Note: Descriptions are shown in the official language in which they were submitted.
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HOECHST AKTIENGESELLSCHAFT Dr. KH/gm HOE 87/F 039
Spec;f;cation
A formu~ation for extending the shelf life of food
products, ~ed;caments and cosmetic products
It is kno~n to use preservatives such as~ for example,
sorb;c acid, benzoic acid, propionic acid, formic acid,
p-hydroxybsnzoic acid, s~licylic acid and sulfurous ~cid
to extend the shelf life of f30d products~ medicaments
and cosmetic products. In the preservation of food pro-
ducts in practic~ the effect of the sa;d preservatives is
often intensi~ied by additives which low~r the ~ater acti-
~ity or the pH of the food products~ such as qodium chlo-
ride, sugar9 acetic~acid or other edible acids. This may
result in Lhanges in the taste of the food products, ~hich
are no~ al~ays des;red~ In the case of mayonnaises and
marinated fish and other delicatessen products, the
consumer prefers the products to be adjusted to a mild
flavor, with io~ additions of salt and vinegar. However,
there is the risk in the higher pH rang~ that the gro~th
of some bacterîa;will be intensified and even that favor-
~ble ~ivin~ conditions for pathogen;c or~anisms are setup. Cases of food poisoning conn~cted ~ith the consump-
tion of only ~eakly acidified products repQatedly occur,
because the antimicrobiaL action of many preservatives
decreases ~arkedly ~ith increasing pH.
Enzy~es havin~ N~acetyl~uramidase activity such as, for
example, lysozyms can like~ise be used ~or the preservation
of foo~ products, for example mîlk~ meat and fîsh products.
The growth of ~icroorganisms in milk products is preven-
ted by preservation ~ith lysozy~e using th~ process in
Britîsh Patent 2,014,032. The use of lyso2y~e-~ike pro-
ducts ~rom secretions of animals and plants for preventing
late bubbLe for~ation in various types of cheese is des-
cribed in French P3tent 8,003,321~
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Ho~e~er, products of these types can be obta;ned not onLy
fro~ plants and an;mals but also fro~ Streptomycetes, as
is evident from Ger~an OffenlegungssGhriften 2,011,935,
2,040,444, 2,146,597 and 3,440,735~
S Ie ha~ no~ been found, surprisingly~ that combining the
non-en~ymatic preservatives with enzymes ~hich have N-
acetylmuramidase activity results in a syner~ism of action
~hich is distinguished by the fact that~ in particular,
food product~ ~edicaments and cosmetic products can be
10 preserved ~ith the mixture for comparatively longer than
by using the individual components.
Henc~ the invention relate~ to:
1~ A for~ulation containing one or mor~ non-enzymat;c
preservatives and one or ~ore enzymPs having N-acetyl-
mura~idase activity in a ratio by ~e;ght of 1 ~o 100t~ 1~0 to 1.
2. A method of preserving goods using the formulation
defined under lo
3. A proce~s for the preparation of th~ formulat;on de-
fined under 109 ~hich comprises convertin~ the non-
enzy~atic pres~rvative aod th~ enzyme having N-acetyl-
mur~idase activity into a suitable application form.
The invention is describ~d in detail, espec;alLy in its
preferred e~bodiments, hereinafter. Th~ invention is
25 aLso d~f;ned in the patent clai~s.
Examples of su;table preservatives used according to the
invention as active substances in the ~ormulation ar~
sorbic acid, benzoic acid, propionic acid, formic acid,
p-hydroxybenzoic acid9 salicyLic acid and sulfurous acidr
30 as ~ell as their salts. Particularly good results can be
obgained uith benzsic acid or sorbic acid or the;r salts.
The second active component in the formulat;on is an
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enzyme having N-acetylmuramidase activity and having the
abil;ty to lyse bacteriaL cells and to prevent further
growth of the microorganisms. Examples o~ enzymes wh;ch
are suitable and preferred are lysozyme, in particular
S from hen's egg white, or bacteriolytic enzyme products
from Str~ptomycetes, in particular from Streptomyces
coelicolor DSM 3030 and its mutants and variants. The
enzyme product from DSM 3030 is readily obtained by the
process described in German Offenlegungsschrift 3,440,735.
In this process, Strepto~yces coelicolor DSM 3030 is cul-
tivated in a fermen~ation medium with the addition o~
sugarbeet molasses in an a~ount of S to 50 9, preterably
10 to 20 9, per liter of culture medium. A further in-
crease in the yield is achieved by addition to th~ culture
medium of calciu~ ions in the form of readily soluble,
non-toxic calcium salts, preferably in the form o~ calcium
chloride9 ~hich is reasonably priced. A caLGium ion con-
centration of D.05 to 1 molar is advantageous, and concen-
trat;ons of 100 to 500 mmol/l are particularly preferred~
for example 1n the form of addition of 0~2 to O.5X by
weight of calcium chloride dihydrate.
The active substances can be added si~ultaneously or suc-
cessiv~ly to food products such as, for example, meat and
meat products, fish, srustacean, shellfish and mollusc pro-
ducts, edible gelat;nous coating compusitions for ~eatproducts, delicates~an products, liquid egg and liquid egg
yolk, vegetable and fruit products, alcohol;c and non-
alcoholic beverage~, milk and milk products, fil~ings for
pastries and bakery products~ confectionery and candies,
33 as ~ell as pharmaceutical and cosmetic products, animal
feeds, tobacco, tobacco products and packaging materials.
Depending on the intended u e, one or mor~ enzymes having
N-acetylmuramidase activity are mixed with at least one of
the non-enzy~atic preservatives in a ratio by ~eight of
1 to 100 to 10~ to ly pr~erably 1:50 to 50:1, and added,
by conv~n~ional processes, to the product to be preserv~d.
The amount of the m;xture added is of an order such ~hat
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0.01 to Z000 U of the enzyme having N-acetylmuram;dase
ac~ivity, preferably 0.1 to 1000 U, are contained in each
mg of the product to be preserved.
The rom~ination of chem;cal preservatives and enzymes hav-
ing ~uramidase activity results in a synergism of action.
This has a particularly advantageous effect on products
~ith a mild pH, which are particularly favored by consumers.
For example~ the action of the comb;nation in a pH range
bet~een 3 and 6~ preferably 4~5 to 5.5, aga;nst micro-
organisms vh;~h spoil food products and are pathogenic isbetter than that of the individual components aLone. The
advantage of the mixture is that the con~ent of che~ical
preservat;~es in food products, cosmetics, pharmaceut;cals
etc can be reduced whiLe simultaneously extending the shelf
Life7 It is self-evident that the combination of act;ve
substances ~ccording tv th~ invention can or shouLd be
used only in the Logical case where the products to be
preserved have been manufacSured under satisfactory hygie-
nis conditions and have a low initial organism count.
2~ The invention is explained further in the exa~ples which
fo~low. Unle~s othervise indicated, persentage data relate
t~ ~eight.
Ex~p~e 1
InvestigaSion of the shelf life of salad mayonnaise
A salad mayonnaise of the folLowing recipe was prepared:
~il 5~.0 %
water 31.1 %
starch 3.1 %
egg yolk 4.6 %
30 sals 1.0 %
sugar 4.0 X
guar gu~ 0.2 %
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The pH of the sal~d mayonna;se ~as adjusted to pH 4.5 ~ith
acetic acid. The mixture ~as div;ded into four batchesr
one of ~hich was Left unaltered (I). The add;tions to the
other portions ~ere 100 U of lysozyme pçr mg (II), 50 U of
Lysozyme per ~9 ~ 0.15 X of potassiu~ sorbate (III) and .
0,2 X of potassium sorbate ~IV~ in each case.
The mixtures were s~ored ;n closed jars at a temperature
of ~10Co
The she~f life vas a~s~ssed on the basis of a~terations
10 percept;b~e with th~ senses and of the total organism count.
Added preservativeShelf lîfe Edays~
I Control test 6
II 100 U of ~ysozyme 12
III 50 U of lysozym~tmg ~ 0O15 %
o~ po~assiu~ sorbate 1~
IV 0O~ X of potassium sorbate 14
Determination of the activity of bacteria ly~ing ~ p~ct:
0.2 ~ of samples containing basteria-lysing en~yme pro-
duct are pipetted to 2.8 ~l of a suspens;on o~ 0.2 mg of
Microcoecus luteus ATCC 4698 ~oehring~r ~annheim) per 0l
of 0.1 M sodium ace~ate buffer (pH 5.0), and the decrease
in turbidiey is determin~d at 25~C by ~easuring the e~-
tinction at 450 n~. 1 un;t is detined ~s the decrease in
extinetiQn by 0.001 photomete~ scale units per 0inute.
Exa~pLe 2
InJestigation of th~ she~f ~ife of mayonnaise
A ~ayonnaise o~ the follo~;ng resipe was prepared:
oil ~.0 ~
30 ~ater10.0 X
sugar4.0 X
~ust~rd 2.0 Z
5~ 2~ X
Th~ pH of the ~ayonnaise ~as adjusted to 4.5 ~ith acetic
acidO The ~ayonnaise was tr~ated~ stored ~nd ~ssess@d in
analogy to Example 1O The she~f liv2s emerged ~s follo~s.
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Added preservative SheLf life ~days~
I Control test
II 100 U of lysozyme/mg 1
III 50 U of lysozyme/mg + 0.15 %
of potassium sorbate 23
IV 002 % of potassium sorbate 17
Exa~pLe 3
lnvestigation of the shelf life of a meatlmayonna;se
product
The salad ~ayonnaises I-IV prepared ;n Example 1 ~ere each
mixed with 40 ~ by wei~ht of thinly sL;ced meat sausage and
20 % by weight of thinly sliced gherkins from one produc-
tion batch. The meat/mayonllaise produc~s were treated,
stored and as essed in analogy to Exa~ple 1. The follow-
ing shelf lives emerged:
Added preservative Shelf life ~days]
I Control ~est 3
II 40 U o~ Lyso2yme/mg 6
III 20 U of lysozyme/mg ~ 0~06 X
of p~tassium sorbate 11
IV 0.08 X of potassium sorbate 8
E~a~ple 4
Investigation of the shel~ life of salad dressing
A salad dressing of the follo~ing recipe ~as prepared:
~ater 34.1 %
oiL 46.1 %
sugar 14.5 X
~ustard 3.0 %
salt 2.1 %
30 xan~han 0.2 X
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The pH ~as adjusted to 4.5 with acetic as;d. The salad
dressing ~as treated, stored and assessed in analogy to
Example 1. The shelf lives emerged as follows:
Added preservativeShelf life Cdays]
5 I Control test 6
II 100 U of lysozyme/mg 10
III 50 U of lysozyme/mg ~ 0~15 %
of potassium sorbate 17
IV 0.2 X of potassium sorbate 12
Exa~ple 5
Investigation of the shelf life of a cream filling
A cake cream of the following recipe was prepared:
sugar 51.2 %
glucose syrup 25.6 %
15 uater 27.0 X
functionalized protein 0.7 X
citric acid 0.4 X
agar agar 0.07 g
flavorings 0.03 X
The cream ~as divided into four batches, one of which was
left unaltered (I~. The additions to the other portions
~ere 100 U of lysozyme per mg (II)~ 50 U of lysozyme per
m~ t a~15 x of potassium sorbate tIlI) and 0.2 % of potas-
sium sorbate (IV~ in each case.
The mixtures ~ere stored in closed jars at a temperature
of ~10C.
The shelf lif@ was ~ssessed on the basis of ait~rations
perceptible ~ith the senses and of the total organism
count.
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Added preservatiYe Shelf life Cdays~ :
I Control test 5
II tO0 U of lysozyme/mg 9
III 50 U of lysozyme/mg + 0.15 %
of potassium sorbate 15
IV 0.2 % of potassium sorbate 11
Exa~p~e 6
Investigation of the shelf life of a foamed fruit dessert
product
A fruit dessert product of thQ follawing recipe was pre-
pared: ~
~ater ~: 74.6~X
icin~:su~ar 18~B X
modified starch ~ 3.7 %
15 instant gelatine 1~2 %
fruit po~der ~ ~ 0.7 %
citric acid 0.4 %
delta-glucono~actone ~ 0.3 %
: ~sodium bicarbonate : : 0.1 X
2Q: ~unct;onalized protein ~ 0.1 %
flavorings :0.1 X
The fruit dessert product ~as divided ;nto four batchesr
one of ~hich was le~t unaltered (I). The additions to
the other psrtions were 100 U of: lysozyme per m~ (II),
50 U of lysozyme per mg ~ 0.15 % of potassium sorbate
(~II) and 0.~ % of potassium sorbate (IV) in eaeh:case.
The mixtures ~ere stored in closed jars at a tempera-
ture of ~10C.
.
The sheLf life was assessed on the basis of alterations
pereeptible ~ith the senses and of the total organism
count.
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Added preservative Shelf life Cdays]
I Control test 6
II 100 U of lysozyme~mg 10
IIl SD U of lysozyme/mg ~ 0.15 %
of potassium sorbate 16
IV 0.~ % of potassium sorbate 11
Exa~p~e 7
Investigation of the shelf life of a moisturi~ing cream
A moisturizing cream with the follo~ing formulation was
prepared:
liquid p~raffin ~G~rman Pharmacopeia)10.0 %
vitamin oil 5,0 X
petrolatum ~German Paharmacopeia) 5.0 X
~Hostacerin T~3 (fatty alcohol polyglycol
15 ethers, emuLsifier~ 5.U X
RHostaphat K~ 340 N (organic phosphoric
acid derivative, emulsi~ier) 4.0 %
pa~mitic acid S.0 %
c~tyl alcohol ~German Phar~acopeia) 1.0 X
~ter 60.0 %
~Hydr~viton (humectant) 5.0 %
The cream ~as divided into four batches, one of which ~as
left unaltered SI)~ During the preparation of creams lI-
IV~ the ~ollo~ing additions were metered into the aqueous5 phase. cream It 250 U of lysozy~e/mg
cream III 150 U of ~ysozyme/mg ~ 0.2 % of
sorbic acid
cream IV 0.3 % of sorbic acid
The cre3ms ~ere stored in close~ containers at room tem30 perature~
The shelf life was assessed o~ the basis of aLterations per-
ceptibLe with the senses and of the total organism coun~
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Added preservative SheLf Life
Cd~ys~
I Control test Y
II 250 U of lysozyme/mg 14
III 150 U of lysozyme/mg ~ D.2 %
of sorbic acid 30
IV 0.3 X of sorbic acid 19
Ex~3ple 8
Investigation of the shelf l;fe of a geL
A pharmaceutical gel with the follo~ing formulation was
10 preparedo
~ater ~7 Z
hydroxyethylcellulose tTylose H 1000) 3 ~
The prepared gel ~as divided into four ba~ches, of which
one ~eL (I) remained untreated. The ~ollowing additives
15 ~ere ~et~red ;nto geLs II~
gel XI 250 U of ~ysozyme per mg
gel III 150 U of lysozyme per mg ~ 002 % of sorbic acid
ge~ IV 0~3 % of sorbie acid.
The ~els ~ere stored in closed containers at room tempera-
2~ ture.
Th~ sheLf ~ife Yas assessed on the basis of ~lterations
perceptible with the senses and of the total organis~
count.
Added preservative Shelf Life
Cdays~
25 I Control test 7
II 250 U of lysozyme~mg 1
III 150 U of Lysozy~e/~g ~ 0.2 X of
sorbic acid 35
IV 0.3 X of sorbic acid 22
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Exa~ple 9
Investigation of the shelf life of liquid rennet
Commercially available liquid rennet preserved ~ith 1.1 Z
benzoic acid was used for the investigation of the shelf
life. The l;qu;d rennet was stored ;n closed containers
at room temperature without and with the addition of
50,000 U of lysozyme per ml.
The shelf life was assessed on the basis of alterat;ons
perceptible with the senses and of the total organism
counta
Added preservative Shelf Life
Cdays~
1~1 X of benzoic acid 16
50,000 U of lysozyme/ml ~
1~1 % of benzoic acid 33
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