceding step 2 in 180cc of methylene chloride was cooled to 0%°C and 245cc trifluoro acetic acid added. This solution was then stirred for an hour and 30 minutes at room temperature.

After this time the product solution was evaporated to dryness to obtain an oily residue. This oil was triturated with ether to give a solid. This solid was accumulated by filtering and washed three times with ether.

The solid salt obtained is dissolved in a 5% sodium bicarbonate solution and the pH adjusted to 5.0. The now product as a zwitterion precipitate is collected by filtration and washed in ice cold water. The product is extremely soluble and some water should be evaporated before further filtration. Upon drying the final dipeptide is obtained in 84% yield as a white crystalline solid M P 174-175°C.

EXAMPLE 1-C

PREPARATION OF ISOPROPYL AND ISOBUTYL ESTER ANALOGS OF

THE DIPEPTIDES OF PRECEDING EXAMPLE 1-B

In a similar procedure as that detailed above for the preparation of the esters described, the substitution of the stoichiometric quantities of the corresponding isopropyl and isobutyl alcohols will yield and corresponding esters of the dipeptide. These products will have enhanced sweet taste as compared to the lower alkyl esters.

As a general rule , the thermal stability of the molecule is believed to be obtained from the presence of the cyclopropyl "bridge" found in the 1-aminocyclopropane carboxylic acid component of the dipeptide while its relative intensity of sweetness derives from the weight of the alkyl ester group attached to molecule. Hence, lower alkyl esters of at least 3 carbons in chain length either normal or branched, and either cyclic or acyclic or combinations of the two are believed to result in dipeptides which are both sweet the thermally stable at the temperatures required for cooking and baking of cakes, pies and other foods. EXAMPLE 2-A

PREPARATION OF THE SWEETENER COMPLEX

In a suitable mixer of the Banberry type, dry blend 10 parts of the peptide product of Example 1 with 1 with 90 parts of a pulverulent dried exudate of the Acacia tree found in Syria, commonly referred to as gum acacia or gum arabic. Both ingredients are water soluble white crystalline 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 sweetener ingredient employed as a replacement for sugar in the following Example 3, which involves the formation of a baking formulation intended for the preparation of a naturally tasting yellow cake. This cake differs from prior cakes in a notable respect-it contains no sucrose.

Prior to the inclusion of the new sweetening composition in the cake formulation, it was heat tested by heating a putty-like paste of the composition to 170°C for 30 minutes to determine if it was degraded and lost any of its sweetness, and to determine if the peptide profiles of the NMR recording underwent any substantial alteration. These tests are even more stringent than the baking conditions of the cake. Test results indicate that no discoloration or bitter taste of decomposition products occurred to alter the sweet character of the peptide. EXAMPLE 2-B

In a manner similar to Example 2-A, blend and complex the dipeptide product of Example 1-B with an equivalent amount of gum acacia to obtain a sweetener comples containing a butyl ester rather than a propyl ester of the dipeptide component.

EXAMPLE 3-A

PREPARATION OF A CAKE FORMULA EMPLOYING A COMPLEX OF GUM ACACIA AND DIPEPTIDE OF EXAMPLE 1

A cake mix recipe for standard yellow cake taken from page 67 of Chapter 4 of the Better Homes and Gardens Cookbook, 1972, printed by Better Homes and gardens Magazine, New York, New York can be altered to substitute the new sweetener complex of Example 2-A for the sugar ingredient of the recipe. The new cake formula hence is as follows:

YELLOW CAKE FORMULA Ingredient Amount in Grams

Corn Oil Margarine 141.7

Sweetener 208 S 340.5

Peptide (30.4)

Stabilizer (310.1)

Eggs (2) 23.0

Whole Milk 283.0

Sodium Bicarbonate 1.1

Vanilla Extract 0.28

Cake Flour 679.2

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 moderate speed until it is fluffy.

The dry ingredients, cake flour, sodium bicarbonate, and salt are also mixed and sifted. They are then added slowly to the creamed mixture in several equal amounts with intermittent addition of whole milk and beating for 3 minutes after each addition. Beat the entire mixture as a dough briskly for about 1-2 minutes. Place the doughy batter into a pie greased and lightly floured 9 x 1½ inch round cake pan and place into an oven pie heated to a bake temperature of 350°F.

Bake the batter for from 30-35 minutes at the 350°F constant temperature to obtain a browned cake. Take out of the oven and cool for about 10 minutes before removing the cake from the pan.

Cool to room temperature and to obtain a tasty sweet cake with no sucrose or calories derived therefrom.

EXAMPLE 3-B In a manner similar to Example 3-A, one can prepare a cake formula by substituting the sweetener component of either Example 1-A, 1-B or 2-B in equal amount for the sweetener from Example 2-A referred to therein to obtain a satisfactory sweet tasting cake food product. In a manner similar to the above cake mixtures, confectionary products, such as hard candies have been prepared as examples of other sweet tasting food products.

EXAMPLE 4 PREPARATION OF A CAKE FORMULA EMPLOYING A COMPLEX OF GUM TRAGACANTH AND DIPEPTIDE OF EXAMPLE 1

Repeat the procedure for Examples 2 and 3, except to substitute in Example 2, 90 parts by weight of gum tragacanth (a water soluble hydrocolloidal polysaccharide gum) for gum acacia of Example 2. The complex will maintain its sweet character during the baking of the cake as in Example 3. The manner and the desirable result will be the same. The edible sweeteners of the present invention are particularly useful as heat stable sweeteners for baking pies and cakes, breads, and other foods which must be heated to temperatures of the order of 350-360°F

The edible sweeteners of the present invention are particularly useful as stabilized sweeteners for fruit juices, fruit preparations, canned vegetables and fruits, dairy products such as egg products, milk drinks, ice cream, syrups, chocolate syrups and bars, candy, icing and dessert toppings, meat products and especially carbonated and non-carbonated beverages. Although the value for R in the general structure indicates that lower alkyl groups, such as, normal or isopropyl or butyl or isobutyl groups are preferred, other alkyl functions as well as amine functions, sulfate and sulfonate salts and alkaryl groups, such as benzyl, may be considered as modifications which may still further stabilize the peptide or increase its sweetening effects. These can be considered functional equivalents

The following claims define the Invention sought to be patented.

Claims

AMENDED CLAIMS

[received by the International Bureau on 05 May 1986 (05.05.86); original claim 7 amended; other claims unchanged (1 page)]

7. (Amended) A heat stabilized dipeptide of the structure:

wherein R is [a butyl or isobutyl or] a propyl group.