Broad spectrum prevention and removal of microbial contamination of food by quaternary ammonium compounds

BACKGROUND OF THE INTENTION

1.

Field of Invention

0001The present invention relates generally to a method of preventing the growth of a broad range of microorganisms on food products. More specifically, the present invention relates to a method of using quaternary ammonium compounds (QACs) for preventing the growth of a broad spectrum of microorganisms on seafood products for example, fish and shellfish and any other seafood products that can be treated utilizing the aqueous treatment methods of the present invention without detrimentally affecting the appearance, texture, and quality of the seafood. More specifically, the present invention relates to a method of using QACs to inhibit the attachment of, to remove, and to prevent the growth of microorganisms on seafood products. Particularly, the use relates to the effect of QACs on microorganisms that can cause foodborne contamination. More particularly, these microorganisms include microorganisms from the genus <i>Staphylococcus, Campylobacter, Arcobacter, Listeria, Aeromonas, Bacillus, Salmonella</i>, non-toxin-producing <i>Escherichia</i>, and pathogenic toxin-producing <i>Escherichia</i>, such as 0157:H7. More particularly, the present invention relates to an improved treatment method of spraying QACs on the food products to prevent broad spectrum microbial growth on these products. The present invention also makes use of a formulation of QACs that makes the treatment method more amenable for commercial use in a food processing plant.

2.

Description of the prior art

0002Prevention of foodborne illnesses by microbial contamination is of major concern to the food processing industry, regulatory agencies, and consumers. A recent report from the Food Safety &amp; Inspection Service (PSIS) of the United States Department of Agriculture (Federal Register, February 3, 1995) estimates that over 2 million cases of foodborne illnesses are produced annually by microbial contamination in the United States, with an associated cost of over $1 billion. Foodborne microbial contamination occurs both prior to entry into the processing facility, and by cross-contamination in the processing environment. The FSIS has instituted new Hazard Analysis and Critical Control Point (HACCP) requirements to reduce the occurrence and number of foodborne pathogens. These regulations must be met by food processors. Although the means of achieving this microbial reduction is left to the discretion of the processor, FSIS expects that antimicrobial treatments will be an important component of HACCP plans. The treatment methods of the present invention, which employ aqueous formulation of QACs, are useful in meeting the HACCP requirements.

0003In their efforts to provide a product completely free of microbial contamination, poultry and meat processors have encountered major difficulties in removing microorganisms that adhere or attach vigorously to poultry and meat tissues intended as food products. If contaminating microorganisms do not attach to the surface of the food, they can be easily rinsed off. However, the microorganisms that become strongly attached cannot be removed by rinsing and are quite resistant to removal by chemical or physical means.

0004Several chemical and physical methods have been proposed to reduce microorganisms in meat products, such as the use of chlorine or chlorine dioxide, ozone, hydrogen peroxide, lactic acid, sodium carbonate, trisodium phosphate, and electrical stimulation. Generally, these methods have shown limited effectiveness in reducing microbial contamination and may affect the physical appearance of the meat products.

0005<i>Salmonella typhimurium</i> contamination has been of special concern to the poultry processing industry because the organism is often present on live birds. Poultry processors have had great difficulty in removing microorganisms, such as <i>S. typhimurium,</i> that attach or adhere to poultry tissues. A variety of chemical and physical approaches have been suggested for use during poultry processing to eliminate <i>S. typhimurium</i> contamination of carcasses and minimize cross-contamination among carcasses. Trisodium phosphate (TSP) has been utilized in poultry processing for suppressing <i>S</i>. <i>typhimurium;</i> however, studies report conflicting results on the efficacy of TSP against <i>Salmonella.</i> As a result of its water solubility, TSP can be washed off of the poultry and thus, cannot inhibit attachment of microorganisms.

0006<patcit id="pcit0001" dnum="US5366983A"><text>U.S. Patent No. 5,366,983</text></patcit> discloses a method for removing or preventing <i>Salmonella</i> contamination of meat products by treatment with an effective amount of an aqueous solution of a QAC. Specifically, quaternary ammonium cationic surfactants, such as alkylpyridinium, particularly cetylpyridinium chloride (CPC) and cetylpyridinium bromide (CPB) were effective in removing <i>S. typhimurium</i> from poultry. This patent, however, does not disclose that QACs have a broader antimicrobial spectrum against any other genuses of food contaminating microorganisms than <i>Salmonella</i>. Further, it does not suggest that this treatment method would be effective on food products other than meat.

0007Food substances differ chemically and physically by virtue of their protein content, porosity, lipophilicity, surface pH, water permeability, surface area, and surface net electrical charge. Porosity of food could be important in the sequestration of bacteria whereas a tough, impermeable integument on a food substance could reduce bacterial contamination of the food. All of these chemical and physical differences among food products make it difficult to predict whether one antimicrobial agent's success on meat products would suggest success on other food products, such as fruit, vegetables, and seafood.

0008For example, CPC is known to bind to proteins; however, if the antimicrobial efficacy of CPC on food products were due in large part to the protein binding then the present method for treating non-proteinaceous fruits and vegetables would not have been expected to be successful.

0009Increasingly, foodborne illnesses caused by other pathogenic and spoilage bacteria than <i>Salmonella</i> have become a problem for food processors. A list of these bacteria with the products in which they have been identified is presented in Table 1: <tables id="tabl0001" num="0001"><table frame="all"><title><b>TABLE 1</b></title><tgroup cols="6"><colspec colnum="1" colname="col1" colwidth="40mm" /><colspec colnum="2" colname="col2" colwidth="15mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><colspec colnum="4" colname="col4" colwidth="12mm" /><colspec colnum="5" colname="col5" colwidth="19mm" /><colspec colnum="6" colname="col6" colwidth="18mm" colsep="0" /><thead><row><entry namest="col1" nameend="col6" align="center" valign="top"><b>INCIDENCE OF PATHOGENIC AND SPOILAGE BACTERIA</b></entry></row><row><entry valign="top">Microorganism</entry><entry valign="top">Poultry</entry><entry valign="top">Beef</entry><entry valign="top">Pork</entry><entry valign="top">Pathogen</entry><entry valign="top">Spoilage</entry></row></thead><tbody><row><entry><i>Aeromonas hydrophila</i></entry><entry>X</entry><entry>X</entry><entry align="center">X</entry><entry /><entry>X</entry></row><row><entry><i>Arcobacter butzleri</i></entry><entry /><entry>X</entry><entry>X</entry><entry>X</entry><entry colsep="1" /></row><row><entry><i>Bacillus cereus</i></entry><entry>X</entry><entry>X</entry><entry>X</entry><entry>X</entry><entry colsep="1" /></row><row><entry><i>Campylobacter</i> jejuni</entry><entry>X</entry><entry>X</entry><entry>X</entry><entry>X</entry><entry colsep="1" /></row><row><entry><i>Escherichia coli 0157:H7</i></entry><entry>X</entry><entry>X</entry><entry>X</entry><entry>X</entry><entry colsep="1" /></row><row><entry><i>Listeria monocytogenes</i></entry><entry>X</entry><entry>X</entry><entry>X</entry><entry>X</entry><entry colsep="1" /></row><row><entry><i>Salmonella typhimrium</i></entry><entry>X</entry><entry>X</entry><entry>X</entry><entry>X</entry><entry colsep="1" /></row><row><entry><i>Staphylococcus</i> aureus</entry><entry>X</entry><entry>X</entry><entry>X</entry><entry>X</entry><entry colsep="1" /></row></tbody></tgroup></table></tables>

0010Among these contaminating microorganisms listed in the table, <i>Escherichia coli</i> 0157:H7 is of special concern because of its virulence, severity of the illness produced, and associated mortality. <i>E</i>. <i>coli</i> 0157:H7 produces strong "shiga-like" toxins that lead to blood clotting abnormalities, kidney failure (hemolytic uremic syndrome), and death. Even if recovery from the acute illness is complete, 15-30% of infected people with hemolytic uremic syndrome will have evidence of chronic kidney disease. The risks associated with contamination with <i>E. coli</i> 0157:H7 are compounded by its reported resistance to antibiotics. In 1993, between 8,000-16,000 cases of foodborne illnesses were produced by <i>E</i>. <i>coli</i> 0157:H7 with an estimated cost of between 0.2 and 0.5 billion dollars.

0011Another virulent food contaminant, <i>Listeria monocytogenes</i> has been found in meat, vegetables, and various milk products; and may cause sepsis, meningitis, and disseminated abscesses. <i>L. monocytogenes</i> is a cold tolerant microorganism capable of growing under refrigeration. In 1993, about 1,700 cases of foodborne illness were produced by <i>L. monocytogenes</i> with an estimated cost of between 0.1 and 0.2 billion dollars.

0012Another microorganism of concern in the food industry is <i>Aeromonas hydrophila</i> which causes spoilage in the food and meat processing industry and reduces the shelf life of these products.

0013Presently, there are no known microbicidal compounds which are effective at preventing and removing contamination in a broad range of food products against a broad spectrum of gram positive, gram negative, aerobic, facultative anaerobic, and microaerophilic microorganisms. The present inventors have determined that QACs are effective against a broad spectrum of different microorganisms which produce foodborne illnesses when they become attached to a broad range of food products. This sensitivity of a broad spectrum of pathogenic microorganisms could not have been predicted.

0014Sensitivity of a microorganism to a particular antimicrobial agent is not predictive of the sensitivity of other microorganisms to the same agent. It is believed that antiseptics or germicides have a continuous spectrum of activity but the relative susceptibilities of different microorganisms must be considered. For example, the germicide, hexachlorophene is primarily effective against Gram positive microorganisms, and cationic antiseptics are not effective against sporulating organisms. Some Gram negative microorganisms, such as <i>Pseudomonas cepacia</i>, have been known to grow in solutions of the drug, benzalkonium chloride. Other bacteria have been known to be capable of growing in 70% ethanol (<nplcit id="ncit0001" npl-type="b"><text>Harvey, S.C., Antimicrobial Drugs in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., pp. 1163-1241 1990</text></nplcit>).

0015In regard to the treatment of food products, it has been reported that Listeria is more resistant to the action of TSP than <i>Salmonella</i> or <i>E. coli</i> (<nplcit id="ncit0002" npl-type="s"><text>Somers, E.B. et al., Int. J. Food Microbiol., 22:269-276, 1994</text></nplcit>). Further, (<nplcit id="ncit0003" npl-type="s"><text>Breen et al., J. Food Sciences, 60:1991-1996, 1995</text></nplcit>) demonstrated that TSP is much less effective in inhibiting <i>Salmonella</i> growth than it is in detaching this organism. Similarly, TSP has reduced the numbers of <i>E. coli</i> 0157:H7 on chicken carcasses but is ineffective in inhibiting the cross-contamination of this microorganism to other chickens.

0016The present invention shows that QACs are effective against <i>E. coli</i> 0157:H7 in suspension in liquids, in reducing the numbers of this bacteria when it is attached to food products, as well as in inhibiting the attachment of this bacteria to food products. It has been reported that <i>E. coli</i> 0157:H7 shows resistance towards broad spectrum antimicrobial agents, such as tetracycline, streptomycin, sulfisoxazole (<nplcit id="ncit0004" npl-type="s"><text>Kim et al., J. Infect. Dis., 170:1606-1609, 1994</text></nplcit>) and oxytetracycline (<nplcit id="ncit0005" npl-type="s"><text>Ciosek et al., Med. Weter. 40:335,338:1984</text></nplcit>), whereas these same agents are very active against regular non-toxin-producing strains of <i>E. coli.</i>

0017Clearly the effectiveness of an antimicrobial agent or biocide against a particular microorganism cannot be predicted based upon its effectiveness against a different microorganism. There are many factors to consider, such as microbial characteristics which may play a role in the effectiveness of an antimicrobial agent against a particular microorganism. These characteristics include but are not limited to: (1) the degree of glycocalyx formation by a given species of attached microorganism, (2) the presence of a lipopolysaccharide- and phospholipid-containing cell envelope in gram negative bacteria, (3) the presence of lipoprotein as in most enteric bacteria and <i>Pseudomonas</i>, and (4) the presence of porin protein channels, for example in <i>E</i>. <i>coli</i> and <i>salmonella</i> (<nplcit id="ncit0006" npl-type="s"><text>Fulton et al., Structure in Medical Microbiology, 3rd Ed., pp. 37-54, 1991</text></nplcit>).

0018<nplcit id="ncit0007" npl-type="s"><text>Kim, J.W. et al.: "Cetylpyridinium Chloride (CPC) treatment on poultry skin to reduce attached Salmonella", J. Food Prot., 59(3), March 1996, pages 322-326</text></nplcit>; <nplcit id="ncit0008" npl-type="s"><text>Database FSTA, International Food Information Service (IFIS), Frankfurt a/Main, Germany, 1995, "Reduction of Salmonella typhimurium on chiken carcasses using pre-chill spraying</text></nplcit>", Acc. No. 95-1-12-s0149; <nplcit id="ncit0009" npl-type="s"><text>Yanbin, Li, et al.: "Pre-chill spray of chicken carcasses to reduce Salmonella typhimurium", J. Food Sci. 62(3), March 1997, page 605</text></nplcit>; and "<nplcit id="ncit0010" npl-type="b"><text>Effects of spraying temperature, pressure and time on reduction of Salmonella typhimurium on chicken skins using pre-chill chemical spraying", 1996 IFT Annual Meeting book of abstracts, USA 1996, page 60 </text></nplcit> disclose the use of cetylpyridinium chloride (CPC) for preventing or removing microbial contamination in chicken skin or chicken carcasses by application of a CPC solution by immersion or spraying.

0019<nplcit id="ncit0011" npl-type="s"><text>Similarly, Database BIOSIS, Biosciences Information Service, Philadelphia USA, August 1996, Compadre, Cesar M. et a/.: "Cetylpyridinium chloride inhibits and reduces attachment of Escherichia coli 0157:H7 and Salmonella typhimurium to meat</text></nplcit>", Acc. No. PREV199699170542 discloses the use of cetylpyridinium chloride for preventing attachment of <i>E. coli</i> and <i>Salmonella typhmurium</i> to "meat and other food products".

0020The food processing industry is in need of a more effective process for the prevention of growth of a broad range of contaminating microorganisms on many different food products. This is especially true for microorganisms which are attached to the surfaces of food. As a result of increasing numbers of illnesses caused by foodborne pathogenic microorganisms, the food processing industry now requires more effective processes for the removal and prevention of a broader spectrum of microorganisms, and particularly for pathogenic microorganisms, such as, toxin-producing <i>Escherichia</i>, i.e., <i>E</i>. <i>coli</i> 0157:H7, which are known to cause serious human diseases as a result of food contamination. The present inventors have provided a method of preventing the growth of microorganisms in liquids associated with food products, an important goal in preventing cross-contamination inside the processing plant; in removing attached microorganisms from food products, in inhibiting the attachment of microorganisms to the food products; and in preventing the growth of microorganisms that remain attached to the food products. Further, the method of the present invention can easily be adapted for use in a food processing plant.

0021Additionally, a concentrated QAC formulation is disclosed for use in dilution to a working solution for use in the present method. A formulation for use in the present invention contains solubility enhancing components which may also result in longer contact times of the formulation with the food product.

<u style="single">SUMMRY OF THE INVENTION</u>

0022The present invention relates to a method for preventing the growth of microorganisms on seafood comprising: <ul id="ul0001" list-style="none" compact="compact"><li>contacting said food product with approximately 50 to 20,000 parts per million of an aqueous solution of a quaternary ammonium compound for less than 90 seconds to prevent the growth of the microorganisms on the seafood, wherein said quaternary ammonium compound is selected from the group consisting of an alkylpyridinium salt represented by the structural formula (I): <chemistry id="chem0001" num="0001"><img file="EP1595457B1_D0001.tif" /></chemistry></li></ul> wherein n is 9-21; and X is a halide, a tetra-alkylammonium salt represented by the structural formula (II): <chemistry id="chem0002" num="0002"><img file="EP1595457B1_D0002.tif" /></chemistry> wherein n is 9-21; R is selected from the group consisting of CH₃ and C₂H₅; and X is a halide, and an alkylalicyclic ammonium salt.

0023The present invention provides a method for the prevention of growth of a broad spectrum of microorganisms on seafood products. The prevention of growth of microorganisms on seafood products is intended to provide a food product that is devoid of or contains minimal numbers of viable microorganisms that could cause illness in humans or animals or spoilage of the food product prior to ingestion. The prevention of growth of microorganisms on food products is intended to include but is not limited to the following mechanisms: (1) removal of attached microorganisms from the food products; (2) inhibition of attachment of microorganisms to the food products; (3) killing or inactivation of attached microorganisms on the food products; and (4) killing or inactivation of microorganisms which are not attached to the food product but which are present in liquids associated with the food products during processing; such as in chill tanks.

0024The microorganisms which are susceptible to QACs include microorganisms from the genus <i>Staphylococcus, Campylobacter, Arcobacter, Listeria, Aeromonas, Bacillus, Salmonella</i>, non-toxin-producing <i>Escherichia</i>, pathogenic toxin-producing <i>Escherichia</i>, and other foodborne microorganisms which are capable of causing microbial foodborne contamination of food for human or animal consumption.

0025Additional microorganisms which are also susceptible to QACs are fungi, such as, <i>Aspergillus flavum</i> and <i>Penicillium chrysogenum</i>, and parasites, such as <i>Entamoeba histolytica</i>.

0026The composition of the present invention comprises a microbial·growth inhibiting effective amount of QAC in an aqueous solution. QACs of the present invention are effective in preventing the growth of a broad spectrum of pathogenic and spoilage microorganisms. QACs, particularly cetylpyridinium chloride (CPC), are especially effective to prevent the growth of a broad spectrum of microorganisms on a broad range of food products.

0027The present invention has an important application in the food processing industry, as well as for home use. QACs are readily available and the cost of carrying out the method of the present invention is not expensive as compared to existing antimicrobial processes. Unlike existing treatments using, for example, TSP, the use of QACs does not alter the appearance, color, taste, or texture of the food product. A range of concentrations of QACs are effective in preventing broad spectrum microbial growth on food products. QACs are safe as shown by the lack of mutagenicity of CPC using the Ames assay. Further, CPC is already approved for human use in products for oral ingestion in preparations, such as Cepacol^® lozenges which are orally ingested in amounts up to 20 mg per day.

0028The present invention also enables a formulation of QAC for use with the present method for the treatment of seafood products, in which for example, CPC is formulated with solubility enhancing agents, such as ethyl alcohol and/or glycerin.

0029The present invention is directed to an improved method of contacting seafood products with the QAC for a period of time of less than 90 seconds, even as short as 20 to 90 seconds, which results in significant prevention of growth of microorganisms on the seafood products.

0030The invention also includes an improved method of contacting QACs with seafood products by spraying the compound on the seafood product. The spraying method can be performed using the QAC in solution in water or using the new formulation with the QAC formulated with solubility enhancing agents.

0031Further, the method of the present invention can optionally include a determination step prior to contacting the seafood product with the QACs to determine the presence of microorganisms on the food before treatment. Any conventional methods for rapidly determining the presence of microorganisms can be utilized as the determination step, which for example, includes PRC and immunoassays.

0032Additionally, the method of the present invention can optionally include a step to determine the presence of QACs on the surface of the food product after contact with the QACs. This determination can be performed immediately after the contacting step or after several washing steps. For example, the QAC can be extracted from the tissues of the food in a form suitable for high performance liquid chromatography (HPLC) analysis. The method comprises ethanol extraction of the food tissue followed by solid-phase extraction using a weak cationic exchange column that selectively separates QACs from other compounds in the matrix that would otherwise interfere with the HPLC analysis. The RPLC assay for quantitation of QAC residues employs a reverse phase cyano column and uses a QAC analog as an internal standard.

<u style="single">BRIEF DESCRIPTION OF THE DRAWINGS</u>

0033<ul id="ul0002" list-style="none" compact="compact"><li><figref idref="f0001">Fig. 1</figref> is a bar graph showing the reduction of viable microorganisms on catfish skin after treatment with CPC in 5% aqueous glycerin on non-selective media.</li><li><figref idref="f0002">Fig. 2</figref> is a bar graph showing the reduction of viable <i>S</i>. <i>typhimurium</i> on catfish skin after treatment with CPC in 5% aqueous glycerin on selective media.</li></ul>

<u style="single">DETAILED DESCRIPTION OF THE INVENTION</u>

0034The present invention is based upon the determination that QACs can be used to treat a broad range of food products to reduce a broad spectrum of foodborne microbial contamination on these products. The present invention is also based upon the finding that QACs are effective in removing, killing, inactivating and inhibiting the attachment of a broad range of foodborne pathogenic microorganisms to food products. These microorganisms include but are not limited to bacteria belonging to the genuses, <i>Salmonella, Staphylococcus, Campylobacter, Arcobacter, Listeria, Aeromonas, Bacillus</i>, non-toxin-producing <i>Escherichia,</i> and the virulent toxin-producing <i>Escherichia</i> strains, such as <i>E. coli</i> 0157:H7; fungi, such as <i>Aspergillus flavus</i> and <i>Penicillium chrysogenum</i>; and parasites, such as <i>Entamoeba histolytica</i>.

0035The compositions used in present invention comprise an effective amount of the QAC in an aqueous solution.

0036Alkylalicyclic ammonium salts are represented by the structural formula (III): <chemistry id="chem0003" num="0003"><img file="EP1595457B1_D0003.tif" /></chemistry> wherein n is 9-21; Z is 4-5; R is selected from the group consisting of CH₃ and C₂H_n; and X is a halide.

0037A variety of QACs, all of which are cationic surface-active agents; i.e., surfactants, were evaluated for their effectiveness in removing attached microorganisms from various foods as well as in inhibiting the attachment of the microorganisms. Of the QACs studied, cetylpyridinium chloride (CPC) was the most effective and will be utilized in the examples set forth below but it not intended to limit the use of QACs to CPC within the meaning of the present invention because other members of QACs also have similar properties against the foodborne pathogenic microorganisms.

0038The present invention is further based on the determination that the contact time of QACs with the seafood products in the immersion process can be reduced to 1 minute yet still result in significant inhibition of microorganism attachment, for foodborne microorganisms including <i>Salmonella</i>, which is a significant improvement and a commercial advantage in the industrial use of this process.

0039The present invention is also based on the determination that a new method of spraying QACs under various pressures on the food products for 20 to 90 seconds significantly reduces the viable foodborne microorganisms on these products.

0040The present invention is also based on the determination that a new formulation of QACs in solutions containing varying concentrations of at least one solubility enhancing agents, such as ethyl alcohol or glycerin, is useful for treatment of food products. This is particularly so when the QAC solutions are to be used in combination with salt, are highly concentrated, or will be subject to cold temperatures, such as inside food processing plants. This new formulation allows concentrated QAC to be stored and then easily diluted for use in the antimicrobial treatment process in the processing plant rather than requiring powder QACs to be mixed prior to using. This new formulation of QACs provides an easy to use concentrate which is advantageous for industrial use. This new QAC formulation can be used in both the standard immersion method or the new spraying method.

0041The above described aspects of the present invention are described in detail below with reference to <figref idref="f0001">Figs. 1</figref>-5.

0042The examples set forth below serve to further illustrate the present invention in its preferred embodiments, and are not intended to limit the present invention. The examples utilize poultry, beef, catfish, broccoli, and grapes as the food products treated in the method, but it is intended that the treatment of other food products which would not be adversely affected by the treatment process are also intended to be encompassed by the present invention.

<u style="single">EXAMPLES</u>

0043The microorganisms utilized in the following examples are as follows: <i>Staphylococcus aureus</i> ATCC 29213, <i>Campylobacter jejuni</i> ATTC 29428, <i>Escherichia coli</i> (non-toxin producing strain) ATCC 25922; <i>Escherichia coli</i> 0157:H7 (toxin-producing strain) ATCC 43895, <i>Arcobacter butzleri</i> ATCC 49616, <i>Listeria monocytogenes</i> ATCC 49594, <i>Aeromonas hydrophila</i> ATCC 49140, <i>Bacillus cereus</i> ATCC 49063, <i>Salmonella typhimurium</i> ATCC 14028 and NCTC 12023, and commercially available cultures of <i>Aspergillus flavus</i> and <i>Penicillium chrysogenum</i>.

Example 1

Bactericidal Activity of Quaternary Ammonium Compounds in Suspension Cultures (Not attached to meat products)

Minimum inhibitory Concentration (MIC) of Quaternary Ammonium Compounds

0044Minimum inhibitory concentrations (MIC) for QAC were determined in Mueller Hinton broth (BBL Microbiology System) using the macrodilution method established by the 1987 National Committee for Clinical Laboratory Standards. Experiments were conducted by 16 hour incubation at 37°C for <i>Staphylococcus aureus</i>, <i>Escherichia coli</i> 0157:H7, <i>Listeria monocytogenes</i>, and <i>Salmonella typhimurium</i>. For <i>Aeromonas hydrophila</i>, and <i>Bacillus cereus</i> incubations were performed at 30°C . MIC were determined by the lowest dilution with no visible turbidity. Table 2 shows the data from the above experiment: <tables id="tabl0002" num="0002"><table frame="all"><title><b>TABLE 2</b></title><tgroup cols="7"><colspec colnum="1" colname="col1" colwidth="38mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="18mm" /><colspec colnum="4" colname="col4" colwidth="18mm" /><colspec colnum="5" colname="col5" colwidth="26mm" /><colspec colnum="6" colname="col6" colwidth="23mm" /><colspec colnum="7" colname="col7" colwidth="31mm" /><thead><row><entry namest="col1" nameend="col7" align="center" valign="top"><b>MINIMAL INHIBITORY CONCENTRATION (MIC)</b></entry></row><row valign="middle"><entry align="center">Cetylpyridiniumchloride (CPC) µg/mL</entry><entry align="center">CPC vs <i>E</i>. <i>coli</i> 0157:H7</entry><entry align="center">CPC vs <i>B</i>.<i>cereus</i></entry><entry align="center">CPC vs <i>S</i>.<i>aureus</i></entry><entry align="center">CPC vs <i>S.typhimurium</i></entry><entry align="center">CPC vs <i>A.hydrophila</i></entry><entry align="center">CPC vs <i>L.monocytogenes</i></entry></row></thead><tbody><row><entry>125</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry></row><row><entry>62.5</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry></row><row><entry>31.25</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry></row><row><entry>15.63</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry></row><row><entry>7.81</entry><entry align="center">+</entry><entry align="center">-</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry></row><row><entry>3.91</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry></row><row><entry>1.96</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry></row><row><entry>0.98</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry></row><row><entry>0.50</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry></row><row><entry>0.25</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">-</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry></row><row><entry>0.00</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry><entry align="center">+</entry></row><row><entry namest="col1" nameend="col7" align="left">(-) No growth (+) Growth MICs were obtained by the macrodilution broth method (National Commitee for Clinical Laboratory Standards).</entry></row></tbody></tgroup></table></tables>

Minimum bactericidal concentration (MIC) of Quaternary Ammonium Compounds

0045Minimum bactericidal concentrations (MBC) for QAC towards <i>Campylobacter jejuni</i> and <i>Arcobacter butzleri</i> were determined in Mueller Hinton broth (BBL Microbiology System) using the macrodilution method established by the 1987 National Committee for Clinical Laboratory Standards. Experiments were conducted by microaerophilic incubation at 37°C for 48 hours. An aliquot of each dilution was pour plated in agar and incubated in microaerophilic conditions at 37°C for 48 hours. MBCs were determined as the lowest dilution with no growth. Table 3 shows the data from the above experiment: <tables id="tabl0003" num="0003"><table frame="all"><title>TABLE 3</title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="69mm" /><colspec colnum="2" colname="col2" colwidth="48mm" /><colspec colnum="3" colname="col3" colwidth="43mm" /><thead><row><entry namest="col1" nameend="col3" align="center" valign="top">MINIMAL BACTERICIDAL CONCENTRATION (MBC)</entry></row><row><entry align="center">Cetylpyridinium Chloride, µg/mL</entry><entry align="center" valign="top">CPC vs <i>Campylobacter jejuni</i></entry><entry align="center" valign="top">CPC vs <i>Arcobacter butzleri</i></entry></row></thead><tbody><row><entry align="char" char="." charoff="8">125</entry><entry align="center">-</entry><entry align="center">-</entry></row><row><entry align="char" char="." charoff="8">62.5</entry><entry align="center">-</entry><entry align="center">-</entry></row><row><entry align="char" char="." charoff="8">31.25</entry><entry align="center">-</entry><entry align="center">+</entry></row><row><entry align="char" char="." charoff="8">15.63</entry><entry align="center">-</entry><entry align="center">+</entry></row><row><entry align="char" char="." charoff="8">7.81</entry><entry align="center">-</entry><entry align="center">+</entry></row><row><entry align="char" char="." charoff="8">3.91</entry><entry align="center">+</entry><entry align="center">+</entry></row><row><entry align="char" char="." charoff="8">1.96</entry><entry align="center">+</entry><entry align="center">+</entry></row><row><entry align="char" char="." charoff="8">0.98</entry><entry align="center">+</entry><entry align="center">+</entry></row><row><entry align="char" char="." charoff="8">0.50</entry><entry align="center">+</entry><entry align="center">+</entry></row><row><entry align="char" char="." charoff="8">0.25</entry><entry align="center">+</entry><entry align="center">+</entry></row><row><entry align="char" char="." charoff="8">0.00</entry><entry align="center">+</entry><entry align="center">+</entry></row><row><entry namest="col1" nameend="col3" align="left">(-) No growth (+) Growth MBCs were obtained by the macrodilution broth method (National Committee for Clinical Laboratory Standards).</entry></row></tbody></tgroup></table></tables>

0046The MIC and MBC data shows that CPC is effective against a broad range of microorganisms.

Activity of Quaternary Ammonium Compounds in Planktonic Cells

0047A 16-hour culture of each of <i>E. coli</i> 0157:H7 in trypticase soy broth was centrifuged (15,000 rpm, 10 min, 4°C). After removal of the supernatant, the pellet was washed with 10 ml 0.04M potassium phosphate buffer (PPB, pH 7.0), and suspended in PPB to a final suspension of 1-2 x 10⁹ cells/ml. Aliquots (1.0 ml) were centrifuged (14,000 rpm, 3 min), and the supernatants were removed. Each pellet was suspended in either 1 ml of an aqueous solution of various concentrations (100-1,000 µg/ml) of test composition (CPC) or 1.0 ml of PPB, vortexed (30 sec), incubated for 1 min at 25°C, and centrifuged (14,000 rpm, 3 min). After removal of the supernatant, each pellet was suspended in 0.5 ml PPB. Cells from each sample were counted using duplicate 0.05 ml aliquots and standard serial dilution techniques on trypticase soy agar, and the data recorded as mean colony-forming units (CFU)/ml.

0048The results of the above experiment show complete reduction of viable <i>E. coli</i> 0157:H7 in suspension was achieved at all concentrations of CPC tested (100, 250, 500, and 1000µg/ml). The results of this experiment are particularly significant for the prevention of cross contamination with <i>E. coli</i> 0157:H7 in industrial processing of meat. As discussed above, this strain of toxin-producing <i>E. coli</i> shows resistance to many broad spectrum antimicrobial agents. These results provide evidence that treatment of meat products with QAC will prevent one contaminated piece of meat from contaminating other uncontaminated pieces because the QAC will kill the organism in the liquid which is the transfer agent responsible for the cross contamination.

Example 2

Effects of Quaternary Ammonium Compounds on the Reduction of viable <i>Salmonella typhimurium</i> attached to Catfish. Black Grapes, and Broccoli

0049The effects of CPC on the reduction of viable <i>S. typhimurium</i> on catfish, black grapes, and broccoli were studied. Test solutions comprised various concentrations of CPC (Sigma Chemical Co., St. Louis, MO) in 5% (v/v) glycerin in 0.008 M, pH 7.2 phosphate buffered saline (PBS). The solutions were prepared by dissolving the appropriate amounts of CPC in the glycerin-PBS mixture.

0050Food samples were small intact mushrooms, small intact black grapes, broccoli florets, and catfish skin squares (2.5 x 2.5 cm) excised from unprocessed, freshly thawed catfish. The fruit and vegetables were purchased from a local grocery, while the fish was shipped frozen from a local catfish supplier. The source of <i>S. typhimurium</i> was ATCC strain # 14028 or NCTC strain # 12023).

0051All colony counts were performed using Salmonella-selective XLD agar (DIPCO, Detroit, MI) plates. Additionally, in the catfish experiments, total aerobic colony counts were performed using a non-selective medium, tryptic soy agar (TSA:DIFCO, Detroit, MI). <i>Salmonella</i> storage was on TSA.

0052Inoculum preparation for <i>S. typhimurium</i> was performed as described in Example 7 above. Food samples were placed in each well of six-well tissue culture plates. The samples were then inoculated with 5 ml of PBS containing 1 to 2 x 10⁶ CFU of <i>S. typhimurium</i> per ml, with the exception of the background control group that was treated only with 5 ml of PBS. Culture plates with the food samples were incubated (30 min., 35°C), and then the incubating solution was removed by aspiration. The inoculated samples were treated with 5 ml of the test solution. Sets of three food samples were used for each concentration of test solution, including one set in which the food samples were treated only with 5 ml of 5% (v/v) glycerin in PBS (0 concentration). The plates were incubated at 25°C with shaking (100 rpm) for 3 min. After incubation, each food sample was prepared and placed in a plastic bag for use with the Stomacher^® 400 laboratory blender as described in Example 7 above. A corner of the bag was aseptically cut and the entire contents were transferred to a sterile centrifuge tube, which was then spun for 10 min (12,000 rpm, 20°C). The pellet was resuspended in 5 ml 0.1% (w/v) peptone/water. One ml of the appropriate dilution was pour plated onto XLD agar for the grape and broccoli experiments and onto both XLD and TSA agar for the catfish in triplicate. After incubation at 37°C for 24 hour, colonies were counted, corrected for dilution, and reported as CFU/skin for catfish and as CFU/gram for the other food samples. The results of these experiments are shown in <figref idref="f0001">Figs 1</figref> and <figref idref="f0002">2</figref>. As the catfish were not irradiated, <figref idref="f0001">Fig. 1</figref> shows the total aerobic bacterial count on non-selective media whereas <figref idref="f0002">Fig. 2</figref> shows only <i>Salmonella</i> counts.

Example 3

Effect of Quaternary Ammonium Compounds on Foodborne Fungi

0053This study tested the effect of CPC on foodborne fungi. Slant cultures of <i>Aspergillus flavus</i> and <i>Penicillium chrysogenum</i> were streaked onto a potato dextrose agar (PDA) plates. Thirty minutes after inoculation or 24 h after inoculation (and incubation at room temperature, two round filters (7 mm in diameter) were put on the surface of each plate. CPC solutions of 200 ppm, 1000 ppm, 5000ppm, and 25,000 ppm or distilled and deionized (DD) water were added to the filters, 10µl per filter. All plates were incubated lid side up at room temperature for 48 hours. The diameters of the inhibition rings were measure. The results presented below show that CPC is effective against foodborne fungi. <tables id="tabl0004" num="0004"><table frame="all"><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="44mm" /><colspec colnum="2" colname="col2" colwidth="36mm" /><colspec colnum="3" colname="col3" colwidth="32mm" /><thead><row><entry namest="col1" nameend="col3" align="left" valign="middle"><b>Effect of CPC on <i>Aspergillus flavus2</i></b></entry></row><row><entry align="center" valign="middle">concentration of CPC (ppm)</entry><entry namest="col2" nameend="col3" align="center" valign="middle">Inhibition Ring (mm)</entry></row><row><entry align="center" valign="middle" /><entry align="center" valign="middle">Immediate Treatment</entry><entry align="center" valign="middle">Delayed Treatment</entry></row></thead><tbody><row><entry align="right" valign="middle">25,000</entry><entry align="center" valign="middle">1.63</entry><entry align="center" valign="middle">1.00</entry></row><row><entry align="right" valign="middle">5,000</entry><entry align="center" valign="middle">2.00</entry><entry align="center" valign="middle">0.92</entry></row><row><entry align="right" valign="middle">1,000</entry><entry align="center" valign="middle">0.38</entry><entry align="center" valign="middle">1.00</entry></row><row><entry align="right" valign="middle">200</entry><entry align="center" valign="middle">0.25</entry><entry align="center" valign="middle">0.33</entry></row><row><entry align="right" valign="middle">0</entry><entry align="center" valign="middle">0</entry><entry align="center" valign="middle">0</entry></row></tbody></tgroup></table></tables><tables id="tabl0005" num="0005"><table frame="all"><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="44mm" /><colspec colnum="2" colname="col2" colwidth="36mm" /><colspec colnum="3" colname="col3" colwidth="32mm" /><thead><row><entry namest="col1" nameend="col3" align="left" valign="top"><b>Effect of CPC on <i>Penicillium chrysogenum</i></b></entry></row><row><entry align="right" valign="top">Concentration of CPC (ppm)</entry><entry namest="col2" nameend="col3" align="center" valign="top">Inhibition Ring (mm)</entry></row><row><entry align="right" valign="top" /><entry align="center" valign="top">Immediate Treatment</entry><entry align="center" valign="top">Delayed Treatment</entry></row></thead><tbody><row><entry align="right">25,000</entry><entry align="center">4.13</entry><entry align="center">1.83</entry></row><row><entry align="right">5,000</entry><entry align="center">3.38</entry><entry align="center">1.92</entry></row><row><entry align="right">1,000</entry><entry align="center">1.00</entry><entry align="center">1.67</entry></row><row><entry align="right">200</entry><entry align="center">0</entry><entry align="center">1.17 0</entry></row><row><entry align="right">0</entry><entry align="center">0</entry><entry align="center">0</entry></row></tbody></tgroup><tgroup cols="3" rowsep="0"><colspec colnum="1" colname="col1" colwidth="44mm" /><colspec colnum="2" colname="col2" colwidth="36mm" /><colspec colnum="3" colname="col3" colwidth="32mm" /><tbody><row><entry namest="col1" nameend="col3" align="justify">CPC is effective against foodborne fungi tested.</entry></row></tbody></tgroup></table></tables>

Formulations of Quaternary Ammonium Compounds

0054When using a composition in an industrial process, it is preferable to work with only small volumes of liquid concentrates rather than large volumes of liquid solutions. A formulation of QAC has been developed that allows QAC concentrations up to 1000-fold greater than those presently available in formulation made in water alone. This formulation which contains at least one solubility enhancing agent provides a soluble concentrate for easy dilution to the final concentration for use in large scale industrial processing. The solubility enhancing agent functions to maintain the solubility of the QAC so that it does not precipitate out of solution. Any compatible solubility agent can used but ethyl alcohol or glycerin are preferred. The formulation can contain ethyl alcohol, glycerin or both. This formulation contains approximately 100,000 ppm to about 300,000 ppm QAC, approximately 0% to about 49% ethyl alcohol and approximately 0 to about 20% glycerin in water. A preferred formulation contains approximately 150,000 ppm to about 250,000 ppm QAC, approximately 10% to about 40% ethyl alcohol and approximately 0.5 to about 10% glycerin in water. More preferably, the ethyl alcohol concentration can range from approximately 15% to about 30% and the glycerin concentration can range from approximately 0.5 to about 5%. Preferably, this formulation contains approximately 200,000 ppm QAC, approximately 20% ethyl alcohol, and approximately 1% glycerin. This formulation is particularly useful as a concentrate to be added to the storage tanks for use in immersion treatment of food products with QAC but it is also useful in a spraying method at a final concentration of approximately 5,000 ppm QAC.

0055A second formulation was developed to increase the contact time of a QAC solution on food products during processing, particularly when delivered by a spraying method. This formulation potentially allows a longer contact time of the QAC with the product without any additional steps which would increase the processing time. The formulation by virtue of its properties potentially increases the antimicrobial effectiveness of the process. This formulation preferably contains approximately 50 ppm to about 20,000 ppm QAC, and at least one of a solubility enhancing agent selected from approximately 0 to about 10% ethyl alcohol, and approximately 0 to about 20% glycerin or both. More preferably this formulation contains approximately 50 ppm to about 5,000 ppm, approximately 0 to about 10% ethyl alcohol, and approximately 1.0% to about 10% glycerin in water. Most preferably this formulation contains approximately 500 ppm to about 5,000 ppm QAC, 0 to 10% ethyl alcohol, and approximately 1.0 to about 5% glycerin in water and more preferably approximately 1.0 to about 3% glycerin.