Note: Descriptions are shown in the official language in which they were submitted.
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AN ANTIBACTERIAL COMPOSITION
For Control of Gram Positive Bacteria in Food Applications
Background of the Invention
1. Field of the Invention
The present invention discloses a process for inhibiting or retarding the
outgrowth of
bacteria on food products by treatment with a composition which includes one
or more
hops acid extracts or modified hops acid extracts plus one or more safe and
suitable
gram positive bacteristatic or bactericidal preparations from the
lantibiotics, pediocin,
lacticin class bacteriocin and/or lytic enzyme categories. More specifically,
the process
comprises using as an ingredient or applying to a food surface a composition
including
nisin, and/or lysozyme and beta hops acids in order to reduce or eliminate
gram
positive spoilage or pathogenic bacteria, and, most especially, all strains of
the harmful
pathogen Listeria monocytogenes. An important public health concern is the
ability of
pathogenic listerial species, especially Listeria monocytogenes, to grow at
commercial
refrigeration temperatures at which processed foods are normally stored for
long
periods of time. This ability to grow under standard conditions of
distribution makes
Lisferia monocytogenes one of the top public health risks associated with raw
and
processed foods today. Any new antimicrobial system must be effective in
commercial
food systems, with formulation and temperature conditions reflecting actual
practices.
The new compositions of this patent are effective in a variety of foods,
especially at the
refrigerated storage and handling temperatures typical of foods at risk for
listerial
contamination.
2. Technology Description
The extent of food borne infections and intoxications in the United States was
quantitatively documented in the CAST report of 1994 (Foodborne Pathogens:
Risks
and Consequences. Task Force Report No. 122, Council for Agricultural Science
and
Technology, Washington D.C.), as well as being extensively characterized in
the past
few years due to better reporting systems and programs (CDC. 1988c. 1997 Final
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FoodNet Surveillance report. U.S. Department of Health and Human Services,
October, 1998). In order to reduce the prevalence of listeriosis and other
food borne
infections, a wide variety of research has been conducted to develop
compositions
which function as food grade anti-bacterial ingredients. Individual compounds
have
been disclosed in this research, with little if any commercial benefit or use,
primarily
because single compounds typically lack the efficacy or are too costly to use
in food
processing and formulations. At this time, there is still a need for better
control of
gram positive pathogens such as Listeria monocytogenes, Staphylococcus aureus,
Bacillus cereus, Clostridium botulinum, C. pen'ringens, and the like, which
pose
significant health risks to consumers. In addition, other gram positive
spoilage
bacteria such as lactobacilli, streptococci, bacilli, enterococci, and
micrococci species
are known to cause spoilage, though not normally illness, and are often the
principal
agents in reducing the shelf life and freshness of selected foods.
Both pathogenic and spoilage bacteria can occur in raw food materials, but
heat
processing tends to reduce bacterial loads dramatically. After processing,
most foods
are at risk for recontamination prior to packaging, distribution, and final
consumption,
when they may be exposed to pathogens in the food handling environment. Even
in
the cleanest processing facilities, selected pathogens may contaminate the
already
processed foods, usually at very low levels. In the case of cold tolerant
pathogens,
primarily various listerial species, they may then grow unchecked on the food
during
distribution and storage until final consumption. The more such pathogens grow
in a
food product, the higher the risk of infection among consumers of that food
product.
This is a special concern for ready to eat meats and dairy products, as such
foods are
not heated or processed again by the user prior to consumption. In such cases,
the
most likely risk is from Listeria species that grow well under refrigeration.
Consumption of elevated levels of any pathogen is recognized to increase the
risk of
infection, especially among infants, the elderly, pregnant women, and any
immune
compromised individuals.
In 1998, it is estimated that there were approximately 500 deaths in the U.S.
caused by
listeriosis presumably contracted from foods. Among major food pathogens,
listeriosis
has the highest mortality, exceeding 20% according to Meade, et al. (Food-
related
illness and death in the United States, CDC 5:5, Sept-Oct 1999). In light of
the risk
and the large social cost, an urgent need for systems to prevent listerial
growth in
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foods is recognized by U.S public health agencies, the Food and Drug
Administration
(FDA), and the United States Department of Agriculture (USDA). The subject of
this
invention is a novel, food grade (Generally Recognized as Safe), flavor
neutral
composition that targets Listeria in actual food systems, especially in
processed, ready
to eat meat products. The desired effect of such a composition is to kill or
reduce the
levels of Listeria bacteria in foods that are at risk for post processing
survival or
contamination by such species. In addition, risks associated with other gram
positive
bacteria, including the above mentioned spoilage types, as well as less common
strains of the Corynebacteria, Diplococci, Mycobacteria, Streptococci, and
Streptomyces genuses have also been noted as problems in food products and may
benefit from such a process or composition.
In 1992 and 1993, U.S. Patent Nos. 5,096,718 and 5,260,061 disclosed the use
of
metabolites of propionic acid bacteria in certain foods to increase the shelf
life of
treated food products. These metabolites demonstrate enhanced efficacy against
gram negative bacteria but, unfortunately, are not effective against gram
positive
bacteria.
U.S. Patent No. 5,217,950 suggested the use of nisin compositions as
bactericides.
Nisin is a lantibiotic, more specifically, a polypeptide with antimicrobial
properties which
is produced in nature by various strains of the bacterium Lactococcus lactis.
Nisin is
indeed primarily effective against gram positive bacteria; however, the common
gram
positive pathogen Listeria monocytogenes is more resistant to nisin than most
other
species of gram positive bacteria. The need to enhance the activity of nisin
against
Listeria monocytogenes is well recognized and accounts for the fact that nisin
by itself
is not used as an antilisterial agent commercially. The 5,217,950 patent
therefore
suggests the combination of a chelating agent, such as disodium
ethylenediaminetetraacetic acid (EDTA) or other acetate salts or citrate salts
with nisin
to effect a broader range of activity against both Listeria species as well as
selected
gram negative bacteria.
U.S. Patent Nos. 5,573,797; 5,593,800 and 5,573,801 disclose antibacterial
compositions which include a combination of a Streptococcus or Pediococcus
derived
bacteriocin or synthetic equivalent antibacterial agent in combination with a
chelating
agent. The composition is applied to the surface of the food to be treated
either by
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direct application or by incorporating the composition onto a flexible film
casing which is
placed into intimate contact with the food surface. The chelating agent binds
free
divalent cations in the outer membrane of gram negative cells, improving
permeability
to the antibacterial agents. In the case of the 5,573,801 patent, the
application of nisin
alone to the surface of cooked meats is disclosed, but the efficacy of this
single
ingredient is so limited that it has not been employed commercially.
U.S. Patent No. 5,458,876 suggests the combination of a lantibiotic (such as
nisin) with
lysozyme as an antibacterial. In this case, lysozyme breaks down the cell wall
and
weakens the structural integrity of the target cell so that the antibacterial
agent
becomes more effective in damaging or killing the bacterial cell. In
particular, this
combination proves to be effective in improving the antibacterial efficacy of
nisin
against Listeria monocytogenes, yielding a significant reduction, though not a
complete
elimination, of listeria at safe and suitable levels of use.
EP 0 466 244 discloses a composition having improved antibacterial properties
comprising a mixture of at least one of each of the following groups of
compounds: (I) a
cell wall lysing substance or a salt thereof , (II) an antibacterial compound
and (III) an
adjuvant selected from organic acids acceptable for use in food products or
preparations for cosmetic use or personal hygiene or salts of these acids,
phosphates
and condensed phosphates or the corresponding acids, and other sequestering
agents. Preferably (I) is lysozyme, (II) may be a bacteriocin (e.g. nisin or
pediocin),
and (III) may be acetic acid, sodium diacetate, lactic acid, citric acid,
propionic acid,
tartaric acid, orthophosphates, hexametaphosphates, tripolyphosphates, other
polyphosphates or sequestering agents containing substituted or non-
substituted
amino groups, for example EDTA.
EP 0 453 860 suggests the combination of nisin with a phosphate buffer
effective at a
pH of between 5.5 and 6.5 to eradicate gram negative bacteria from surfaces.
WO 97/23136 suggests a bacterial decontamination method which involves
treatment
with a solution of low concentration alkali metal orthophosphate combined with
either
osmotic shock and/or lysozyme in solution and/or nisin in solution. This
reference
tested the combination of low concentrations of trisodium orthophosphate with
lysozyme against certain bacteria on lettuce leaves or chicken skin, and the
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combination of low concentrations of trisodium orthophosphate with nisin
against
certain bacteria on chicken skin.
The published Australian patent application AU-A-18604/88 discloses the use of
bacteria lysing enzyme products with N-acetylmuramidase, e.g. lysozyme,
together
with non-enzymatic preservatives for preserving foodstuffs. Non-enzymatic
preservatives mentioned in this publication are complexing agents such as
citric acid
and EDTA, amino acids, particularly amino acids, such as cysteine, alanine,
tyrosine
and glycine and nucleosides and nucleotides such as inosine 5'-inosine
monophosphate or phosphates such as tetrasodiumpyrophosphate (diphosphate),
sodium tripolyphosphate (triphosphate) and polyphosphate or reddening agents
such
as alkali metal nitrates.
U.S. Patent No. 5,286,506 discloses the use of the fat soluble beta acids
extract of
hops for their bacteriostatic effects against Listeria monocytogenes in foods
at 6 to 50
ppm by weight of the food. In addition, U.S. Patent Nos. 5,370,863 and
5,455,038
suggest that certain hops acid derivatives that are chemically hydrogenated
may have
antibacterial activity against listeria species. However, these extracts are
not food
grade (GRAS) and are not allowed for use in foods outside of brewing.
Finally, Johnson et al disclose in the International Journal of Food
Microbiology 33
(1996) 195-207 that hops acids and hops acid derivatives have limited efficacy
against
listerial species in fat containing foods such as cheeses, meats, sauces, and
dressings,
presumably due to the migration or entrapment of the beta acids into the fat
emulsion
and their subsequent unavailability for inhibition of bacterial growth in the
aqueous
portion of the food emulsion. The problem of the lack of activity of hop beta
acids in fat
containing foods has prevented them from being used commercially as natural
antimicrobial agents for control of listeria or other gram positive pathogens.
To the extent necessary for completion of this patent application, all of the
above cited
references are expressly incorporated by reference.
In light of the above teachings, there still exists a need in the art for a
method for
treating foods with bactericidal compositions that are active at reasonable
usage levels
in common food applications that are at risk for gram positive pathogens. More
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specifically, there exists a need for effective treatments that can be
conveniently
integrated into existing processing protocols for these products. Finally,
there exists a
need for more complete and effective reduction, or even elimination, of
harmful gram
positive pathogens by use of safe, suitable, and cost effective levels of food
grade anti
s microbial ingredients such as nisin, lysozyme, and hops acids.
Brief Summary of the Invention
It is now discovered, quite surprisingly, that a composition which has a first
component
that includes one or more gram positive bacteristatic or bactericidal
compounds from
one or more of the following classes of materials: lantibiotics, pediocin,
and/or lacticin
class bacteriocins, or lytic enzymes, and a second component which includes
one or
more natural hops acids or hops resins or derivatives thereof provides
excellent
antibacterial properties, especially against potentially harmful bacteria of
the listeria
genus, by dramatically surpassing the antibacterial efficacy of any of the
individual
components or of previously published compositions.
One embodiment of the present invention comprises an antibacterial composition
containing as a first component: (a) one or more gram positive bacteristatic
or
bactericidal compounds from one or more of the following classes of materials:
lantibiotic, pediocin, and lacticin class bacteriocins, and/or lysozyme, a
natural enzyme
from egg white; and as a second component (b) one or more hops acids or hops
acid
derivatives or hops resin or hops resin derivatives. Particularly preferred is
a
composition containing a lantibiotic bacteriocin, lysozyme, and beta hops acid
extract.
In another embodiment, the present invention provides a method for retarding
growth,
reducing viable numbers, or totally eliminating gram positive bacteria, and
especially
Listeria monocyfogenes, in food products comprising the step of treating the
surfaces
of said food product with an effective amount of a composition comprising as a
first
component: (a) one or more gram positive bacteristatic or bactericidal
compounds
from one or more of the following classes of materials: lantibiotics, pediocin
and
lacticin class bacteriocins, or lytic enzymes; and as a second component (b)
one or
more hops acid or hops acid derivatives or hops resin or hops resin
derivatives.
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It is an object of the present invention to provide a process for treating
food products in
order to protect against harmful bacteria and in order to maintain the
antibacterial
activity of the composition even on or in a fat containing food.
An additional object of the present invention is to provide a novel
composition having
substantially greater antibacterial properties than previously observed for
the individual
components of the composition.
These, and other objects, will readily be apparent to those skilled in the art
as
reference is made to the detailed description of the preferred embodiment.
Detailed Description of the Preferred Embodiment
In describing the preferred embodiment, certain terminology will be utilized
for the sake
of clarity. Such terminology is intended to encompass the recited embodiment,
as well
as all technical equivalents which operate in a similar manner for a similar
purpose to
achieve a similar result.
The present invention provides a novel antibacterial composition and its use
in a
process for reducing, retarding, or totally eliminating harmful bacteria from
food
surfaces, even in fat containing foods.
The novel antibacterial composition comprises: (a) one or more gram positive
bacteristatic or bactericidal compounds from one or more of the following
classes of
materials: lantibiotics, pediocin and lacticin class bacteriocins, or lytic
enzymes; and (b)
one or more hops acid extracts or hops acid derivatives or hops resin or hops
resin
derivatives.
The first component of the inventive composition is one or more compounds
having
bacteristatic or bactericidal activity against gram positive bacteria. Such
compounds
preferably include, but are not limited to lantibiotic, pediocin, and lacticin
class
bacteriocins and/or lysozyme from egg white, shellfish, or other natural
sources.
Combinations of more than one compound having bacteristatic or bactericidal
activity
against gram positive bacteria (e.g., nisin and lysozyme) are specifically
contemplated
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as falling within the more preferred scope of the first component of the
present
invention.
A first class of gram positive bacteristatic compounds comprises a
lantibiotic. The term
"lantibiotics" was coined by Schnell et al. (1988. Nature 333:276-278) to
describe a
group of bacteriocins including nisin which contain the amino acid lanthionine
and other
"non-protein" amino acids. The common properties of these bacteriocins are
reviewed
by Kellner et al. (1988. Eur. J. Biochem 177:53-59) wherein they note that ".
. .
polycyclic polypeptide antibiotics possess a high content of unsaturated amino
acids
(dehydroalanine, dehydrobutrine) and thioether amino acids (meso-lanthionine,
(2S,3S,6R)-3-methyllanthionine). Furthermore, lysinoalanine, 3-hydroxyaspartic
acid
and S-(2-aminovinyl)-D-cystine are found in some members." Members of this
group
include nisin, subtilin, pep 5, epidermin, gallidermin, cinnamycin, Ro09-0198,
duramycin and ancovenin. These ribosomally synthesized peptide antibiotics
contain
from 19 to 34 amino acids and are produced by various microbes including
Staphlococcus species, lactic acid bacteria, Bacillus species and Streptomyces
species. In addition to their unique composition of non-protein amino acids,
they can be
distinguished from other polypeptide antibiotics on the basis of their
specificity.
Bacteriocins in general, and the lantibiotics in particular, are characterized
by a very
narrow spectrum of action. Thus, only a few species of bacteria are sensitive
to a
particular bacteriocin at practical, safe and suitable concentrations. At
normal, legally
permitted levels of use in foods (eg., up to 12.5 ppm of pure nisin in a food
system),
such bacteriocins tend to have only bacteristatic (ie, growth inhibiting)
properties at
best. This is in contrast with other broad spectrum polypeptide antibiotics,
such as
polymyxin B1 which are actively bactericidal against a wide range of bacteria,
even at
very low levels of use, as well as the "lytic peptides" discussed by Jaynes et
al., in
published international application WO 89/00194, which are active against most
bacteria, yeasts and even mammalian cells.
Nisin is a ribosomally coded peptide which occasionally occurs as a dimer with
a
molecular weight of about 7000. It contains several unusual amino acids
including
beta-methyllanthionine, dehydroalanine, and lanthionine among its total of 34
amino
acids. There are five unusual thio-ether linkages in the peptide which
contribute to its
stability in acid solutions. Nisin is one of the most thoroughly characterized
bacteriocins, and shares remarkable homology of structure and action with
other
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lantibiotics, for example Subtilin and epidermin [Buchman et al 1988. J. Bio.
Chem. 263
(31 ):16260-16266]. Recent reviews of nisin, its physical properties and uses
include
"Bacteriocins of Lactic Acid Bacteria", T. R. Klaenhammer, 1988. Biochimie
70:337-
349, "Nisin", A. Hurst, 1981. Avd. Appl. Microbiol. 27:85-121, and U.S. Pat.
No.
4,740,593. Nisin is the collective name describing several closely related
substances
which exhibit similar amino acid compositions, and some limited range of
antibiotic
activity. This phenomenon is discussed by E. Lipinska in "Antibiotics and
Antibiosis in
Agriculture" (M. Woodbine, Ed.) 1988, pp. 103-130.
The use of nisin to combat L. monocytogenes has been reported by M. Doyle;
"Effect
of Environmental and Processing Conditions on Listeria Monocytogenes", Food
Technology, 1988.42(4):169-171. This reference describes the initial
inhibition of the
organism's growth (for about 12 hours) and reports that L. monocytogenes may
grow
at a pH level as low as 5.0 and is resistant to alkaline pH with the ability
to grow at pH
9.6.
Nisin is commercially available from Rhodia Inc. in a standardized 2.5 weight
percent
preparation under the trademark NovasinTM. Lantibiotic containing protein may
also
be present as a low level fermentation by-product in certain varieties of
cheddar or
American cheese and in the fermented skim milk product known as MICROGARD~
MG300 . In practice the lantibiotic is added to the food product in amounts
between
about 1 to about 100 ppm (by weight of solution used for treatment) of active
ingredient
(nisin), with preferred levels of 1 to 12.5 ppm, based on safety and
suitability of use in
foods.
As alternatives to use of lantibiotics in the preferred combination, it is
also known that
use of a Pediococcus bacterial metabolite, specifically pediocin, as a
substitute can
yield efficacious results. Though the pediocins are not yet approved for use
in foods,
they may yet be accepted for commercial application in the future. In
addition, the new
class of streptococcal bacteriocins called lacticins, especially Lacticin 3147
as
described in Irish Patent Application No. 980500, should produce similar
activity
against gram positive bacteria. Like the lantibiotics, both pediocins and
lacticins are
known to have bacteristatic activity primarily against a limited range of gram
positive
bacteria.
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A second class of gram positive bactericidal proteins comprises the lytic
enzymes
especially lysozyme, most commonly derived from egg albumin in a food grade
extraction process, but also available from arctic scallops, human milk,
tears, and other
natural sources. When lysozyme is used as an antimicrobial, it is added to the
food
product in amounts between about 20 to about 500 ppm (by weight of solution
used for
treatment), more preferably between about 50 to about 200 ppm, primarily to
inhibit
Clostridium tyrobutyricum in ripened cheeses. Lysozyme is not bactericidal at
these
levels against other gram positive bacteria, but it has been used at higher
levels
(greater than 1000 ppm, typically 2000 ppm or more) to remove the cell wall
from a
wide range of gram positive bacteria.
Lysozymes (Muramidase; mucopeptide N-acetylmucamoylhydrolase; 1,4-.beta.-N
acetylhexosaminodase, E.C. 3.2.1.17) are mucolytic enzymes which have been
isolated from various sources and are well characterized enzymes. First
discovered in
1922 by W. Fleming, egg white lysozyme was among the first proteins sequenced,
the
first for which a three dimensional structure was suggested using x-ray
crystallography
and the first for which a detailed mechanism of action was proposed. Its
antimicrobial
activity against gram positive bacteria is well documented, for example by V.
N. Procter
et al in CRC Crit. Reviews in Food Science and Nutrition, 1988, 26(4):359-395.
The
molecular weight of egg white lysozyme is approximately 14,300 to 14,600, the
isoelectric point is pH 10.5-10.7. It is composed of 129 amino acids which are
interconnected by four disulfide bridges. Similar enzymes have been isolated
and
characterized from other sources including such diverse producers as
Escherichia coli
bacteriophage T4 and human tears. Despite slight differences (for example, the
human
lysozyme has 130 amino acids) the capacity for hydrolysis of acetylhexosamine
polymers remains essentially the same. Accordingly, for purposes of this
invention, the
term lysozyme is intended to include those cell wall or peptidoglycan
degrading
enzymes which have the ability to hydrolyze acetylhexosamine and related
polymers.
Lysozyme is known to kill or inhibit the growth of bacteria and fungi, and is
used in
Europe to control the growth of the spoilage organism Clostridium
tyrobutyricum in a
wide variety of cheeses. It has also been proposed for use in a variety of
other food
preservation applications and has been reported to inhibit the growth of (and
in some
cases kill) Listeria monocytogenes (Hughey et al, 1987, Appl. Environ.
Microbiol
53:2165-2170). Lysozyme derived from egg albumin with an activity of about
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Shugar units/mg is commercially available from Rhodia under the trademark
NovaGARDTM.
In summary, the first component of the novel composition is preferably the
previously
disclosed combination of lantibiotics and lytic enzymes, especially the more
preferred
combination of nisin and egg white lysozyme.
The second component of the novel composition is one or more hops acid
extracts or
hops acid derivatives or hops resins or hops resin derivatives or combinations
of some
or all of these. The bitter components of the hops used in beer making,
particularly the
beta-acids, have now been found to be useful as bactericidal agents in food
products,
particularly in combination with the above mentioned bacteristatic and/or
bactericidal
components. The most prevalent groups of bitter acids contained in hops are
the
alpha-acids and the beta-acids, also referred to as humulones and lupulones,
respectively. Both contribute bitterness to beer, but the alpha-acids are much
more
bitter than the beta-acids and not desirable for use in most food products.
Producers of
hops extracts isolate the alpha and beta acids commercially by various
chromatographic methods and have recently developed a technique to separate
the
two acid fractions using liquid carbon dioxide under supercritical conditions.
A by-
product of the operation is a product which contains approximately 61 weight
percent
beta-acids, the remainder consisting essentially of hops resins. This by-
product can
be standardized with malto dextrin or other food grade carrier, spray dried,
and used
as an antibacterial food ingredient. A preferred beta hops acids composition
is
commercially available as a natural flavor extract containing 1 weight percent
beta
hops acids.
The alpha-acids contained in hops are commonly known as humulone, cohumulone
and adhumulone, white the beta-acids contained in hops are commonly known as
lupulone, colupulone and adlupulone. Chemically modified derivatives of hops
acids or
hops resins which have demonstrated antibacterial properties such as
hexahydrocolupulone and tetrahydroisohumulone, as disclosed in U.S. Patent No.
5,455,038, are specifically contemplated for use in association with the
present
invention. Also considered as specifically contemplated for use in association
with the
present invention is the use of the acid salt forms of the hops acids or hops
resins.
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In practice, the hops acid or resin or derivatives thereof is added to the
food product in
amounts between about 0.1 to about 50 ppm of active ingredient (by weight of
solution
used for treatment), more preferably between about 0.40 to about 20 ppm.
Other additives which can be present in the inventive composition include, but
are not
limited to the following materials: other antibacterial, such as chitosan or
its
derivatives, and/or chelating agents, natural or synthetic seasonings,
essential oils,
and/or flavors, dyes and/or colorants, vitamins, minerals, nutrients, enzymes,
binding
agents such as guar gum and xanthan gum and the like. In particularly
preferred
embodiments, guar gum is present in the inventive composition to aid in the
binding of
the antimicrobial components to the food surface being treated. The addition
of these
materials is not considered critical to the success of the present invention
and would be
considered within the skill of the artisan.
The antimicrobial composition of the present invention may be used in
connection with
any food product which is susceptible to microbial degradation. These include,
but are
not limited to fruits and vegetables including derived products, grain and
grain derived
products, dairy foods, meat, poultry, and seafood. In particularly preferred
embodiments, the composition is used in connection with meat, poultry and/or
seafood,
more particularly with fat containing cooked meats such as hotdogs, sausages,
roast
beef, turkey, corned beef and deli meats.
To reduce the amount of bacteria on a food surface, the novel composition is
simply
applied to the food surface either before or after cooking. In practice the
application of
the composition of matter to the food surface may either be a direct
application or an
indirect application. The use of the term "food surface" is defined to include
any and all
internal or external surfaces of the food product being treated.
The composition according to the present invention is most readily used by
applying it
on the exterior surface of a blended food product, such as a hot dog or
bologna, or of a
solid food, such as a piece of roasted beef, so as to minimize loss of
activity in the fat
phase of the food. The composition may alternatively be included in the
emulsion or
raw ingredients of a food such as sauces or salsas, before or after cooking,
or to the
interior of solid products, such as hams, by injection or tumbling. In still
other
embodiments, the composition may be applied as a marinade, breading, seasoning
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rub, glaze, colorant mixture, and the like, the key criteria being that the
antimicrobial
composition be available to the surface subject to bacterial degradation. In a
preferred
embodiment, the composition may be indirectly placed into contact with the
food
surface by applying the composition to food packaging materials or casings and
thereafter applying the packaging to the food surface. The use of surface
treatment
strategies, whether direct or indirect, benefits from the minimization of loss
into the fat
phase of the fat containing food product. The bacteristatically or
bactericidally optimum
effective amount to be used will depend on the composition of the particular
food
product to be treated and the method used for applying the composition to the
food
surface, but can be determined by simple experimentation.
In a preferred embodiment of this invention, the antibacterial composition
comprises
from about 38.5 to 99.8 parts by weight of the first component, which includes
at least
one bacteristatic or bactericidal compound selected from the group consisting
of
lantibiotics, pediocin, lacticin class bacteriocins, and lytic enzymes; to
about 61.5 to 0.2
parts by weight of the second component, which includes at least one compound
selected from the group consisting of hops acids, hops acid derivatives, hops
resins,
and hops resin derivatives; all parts by weight being based on the total
weight of the
first and second components of the composition.
In a more specifically preferred composition embodiment of this invention
wherein the
first component comprises two compounds, it is preferred that the composition
comprises, as a first component, from about 1.0 to 2.5 parts by weight
lantibiotic, and
from about 37.5 to 97.3 parts by weight lytic enzyme; and as a second
component,
from about 61.5 to 0,2 parts by weight of at least one compound selected from
the
group consisting of hops acids, hops acid derivatives, hops resins, and hops
resin
derivatives; all parts by weight being based on the total weight of the first
and second
components of the composition.
The following non-limiting examples illustrate the broad range antimicrobial
compositions which constitute the present invention.
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Example 1
As shown in Table 1, two gram positive bacterial strains are tested in
Trypticase soy
broth, pH 6.0 at 30°C for 48 hours to show the inhibitory effect of the
NovasinTM nisin
preparation, lysozyme and beta hops acid either alone or in combination. The
test
demonstrates the surprisingly synergistic effect of NovasinTM, lysozyme and
beta hops
acid (BHA). Table 1 shows that at the same concentration, the combination of
NovasinTM, lysozyme and BHA has a significantly better inhibition than the use
of each
component alone or in two component combinations. The three component
combination demonstrates a 5 log reduction of both strains while only 1 to 3
log
reduction is observed for any single component treatment or two component
treatment.
Table 1
CFU/m 1
Treatment L. alimentarius L. monocytogenes
Control 3.1 x I Oe8 1.4 x 10e8
Novasin (NS) 50 ppm 5.3 x 10e6 7.1 x 10e7
Lysozyme (LY) 50 ppm 6.3 x 10e6 2.5 x 10e7
Beta hops acid (BHA)* 5 1.2 x 10e8 6.2 x 10e6
ppm
NS SO ppm + LY 50 ppm 2.5 x 10e5 4.1 x 10e5
LY 50 ppm + BHA 5 ppm 5.6 x 1 Oe6 6.3 x 10e6
NS 50 ppm + BHA 5 ppm 3.5 x 10e5 5.2 x I
Oe5
NS 50 ppm, LY 50 ppm , BHA 2.8 x I Oe3 3.1 x I
5 ppm Oe3
*In this specification, BHA means beta hops acids.
Example 2
To demonstrate in vivo efficacy of the inventive composition, hot dogs are
inoculated
with Listeria monocytogenes. The hot dogs are dipped into suspensions
containing
either (1) NovasinTM, lysozyme and BHA; or (2) NovasinTM and BHA, and then
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inoculated with L. monocytogenes on the surfaces. The hot dogs are then packed
in
sterile bags and kept at 10°C for 13 days. On each sampling day, the
hot dogs are
rinsed with sterile saline and the resins are plated on Listeria select agar
to obtain a
Listeria count. Enrichment is performed for the hot dogs by transferring 1 ml
of the
rinse to BHI broth and incubating for 24 hr, followed by plating on Listeria
selective
agar. The treatments that have a 0 count on Listeria select agar after
enrichment are
considered as negative and reported as a fraction of total treated samples.
Table 2 shows that while NovasinTM and BHA dipping significantly reduces the
initial
levels of Listeria monocytogenes on hot dogs, the three component combination
reduces the levels of Listeria monocytogenes to practically undetectable
levels, either
by direct plating, or by enrichment recovery techniques. In this three
component
combination there is no observed survival of viable Listeria monocytogenes, as
confirmed by the negative results (3/3) after sensitive enrichment techniques
that are
used to recover low levels of damaged cells. The finding that all of the
samples treated
with the novel three component combination were negative for Listeria
monocytogenes
even after enrichment is the most unexpected finding of this application. No
such
finding has previously been reported using any one of these antimicrobial
ingredients
at the safe and suitable usage levels described herein.
Table 2
CFU/ hot
dog
Treatment day-0 day-2 day-13 Negative after
Enrichment
Control 5x 10e4 1.1 x 10e5 9.2 x 10e6 NA
NovasinTM + BHA 5 x 10e4 30 25 1/3
NovasinTM + BHA+ lysozyme 5 x 10e4 <10 <10 3/3
Example 3
As shown in Table 3, two gram positive spore formers of the Bacillus genus
were
tested in Trypticase soy broth, pH 6.0 at 30C for 48 hours to show the
inhibitory effect
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of the Novasin, lysozyme and beta hops acid either alone or in combination.
The test
demonstrates the surprisingly synergistic effect of the Novasin, lysozyme and
beta
hops acid combination against spore formers. Table 3 shows that at the same
concentration, the combination of Novasin, lysozyme and BHA shows
significantly
better inhibition than any ingredient alone or with only two components out of
three.
The three way combination gives a complete kill of all the inoculated spores,
while
significant but not complete elimination is observed for single component or
two
component treatments. Thus, it is here conclusively demonstrated that a
composition
of Novasin, lysozyme and BHA demonstrates synergistic bactericidal activity
against
spore forming bacteria.
Table 3
Treatment CFU/ml
B. subtilis B. cereus
Control 2.5 x 10e8 1.1 x 10e8
50 ppm Novasin 3.2 x 10e4 6.4 x 10e6
1 ppm BHA 360
5 x 10e3
100 ppm Lysozyme 1.2 x 10e8 1.1 x 10e8
50 ppm Novasin+1 ppm BHA 40 950
50 ppm Novasin, 1 ppm BHA
and 50 ppm Lysozyme <10 <10
Example 4
Efficacy of the inventive composition for cooked hams is demonstrated in Table
4.
Cooked hams were inoculated with Listeria monocytogenes on the surface, and
then
topically treated by spraying the surface with a solution of either NovasinTM,
lysozyme
and BHA (Treatment A); or lysozyme and BHA (Treatment B); . The hams were then
shrink wrapped and vacuum packed in sterile bags and stored at 40F (4°
C) for 60
days. On each sampling day, the hams were rinsed with sterile buffer and the
resins
were plated on Listeria select agar to determine the viable Listeria count.
Enrichment
methods were performed for samples with Listeria count below detectable plate
count
levels by transferring 1 ml of the rinse to brain heart infusion (BHI) broth
and incubating
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for 24 hr, followed by plating on Listeria selective agar. Treatments that had
a negative
(less than 1/m1) count on Listeria select agar after enrichment are considered
as
negative and reported as a fraction of total treated samples.
Table 4 shows that the three component combination reduced the inoculated
Listeria
monocytogenes to undetectable levels, either by direct plating, or by
enrichment
recovery techniques while the control group increased 10,000 fold (4 logs).
The two
way "lysozyme plus BHA" combination showed inhibition but was not as
bactericidal.
In the three component combination there is no observed survival of viable
Listeria
monocytogenes, as confirmed by the negative results after enrichment, a
technique
that is used to recover very low levels of viable or damaged cells. Therefore,
the data
demonstrate that topical application of the preferred three component
composition can
completely eliminate Listeria monocytogenes in a high fat processed food such
as
cooked ham.
Table 4
Listeria counts CFU/ ham pack
Day Control Treatment A Treatment B
1 250 0 50
7 280 0 200
15 290 0 160
6100 0 200
45 6600 0 4600
25 60 20000 0 5500
Example 5
Efficacy of the inventive composition for protecting wieners from Listeria.
monocytogenes is demonstrated in Table 5. The composition of Novasin and BHA
was
delivered onto the surface of wieners by incorporation of the composition into
the
cellulosic casing that contains the hotdog emulsion during cooking. After
cooking, the
casings were peeled off, and then the finished wieners were surface inoculated
with
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Listeria monocytogenes and vacuum packed in sterile bags. The packaged,
inoculated
wieners were stored at 40°F (4 C) for over 60 days, with fresh packages
opened for
each sampling point. At each sampling, the wieners were rinsed along with
their
package using sterile buffer and the buffer solutions were then plated on
Listeria select
agar to determine the viable Listeria count.
Table 5 shows that the two component combination inhibited growth of Listeria
monocytogenes for more than 61 days, and resulted at the end of incubation in
a
greater than 4 log (10,000 fold) Listeria reduction compared to the controls.
The data
demonstrate that casing delivered application of the preferred two component
composition prevents the outgrowth of Listeria monocytogenes on wieners and
may be
a commercially practical way to deliver the composition and thereby improve
the safety
of wieners and sausages. More importantly, the surface application method
permits
the activity of the components to be fully realized, even in the high fat
environment of a
typical hot dog emulsion. In contrast, antimicrobial activity is dramatically
reduced
when the composition is employed as an ingredient within the hotdog emulsion
prior to
cooking (data not shown), consistent with the observations by previous authors
that
demonstrate the loss of nisin activity (Muriana, P.M. and Kanach, L, Use of
Nisaplin to
Inhibit Spoilage Bacteria in Buttermilk Ranch Dressing, J. Food Protection,
Vol. 58, No
10, 1995) and hops acid activity (Johnson et al., 1996) in foods containing
significant
fat levels. Therefore, the disclosed method of surface application reveals a
surprising
way to maximize efficacy of the inventive composition in foods containing
elevated fat
levels (more than 4% w/w).
Table 5
Listeria counts loq CFU/
package
Days Wiener made with control casing Wiener made with treated casing
0 2.72 2.72
7 2.51 1.8
27 3.66 1.92
33 4.3 1.84
4.34 2.23
35 54 5.48 3.43
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61 6.83 2.08
Having described the invention in detail and by reference to the preferred
embodiments thereof, it will be apparent that modifications and variations are
possible
without departing from the scope of the appended claims.
19
