CA1261855A - Derivatives of d-amino acids having anti-microbial properties - Google Patents

Derivatives of d-amino acids having anti-microbial properties

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Publication number
CA1261855A
CA1261855A CA000506396A CA506396A CA1261855A CA 1261855 A CA1261855 A CA 1261855A CA 000506396 A CA000506396 A CA 000506396A CA 506396 A CA506396 A CA 506396A CA 1261855 A CA1261855 A CA 1261855A
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Prior art keywords
group
sorbyl
acid
tryptophan
alanine
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CA000506396A
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French (fr)
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Alenka Paquet
Khalil Rayman
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Canada Minister of National Health and Welfare
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Canada Minister of National Health and Welfare
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/49—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a carbon atom of an acyclic unsaturated carbon skeleton
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
    • A01N43/38—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B4/00—General methods for preserving meat, sausages, fish or fish products
    • A23B4/02—Preserving by means of inorganic salts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B4/00—General methods for preserving meat, sausages, fish or fish products
    • A23B4/02—Preserving by means of inorganic salts
    • A23B4/027—Preserving by means of inorganic salts by inorganic salts other than kitchen salt, or mixtures thereof with organic compounds, e.g. biochemical compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B4/00—General methods for preserving meat, sausages, fish or fish products
    • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
    • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
    • A23B4/20—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3481—Organic compounds containing oxygen
    • A23L3/3499—Organic compounds containing oxygen with doubly-bound oxygen
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3526—Organic compounds containing nitrogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C07C233/46—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/47—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
    • C07C323/59—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton with acylated amino groups bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D209/20—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals substituted additionally by nitrogen atoms, e.g. tryptophane

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Abstract

TITLE

DERIVATIVES OF D-AMINO ACIDS HAVING ANTI-MICROBIAL PROPERTIES

INVENTORS

Alenka Paquet Khalil Rayman ABSTRACT OF DISCLOSURE

N-acyl-D-amino acid derivatives having anti-microbial activity, particularly against organisms such as Clostridium botulinum have been developed. These compounds may be used in conjunction and in admixture with other food additives.

Description

8~ . - 2 - 32~7-179 FIELD ON THE INVENTION

This invention relates to means of controlling growth of Clostridiu~
botulinum in manufactured or processed foods using N-acyl-D-amino acid derivatiyes, preferentially in combination with the minimum amounts of sodium nitrite necessary to produce satisfying color and taste.

BACKGROUND TO THE INVENTION

This inven~ion relates to means to control growth of Clostridium botulinum in certain food products. There have been numerous methods'designed to control botulinum bacteria r_ Mechanism of antim;crobial effect of various food preservatives (Ed. N. Molin), Almquist and Wiksel, Stockholm 1964. p.
1]. Sodium nitrite has been widely used for this purpose. ~Gray, J.I., and C.J. Randall. J. Food Protection 42, 168 (1979);. In the Health Effects of Nitrate, Nitr;te and N-Nitroso Compounds, Study by the Comnittee on Nitrite and Alternative Curing Agents in Foods Part 1. ~lationai Academy Press.
Washington, D.C. 1981].
The possibil~ity of formation of carcinogenic~nitrosamines during cooking of nitrite cured meats led to a search for alternatives to nitrite or an adjunct which would reduce the nitrite content in meats. Some of the~
promising alternatives incl~ude irradiation [Rowley, n~B~ et al.~ J. Food 5ci.
48, 1016, (1982~ use~of Lactobacil~lus;cultures to reduce product pil rTanaka, N. et al. J.~Food Protect;on~43,~450,~(1980)] use of sulfur dioxide [Tompkin, R.B. et al.~ App1.~Environ.~Mi~crobiol, 39, 1096, ;. ~

:

~6~

(1980)] use of sodium hypophosfite ~Banner, R.J. Food ~nglneering 53 130, (1980)] and fumarate esters ~huntanen, C.N. 41st Annual Meeting of Institute of Food Technologlsts. June 1981. Atlanta Ga.) One of the most efficient agents proposed as a partial sodium nitrite replacement is potassium sorbate or sorbic acid [Sofos, J.N. and Busta, F.F. Food Technol. 24, 244 (1980), Busta, F.F. Food Technol. of Australia 34, 529 (1982)], Blocher, J.C. et al, J. Food Sci. 47, 405 (1981)]. Ueno, R.N. et al. U.S. Pat.
4,299,852 (1981), Ueno, R.N. et al, U.S. Pat. 4,342,789 (1982).
However, recent reports show that mutagenicity was detected in some food preparations aured with the sorbic acid-sodium nitrite mixtures [Khoudokormoff, B. International Symposium on the Chemical Toxicology of Food. June 1978. Milan, Italy.
Khoudokormoff, B. Fed. Cosmet. Toxicol. 19, 405-407 (1981)].
Reports on mutagenicity of sorbic acid itself in wine and curing brines have also emerged [Lafout, M.G. and Lafout, S.P. Med. Nutr.
15, 195 (1979)]. There is, ther~efore a need for the preservatives which do not have these side effects.
This invention provides N-acylamino acids o~ the formula, X - CO - NH - Y
wherein X, when taken in conjunctlon with the CO group, is an acyl moiety and Y, when taken in conjunction with the NH group, is a D-amino acld or glycine moiety,~or a food~tuff acceptable salt thereof, other than glycyl D-alanine, aaetyl D-tryptophan, acetyl D-methionine, acetyl D-valine~and acetyl D-alanine.
Preferably X does not contain any NH2 ~roups and ~B- :

. . ; .

~6~
632~7-17g preferably the carbon atom of the carbonyl group is attached to another carbon atom. X may be a saturated moiety, so that, for example, ~CO may be lauroyl, myristoyl or palmitoyl, or X may be unsaturated so that XCO ls, for example, sorbyl.
X is preferably, selected ~rom the group consisting of sorbyl or fatty acyl (C8-C24). Y is preferably an a-D-amino acid and suitable examples can be selected form the group consisting of D-alanine, D-tryptophan, D-methionine, D-valine and D-aspar~ic acid.
This invention also provides salts of such compounds suitable for use in foodstuf~s. Mention is made of alkali metal and alkaline earth metal salts, of which sodium and potassium salts are generally preferred.
Compounds of the above referenced type may be used in combination with a ~ood product and when so employed preferably present at a concantration of at least 2,600 ppm by weight. Such compounds may be used in combination with food grade nitrltes.
The invention therefore also provides a;~composition according to claim 26 wherein the active ingredient comprises N-sorbyl-D-tryptophan, N-so~rbyl-D-alanine, N-sorbyl-D-valine, N-sorbyl-D-methionlne, N-sorbyl-D-aspartic acld, Palmltoyl-D-~ryptophan, Myrlstoyl-D-aspartic~acid or Hexanoyl-D-alanine.
These compounds may;further be used~ in~Dethods for control of microorganisms in ~ood products, particularly mea~-containing products ~and especially red meat-containing products.
Such a method compris~s incorporating at least about~2600 ppm of such a compound to suah a food produc~. In such a compound the ~ ; 4 ~6~
63247-17g X - C0 group is preferably sorbyl and the NH - Y group is preferably D-tryptophan. The compound is preferably, combined with addition of 60 ppm o~ sodium nitrlte ~o enhance ac~ivity and to maintain desirable colour and taste for consumers satis~action.
The above compound has additional advantage of being stable towards intestinal peptidases which suggests that such food additive will not be metabollzed.
Preferred means for practising the invention comprise:
(a) acylation of D-amino acids by sorbic acid, succinimidyl esters or any other active ester such pentachlorophenyl, penta~luorophenyl, or by sorbyl chloride or sorbic anhydride.
(b) recovering the N-sorbylamino acid as product.
(c) incorporatlng the~derivative or the salt (sodium or potassium thereof) optionally in combination with sodium nitrite, in to a desired~processed or manufactured food product such food product belng for ex~ample sausages,~canDed m~lnced~meat~products, corned beef, luncheon meats, meat products commlnuted and stuffed into casings. ;
It can be~se;en~that the~ present ~lnv~entlon descrlbes~the advantageous use of non-mutagenic amino acid derivatives of sorbic acid as potent food~preservatlves.~Such use represents replacement of the applicatlon of high~levels of sodlum ni~trite~
(150 ppm) or the pote~ntlally~mutagenic~mlxture~of ~ 4a~

- : :

~: , : ~ : -potassium sorbate and sodium nitrite. The named compounds are easily and inexpensive~y obtainable, stable at any pH or temperature in most food applicat;ons where preservation against Clostridium botulinum outgrowth is required.

DETAILED DESCRIPTION OF THE INVENTION
CHEMICAL SYNTHESES

Material and methods Melting points (uncorrected) were taken by the capillary method. The NMR
spectra were recorded on a Yarian T-60 spectrometer. Optical rotations were determined on Perkin-Elmer 141 polarimeter. Amino acids were purchased from Sigma Chemical Company (Sainl; Louis, Missouri, U.S.A.). Characterizing data for some compounds of the invention are given in Table 1.
Succinimidyl sorbate To a suspension of N-hydroxysuccinimide (36.87g, 0.32~mole) and sorbic acid (35.92g, 0.32 mole) in methylene chioride (200mL) was added l-ethyl-3-(3-dimethylaminopropyl) carbodi1mide (61.42g, 0.32 mole) dissolved in methylene chloride (140mL). The mixt~re was stirred at room temperature for 3 hours and then kept overnight at 0C. The mixture was extracted 3 times with 10% citric~ acid, 3 times with saturated~sodium bicarbonate~so1ution and with water till neutrality. The semicrystalline~residue (66.0g, 9~.6%) was recrystallized~from methylene chloride-diethyl ether giving 55.0 and 5.29 (91.4%) of the crystal~line title compound. M.p. 104C. Analysis calculated for CloHl1 ~04(2n9.18): C~, 57.4,~H,~5.3; N. 6.7. Found: C, 57.0; H, ::

:
::: ~: :
.
:
:: :~
:
. . ~

5.5, N. 6.5%.
NMR, : 7.46 (lH, m, C3), 6.33 (2H, m, C4 and C5), 5.93 (lH, d, C2), 2.88 (4H, -s, 2CH2 succinimidyl moiety), 1.9 (3H, d, CH3) (CDC13).

Sorbyl-D-tryptophan To a suspension of D-tryptophan (20.439, 0.1 mole) and sodium bicarbonate (12.69, 0.15 mole) in water (200mL) and acetone (lOOmL) was added succinimidyl sorbate (20.92g, 0.1 mole) in acetone (lOOmL) in 3 portions. The reaction mixture was stirred at room temperature overnight, acidified to pH 4.5 and acetone removed by distillation. The pH was adjusted to 2, the crystalline product was separated by filtration, washed with distilled water (till neutrality of the filtrate) and recrystallized from the mixture of ethanol with water giving 25.929 (87%) of the title compound.

DEMONST MTIO~ OF UTILITIY OF THE COMPOUNDS

Materials and Methods Organism Five type A strains of Clostrldium botulinum, 6A, 17A, 62A, CK2A, and 317121A, and five type B strains, lBl, 368B,~462B, 13982B and MRB were used throughout these studles. Spores were prepared by the method of Schmldt et al. ~J. Food Sci. 27, 77 (1962)3 and enumerated on Wynne agar supplemented with egg yolk ~Hauschild, A.H.W.~et al. J. Food Prot. 45, 500 ~1982)3.
Meat Slurries ~ ~ ;
To fresh minced pork meat ~two parts of 3% NaCl solution were added to ~: : : : .` ~ : ::

::

:

, `` ~ _ 7 _ obtain a final concentration of 2~ brine. Compounds to be tested were added in powdered form as potassium salts to the slurries in final amounts of .
0.26%. A mixture of approximately 200 botu1inum spores were added per lmL of slurry unless indicated otherwise. The spore mixture compr;sed an approximately equal number of each of the five type A and five type B strains of C. botulinum. The meat slurry was blended for one minute, the pH adjusted (values indi~cated at the tables), and blended for an additional minute. Then the slurry was dispensed in 10 ml volumes to sterile 16 X 150mm test tubes, processed to 70C, cooled and sealed as described by Rayman et al ~App.
Environ. Microbiol. 41, 375 (1981?]. Five tubes of slurries were abused by incubating at 25C for 56 days or until growth was observed as evidenced by gas production.
Antibotulinal effect of N-acyl-D-amino acids A series of N-acyl-D-amino acids were tested using the above described technique in the presence or absence of 60 ppm of sodium nitrite: it can be seen that derivatives of D-tryptophan in conjunction with 60 ppm of sodium nitrite~ as well as myristoyl-D-aspartc acld and glycyl-D-alanine exhibited the highest inhibition. The results are presented in Table 2.
:
Comparison of the activity of sorbyl-D-tryptophan with its L-isomer ~ Meat slurries were treated with 0.26~ of either sorbyl-D-tryptophan or sorbyl-L-tryptophan converted into potassium salts. The results in Table 3 indicate superior inhibltion of spore outgrowth;by the N-acyl-D-amino acid derivative; sorbyl-D-tryptophan inhibited outgrowth over the entire 56 days of abuse, whereas, its L-isomer inhibited outgrowth for an average of 11 days which was only sllghtly l~onger than~60 ppm nitrite a1one or in combination with 0.26X D-tryptophdn~

.

L8~

Comparison of the activity of sorbyl-D-tryptophan with control experiments.
Results of the control experiments carried out with 150 ppm of sodium `
nitrite and 0.26~ of potassium sorbate are shown in the Table 4.
Effect of pH on inhibition of spore outgrowth Sorbyl-D-tryptophan which showed the greatest inhibition of spore outgrowth, was selected for further testing. This compound at a final concentration of 0.26% was added individually to inoculated pork slurrieS
containing 60 ppm nitrite and adjusted to pH 5.8 or 6Ø The results in Table S indicate a decrease in effectiveness of the compound to inhibit spore outgrowth as the pH increased.
Contribution of D-tryptophan to inhibitory activity The sorbic acid and D-tryptophan moieties contribute 32.46% and 59.12% of the weight respectively to sorbyl-D-tryptophan. The experiments shown in Table 6 were performed to determine whether the moieties separately and in proportions equivalen~ to those present in the N-acylamino acid compound ~ould effect the same degree of spore outgrowth inhibition as the derivatized amino acids. The results indicate a reduction in the inhibitory effects of the individual moieties applied as~potassium salts in such final concentration in the slurries which corresponded to their molar proportions in the N-acylamino acid molecule and urplylng altogether 0.26% of the growth 1nhibttor.

.

85~

g EXAMINATO~ OF MUTAGENIC
POTENTIAL OF THE COMPOU~IDS
UNDER STUDY

Compounds of this type with this utility should be examined for mutagenic potential as part of the process of selection of a preferred co~pound or set of compounds.
Materials and methods .
The salmonella/microsome assay was used for this study according to Mason, D.M, and Ames, B.N. ~ut. Research 113, 173, 1983. A modification of the standard pla`te incorporation assay3 the so-called "preincubation" assay was also employed. In this method the bacteria and test chemicals were preincubated for 20 minutes at 37C before plating. Salmomella typhimurium strains TA 97, TA 98 and TA lOO were used.~ For metabollc activdtion, liver homogenates prepared from rats induced with~aroclor 1~254 were~employed.
Results are expressed dS average number of revertant colonies~per plate~
calculated from duplicate assay plates.
The results of the mutagenicity as~says on the test compounds are preseoted in Tables 7a,~7b. AIl~ test substances~gave negatlve~responses,~l.é., no increases in the number of revertants~;above the~negative controi jsolvent~
wère observed. ;Table 7c shows the positive mutd~gens empl~oyed~with each~as~say~
and their mutagenic activlties.

: ~ ~

a~ ~ 01 ~
a~ D C
,ca ~ , ~_ ~ cn O O
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_able 3 Ant~botul~nal effect of N-acyl-D-am~no ac1d and N-acyl-L-amino ac~d der~vatives*
Am~no acld N1tr~te Average no. of days der~vative *** (60ppm~ for ~rowth **
None - 5 None + 7 Sorbyl-L-tryptophan + ll Sorbyl-D-tryptophan + >56 D-tryptophan + 8 * Pork slurrles ~noculated with approx1nlately lO0 mixed botul~num spores per ml conta~ned 2% NaCl and~processed to 70C pH 5.8.
** F~ve tubes of slurry were used for each treatment.
*** In the form of potass~um salt and ln the f~nal concentrat~on of 0.26%.

.
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~'~G1855 Table 4 Comparison of the activity of sorbyl-D-tryptophan, potassium sorbate and 150 ppm of sodium nitrite on inhibition of spore outgrowth.

Compound tested* Range of days Av. no. of days _ _for growth __ for_qrowth **
Sorbyl-D-tryptophan 11- >56 27 (6) ***
~otassium sorbate 9- >56 18 (10) 150 ppm of sodium nitrite - 22- >56 39 (1) * Pork slurries inoculated with app~oximately 200 mixed botulinum spores per 1 ml. contain 2~ NaCl and, except - for the 150 ppm nitrite treatment, 60 ppm of sodium nitrite and 0.26~ of the compound under test.

** To calculate number of days, >56 was taken as 56 days for growth to occur.
*** Numbers in parentheses represent the number of e~periments from which the average number of days was calculated.

:

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- 15 ~ 8~ -Table 5 Compar~son of the activ~ty of sorbyl-D-tryptophan, potass~um sorbate and 150 ppm of sod~um n1tr~te at d~fferent pH values on ~nhlb~t~on of spore outgrowth.

Compound tested *pH of pork Range of days Av no. of days slurry for growth for qrowth **
Sorbyl-D-tryptophan 5.8 11-~56 27 ~6)~**
6.0 6-41 18 ~4) Potass~um sorbate 5.8 9->56 18 (10) 6.0 7-12 10 (2) 150 ppm nltrlte 5.8 22-~56 39 (1) 6.0 20->56 30 (1) * Pork slurr~es ~noculted w1th approx~mately 200 m~xed botullnum spores per 1 mL contalned 2% NaCl and, except for the 150 ppm n1tr~te treatement,:60 ppm nitr~te and 0.26~ of a compound.
*~ To calculate average number of days, >56 was taken as 56 days for growth to occur.
~* Numbers ~n parentheses represent the number of exper1ments from whlch the average number of days was calculated.

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Table 6 Effect of N-acylamlno ac~d der~vat~ves and ~ndlv~dual constltuents thereof on ~nh~b1t~on of spore outgrowth *

Compound tested **~ Range of days for . Av. no. of days _ __ for growth for growth **
Sorbyl-D-tryptophan 11-~56 24 Potass~um sorbate 8-13 '0 Potasslum sorbate (o.lo%) plus D-tryptophan (0.16%) 8-11 10 * Pork slurr~es ~noculated w1th approx~mately 200 m~xed botul~num spores per ml conta~n 2% NaCl and 60 ppm n1tr~te pH 5.8.
** See footnote to Table 3.
*** The ftrst two compounds were used at a f1nal concentrat1On of 0.26%.

:

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~i3L855 Table 7a Mut~qenic Assa~/ of SorbY~ trYptophan . _ . . ~
~oncenlration l~e~/ertants/p~late ~plate lA 97 S --------- ~~S9--~---~~-~ S-9~

. _ ~ample 1 standard assay 0 water 138 242 23 27 118 127 500 122 252 24 30 13~ 115 lO00 122 269 32 25 117 112 2000 119 2~6 23 27 111 109 Sample 2 standard assay 0 water 137 154 24 41 lh7 136 S00 131 1~2 33 3~ 1~6~ 132 lO00 119 131 23 39 1~0 145 ~000 13~ 15~ 24 : 3~ 157 133 2500 110 139 35 3~ 157 126 : pr~e~nruha~t~on:assay 0 water : 116 123 31 38 153 144 500 i14 118 ~ 24 : 39 158 146 looo lo n 125 2~5:: ~39: ~ 150 148 2000 ~ 139 24 : 4~ 50 1~0 2500 99 117 ~ 29 ~ 41 ~157 157 Prelnellbat1On - 20 ~n at ~I~
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Table 7b Mutagenic Assay of Potassium Sorbate and Glycyl-D-alanine Concentration Revertants/plate ~g/plate TA 97 +S9 +S9 K+ sorbate 0 water 138 286 28 27 118 127 1.000 166 292 19 19 116 105 5.000 218 274 15 24 99 92 gly-D-ala lO0 145 248 31 31 110 130 l.000 135 322 23 22 147 130 5.000 113 17~ 18 24 :123 139 :

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Table 7c __, Mutagenic Assay of Positive Control Substances Chemical Concentration Bacterial Metabolic Revertant/
~g/plate strain activation plate sodium azide10 TA 100 1706
4-nitro-1.2-phenylenediamine 10 TA 97 - 835 2-nitrofluorene 100 TA 98 _ 2055 Benzo(a)pyrene 5 TA 97 + 589 TA 98 + 417 :TA:100 + 361 For metabolic activation aroclor induced rat liver homogenate was used. ~ :
From these results, it is concluded that potassium sorbate, glycyl-D-aIan:ine,~sorb~l-D-tryptophan, are not mutagenic in the Salmonella7microsome assay.

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Claims (29)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An N-acylamino acid of the formula:
X-CO-NH-Y
wherein X when taken in conjunction with the CO group, is an acyl moiety and Y when taken in conjunction with NH group, is a D-amino acid or glycine moiety, or a foodstuff acceptable salt thereof, other than glycyl D-alanine, acetyl D-tryptophan, acetyl D-methionine, acetyl D-valine and acetyl D-alanine.
2. The compound of claim 1 wherein the acyl moiety has from 8 to 24 carbon atoms.
3. The compound of claim 1 wherein XCO is selected from the group consisting of sorbyl-, lauroyl-, and myrisotyl-, and palmitoyl-groups and Y, together with the NH group, is selected from the group consisting of D-alanine, D-tryptophan, glycine, D-valine and D-aspartic acid.
4. A composition comprising an N-acylamino acid of the formula:
X-CO-NH-Y
wherein X when taken in conjunction with the CO group, is an acyl moiety and Y when taken in conjunction with the NH group is a D-amino acid or glycine moiety, or a suitable salt thereof, in combination with a selected food product.
5. The composition of claim 4 wherein at least 2600 ppm of the N-acylamino acid is present.
6. A composition comprising an N-acylamino acid of the formula:
X-CO-NH-Y
wherein X, when taken in conjunction with the CO group, is an acyl moiety and Y, when taken in conjunction with the NH group is a D-amino acid or glycine moiety, or a suitable salt thereof, in combination with a food grade nitrite.
7. A food product in combination with the composition of claim 6 wherein at least about 2600 ppm of the N-acyl-D-amino acid is present.
8. The food product of claim 7 wherein at least about 60 ppm of food grade nitrite is present.
9. The compound of claim 1 in salt form with sodium or potassium.
10. N-sorbyl-D-tryptophan.
11. N-sorbyl-D-alanine.
12. N-sorbyl-D-valine.
13. N-sorbyl-D-methionine.
14. N-sorbyl-D-aspartic acid.
15. Palmitoyl-D-tryptophan.
16. Myristoyl-D-aspartic acid.
17. Hexanoyl-D-alanine.
18. A method for control of microorganisms in a food product comprising incorporating an effective amount of an N-acyl-amino acid of the formula:
X-CO-NH-Y
wherein X, when taken in conjunction with the CO group, is an acyl moiety and Y, when taken in conjunction with the NH group is a D-amino acid or glycine moiety, or a suitable salt thereof, to said food product.
19. The method of claim 18 wherein said food product is a meat-containing product.
20. The method of claim 19 wherein said meat containing product is a canned minced meat product.
21. The method of claim 19 wherein the said meat product is a comminuted meat product stuffed in casings.
22. The method of claim 19 wherein the said meat product is sausage.
23. The method of claim 19 wherein said meat-containing product is a red-meat containing product, the compound is selected from the group having an acyl moiety selected from sorbic acid or a fatty acid having a C8-C24 chain and a D-amino acid moiety selected from D-alanine, D-tryptophan, D-methionine, D-valine and D-aspartic acid.
24. The method of claim 22 wherein said N-acylamino acid is incorporated in combination with a food grade nitrite.
25. A process for preparing an N-acyl-D-amino acid according to claim 1 which comprises acylating of a selected D-amino acid.
26. A food- or feed-acceptable composition comprising an N-acylamino acid of the formula:
X-CO-NH-Y
wherein X when taken in conjunction with the CO group, is an acyl moiety, and Y when taken in conjunction with the NH group is a D-amino acid or glycine moiety, or a food- or feed-acceptable salt thereof, as an active ingredient, in combination with a further active ingredient or a food- or feed-acceptable diluent or carrier.
27. A composition according to claim 26 wherein XCO is selected from the group consisting of sorbyl-, lauroyl-, and myrisotyl-, and palmitoyl-groups and Y, together with the NH
group, is selected from the group consisting of D-alanine, D-tryptophan, glycine, D-valine and D-aspartic acid.
28. A composition according to claim 26 wherein in the active ingredient the acyl moiety is selected from sorbic acid or a fatty acid having a C8-C24 chain and a D-amino acid moiety selected from D-alanine, D-tryptophan, D-methionine, D-valine and D-aspartic acid.
29. A composition according to claim 26 wherein the active ingredient comprises N-sorbyl-D-tryptophan, N-sorbyl-D-alanine, N-sorbyl-D-valine, N-sorbyl-D-methionine, N-sorbyl-D-aspartic acid, Palmitoyl-D-tryptophan, Myristoyl-D-aspartic acid or Hexanoyl-D-alanine.
CA000506396A 1986-04-11 1986-04-11 Derivatives of d-amino acids having anti-microbial properties Expired CA1261855A (en)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5053424A (en) * 1989-07-31 1991-10-01 Sigma-Tau Industrie Farmaceutiche Riunite S.P.A. 2-(n-acyl)amino-6,7-dimethoxy tetralines, and pharmaceutical compositions having antihypertensive activity containing same
FR2672598A1 (en) * 1991-02-11 1992-08-14 Adir NOVEL N-MYRISTOYLTRANSFERASE INHIBITORS, PROCESS FOR THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM
WO2006021588A1 (en) 2004-08-27 2006-03-02 Purac Biochem Bv Antibacterial agent for preserving fresh meat
US7465471B2 (en) 2003-03-21 2008-12-16 Novus International, Inc. Palatability of aquaculture feed
US8404714B2 (en) 2008-01-04 2013-03-26 Novus International, Inc. Combinations to improve animal health and performance
US8465761B2 (en) 2007-07-03 2013-06-18 Novus International, Inc. Piglet feed rations having low levels of fermentable carbohydrates
US8691843B2 (en) 2006-07-12 2014-04-08 Novus International, Inc. Antioxidant combinations for use in ruminant feed rations
WO2023198048A1 (en) * 2022-04-12 2023-10-19 元素驱动(杭州)生物科技有限公司 Preparation methods for n-acetyl-d-amino acid, d-amino acid, and d-amino acid derivative.

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5053424A (en) * 1989-07-31 1991-10-01 Sigma-Tau Industrie Farmaceutiche Riunite S.P.A. 2-(n-acyl)amino-6,7-dimethoxy tetralines, and pharmaceutical compositions having antihypertensive activity containing same
FR2672598A1 (en) * 1991-02-11 1992-08-14 Adir NOVEL N-MYRISTOYLTRANSFERASE INHIBITORS, PROCESS FOR THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM
EP0499521A1 (en) * 1991-02-11 1992-08-19 Adir Et Compagnie Inhibitors of N-myristoyltransferase, their preparation process and pharmaceutical compositions containing them
US7465471B2 (en) 2003-03-21 2008-12-16 Novus International, Inc. Palatability of aquaculture feed
WO2006021588A1 (en) 2004-08-27 2006-03-02 Purac Biochem Bv Antibacterial agent for preserving fresh meat
US8691843B2 (en) 2006-07-12 2014-04-08 Novus International, Inc. Antioxidant combinations for use in ruminant feed rations
US8465761B2 (en) 2007-07-03 2013-06-18 Novus International, Inc. Piglet feed rations having low levels of fermentable carbohydrates
US8404714B2 (en) 2008-01-04 2013-03-26 Novus International, Inc. Combinations to improve animal health and performance
WO2023198048A1 (en) * 2022-04-12 2023-10-19 元素驱动(杭州)生物科技有限公司 Preparation methods for n-acetyl-d-amino acid, d-amino acid, and d-amino acid derivative.

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