Note: Descriptions are shown in the official language in which they were submitted.
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~
63247-179
(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~
- : :
~: ,
: ~ : -
-- 5 --
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,
::
:
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.
:
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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
~: : : : .` ~ : ::
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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~
-- 8 --
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.
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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
- 14 - 63247-179
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.
:
:
: ~ : : : : :: :
- 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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- 16 -
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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- 17
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
100 127 179 27 27 132 113
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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- 18 - 63247-179
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
100 136 284 29 27 114 109
500 128 3I9 24 30 112 120
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
500 141 180 30 25 105 120
l.000 135 322 23 22 147 130
5.000 113 17~ 18 24 :123 139
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- 19 - 63247-179
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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