A concentrated solution of quaternary ammonium compounds and methods of use

A NON-FOAMING CONCENTRATED SOLUTION OF QUATERNARY AMMONIUM COMPOUNDS AND METHODS FOR THEIR USE

Background of the invention:

This application is a partial continuation of US Application No. 08/840288, filed April 14, 1997, which is a partial continuation of US Application No. 08/631578 filed April 12, 1997. 1996, currently US Patent No. 5,855,940, both of which are incorporated herein by reference in their entireties.

1-Field of the invention:

The present invention relates to a solution comprising a concentrated amount of a quaternary ammonium antimicrobial compound (QAC). The QAC concentrate of the present invention uses GRAS (generally recognized as safe) components to obtain a true solution, not an emulsion, of QAC. This concentrated QAC solution is prepared in combination with at least one solubility-enhancing agent and is useful for preparing solutions to be diluted to a useful final concentration for use in industrial food processing or home food preparation. , and on surfaces related to food processing.

The present invention relates in general to a solution comprising a concentrated amount of an antimicrobial QAC and at least one solubility enhancing agent suitable for use in methods of preventing the growth of a wide range of microorganisms on food products and on them, as well as on the surfaces that come into contact with them in the domestic or industrial environment. More specifically, the present invention relates to an invention comprising a concentrated amount of an antimicrobial QAC and at least one solubility enhancing agent, suitable for use in a method for preventing the growth of a broad spectrum of microorganisms on surfaces and within food products, by contacting said food products, such as meat products, for example poultry, beef, pork, lamb, venison and other grocery products from butchers; seafood, for example fish and shellfish; fruit; vegetables; dairy products; pet food or snacks, such as those made from animal meat, skin, and parts which may include pig ears, rawhide, and jerky; and other food products that can be treated using the aqueous treatment methods of the present invention without detriment to the appearance, texture, and quality of the food. More specifically, the present invention relates to a solution comprising a concentrated amount of an antimicrobial QAC suitable for use in a method for inhibiting adherence, killing, and or preventing the growth of microorganisms in food products. Particularly, the use of the solution comprising a concentrated amount of an antimicrobial QAC relates to the effect of QACs on microorganisms that can cause contamination-bearing foods. More particularly, these microorganisms comprise microorganisms of the genus Staphylococcus, Streptococcus, Campylobacter, Arcobacter, Listeria, Aeromonas, Badilus,

Salmonella, non-toxin-producing Escherichia, pathogenic toxin-producing Escherichia, such as 0157:H7. More particularly, the present invention relates to an improved treatment method for applying diluted QAC to food products, by any procedure, but preferably comprises spraying or nebulizing diluted QAC onto food products, to prevent the growth of a broad spectrum of microorganisms on the food. products, where the QAC application time can be as short as at least one tenth of a second. This short application time of diluted QAC is particularly useful in a commercial or industrial setup.

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2-Description of previous techniques:

The prevention of diseases caused by microbial contamination of food is of special interest for the food processing industry, for regulatory bodies and for consumers. A recent report from the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (Federal Register, February 3, 1995), estimates that more than two million cases of illnesses caused by food carrying microbial contamination, with an associated cost of more than one billion dollars. Microbial contamination carried in foods occurs before the latter enters processing devices, and through cross-contamination in the processing environment. FSIS has established new Hazard Analysis and Critical Control Point (HACCP) requirements to reduce the occurrence and number of foodborne pathogens. Food processors must comply with these regulations. Although the means to achieve this microbial reduction is at the discretion of the processor, FSIS expects that antimicrobial treatments will be an important part of HACCP plans. The treatment methods of the present invention, which use aqueous formulations prepared from concentrated QACs, are useful in meeting HACCP requirements.

In efforts to provide a product completely free of microbial contamination, poultry and meat processors have encountered significant difficulties in removing strongly adhering or attached microorganisms to poultry and meat tissues for use as food products. If the contaminating microorganisms do not attach to the surface of the food, they can be easily washed away. However, strongly adhering microorganisms cannot be washed away and are quite resistant to removal by chemical or physical methods.

Various 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. In general, these methods have shown limited effectiveness in reducing microbial contamination and may affect the physical appearance of meat products.

Salmonella typhimurium contamination has been of particular interest in the poultry processing industry because the organism is often found in live birds. Poultry processors have had great difficulty removing microorganisms, such as S. typhimurium, that attach or adhere to bird tissue. To eliminate S. typhimurium contamination from birds and to minimize cross-bird contamination, a number of chemical and physical approaches have been suggested for use in poultry processing. In poultry processing, trisodium phosphate (TSP) has been used to kill S Typhimurium·, however studies give conflicting results regarding the efficacy of TSP against Salmonella. As a result of its solubility in water, TSP can be washed out of birds and consequently does not inhibit the adherence of microorganisms.

US Patent No. 5,366,983, incorporated herein by reference, discloses a method of eliminating and preventing Salmonella contamination of meat products by treating an aqueous solution of QAC. Specifically, cationic quaternary ammonium surfactants such as alkylpyridinium, particularly cetylpyridinium chloride (CPC) and cetylpyridinium bromide (CPB) were effective in killing S. typhimurium from birds. This patent, however, does not disclose that QACs have a broader antimicrobial spectrum, against any other genus of non-/Y contaminating microorganisms.

Salmonella. Furthermore, it does not suggest that this method of treatment is effective on food products other than meat. Furthermore, it does not suggest that very short QAC application times can be used that still provide effective antimicrobial treatment. It also does not suggest concentrated QAC solutions, as disclosed herein, which are particularly useful for preparing dilute QAC solutions.

Food substances differ chemically and physically by virtue of their protein content, porosity, lipophilicity, pH, water permeability, surface area, and surface electrical charge network. The porosity of the food could be important in terms of bacterial sequestration, whereas an impermeable integument of the food substance could reduce bacterial contamination of the food. All these chemical and physical differences between food products make it difficult to predict that the success of an antimicrobial agent on meat products will mean that it will be successful on other food products, such as fruits, vegetables, seafood, dairy products. and pet food and snacks.

For example, QAC and CPC are known to bind to proteins, however, if the antimicrobial efficacy of CPC on food products is largely due to its protein binding, it should therefore not be expected that the mentioned method be successful for treating non-protein fruits and vegetables.

Increasingly, foodborne illnesses due to other pathogenic and spoilage bacteria besides Salmonella have become a problem for food processors. The table presents a list of these bacteria and the products where they have been identified.

<td>Microorganism</td><td>Birds</td><td>cattle</td><td>Pig</td><td>Pathogen</td><td>Deterioration</td>

<td>Aeromonas hydrophila</td><td>X</td><td>X</td><td>X</td><td></td><td>X</td>

<td>Arcobacter butzleri</td><td></td><td>X</td><td>X</td><td>X</td><td></td>

<td>bacillus cereus</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td>

<td>Campylobacter jejuni</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td>

<td>Escherichia coli 0157:H7</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td>

<td>listeria monocytogenes</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td>

<td>Salmonella typhimurium</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td>

<td>Staphylococcus aureus</td><td>X</td><td>X</td><td>X</td><td>X</td><td></td>

Among these contaminating microorganisms, listed in the table, the

Escherichia coli 0157:H7 is of special interest due to its virulence, the severity of the disease produced, and the associated mortality. The Escherichia

coli 0157:H7 produces “shiga-like” toxins that cause blood clotting abnormalities, kidney failure (hemolytic-uremic syndrome), and death. Although recovery from the acute disease is complete, 15-30% of people infected with hemolytic-uremic syndrome will develop chronic kidney disease. The risks associated with contamination with E coli 0157: H7 are due to their resistance to antibiotics. In 1993 between 8000-16000 foodborne illnesses were due to E coli 0157:H7, with an estimated cost between 0.2 and 0.5 billion dollars.

Another virulent food contaminant, Listeria monocytogenes has been found in meat, animals, and various dairy products, and can cause sepsis, meningitis, and disseminated abscesses. Listeria monocytogenes is a cold-tolerant microorganism, capable of growing under refrigerated conditions. In 1993, there were about 1700 cases of foodborne illnesses due to Listeria monocytogenes, with an estimated cost between 0.1 and 0.2 billion dollars.

Another microorganism of interest in the food industry is the

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Aeromonas hydrophila that causes spoilage in the food and meat processing industry, and decreases the shelf life of these products.

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Currently, there are no effective bactericidal compounds to prevent and eliminate contamination of a wide range of food products against a broad spectrum of gram positive, gram negative, aerobic, facultative anaerobic, and microaerophilic microorganisms. The inventors of the present invention have proven that QACs are effective against a wide spectrum of different microorganisms that cause foodborne illness, when they adhere to a wide range of food products. This sensitivity of a broad spectrum of pathogenic microorganisms could not have been predicted.

The susceptibility of a microorganism to a particular microbial agent cannot predict the susceptibility of other microorganisms to the same agent. Antiseptics or germicides are believed to have a continuous spectrum of activity, but relative susceptibilities to different microorganisms must be considered. For example, the germicide Hexachlorophene is basically effective against Gram-positive microorganisms, and cationic antiseptics are not effective against sporulating microorganisms. Some Gram negative microorganisms, such as Pseudomonas cepacia, have grown in solutions of the drug benzalkonium chloride. Other bacteria have been able to grow in 70% ethanol (Harvey, SC, Antimicrobial Drugs in Reminqton's Pharmaceutical Sciences. 18^to Edition, Marck Publishing Co, pages 1163-1241 1990).

Regarding the treatment of food products, Listeria has been reported to be more resistant to the action of TSP than salmonella or E coli (Somers, EB et al, Int J Food Microbiol. 22:269-276,1994). Furthermore (Breen et al, J Food Sciences, 60:1991-1996,1995) demonstrated that TSP is much less effective in inhibiting the growth of Salmonella than in shedding this organism. Similarly, the TSP reduced the amount of E coli

0157: H7 in chicken carcasses, but it is not effective in inhibiting cross-contamination of this microorganism to other chickens.

The present invention shows that QACs are effective against E coli 0157:H7 in liquids, reducing the amount of this bacterium when it is attached to food products, as well as inhibiting the adherence of this bacterium to food products. E coli 0157:H7 has been reported to exhibit resistance to broad-spectrum antimicrobial agents such as tetracycline, streptomycin, sulfisoxazole (Kim et al, J Infect Dis, 170:1606-1609,1994) and oxytetracycline (Ciosek et al, Med Weter 40: 335-338,1984), whereas these same agents are highly active against common non-toxin-producing strains of E coli.

Certainly, the effectiveness of an antimicrobial or biocidal agent against a particular microorganism cannot be predicted based on its effectiveness against a different microorganism. There are many factors to consider, such as microbial characteristics, which play a role in the effectiveness of an antimicrobial agent against a particular microorganism. These features may include, without limitation: (1) the degree of glycocalyx formation by given species of attached microorganisms, (2) the presence of a cell envelope containing lipopolysaccharides and phospholipids in Gram-negative bacteria, (3) the presence of a lipoprotein, as in most of Enterobacteria and Pseudomonas, and (4) the presence of communication channels, for example in E coli and Salmonella (Fulton et al, Structure in Medical Microbiology. 3^to edition, pages 37-54, 1991).

The food processing industry, as well as home food preparation, in restaurants or institutions, requires more effective products and processes to prevent the growth of a wide range of microorganisms that contaminate many products.

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food and surfaces that come into contact with food products and food juices or liquids. This is especially true for microorganisms that adhere to food surfaces. As a result of an increasing number of illnesses due to 5 foodborne pathogenic microorganisms, the food processing industry now requires more effective processes for the elimination and prevention of a broader spectrum of microorganisms, particularly for pathogenic microorganisms. , such as toxin-producing Escherichia coli, i.e. E coli 0157: H7, €10 Known to cause serious human illness as a result of food contamination. The present invention provides a composition comprising a concentrated solution of QAC and at least one solubility enhancing agent, and methods for preventing the growth of microorganisms on and in food, as well as in liquids and food-related surfaces. food products and their preparation.

This prevention method is an important goal for the prevention of cross-contamination from infected food products, for the elimination of adherent microorganisms on food products, for the inhibition of adherence of microorganisms on food products r ^20 and for the prevention of the growth of microorganisms that remain attached to food products. Furthermore, the method of the present invention can be easily adapted for use in a food processing plant.

Furthermore the present invention provides compositions comprising a solution comprising a concentrated amount of QAC in combination with at least one solubility enhancing agent or solvent. This concentrated QAC solution of the present invention provides a stock solution from which dilute QAC compositions can be prepared for the treatment of food products and surfaces related to food processing and preparation, including the bodies of animals to from which food products are prepared. For example, to increase the safety of processing milk and milk products, the udders of dairy cows can be treated prior to milking with a dilute solution of the concentrated QAC solution. In addition, a dilute solution of QAC with the components described herein, or in combination with other components recognized as effective for human and pet hand and body washing, may be useful. Concentrated QAC solutions are useful for preparing dilute working solutions for use in the present method. The formulations of the present invention contain solubility enhancing components, which allow more concentrated QAC compositions to be prepared.

US Patent No. 5,405,604 discloses a concentrated mouthwash, methods of use, and methods for making the mouthwash. The mouthwash is composed of a concentrated composition in the form of oil-in-water emulsions, consisting essentially of a QAC of between approximately 0.05% and approximately 10%; between about 30% and about 85% of a solvent that acts as a flavor oil carrier, where the solvent is propylene glycol, polyethylene glycol, and mixtures thereof; between about 0.2% and about 9.0% of a flavoring oil; and water. The composition of the present invention differs from the mouthwash composition by containing more than 10% QAC, by being a true homogeneous solution rather than an emulsion, and by not containing flavoring oils.

WO 98/03066 discloses an antimicrobial composition, methods for its preparation and methods for its use. The composition is made up of a subcomponent a) a substituted or unsubstituted C1.C4 monocarboxylic acid, at 50-99% by weight, and a subcomponent b) a €10 compound^wtwenty Microbiocidal or microbiostatic cationic organic nitrogen, in approximately 0.1-50% by weight. The composition of the present invention differs from the composition of WO 98/03066, in that it contains a solubility enhancing agent, whereas WO 98/03066 does not. The present invention differs from WO 98/03066, in that it does not contain an organic acid, such as a monocarboxylic acid, and in that it specifically does not contain a C1-C4 monocarboxylic acid which is the primary component of WO 98/03066. 03066. The disclosure of the patent WO 98/03066 refers that the efficacy of an unsubstituted C1-C4 monocarboxylic acid containing compositions against Salmonella can be increased by adding a cationic organic nitrogenous compound. This invention supports the theory that a microbicidal cationic nitrogenous compound is more apt, in terms of its effect, on microbes damaged by C1-C4 carboxylic acids. In addition, the compositions of the present invention may contain an additional organic acid that mixes with the cationic organic nitrogenous compound to form an "ancat" or "catan" compound, not present in the composition of the present invention.

Summary of the invention

The QAC concentrated solution of the present invention provides a concentrated antimicrobial solution that can be readily diluted to form a solution that is contacted with food products and food-related surfaces, including live or dead animal parts, in the case of food products. food obtained from animals. The concentrated QAC solution of the present invention comprises a QAC and at least one solubility enhancing agent. Preferably the QAC is in a concentration greater than about 10% by weight. The concentrated solution is diluted to obtain a dilute amount of QAC effective to inhibit growth, in an aqueous solution with the dilute solubility enhancing agent. The 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 in preventing the growth of a broad spectrum of microorganisms that act on a wide range of food products.

The present invention provides a method for preventing the growth of microorganisms in food products, which comprises contacting a food product with an amount of QAC effective to prevent the growth of a broad spectrum of microorganisms in food products, the application time of the compound on food item j for at least a fraction of a second. The purpose of preventing the growth of microorganisms in food products is to provide a food product free or with minimal amounts of viable microorganisms that could cause disease in humans or animals, or spoilage of the food product prior to ingestion. Prevention of the growth of microorganisms on food products includes, but is not limited to, the following mechanisms: (1) elimination of adherent microorganisms on food products, (2) inhibition of adherence of microorganisms on food products, (3) ) killing or inactivation of microorganisms in food products; and (4) the killing or inactivation of microorganisms not attached to the food product, but present in liquids associated with the food products during processing, such as in cooling tanks, or present on related surfaces. with the preparation of food, liquids that remain on said surfaces, such as countertops, cutting boards and sinks, and equipment used in food preparation and food sanitization.

The microorganisms that are included in the field of the present invention are those microorganisms susceptible to QACs, plus

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specifically microorganisms of the genus Staphilococcus, Streptococcus,

Campylobacter, Arcobacter, Listeria, Aeromonas, Bacillus, Salmonella,

Non-toxin-producing Escherichia, pathogenic toxin-producing Escherichia, and other microorganisms capable of causing food carriers of bacterial contamination in food for human or animal consumption.

Other considered microorganisms, which are also susceptible to QACs, are Aspergillus flavum and Penicillium chrysogenum, and parasites such as Entamoeba histolytica.

The present invention has important application in the food processing industry, as well as in home food preparation, and in institutions. QACs are readily available and the cost of carrying out the method of the present invention is inexpensive compared to existing antimicrobial growths. Unlike existing treatments that use, for example, TSP, the use of QACs does not alter the appearance, color, flavor or texture of food products. A range of QAC concentrations are effective to prevent the growth of a broad spectrum of microbials in food products. QACs are tested for mutagenicity by the Ames test. The preferred QAC of the present invention, CPC, was shown to be non-mutagenic by the Ames test. In addition, CPC is already approved for human use in oral preparations such as Cepacol17 tablets, which are taken orally in an amount of up to 20 mg per day.

The present invention also aims to improve a method for contacting food products with QAC, where the application time of the QAC on the food products is at least a fraction of a second, and which can range from about 0.1 seconds and approximately 5 seconds. A range of about 1 to 2 seconds can also be used. It is important that the application time of the QAC is sufficient to significantly prevent the growth of microorganisms in food products.

The present invention also encompasses an improved method of contacting QAC with food products by spraying or misting the compound onto the food products. The spray or mist method can be performed using a water-diluted QAC solution or using the new QAC concentrate formulation with at least one solubility-enhancing agent, or using the water-diluted QAC concentrate formulation. Direct spraying or misting of the concentrate is possible if the QAC percentage of the concentrate is suitable for use in food products.

The present invention is intended to encompass any method that contacts the QAC solution with a food product, by any direct method, including spraying, misting, dipping, and soaking.

But the present invention also encompasses any method of contacting the QAC solution with food by indirect procedures, such as applying the concentrated or diluted QAC solution to food processing equipment or preparation surfaces. that are in contact with food products during processing, preparation, storage and/or packaging.

In addition, the method of the present invention may optionally include a step to determine the presence of microorganisms in food prior to treatment, prior to contacting the food product with the QACs. Any conventional method for easily determining the presence of microorganisms, such as PCR and immunoassay, can be used as the determination step.

Furthermore, the method of the present invention optionally comprises a step for determining the presence of QACs on the surface of food products after having been in contact with the QACs. This determination is made immediately after the commissioning step.

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c^wtwenty contact or after several washing steps. For example, QAC is extracted from food tissues in a manner suitable for analysis by high performance liquid chromatography (HPLC). This method involves ethanol extraction on food tissues followed by solid phase extraction using a weak cation exchange column that selectively separates QACs from other matrix compounds that would otherwise interfere with HPLC analysis. The HPLC assay for the quantification of QAC residues employs a reverse phase cyano column and uses an analogous QAC as internal standard.

Brief description of the drawings

Figure 1 is a bar graph showing the inhibition of E coli 0157:H7 adherence of a bovine vacuum section after CPC treatment.

Figure 2 is a bar graph showing the decrease in viable microorganisms on catfish skin after treatment with 5% CPC in aqueous glycerin in non-selective media.

Figure 3 is a bar graph showing the decline in catfish skin S Typhimurium selective media after treatment with 5% CPC in aqueous glycerin.

Figure 4 is a bar graph showing the decrease in viable S. typhimurium on black grapes after treatment with 5% CPC in aqueous glycerin.

Figure 5 is a bar graph showing the decrease in S Typhimurium in broccoli after treatment with 5% CPC in aqueous glycerin.

Detailed description of the invention

The present invention is based on the determination that QACs are useful for treating a wide range of food products in order to diminish a broad spectrum of bacterial contamination carried on food and on surfaces associated with the processing and preparation of these food products. The present invention is also based on the finding that QACs are effective in eliminating, killing, inactivating and inhibiting the adherence in food products of a wide range of foodborne pathogenic microorganisms. These microorganisms include but are not limited to bacteria belonging to the genera Salmonella, Staphylococcus, Streptococcus, Campylobacter, Arcobacter, Listeria, Aeromonas Bacillus, non-toxin-producing Escheríchia, and virulent toxin-producing strains of Escheríchia, such as E. coli 0157:H7; fungi, such as Aspergillus flavus and Penicillium Chrysogenumr, and parasites such as Entamoeba histolytica.

The compositions of the present invention comprise an effective amount of QAC in an aqueous solution. In particular, the saturated concentration of the present invention provides an ideal antimicrobial solution for use in industrial applications, where large amounts of dilute QAC solutions are needed for food processing. The saturated solution of the present invention contains a minimal amount of components, GRAS (generally recognized as safe) components and solubility enhancing agents. The

J QAC stock solutions of the present invention confer many advantages in the preparation of dilute QAC solutions. Large amounts of powdered QAC are solubilized in an aqueous solvent containing at least one solubility enhancing agent. It is difficult to prepare concentrated solutions of QAC in water alone, because QAC precipitates out of solution. In fact, it is difficult to solubilize more than about 5 to about 10% QAC, and under some conditions more than 1% QAC, depending on the temperature of the solution, without the aid of solubility enhancing agents. However, the inventors of the present invention have determined that )&lt;?

QAC concentrates if prepared in combination with at least one solubility enhancing agent or solvent, such as an alcohol or a polyglycol. QACs are known cationic surfactants, and as such aqueous solution preparations of QACs have abundant

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foam. However, when the concentrated QAC solution comprises a QAC with a concentration greater than 10% or greater than 15%, and at least one solubility-enhancing agent is used to prepare the diluted QAC solutions, the solubility of the QAC is greatly decreased. abundant foam that usually forms when preparing aqueous solutions of QAC. In the preparation of the concentrate, a minimum of foam is formed, and once the concentrate is prepared, there is no foam. In addition, the concentrate is diluted with a minimum of agitation and therefore with a minimum of foaming. If the concentrated QAC is exposed to low temperatures, the concentrated QAC solution resists precipitation. If frozen, the stock solution of QAC with at least one solubility enhancing agent, the

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itself results in a solution when thawed. If after shipping or storage at room temperature, or freezing, a precipitate of QAC persists, then the temperature of the solution is raised until the precipitate disappears. If necessary, large quantities of QAC concentrate can be heated in large containers or drums on a drum heater. Furthermore, compared to dilute aqueous QAC solutions, concentrated QAC solutions, together with high concentrations of at least one solubility-enhancing agent such as appropriate water-miscible organic solvents, present minimal risk of deterioration or downtime. limited on shelf.

In addition, concentrated QAC solutions increase user safety by eliminating inhalation of QAC dust, which is a problem when handling QAC dust, particularly when handled in large quantities, as it can cause lung, eye, and eye irritation. , throat, nasal or skin. QAC solution concentrate decreases the volume and mass of the solution to be transported and stored during industrial applications of QAC solutions. And, very importantly, when the concentrated QAC solution is diluted in water to prepare diluted QAC solutions for use in food products, the diluted concentrated solutions exhibit very good antimicrobial efficacy.

The present invention is particularly intended for a concentrated solution of QAC comprising a quaternary ammonium in a concentration greater than about 10% by weight, and at least one solubility enhancing agent. The solubility-enhancing agent is any water-miscible organic solvent that enhances the solubility of the QAC powder in an aqueous solution, such that it forms a solution for concentrations greater than 10% by weight. A 10% by weight solution is prepared by weighing 10 grams of QAC and dissolving it in 90 grams of a liquid comprising at least one solubility enhancing agent and water, if necessary, to bring the weight to 90 grams of liquid. . The concentrated QAC solution of the present invention comprises QAC in solution at concentrations greater than about 10% by weight and &lt; preferably at concentrations of about 15% by weight. The QAC concentrated solution comprises QAC in solution at concentrations ranging from greater than about 10% or greater than about 15% by weight, to approximately 60% by weight. Although a QAC concentration of greater than about 60% by weight in the concentrated QAC solution can be used, the useful upper limit is determined by the interaction between the % (or weight) of QAC and the solubility enhancing agents used to prepare the concentrated solution. Specific solubility enhancing agents or combinations thereof can result in concentrated QAC formulations.

Η greater than 60%. It is important to dissolve and solubilize all of the QAC powder before preparing the diluted formulation to treat food products.

Preferably, the QAC is in a concentration between greater than approximately 10%, or greater than approximately 15%, and, approximately 15% by weight, and approximately 50% by weight, and more preferably in a concentration between approximately 10% or about 15% by weight, and about 40% by weight. But it is also useful for the present concentrated solution, the concentration of QAC comprised between approximately 10% and approximately 30% by weight or between approximately 15% and approximately 25% by weight, and within this range the concentration of approximately 20% in weight.

The QAC concentrations of the present invention are described in either concentrations expressed in parts per million (ppm) or as % by weight, where 100,000 ppm equals 10% by weight. Examples use CPC and 15 use both ppm and % to report concentration.

To bring the missing weight of the solution to 100% by weight, the solubility enhancing agent or a combination of these agents is added, and, if necessary, water. In the present invention, a solubility-enhancing agent is considered to be any solubility-enhancing agent that solubilizes QACs at concentrations greater than about 10% by weight; but alcohols are the preferred solubility enhancing agents. In addition, useful solubility enhancing agents are polyglycols, such as polyethylene glycol. The present invention contemplates using one or more of these solubility enhancing agents. More preferably, the alcohol is selected from the group consisting of monohydric alcohol, dihydric alcohol, trihydric alcohol, and a combination thereof. Any of these types of alcohols can be used alone or in combination with one or more of the other types of alcohols, to obtain the desired wt% solubility enhancing agent. If a monohydric alcohol is used, then it is preferred that this type of alcohol is preferably an aliphatic alcohol, more preferably ethyl alcohol. If a dihydric alcohol is used, then a glycol or derivative thereof is preferred. Of the glycols, the most preferred is propylene glycol, which is readily available through a number of distributors. Propylene glycol confers advantages over other alcohols as a solubility enhancing agent at high concentrations of QAC, such as CPC. Trihydric alcohols, such as glycerol or derivatives thereof are also useful as solubility enhancing agents in the present concentrated CPC solutions. The choice of alcohol depends on the final food product with which it is contacted, and is chosen to be compatible with the treatment steps, prior to or after the contact of the QAC with the food product. If a polyglycol is used as a solubility enhancing agent, polyethylene glycol is preferred, and particularly the lower molecular weight species, with an average molecular weight of less than or equal to 600, which are well known and have properties similar to propylene glycol.

If ethyl alcohol is used as solubility enhancing agent, it is present in concentrations up to about 40% by weight. In the present invention, the ranges of ethyl alcohol are between approximately 0.5% by weight and approximately 40% by weight, between approximately 10% by weight and approximately 40% by weight, between approximately 15% by weight and approximately 30% by weight. weight, and within this range is useful in the present invention, the concentration of about 20% by weight.

The concentrated QAC solutions contain at least one solubility-enhancing agent, such as an alcohol up to a concentration of about 70% by weight. More preferably, the alcohol is at a concentration of up to about 60% in

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weight and may range from about 10% by weight to about 60% by weight. The concentration of the solubility enhancing agent varies depending on the weight % of the QAC to be dissolved in solution, as well as the particular intended use for the concentrated QAC solution and dilutions thereof.

Preferably the concentrated QAC solution comprises a QAC concentration of about 40% by weight and at least one alcohol in a concentration of between about 50% by weight and about 60% by weight, with water to make up the remaining % by weight. The preferred alcohol for this solution is propylene glycol. More preferably, the concentrated QAC solution comprises a QAC in concentrations of approximately 40% by weight, and at least one alcohol at a concentration between approximately 55% by weight and approximately 60% by weight, and water present at approximately 5% by weight. . The most preferred concentrated QAC solution comprises QAC at a concentration of about 40% by weight, an alcohol at a concentration of about 57% by weight, and water at about 3% by weight. Again, the preferred alcohol in this solution is propylene glycol.

However, a concentrated solution of QAC comprising QAC at a concentration of about 40% by weight and at least one alcohol at a concentration of up to about 50% by weight, and preferably about 50% by weight, is also useful, however. In this concentrated aqueous solution, the solubility enhancing agent can be a combination of alcohols, such as ethyl alcohol and propylene glycol. But glycerol is also useful as a solubility enhancing agent, alone or in combination with other alcohols or polyglycols. For this purpose, glycerol is useful at a concentration up to and including about 20% by weight, and is also useful at concentrations between about 0.5% and about 10% by weight and within this range, at a concentration of about 1%. Glycerol is useful in methods where propylene glycol is not the alcohol of choice to solubilize the QAC. Another useful concentrated QAC solution comprises a QAC at a concentration of about 20% by weight and at least one alcohol at a concentration of about 50% by weight, such as a combination of ethyl alcohol and propylene glycol, and preferably a combination where each alcohol is approximately 25% by weight.

The useful QAC of the present concentrated QAC solution is selected from the group consisting of alkylpyridinium, tetra-alkylammonium, and alkylalicyclic ammonium salts.

Alkylpyridinium is represented by structural formula (I):

where n is 9-21; and X is a halogen.

Tetra-alquinamonium is represented by the structural formula (II):

«i where n is 9-21; R is selected from the group consisting of CH3 and C2H5; and X is a halogen.

Alkyl-alicyclic ammonium salts are represented by the formula

Υ\ structural (III):

F

<img file="ECSP024309A_D0012.tif" />

where n is 9-21; Z is 4-5; R is selected from the group consisting of CH₃ and C2H5; and X is a halogen.

In relation to their effectiveness to eliminate adhered microorganisms in different foods, as well as to inhibit the adherence of microorganisms, a number of QACs are evaluated, all of them cationic surfactants, that is, surfactants. Of the QACs studied, the most effective is cetylpyridinium chloride, which is used in the examples set forth below, but without this restricting the use of QACs to CPC, within the scope of the present invention, since other members of QAC also have similar properties against foodborne pathogenic microorganisms. QACs that have between 12 and 16 carbon atoms in the long chain sector have the highest antimicrobial activity. The preferred QAC, which is CPC, has 16 carbon atoms in the long chain sector.

The present invention further comprises; diluting the concentrated QAC solution, including at least one solubility enhancing agent, and water if required to obtain the CPC wt%; and contacting this diluted QAC solution with a food product to prevent microbial growth or microbial adherence on the food products. The diluted QAC solution comprises QAC in a concentration up to and including about 1% QAC by weight.

The United States Department of Agriculture considers % by weight as the expression of QAC concentration to treat food products, acceptable in use. The amount of QAC that remains in a particular food product varies according to the different types of food treated and the method of application. The concentrated QAC solution described in the present invention is diluted with water to obtain a dilute QAC solution ranging from about 0.1% up to and including about 1%, but can be increased or decreased depending on the food product treated and the method. used. The concentrated QAC solution, which was prepared on a weight-to-weight basis as described above, is diluted to obtain the desired treatment QAC concentration by volume-to-volume dilution. For example, a 40% stock QAC solution is diluted to a 1% QAC by diluting 2.5 mL of the stock QAC with 97.5 mL of water. In a food processing plant, volume-in-volume dilution is preferred due to its ease of preparation. However, a weight by weight dilution can also be used to prepare dilute QAC solutions, where 2.5 grams of a 40% by weight QAC solution is mixed with 97.5 grams of water, to obtain a QAC solution at 1%. Dilution of QAC from the stock solution also results in dilution of the solubility enhancing agent found in the stock solution. The concentrated QAC solution dilution is useful for contacting the food product by spraying, misting, immersion, or by any other contact method that is suitable for contacting the QAC solution dilution with the food product, including contact indirect, such as contact of food processing or preparation equipment or surfaces that are in contact with food during processing, preparation, storage and/or packaging. The shorter the application time of the QAC solution, the better, particularly for industrial and commercial food processing purposes.

The present invention is further based on the determination that the application time of QACs on food products by spraying or misting processes can be reduced to as low as at least about 0.1 second, while continues to be effective in significantly inhibiting the adherence of microorganisms to foods that carry microorganisms, which is a significant improvement and commercial advantage in terms of the commercial use of this process. Misting or spray application processes allow the application time of the diluted QAC solution onto the food product to be as short as 20 seconds, but more preferably about 10 seconds or less and more preferably about 5 seconds or less. The most preferred QAC application time on the food product is between about 0.1 and about 5 seconds and within this range between about 0.1 and about 2 seconds is also useful, with a preferred range between about 0. 5 seconds and about 2 seconds. It is to be understood that the present invention contemplates application times of the diluted QAC solution as short as physically possible, so long as they result in the inhibition of microorganisms in food products or in liquids and surfaces with which the QAC comes into contact. food product. Furthermore, different time intervals less than 20 seconds are contemplated in the present invention.

In the present method any type of contact method of the

QAC with the food product, as long as it allows short application times. A method using a booth for spraying or misting the food product is useful in the present invention. The machinery used in such booths in the processing stages of a food processing plant can be adapted to reduce the application time to a minimum, while achieving an effective antimicrobial effect on food. All of these short application times, ie less than 20 seconds and as low as 0.1 second, significantly decrease viable foodborne microorganisms in these food products. In addition, a very small amount of dilute QAC solution is necessary for spray or mist treatment; for example, for effective treatment, as low as about 1 ounce of dilute QAC solution, per pound of food product, is useful.

In a poultry processing plant, the present QAC short time application method is useful for treating chickens post-chill after they have been immersed in a cold water cooling bath. Chickens are removed from the cooling bath and treated with the diluted QAC solution of the present invention for an application time of less than about 20 seconds, preferably less than about 10 seconds, more preferably less than about 5 seconds, most preferably less of about 2 seconds, more preferably even less than about 0.1 second. Next, the treated chickens are packaged without subsequent washing or rinsing. However, the optional method may include, if deemed necessary, at least one step of washing the chickens prior to packaging. The optional washing step may include spraying or misting the food product with water or immersing the food product in a container or tank of water.

The aspects described in the present invention are described in detail in the examples below with reference to Figures 1-5.

The examples shown below in their preferred embodiments serve to further illustrate the present invention, without this implying limits to the present invention. Examples use as food products treated by this method, poultry, beef, catfish,

<img file="ECSP024309A_D0013.tif" />

<img file="ECSP024309A_D0014.tif" />

broccoli and grapes but other food products that are not adversely affected by the treatment process may also be treated within the scope of the present invention.

Example 1

Bactericidal activity of quaternary ammonium compounds on suspension cultures (not attached to meat products)

Minimum Inhibitory Concentration (MIC) of Quaternary Ammonium Compounds

Minimum inhibitory concentrations (MIC) were determined for

QAC in a Mueller Hinton culture (BBL Microbiology System) using the macrodilution method established by the National Guidelines Committee for

Clinical Laboratories of 1987. The experiments were carried out by incubating for 16 hours at 37°C for Staphylococcus aureus, Escheríchia coli 0157: H7, Listeria monocytogenes and Salmonella typhimurium. For Aeromonas hydrophila and Bacillus cereus, incubations were carried out at 30°C. MICs were determined through the lowest dilution without visible turbidity. The

Table 2 shows the data from the previous experiment:

TABLE 2

MINIMUM INHIBITORY CONCENTRATION (MIC)

<td>Chloride</td><td>cpc</td><td>cpc</td><td>cpc</td><td>cpc</td><td>cpc</td><td>cpc</td>

<td>cetylpyridinium</td><td>Versus</td><td>Versus</td><td>Versus</td><td>Versus</td><td>Versus</td><td>Versus</td>

<td>. (CPC)</td><td>e coli</td><td>B.</td><td>S</td><td>S</td><td>TO</td><td>L</td>

<td>pg/rnl</td><td> 0157:</td><td>cereus</td><td>aureus</td><td>typhimurium</td><td>hydrophi</td><td>monocytog</td>

<td></td><td>H7</td><td></td><td></td><td></td><td>the</td><td>enes</td>

<td> 125</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 62,5</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Chloride</td><td>cpc</td><td>cpc</td><td>cpc</td><td>cpc</td><td>cpc</td><td>cpc</td>

<td>cetylpyridinium</td><td>Versus</td><td>Versus</td><td>Versus</td><td>Versus</td><td>Versus</td><td>Versus</td>

<td>, (CPC)</td><td>e coli</td><td>B.</td><td>S</td><td>S</td><td>TO</td><td>L</td>

<td>pg/ml</td><td> 0157:</td><td>cereus</td><td>aureus</td><td>typhimurium</td><td>hydrophi</td><td>monocytog</td>

<td></td><td>H7</td><td></td><td></td><td></td><td>the</td><td>enes</td>

<td> 31,25</td><td></td><td></td><td></td><td> +</td><td> +</td><td></td>

<td> 15,63</td><td></td><td></td><td></td><td> +</td><td> +</td><td></td>

<td> 7,81</td><td> +</td><td></td><td></td><td> +</td><td> +</td><td></td>

<td> 3,91</td><td> +</td><td> +</td><td></td><td> +</td><td> +</td><td></td>

<td> 1,96</td><td> +</td><td> +</td><td></td><td> +</td><td> +</td><td></td>

<td> 0,98</td><td> +</td><td> +</td><td></td><td> +</td><td> +</td><td></td>

<td> 0,50</td><td> +</td><td> +</td><td></td><td> +</td><td> +</td><td> +</td>

<td> 0,25</td><td> +</td><td> +</td><td></td><td> +</td><td> +</td><td> +</td>

<td> 0,00</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td><td> +</td>

(-) no growth (-^growth

The MIO were obtained by means of the culture macrodilution method (Committee

National Guidelines for Clinical Laboratories).

<img file="ECSP024309A_D0015.tif" />

Minimum Bactericidal Concentration (MBC) of Quaternary Ammonium Compounds

The minimum bactericidal concentrations (MBC) were determined for

QAC for Campylobacter jejuni and Arcobacter butzleri in a culture of

Mueller Hinton (BBL Microbiology System) using the macrodilution method established by the National Committee for Guidelines for Clinical Laboratories in 1987. Experiments were performed by microaerophilic incubation at 37°C for 48 hours. An aliquot of each dilution was plated on agar and incubated under microaerophilic conditions at 37°C for 48 hours. HE

<img file="ECSP024309A_D0016.tif" />

determined MBCs through the lowest dilution without growth. Table 3 shows the data from the previous experiment:

TABLE 3

MINIMUM BACTERICIDAL CONCENTRATION (MBC)

<td>Cetylpyridinium chloride, pg/mL</td><td>cpc Versus Campylobacter ¡ejuni</td><td>cpc Versus Arcobacter butzleri</td>

<td> 125</td><td></td><td></td>

<td> 62,5</td><td></td><td></td>

<td> 31,25</td><td></td><td> +</td>

<td> 15,63</td><td></td><td> +</td>

<td> 7,81</td><td></td><td> +</td>

<td> 3,91</td><td> +</td><td> +</td>

<td> 1,96</td><td> +</td><td> +</td>

<td> 0,98</td><td> +</td><td> +</td>

<td> 0,50</td><td> +</td><td> +</td>

<td> 0,25</td><td> +</td><td> +</td>

<td> 0,00</td><td> +</td><td> +</td>

<td colspan="3">(-) No growth (+) Growth The MBC were obtained by means of the culture macrodilution method (Committee National Guidelines for Clinical Laboratories).</td>

The MIC and MBC data show that CPC is effective against a wide range of microorganisms.

A 16 hour culture of E coli 0157:H7 was centrifuged over a soybean trypticase culture (15000 rpm, 10 minutes, 4°C). After the removal of the

Activity of quaternary ammonium compounds in planktonic cells supernatant, the pellet was washed with 10 ml of 0.04 μ potassium phosphate buffer (PPB, pH 7.0) and suspended in PPB to a final suspension of 1 to 2 χ10⁹ cells/ml. Aliquots (1.0 mL) were centrifuged (14000 rpm, 3 minutes) and supernatants removed. Each precipitate was resuspended in 1 ml of an aqueous solution of various concentrations (100 to 1000 pg/ml) of the test composition (CPC) or in 1.0 ml of PPB, shaken (30 sec), it was incubated for 1 minute at 25°C, and centrifuged (14000 rpm, 3 minutes). After removing the supernatant, each pellet was suspended in 0.5 ml of PPB. Cells from each sample were counted using duplicate 0.05 ml aliquots and common serial dilution techniques on trypticase soy agar, with data recorded as mean colony forming units (CFU)/ml.

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

Example 2

Effects of quaternary ammonium compounds on the reduction of viable bacteria fixed on chicken skin *7_S

Chicken skins (2.5 x 2.5 cm) were removed from the paw area, sterilized by a 45 KGy dose of irradiation from an electron source, and placed epidermis side up in each cavity of the skin. a six well tissue culture plate. Each piece of skin was inoculated with

5 mL of 0.008 M phosphate-buffered saline (PBS, pH 7.2) containing 6 to 8 x 10³ CFU/ml of bacteria, with the exception of the background control group, which was treated with only 5 ml of PBS. The plates were incubated (30 min, 35°C) and each piece of skin washed (2X, 5ml PBS) to remove loosely bound (unbound) microorganisms. Each £10 inoculated skin was treated with 5 ml of PBS containing CPC. Three pieces of skin were used for each CPC concentration, including one in which the skins were treated with only 5 ml of PBS (0 concentration). The plates were incubated with shaking (100 rpm) for 30 minutes at 25°C. After incubation, each piece of skin was washed (5 ml PBS), placed in a sterile plastic bag containing 80 ml saline or 1% peptone, and homogenized for 2 min using a blender. laboratory (Stomacher7 400, Seward Medical, London, England). Three aliquots of the homogenate (1 ml) were plated and incubated (37°C, 18 to 24 hours). Bacterial colonies were counted, corrected for dilution, and the result reported as CFU/skin.

These studies show the reduction in viable bacteria (Salmonella typhimurium, Staphylococcus aureus, Campylobacter jejuni, Escherichia coti (non-toxin-producing strain) and Escherichia coli 0157:H7) after treatment with concentrations of 50 to 1000 ppm of CPC. Higher concentrations, up to 8000 ppm CPC, were tested against Escherichia coli.

0157:H7 and were found to reduce the amount of attached bacteria to less than 0.1%. These studies show a significant inhibition of the growth of these five bacteria on chicken skins.

Example 3

Effects of quaternary ammonium compounds on the inhibition of bacterial attachment to chicken skins

Chicken skins (2.5 x 2.5 cm) were removed from the paw area, sterilized by a 45 KGy dose of irradiation from an electron source, and placed epidermis side up in each cavity of the skin. a six well tissue culture plate. Each piece of skin was inoculated with 5 ml of 0.008 M phosphate-buffered saline (PBS, pH 7.2) containing CPCs. Three pieces of skin were used for each concentration of test compound, including one in which the skins were treated with only 5 ml of PBS (0 concentration). The plates were incubated with shaking (100 rpm) for various times (1 minute or 10 minutes). The incubation solution was removed by aspiration and the skins were washed (5 ml of PBS) and then incubated for 30 minutes at 25°C with 5 ml of PBS containing 6 to 8 x 10³CFU/ml of bacteria. After incubation, each piece of skin was washed (2X, 5 mL PBS) to remove loosely bound (unfixed) microorganisms, placed in a sterile plastic bag containing 80 mL saline or 1% peptone, and homogenized for 2 min using a laboratory blender (Stomacher7 400, Seward Medical, London, England). Three aliquots of the homogenate (1 ml) were plated and incubated (37°C, 18 to 24 hours). Bacterial colonies were counted, corrected for dilution, and the result reported as CFU/skin.

These studies show inhibition of the attachment of bacteria (Salmonella typhimurium, Staphylococcus aureus, Campylobacter jejuni, Escherichia coli (non-toxin-producing strain) and Escherichia coli 0157:H7) to chicken skins after treatment with concentrations of 50 to 1000 ppm CPC. The data in these studies show that pretreatment of chicken skins with CPC significantly inhibits the attachment of these microorganisms to chicken skins.

Treatment of chicken skin with CPC for only 1 minute results in significant inhibition of S typhimurium binding at concentrations of 500 ppm and 1000 ppm. This shorter contact time of the

QAC with meat products supports the use of shorter contact times than previously reported to be effective.

Generally, immersions in ice tanks can be extended for up to 60 minutes, but the data presented in this paper support the fact that a shorter immersion time can be used and still obtain a significant reduction in the number of viable microorganisms.

The CPC contacting step of the present invention can be carried out for about 20 seconds to about 60 minutes. The present invention also describes useful contact times within this range of less than 10 minutes, and in ranges from about 20 seconds to about 9 minutes, from about 20 seconds to about 5 minutes, and from about 20 seconds to about 90 seconds.

Example 4

Effects of quaternary ammonium compounds on the reduction of viable bacteria fixed on beef tissue r

Squares of beef tissue (2.5 x 2.5 cm) approximately 0.5 cm thick were prepared, sterilized by a dose of 45

KGy of irradiation from an electron source and placed in each well of a six-well tissue culture plate. Each piece of tissue was inoculated with 5 mL of 0.008 M phosphate-buffered saline (PBS, pH 7.2) containing 6 to 8 x 10³ CFU/ml of bacteria, with the exception of the background control group, which was treated with only 5 ml of PBS. Plates were incubated (30 min, 35°C) and each square was washed (2X, 5ml PBS) to remove loosely bound microorganisms. The inoculated squares were treated with 5 ml of PBS containing the CPC. Three pieces of tissue were used for each concentration of test compound, including one in which the squares were treated with only 5 ml of PBS (0 concentration). The plates were incubated with shaking (100 rpm) for 30 minutes at 25°C. After incubation, each piece of skin was washed (5 mL PBS), placed in a sterile plastic bag containing 50 mL saline or 1% peptone, and homogenized for 2 min using a blender. laboratory (Stomacher7 400, Seward Medical, London, England). Three aliquots of the homogenate (1 ml) were plated and incubated (37°C, 18 to 24 hours). Bacterial colonies were counted, corrected for dilution, and the result reported as CFU/sq.

The results of this study show a reduction in viable Escherichia coli 0157:H7 after treatment with concentrations of 50 to 1000 ppm of CPC on beef tissue, with a 62 to 64% reduction in bacteria fixed at 500 and 1000ppm CPC. Example 5

Effects of quaternary ammonium compounds on the inhibition of bacterial attachment to beef tissue

Squares of beef tissue (2.5 x 2.5 cm) approximately 0.5 cm thick were prepared, sterilized by a 45 KGy dose of irradiation from an electron source, and placed in each well. of a six-well tissue culture plate. Each piece of tissue was inoculated with 5 ml of 0.008 M phosphate-buffered saline (PBS, pH 7.2) containing CPCs. Three pieces of skin were used for each concentration of test compound, including one in which the skins were treated with only 5 ml of PBS (0 concentration). The culture plates were incubated with shaking (100 rpm) for 10 minutes at 25°C. The incubation solution was removed by aspiration and the squares were washed (5 mL PBS) and then incubated (30 min, 35°C) with 5 mL PBS containing 6 to 8 x

<img file="ECSP024309A_D0017.tif" />

10³CFU/ml of bacteria. After incubation, each piece of tissue was washed (2X, 5 mL PBS) to remove loosely bound (unfixed) microorganisms, placed in a sterile plastic bag containing 80 mL saline or 1% peptone, and homogenized for 2 minutes using a laboratory blender (Stomacher7 400, Seward Medical, London,

England). Three aliquots of the homogenate (1 ml) were plated and incubated (37°C, 18 to 24 hours). Bacterial colonies were counted, corrected for dilution, and the result reported as CFU/square.

The results of this study show inhibition of binding of £10 Escherichia coli 0157:H7 after treatment with 50 to 1000 ppm of

CPC, with a 76% reduction in the amount of bacteria attached to meat at concentrations of 1000 ppm CPC. Figure 1 shows the results of a separate test using higher CPC concentrations and the same experimental procedure. At 20,000 ppm CPC, the binding of bacteria to meat was completely inhibited.

Example 6

Spraying of pre-chilled poultry meat with 0.1% r-cetylpyridinium chloride

^w A spray test chamber was designed and built for use in a pilot poultry processing plant. The spray test system consisted of a test chamber, a spray water storage tank, a pressure pump, a filter, pressure regulators, a plastic spray chamber with eight nozzles located on four sides, and a manifold. for used water. There were three nozzles in each of the tubes for a spray from the front and the back. One nozzle was used to spray from above and one to spray from below. Chamber dimensions are preferably 3 x 3 x 3 feet. With a high-pressure pump, the pressure could be adjusted between 0 and 140 psi. The distance between the spray nozzles and the chicken meat was 12 to 15 inches. The top nozzle was used to spray into the chicken meat. Spray nozzles with a flattened conical shape (1/8TK-SS1, Spraying Systems Co) were used.

The spray solution in the storage tank was pumped into the pressure regulator and then sprayed through the nozzles in the chamber. In the spray chamber, several spray layers consisting of stainless steel nozzles and tubes were installed, and the chamber was covered with plastic sheeting to prevent chemical drift. A shackle was used to hang a piece of chicken meat in the chamber.

Pre-chilled chicken meat pieces were obtained from a local poultry processing plant. They were removed from the end of an evisceration processing line, transported to the research laboratory, and immediately used for testing. The time elapsed between the processing plant and the research laboratory was less than half an hour. The temperature of the chicken meat was in the range of 32 to 37°C.

Chicken meat was inoculated by spraying 1 ml of S typhimurium at 1 x 10⁶CFU/ml and then incubated at room temperature for 30 minutes. The inoculated chicken meat was washed by spraying it with tap water at 30 psi and 22°C for 5 seconds to remove loosely attached Salmonella cells. Each piece of meat was then hung in the spray chamber and sprayed with one of the test compounds. After spraying, each piece of meat was washed with running water for 20 seconds. Chicken meat was then washed with peptone-buffered water in a plastic bag on an automatic shaker to obtain samples for microbial analysis. Chicken skin color was visually examined by comparing birds treated with test compounds, such as QACs, to untreated birds.

CPC was used at a concentration of 1000 ppm at various spray pressures and durations. The temperature of the dew water was set at temperature ο

ambient, ie, 22°C. Pressures were set at 30, 50, and 120 psi, and durations at 30 and 90 seconds. Three replicates were made for each test.

The reduction for S. typhimurium in each piece of chicken meat was compared between the groups sprayed with test compounds, the water sprayed and the non-sprayed groups.

After the spray treatments, each piece of meat was mechanically agitated with 100 ml of peptone-buffered water (BPW) for 1 minute and the wash water was collected. Samples were diluted, spiked, plated on XLT agar or Petrifilm agar (3M, Inc; St Paul, MN for total aerobic count plates) and incubated for 18 to 24 hours at 37°C. Then, the colony-forming units were counted. The amount of attached bacteria was calculated using a most likely amount technique. Most likely Salmonella counts and total aerobic plate counts were performed on each piece of meat using the wash water samples. An analysis of variance was used to evaluate the experimental data to determine any significant differences between treatment groups and controls (SAS/STAT User's Guide, SAS Institute, Inc, Cary, NC 1993).

The results of this experiment show that 30 and 90 second spraying of a 1000 ppm CPC solution at pressures of 30, 50 and 120 psi causes a significant reduction in the amount of Salmonella on chicken meat. These data show that the spray method is a viable alternative method to the common method of dipping or washing chickens when sprayed for 30 seconds to 90 seconds with a pressure in the range of 30 to 120 psi at a concentration of 0. 1% CPC. It may be possible to use lower CPC concentrations with variable spray pressures within the described range of 30 to 120 psi, or higher, and vary spray times to obtain the most efficient process that results in a significant reduction in microorganisms in the feed. The spray method will be advantageous for use in an industrial process, since many pieces of chicken meat could be sprayed automatically for short periods of time and still achieve a significant reduction in pathogenic bacteria.

Example 7

Concentration and effective time study of the effects of quaternary ammonium compounds on S typhimurium in chicken skin

The effects of CPC on the inhibition and reduction of viable S. typhimurium on chicken skin were studied. Test solutions comprised various concentrations of CPC (Sigma Chemical Co, St Louis, MO) in 5% (v/v) glycerin in 0.008 M phosphate buffered saline, pH 7.2 (PBS). Solutions were prepared by dissolving appropriate amounts of CPC in the glycerin-PBS mixture. Skin squares (2.5 x 2.5 cm) were sterilized from fresh unprocessed chicken feet by an irradiation dose of 45 kGy (electron beam from a focused linear accelerator, Iowa State University). The source of S typhimurium was ATCC strain # 14028 or NCTC strain # 12023. All colony counts were performed on tryptic soy agar (TSA; DIFCO, Detroit, MI) plates. Salmonella stock solution was in TSA. The preparation of inocula was done as follows. A flask containing 50 ml of tryptic soybean culture was inoculated with S typhimurium from a single colony and then incubated (37°C) with shaking (150 rpm) overnight. A one ml aliquot of culture was washed with 9 ml PBS (4800 rpm, 10 minutes) twice. The pellet was resuspended in PBS to obtain a final cell concentration (spectrophotometrically, 420 nm) of 1 to 2 x 10⁶ colony forming units (CFU) per ml.

Chicken skin was removed from the leg area and placed epidermis side up in each well of a six-well tissue culture plate. The skin pieces were inoculated with 5 ml of PBS containing 1 to 2 x 10⁶ CFU of S typhimurium per ml, with the exception of the background control group, which was treated with only 5 ml of PBS. The culture plates with the skin pieces were incubated (30 minutes, 35°C) and the incubation solution was removed by aspiration. The inoculated skins were treated with 5 ml of the test solution. Sets of three skin pieces were used for each concentration of test solution, including one set in which the skins were treated with only 5 ml of 5% (v/v) glycerin in PBS (0 concentration). The plates were incubated at 25°C with shaking (100 rpm) for

1, 3 or 10 minutes. After incubation, each piece of skin was washed with ς10 aspiration (5 mL PBS), placed in a sterile plastic bag containing 50 mL 0.1% (w/v) peptone, and homogenized. for 2 minutes using a stomacher7 400 laboratory blender (Seward Medical Co,

London England). One edge of the bag was cut aseptically and all its contents were transferred to a sterile centrifuge tube which was then centrifuged for 10 minutes (12000 rpm, 20°C). The precipitate was resuspended in 5 ml of 0.1% (w/v) peptone/water. One ml of the appropriate dilution was plated on TSA agar in triplicate and then incubated at 37°C for hours, after which colonies were counted, corrected for dilution, and the result reported as CFU/skin. The results show that the reduction of Salmonella depended on the concentration of CPC and the exposure time. Near 5 log-to decontamination was achieved when treating with 4000 and 8000 ppm CPC solutions for contact times as low as 3 minutes.

Skin squares were placed epidermis side up in each well of a six-well tissue culture dish. The pieces three pieces of skin for each concentration of test solution, including one set in which the skins were treated with only 5 ml of 5% (v/v) glycerin in PBS (0 concentration). The culture dishes with the skin pieces were treated with 5 ml of the test solution. Pools were incubated at 25°C with shaking (100 rpm) for 1, 3, or 10 minutes. The incubation solution was removed by aspiration and the skins washed (5 mL PBS) and then incubated (30 min, 35°C) with 5 mL PBS containing 1 to 2 x 10⁶ CFU of S typhimurium by me. After incubation, each piece of skin was washed with aspiration (5 mL PBS), placed in a sterile plastic bag containing 50 mL of 0.1% (w/v) peptone, and homogenized by 2 min using a stomacher7 400 laboratory blender (Seward Medical Co, London, England). Three aliquots of the homogenates (1 ml) were plated on TSA agar and incubated at 37°C for 24 hours to then count the colonies, correct for dilution and report the result as log™ CFU/skin. The results indicate that prevention of Salmonella contamination by CPC pretreatment also showed a concentration and time dependency. The most marked effects were observed for a pre-treatment of 10 minutes, where a 4.9 logw inhibition of Salmonella binding was verified at a concentration of 8000 ppm. This result is important since the prevention of contamination is of great importance in food processing.

Log-ιο CFU/skin values ​​for controls were within the range of 4.61 to 5.03. Differences between treated and control samples were analyzed using ANOVA followed by Newman-Keuls multiple range analysis and were found to be statistically significant (p&lt;0.01).

In another spray experiment, a 3.3 log-ιο reduction for Salmonella was obtained after a 90 second spray of chicken meat with a 5000 ppm CPC solution.

Example 8

Effects of quaternary ammonium compounds on the reduction of

Viable Listeria monocytogenes attached to chicken skin

The steps of Example 2 were followed, except that L monocytogenes was used to inoculate chicken skin and the media in the plastic bag used in the stomacher 400 contained 0.1% peptone. At concentrations of

CPC of 2000 ppm or greater, a greater than 4 log 10 reduction was observed in

L. monocytogenes.

Example 9

Effects of Quaternary Ammonium Compounds on the Inhibition of the Attachment of Viable Listeria monocytogenes Attached to Chicken Skin

The steps of Example 3 were followed, except that L 10 monocytogenes was used to inoculate chicken skin and the media in the plastic bag used in the stomacher 400 contained 0.1% peptone. The results of this study show an 82% reduction in fixed bacteria at 50 ppm, a 92% reduction at 100 ppm, and a 100% reduction at 500 and 1000 ppm.

Example 10

Effects of quaternary ammonium compounds on the reduction of

Viable Salmonella typhimurium attached to catfish, black grape, and broccoli

The effects of CPC on the reduction of viable S typhimurium on catfish, black grape and broccoli were studied. Test solutions comprised various concentrations of CPC (Sigma Chemical Co, St Louis, 20 MO) in 5% (v/v) glycerin in 0.008 M phosphate buffered saline, pH 7.2 (PBS). Solutions were prepared by dissolving appropriate amounts of CPC in the glycerin-PBS mixture.

Food samples were intact small black grapes, broccoli florets, and catfish skin squares (2.5 x 2.5 cm) separated from 25 freshly thawed unprocessed catfish. The fruits and vegetables were purchased from a local grocery store, while the fish was shipped frozen from a local catfish supplier. The source of S typhimurium was ATCC cell # 14028 or NCTC strain # 12023.

All colony counts were developed using Salmonella selective XLD agar plates (DIFCO, Detroit, MI). Additionally, in catfish experiments, total aerobic colony counts were performed using a non-selective medium, tryptic soy agar (TSA: DIFCO, Detroit, MI). Salmonella storage was at TSA.

Preparation of the S. typhimurium inocula was carried out as described in Example 7 above. Food samples were placed in each well of six-well tissue culture plates. Samples were then inoculated with 5 ml of PBS containing 1 to 2 χ 10⁶ CFU of S typhimurium per ml, with the exception of a baseline control group that was treated with only 5 ml of PBS. The culture plates with the food samples were incubated (30 minutes, 35°C) and then the incubation 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 test solution concentration, including one set in which the food samples were treated with only 5 mL of 5% (v/v) glycerin in PBS (0 concentration). The plates were incubated at 25°C with shaking (100 rpm) for 3 minutes. After incubation, each food sample was prepared and placed in a plastic bag for use with the Stomacher7 400 Laboratory Blender, as described in Example 7 above. One edge of the bag was cut aseptically and the entire contents were transferred to a sterile centrifuge tube which was then centrifuged for 10 minutes (12000 rpm, 20°C). The precipitate was resuspended in 5 ml of 0.1% (w/v) peptone/water. One ml of the appropriate dilution was placed on XLD agar for the grape and broccoli experiments, and on XLD and TSA agar for catfish, in triplicate. After incubation at 37°C for 24 hours, colonies were counted, corrected for dilution, and the result reported as

<img file="ECSP024309A_D0018.tif" />

Ί&gt;1

CFU/skin for catfish and as CFU/gram for the other feed samples. The results of these experiments are shown in Figures 2 through

5. Since the catfish had not been irradiated, Figure 2 shows total aerobic bacterial counts on nonselective media, while Figure 3 shows Salmonella counts only.

Example 11

Effect of quaternary ammonium compound spray on the reduction of viable bacteria in whole chickens c

These experiments evaluated the effect that QAC spraying whole chickens using a commercial spray would have on the reduction of viable bacteria. Bacteria inoculation solutions were made as follows: E coli (ATTC # 25922) was grown in Brain Heart Infusion (BHI) solution for 20 to 24 hours, then diluted to a concentration of 1 in 1000 adding 0.5 ml of E coli culture to 500 ml of physiological saline solution (PSS). S typhimurium was grown in BHI for 20 to 24 hours and then diluted to a concentration of 1 in 5000 by adding 0.1 ml of S typhimurium culture to 500 ml of physiological saline (PSS). The CPC treatment solution was prepared at a concentration of 5000 ppm. Prechill chickens were obtained from a local poultry processing plant for each trial. Chickens were placed in a line of shackles and 1 ml of the inoculation solution was sprayed on the belly of each chicken, and 1 ml on the back. The bacteria were allowed to settle for 30 minutes at room temperature. After attachment, chicks on the shackle line were washed under running water for 20 seconds. The chickens were divided into groups of ten. For each case, there was a group of ten chickens that was sprayed with 5000 ppm CPC and there was a group of ten chickens that was sprayed with plain water only. In the tests with S typhimurium there was also a group that was not sprayed after the inoculation to evaluate the effect of the spray.

For all bacteria, a group of chickens was treated with Johnson™ Wash for 20 seconds at 60 psi with 35 cups of tap water. After washing, the chicks were allowed to rest for 90 seconds and then washed with 20 cups of tap water for 20 seconds at 80 psi. This wash cycle was repeated every two to three times. The interval of each wash was also 90 seconds. Another group of chickens was treated with 5000 ppm CPC for 20 seconds at 60 psi in the Johnson™ wash, then allowed to sit for 90 seconds, and then washed with 20 cups of tap water for 20 seconds at 80 psi. This wash cycle was repeated every two to three times.

After treatment, the chickens were placed in plastic bags and 100 ml of 0.1% peptone-buffered water (BPW) was added to each bag. The bags were shaken mechanically and the lavage was collected using the Most Probable Quantity (MPN) technique. Petrifilm™ was also used for the evaluation of total aerobic counts per plate (TPC). Pre-existing C jejuni (not inoculated) were enumerated by the MPN technique and E coli by Petrifilm™.

The results presented below show that CPC treatment is effective in reducing the amount of C jejuni, E coli and S typhimurium. The wash water from the tests was evaluated with S typhimurium and found that CPC in the wash water reduced Salmonella by 1 log. Thus, the kill data presented below for Salmonella can be reduced by 1 log.

<td colspan="5">BACTERIA PRESENT</td>

<td colspan="2"></td><td>control with water</td><td>5000 ppm of cpc</td><td>reduction in Log₁₀</td>

<td>C JEJUNI</td><td>Test 1</td><td> 2,613</td><td> 0</td><td> 2,613</td>

<td></td><td>test 2</td><td> 2,643</td><td> 0,629</td><td> 2,014</td>

~SZ

Ace

<td>ECOLI</td><td>Test 1</td><td> 1,974</td><td> 0,386</td><td> 1,588</td>

<td></td><td>test 2</td><td> 1,380</td><td> 0,460</td><td> 0,920</td>

<td>S TYPHIMURIUM</td><td>Dewless Control</td><td>control with dew</td><td>5000ppm of CPCs</td><td colspan="2">Reduction in Logi₀ UFC</td>

<td></td><td></td><td></td><td></td><td>Treatment</td><td>Treatment</td>

<td></td><td></td><td></td><td></td><td>no dew</td><td>with dew</td>

<td></td><td></td><td></td><td></td><td>vs. CPCs</td><td>vs. CPCs</td>

<td> 1 (12/02/96)</td><td> 5,342</td><td> 5,039</td><td> 4,295</td><td> 1,047</td><td> 0,744</td>

<td> 2 (12/09/96)</td><td> 5,304</td><td> 4,932</td><td> 1,977</td><td> 3,327</td><td> 2,955</td>

<td> 3(12/16/96)</td><td> 5,001</td><td> 5,154</td><td> 2,606</td><td> 2,395</td><td> 2,548</td>

<td> 4 (01/27/97)</td><td> 4,72</td><td> 4,48</td><td> 1,03</td><td> 3,69</td><td> 3,45</td>

<td> 5 (02/03/97)</td><td> 4,185</td><td> 4,212</td><td> 1,426</td><td> 2,76</td><td> 2,79</td>

Example 12

Effect of quaternary ammonium compounds on food fungi

This study evaluated the effect of CPC on food mushrooms. Slant cultures of Aspergillus flavus and Penidllium chrysogenum were plated on potato dextrose agar (PDA) plates. Thirty minutes after inoculation or 24 hours after inoculation (and incubation at room temperature), two round filters (7 mm diameter) were placed on the surface of each plate. Added 200 ppm CPC solutions,

1000 ppm, 5,000 ppm, and 25,000 ppm, or distilled and deionized (DD) water to filters, 10 μΙ per filter. All plates were incubated lid side up at room temperature for 48 hours. The diameters of the inhibition rings were measured. The results presented below show that CPC is effective against food molds.

<td colspan="3">Effect of CPC on Aspergillus flavus</td>

<td>CPC concentration (ppm)</td><td colspan="2">Inhibition ring (mm)</td>

<td></td><td>immediate treatment</td><td>Treatment delayed</td>

<td> 25000</td><td> 1,63</td><td> 1,00</td>

<td> 5000</td><td> 2,00</td><td> 0,92</td>

<td> 1000</td><td> 0,38</td><td> 1,00</td>

<td> 200</td><td> 0,25</td><td> 0,33</td>

<td> 0</td><td> 0</td><td> 0</td>

<td colspan="3">Effect of CPC on Penicillium chrysogenum</td>

<td>CPC concentration (ppm)</td><td colspan="2">Inhibition ring (mm)</td>

<td></td><td>immediate treatment</td><td>delayed treatment</td>

<td> 25000</td><td> 4,13</td><td> 1,83</td>

<td> 5000</td><td> 3,38</td><td> 1,92</td>

<td> 1000</td><td> 1,00</td><td> 1,67</td>

<td> 200</td><td> 0</td><td> 1,17</td>

<td> 0</td><td> 0</td><td> 0</td>

CPC is effective against tested food molds.

Example 13

Effect of Quaternary Ammonium Compounds on chickens using 5 short application times

In two trials conducted at a broiler chicken processing facility, the Cecureú formulation (0.2 to 0.5% CPC) was used, which is diluted from a CPC concentrate containing CPC (40%), propylene glycol (57%) and water

<img file="ECSP024309A_D0019.tif" />

€10 ^20 (3%), all components on a weight by weight basis, to treat post-chill chickens. In these studies, the final washing booth or "faecal failure" booth, which is located before the accommodation and packaging sections, but before the immersion cooling section, was modified for the application of the CPC formulation. Modifications to the booth included changing the nozzles to allow only small volumes (1 to 6 ounces) of formulation per chicken, and modifying the spray pattern on the chickens to allow full coverage of a maximum surface area. In addition, the entire length of the cabin was extended and cabin evacuation mechanisms were installed. The Cecureü concentrated formulation was either diluted to the correct concentration for use at the point of direct application to the chicken or diluted and held in large containers prior to application. The diluted Cecureú solution was applied to each chicken for about 1.5 seconds. The solution temperature was either room temperature or slightly above or below that, depending on storage conditions.

After treatment of the chickens with the diluted Cecureü solution, the chickens were allowed to drain for approximately 3 minutes before analysis for microbes. Chickens were analyzed using a technique of washing whole chickens in 400 ml of peptone-buffered water. Samples were evaluated for the presence of Campylobacter, Salmonella, and non-toxin-producing E. coli, and aerobic plate counts were performed to estimate total organisms. Control chickens were also tested for these same organisms, but these chickens were collected just prior to the modified fecal failure booth. In both tests, Campylobacter, E coli, and aerobic plaque (total aerobic bacteria) counts were significantly reduced by more than 99%. In both tests, the incidence of Salmonella was significantly reduced to less than 10% positive, while the incidence rates in control chickens with Salmonella were greater, in some cases, than 60%.

The foregoing description of the preferred embodiments of the present invention is presented for illustrative purposes and is not intended to limit the invention to the specific compositions used in the examples, as various modifications to the described invention are possible in light of the above teachings. . The present invention is based on the discovery that QACs prevent and significantly reduce bacterial contamination over a broad spectrum. QAC's concentrated formulation provides many advantages for use on a large scale in a food processing plant. The invention is intended to cover alternatives, modifications, and equivalents that may be included within the scope and spirit of the invention, as defined by the appended claims.