Field of the Invention

The present invention concerns the use of peptide bacteriocins in liquid whole egg products to combat coagulation and other forms of spoilage, and to combat Listeria monocytogenes .

Background of the Invention

Until recently pasteurization processes for liquid whole egg products were relatively mild. While these pasteurization procedures are capable of effectively eliminating Salmonella organisms from eggs they are not capable of destroying other organisms that can spoil egg products held under non-freezing refrigerated temperatures. The shelf life of commercial pasteurized egg products is therefore limited, usually to 7-14 days, and pasteurized egg products require freezing and frozen distribution systems.

Methods of ultrapasteurization of liquid whole egg products have recently been developed by Swartzel, Ball and Samimi . See U.S. Patents No. : 5,105,724; 5,019,408; 5,019,407; 4,994,291; 4,957,759; and 4,808,425. This work has opened the door to other developments in ultrapasteurization technology as well. Ultrapasteurization decreases the number of spoilage microorganisms to levels lower than that obtained with

pasteurization procedures, without sacrificing the functional performance of the product. Ultra¬ pasteurization produces liquid whole egg products with refrigerated shelf lives greatly exceeding that available with conventional pasteurization procedures.

Coagulation of ultrapasteurized liquid whole egg product can occur when the product is stored at abuse temperatures (i.e., temperature > 40°F) for varying periods . Other types of spoilage may occur depending on the temperature and the type of bacteria in the product . In addition to coagulation, spoilage may take the form of changes in pH, changes in viscosity, changes in color or off odor.

Summary of the Invention It has now been found that coagulation in extended-shelf life liquid whole egg products subjected to non-refrigerated temperatures is due to the growth of Bacillus species, and that the addition of lanthionine bacteriocins retards this growth of Bacillus species and therefore combats the coagulation of such products.

A first object of the present invention is a method of making a packaged liquid whole egg product characterized by a preselected refrigerated shelf life of about four weeks to about 36 weeks, wherein a lanthionine bacteriocin is added to said product prior to packaging and in an amount effective to combat Bacillus-induced coagulation of said product when stored at non- refrigerated temperatures.

More particularly disclosed is a method of combatting Bacillus-induced coagulation in a packaged liquid whole egg product characterized by a preselected refrigerated shelf life of about four weeks to about 36 weeks. The method comprises, first, passing a liquid whole egg product as a continuous stream through a pasteurizing apparatus during which the liquid whole egg product is heated for a predetermined time and to a

predetermined temperature. The predetermined temperature and predetermined time are chosen to impart the preselected shelf life to the liquid whole egg product. The liquid whole egg product is then aseptically packaged. A lanthionine bacteriocin is added to the product prior to packaging in an amount effective to combat coagulation of the product when stored at non- refrigerated temperatures .

A second aspect of the present invention is a liquid whole egg product characterized by a preselected refrigerated shelf life of about four weeks to about 36 weeks . The product contains a lanthionine bacteriocin in an amount effective to combat Bacillus-induced coagulation of the product at non-refrigerated temperatures.

Another object of the present invention is a method of combatting the growth of Streptococcus faecalis in pasteurized liquid whole egg product, wherein a lanthionine bacteriocin is added to said product prior to packaging and in an amount effective to combat the growth of S . faecalis in said product.

Still another object of the present invention is a method of combatting the growth of Listeria monocytogenes in ultrapasteurized liquid whole egg product characterized by a refrigerated shelf life of about four weeks to about 36 weeks, when the product is stored under non-refrigerated conditions. The method utilizes lanthionine bacteriocins in amounts effective to combat the growth of Listeria monocytogenes . In a further aspect, this method also includes adjusting the pH of the liquid whole egg product to between 6 and 7.

A further aspect of the present invention is an ultrapasteurized liquid whole egg product with a preselected refrigerated shelf life of about four weeks to about 36 weeks, having a pH of from 6 to 7 and containing a lanthionine bacteriocin in an amount effective to inactivate, and to reduce the rate of growth

of, Listeria in the product at non-refrigerated temperatures.

The foregoing and other objects and aspects of the present invention are explained in detail in the specification set forth below.

Brief Description of the Figures FIGURE 1A shows the effect of three concentrations of nisin on the growth of L . monocytogenes (log CFU/ml) in ultrapasteurized liquid whole egg with a pH of 7.5, stored at a temperature of 4°C. Open circles represent 0 IU/ml nisin; open triangles represent 100 IU/ml nisin; open squares represent 1000 IU/ml nisin.

FIGURE IB shows the effect of three concentrations of nisin on the growth of L . monocytogenes (log CFU/ml) in ultrapasteurized liquid whole egg with a pH of 7.5, stored at a temperature of 10°C. Open circles represent 0 IU/ml nisin; open triangles represent 100 IU/ml nisin; open squares represent 1000 IU/ml nisin.

FIGURE 2A shows the effect of three concentrations of nisin on the growth of L . monocytogenes

(log CFU/ml) in ultrapasteurized liquid whole egg with a pH of 6.6, stored at a temperature of 4°C. Open circles represent 0 IU/ml nisin; open triangles represent 100

IU/ml nisin; open squares represent 1000 IU/ml nisin. FIGURE 2B shows the effect of three concentrations of nisin on the growth of L . monocytogenes

(log CFU/ml) in ultrapasteurized liquid whole egg with a pH of 6.6, stored at a temperature of 10°C. Open circles represent 0 IU/ml nisin; open triangles represent 100 IU/ml nisin; open squares represent 1000 IU/ml nisin.

FIGURE 3A shows the effect of three concentrations of nisin on the mesophilic aerobic plate count (log CFU/ml) of ultrapasteurized liquid whole egg with a pH of 7.5, stored at a temperature of 4°C. Open circles represent 0 IU/ml nisin; open triangles represent 100 IU/ml nisin; open squares represent 1000 IU/ml nisin.

FIGURE 3B shows the effect of three concentrations of nisin on the mesophilic aerobic plate co i.t (log CFU/ml) of ultrapasteurized liquid whole egg with a pH of 7.5, stored at a temperature of 10°C. Open circles represent 0 IU/ml nisin; open triangles represent 100 IU/ml nisin; open squares represent 1000 IU/ml nisin.

FIGURE 4A shows the effect of three concentrations of nisin on the mesophilic aerobic plate count (log CFU/ml) of ultrapasteurized liquid whole egg with a pH of 6.6, stored at a temperature of 4°C. Open circles represent 0 IU/ml nisin; open triangles represent 100 IU/ml nisin; open squares represent 1000 IU/ml nisin.

FIGURE 4B shows the effect of three concentrations of nisin on the mesophilic aerobic plate count (log CFU/ml) of ultrapasteurized liquid whole egg with a pH of 6.6, stored at a temperature of 10°C. Open circles represent 0 IU/ml nisin; open triangles represent 100 IU/ml nisin; open squares represent 1000 IU/ml nisin.

Detailed Description of the Invention An aspect of the present invention is a method of combatting coagulation in liquid whole egg product after processing; the method uses compounds containing bacteriocins . A further aspect of the present invention is a method of restricting the survival and growth of Listeria monocytogenes in liquid whole egg product stored at refrigeration temperatures; the method uses compounds containing bacteriocins. The present invention may be practiced with any suitable method of ultrapasteurization that produces liquid whole egg products having extended refrigerated shelf life.

Disclosed is a method of combatting Bacillus- induced coagulation in a packaged liquid whole egg product characterized by a preselected refrigerated shelf life of about four weeks to about 36 weeks. The method comprises, in a particular embodiment, passing the liquid whole egg product as a continuous stream through a

pasteurizing apparatus, during which the liquid whole egg product is :

(a) heated to a predetermined holding temperature, then (b) maintained at the predetermined holding temperature for a predetermined holding time, then

(c) cooled, and then

(d) aseptically packaged, wherein a lanthionine bacteriocin is added to the product prior to packaging in an amount effective to combat coagulation of the product when stored at non- refrigerated temperatures .

Coagulation in eggs refers to a change from the fluid to the solid or semisolid state, and is known to be caused by a number of factors including heat, mechanical means, salts, acids, alkalis, and other reagents such as urea. See Cunningham, Egg-Product Pasteurization, in Stadelman and Cotterill (Eds.) Ecrg Science and Technology, 249 (1990) . U.S. Pat. No. 5,096,728 to Rapp discloses the addition of organosulfur compounds to egg products prior to pasteurization to reduce coagulation due to heating during the pasteurization process, however, Rapp does not address coagulation after ultrapasteurization due to storage at abuse temperatures. As used herein, "combatting coagulation" refers to reducing the extent of coagulation that would otherwise be experienced.

Disclosed is a method of combatting the survival and growth of Listeria monocytogenes in a packaged liquid whole egg product characterized by a preselected refrigerated shelf life of about four weeks to about 36 weeks. The method comprises, in a particular embodiment, passing the liquid whole egg product as a continuous stream through a pasteurizing apparatus, during which the liquid whole egg product is:

(a) heated to a predetermined holding temperature, then

(b) maintained at the predetermined holding temperature for a predetermined holding time, then

(c) cooled, and then

(d) aseptically packaged, wherein a lanthionine bacteriocin is added to the product prior to packaging in an amount effective and at a pH effective to combat the survival and growth of Lis teria monocytogenes in the product when stored at non- refrigerated temperatures. The predetermined time and predetermined temperature are chosen to impart a preselected shelf life to the liquid whole egg product. As an example, a lanthionine bacteriocin may be added in amounts effective to essentially prevent an increase in the numbers of Listeria monocytogenes organisms in the liquid whole egg product for two weeks when stored at 10°C. Survival and growth of Listeria monocytogenes in liquid whole eggs refers to the continued presence of Listeria monocytogenes and the increase in the numbers of Listeria organisms over time. A method accepted in the art of measuring the presence and increase of microorganisms is to assess the numbers of colony forming units of a given microorganism from a sample (on a growth medium) over time.

Ultrapasteurization refers to processes that decrease the number of spoilage microorganisms to levels lower than obtained with a pasteurization process to thereby obtain a product with an extended shelf life under refrigerated conditions. Representative processing techniques are exemplified in U.S. Pat. Nos. 4,808,425 and 5,019,408 to Swartzel, Ball and Hamid-Samimi, which disclose ultrapasteurization of liquid whole egg products using continuous flow, high temperature, short time pasteurization equipment (the disclosures of all U.S.

Patent references cited herein are to be incorporated herein by reference) . In these processes a continuous stream of liquid whole egg product is heated to a predetermined temperature by contact with a heated surface, maintained at the predetermined temperature for a predetermined holding time, and then cooled and aseptically packaged. U.S. Pat. No. 4,957,760 to Swartzel, Ball and Liebrecht discloses ultrapasteurization of liquid whole egg product in a continuous stream, wherein the product is heated to a first predetermined temperature and maintained at that temperature for a predetermined time, then heated to a second predetermined temperature using steam and maintained at the second temperature for a predetermined time, then cooled and aseptically packaged. U.S. Pat. No. 5,019,407 to Swartzel and Ball discloses a pasteurization process using an egg yolk product stream and an egg white product stream, wherein the egg yolk product stream is heated to a predetermined temperature greater than the highest temperature of the egg white product stream, and product streams are recombined to equilibrate to a second predetermined temperature.

Methods for making extended shelf-life liquid whole egg product are also discussed in U.S. Pat. No. 5,167,976 to Papetti . U.S. Pat. No. 4,971,827 to Huang discloses processing of egg white only product to extend refrigerated shelf life.

It will be readily appreciated that the use of the present invention is not dependent on the method of ultrapasteurization used. Both batch ultrapasteurization and continuous stream methods of ultrapasteurization may be used. It will also be readily appreciated that use of the present invention is not dependent on the method of heating utilized in the ultrapasteurization process. Methods of heating utilized in ultrapasteurization procedures may include, but are not limited to, direct contact with a heated surface { see, e . g. , U.S. Pat. Nos.

4,808,425; 4,957,760; and 5,019,408), steam injection { see, e . g. U.S. Pat. No. 4,675,202) , steam infusion ( see, e . g. , U.S. Pat. No. 4,957,760) , the use of electrical currents ( see, e . g. , U.S. Pat. No. 4,417,132; U.S. Pat. No. 4,739,140) , or the use of microwave energy ( see, e . g. , U.S. Pat. No. 4,853,238) .

As used herein "prior to packaging" means any time prior to aseptic packaging of the egg product, and includes prior to ultrapasteurization of an egg product, during ultrapasteurization of an egg product, and following ultrapasteurization of an egg product but prior to packaging. The addition of lanthionine bacteriocins to egg products, as practiced in the present invention, may occur at any of these times. Examples of whole egg products which can be pasteurized in liquid form by the techniques described above include whole egg, fortified whole egg (whole egg with added yolk) , salt whole egg (e.g., salt 10%) , sugar whole egg (e.g., sugar 10%) , blends of whole egg with syrup solids, syrups, dextrose and dextrins and/or gums and thickening agents, blends of whole eggs with less than 1% sugar and/or salt, scrambled egg mixes (for example, a mix of about 51% egg solids, 30% skim milk solids, 15% vegetable oil and 1.5% salt) , reduced cholesterol egg products and blends thereof, custard blends, and the like. Products which are extremely sensitive to thermal processing and which are particularly suitable for ultrapasteurization by the present invention include, for example, liquid whole egg and blends thereof (less than 2% added non-egg ingredients) , fortified whole egg and blends thereof (24- 38% egg solids, 2-12% added non-egg ingredients) , liquid salt whole egg, liquid sugar whole egg, and other liquid whole egg blends which are 24-38% egg solids and 12% or less of added non-egg ingredients. Terms used herein have their standard meaning in accordance with industry

and regulator usage. See, e.g., 7 C.F.R. §59.570 (b) (1985) .

Ultrapasteurized liquid whole egg products are sold to both retail and institutional markets. The product has a shelf-life of 4-36 weeks, or more preferably 8-36 weeks, if refrigerated. As used herein, the term "refrigerated" means maintained at a temperature below 40°F but above freezing. If stored at abuse temperatures the shelf-life of the product is reduced. Abuse temperatures are defined as those temperatures above 40°F. The product may temporarily be subjected to abuse temperatures during shipping.

Reduced shelf-life in ultrapasteurized liquid whole egg product is noted when stored at abuse temperatures of > 40°F. This reduction in shelf life may manifest as coagulation or as other forms of spoilage. Coagulation is noted to be primarily a temperature abuse problem, in that all product will coagulate when held at elevated temperatures. The abuse time necessary to cause coagulation varies depending on the initial numbers of microorganisms present, the types of organisms present, and the temperature of abuse. Coagulation results in a lowered pH and changed color of the product . Other types of spoilage may or may not occur concurrently with coagulation; other types of spoilage may occur without coagulation. In addition to coagulation, spoilage is considered to include any signs of off color, off odor, or change of pH or viscosity in the product.

Our analyses of coagulated ultrapasteurized liquid whole egg product, as discussed below, indicates that Bacillus-type organisms are the main cause of the problem, primarily B. cereus . B . cereus is a ubiquitous organism that is found in many refrigerated foods; Bacillus strains are common contaminants of raw eggs and may survive pasteurization. See e.g. , Wood and Waites,

Food Microbiology 5:103 (1988) .

The thermal treatments of liquid egg pasteurization processes are designed to inactivate egg- associated bacterial pathogens, especially Salmonella species. In recent years, research efforts have been directed at elucidating the prevalence, survival, and growth characteristics of the bacterial pathogen Listeria monocytogenes in eggs and egg products (Foegeding and Stanley, J. Food Prot . 53, 6-8 (1990)) . Over the past decade, L . monocytogenes has been firmly established as an important food-associated pathogen due to its widespread distribution in nature, its relative resistance to heat and environmental extremes (e.g., salt, nitrite, pH extremes) , and its ability to grow at temperatures as low as 1°C (34°F) (Lovett and Twedt, Listeria pp. 8-11, In J.L. Oblinger (ed.) , Bacteria Associated with Foodborne Diseases, Food Technol . 42, 1- 20 (reprint) (1988) ; Ryser and Marth, (1991) ) .

United States Department of Agriculture- prescribed conventional pasteurization processes for LWE are designed to provide a 9 orders-of-magnitude (9-D) process for inactivation of salmonellae in the average particle (USDA, Egg Pasteurization Manual, ARS 74-48, Poultry Laboratory, Agricultural Research Service, United States Department of Agriculture, Albany, CA (1969)) . Listeria thermal inactivation studies conducted by Foegeding and Leasor (1990) indicate that such conventional minimal pasteurization processes would effect only a 2.1 to 2.7-D inactivation of L . monocytogenes in LWE; thus, the margin of safety provided by conventional pasteurization processes is substantially lower for L. monocytogenes than for most Salmonella species .

Because of the relative heat resistance of L. monocytogenes, its ability to persist in egg processing plant environments, and its ability to grow in LWE at refrigeration temperatures, postprocessing Listeria

contamination of refrigerated LWE is a food safety concern.

Nisin is a bacteriocin with known activity against Bacillus species and against certain strains of Streptococcus, and is widely used as a food additive. Bacteriocins are proteins produced by certain bacteria, and have a lethal effect on other bacteria. Bacteriocins generally have a narrower range of activity than antibiotics and are more potent. Nisin is produced by several strains of the bacterium Streptococcus lactis (also known as Lactococcus lactis) , and can be isolated and concentrated from cultures of Streptococcus lactis or from recombinant microorganisms containing DNA encoding for nisin production. See Cheeseman and Berridge Biochem . J. 65, 603 (1957) ; Bailey and Hurst, Canadian J. Microbiol . 17, 61 (1971) . Nisin is available commercially as NISAPLIN (TM) (Aplin & Barrett Ltd., Dorset, Great Britain) . NISAPLIN (TM) has a standardized activity of 1 x 10 ⁶ IU of nisin/g and contains 25 mg nisin/g. The activity of pure nisin is 40 times that of NISAPLIN (TM) .

Nisin has activity against a range of Gram positive bacteria, particularly the spore formers. Nisin is known to inhibit the majority of spore forming species of Clostridium and Bacillus with the spores being more sensitive than the vegetative cells. Nisin action against spores is sporicidal . It has been demonstrated that heat-damaged spores are more sensitive to nisin than non heat-damaged spores. See Delves-Broughton, Food Technology, 106-113, (Nov. 1990) . A summary of nisin' s properties can be found in Hurst, A. Advances in Applied Microbiology 27:85-123 (1981) .

Nisin is used as a preservative in dairy products such as processed cheese, cream, and milk. U.S. Pats. No. 4,584,199 and 4,597,972. Nisin has also been used to control post-processing contamination in processed meat (US Pat. No. 5,015,487) and in a process

for deacidifying wine (US Pat. No. 5,059,431) . Its use has been disclosed for preventing Clostridium botulinum contamination in canned vegetables, cold meat products and wet fish systems. (U.S. Patent No. 4,597,972) . A summary of the international use of nisin as a food additive may be found in Delves-Broughton, Food Technology, 106-113 (Nov. 1990) .

Nisin is one of a group of lanthionine- containing polypeptides. Lanthionine-containing polypeptides are also known as lantibiotics, and are produced by Gram-positive bacteria of different genera. For a recent review of lantibiotics, see Lantibiotics: A Survey G. Jung (Ed.) 1991.

Lantibiotics which may be useful in the present invention include, but are not limited to, nisin, subtilin (Gross et al . Z. Physiol . Chem . , 354, 810

(1973)) , epidermin (Schnell et al . Nature , 333,276

(1988)) , Pep 5 (Sahl, J ^" . Bacteriol . , 162, 833 (1985)) , gallidermin (Kellner et al, Eur . J. Biochem . Ill , 53 (1988)) , mersacidin (Lantibiotics: A Survey G. Jung

(Ed.) , 1-34) , actagardine (Kettenring et al . , J.

Antibiotics , 53, 1082 (1990)) , cinnamycin (Kessler et al., Helv. Chim . Acta , 71, 1924 (1988)) , duramycin

(Gross, Adv. Exp . Med. Biol . 86b, 131 (1977)) , ancovenin (Wakamiya et al . , Tetrahedron Lett . 26, 665 (1985)) , and Ro09-0198. Some of these compounds have been found to have molecular structures similar to nisin. See Hurst, pp. 85-86; Schnell et al . , Nature 333:276 (1988) ; Sahl, J. Bacteriol . 162;833 (1985) . As discussed in detail below, a nisin preparation in the commercial form of NISAPLIN (TM) from Aplin and Barrett proved to be effective in combatting coagulation and in retarding other forms of spoilage in ultrapasteurized liquid whole egg products stored at abuse temperatures (> 40°F) . The present method uses nisin in a range of from 10 - 10,000 IU/g of liquid whole egg product, and more preferably in the range of from

100-500 IU/g of liquid whole egg product, to combat Bacillus-induced coagulation and other forms of spoilage of ultrapasteurized liquid whole egg product stored at temperatures over 40°F. The method can also be used in pasteurized liquid whole egg products.

Benkerroum and Sandine, J ^" . Dairy Sci . , 71, 3237

(1988) reported that the inactivation of L. monocytogenes

ATCC 7644 (log 3.6 CFU/ml) by nisin (3,700 IU/ml) in trypticase soy broth occurred more rapidly at pH 5.5 and 5.9 than pH 6.5 or 7.0. Harris et al . , J ^" . Food Prot . , 54, 836-840, (1991) , demonstrated that the lethality of nisin (37 to 1850 IU/ml) in BHI agar was increased when the pH of the medium was reduced from 6.5 to 5.5 with HCl or lactic acid. Mohamed et al . , Food antibiotic nisin: Comparative effects on Erysipelothrix and Listeria, In Woodbine, M. (ed.) , Antimicrobials and Agriculture, Butterworths, London (1984) reported that the MICs of nisin against two strains of L. monocytogenes in nutrient broth were reduced from 256 IU/ml to 128 IU/ml when the pH of the medium was reduced from 7.4 to 6.5.

The antibacterial activity of acidulants depends on both the type and concentration of acid rather than on pH alone. Several investigators have demonstrated that when compared at equal initial pH values in laboratory media, the relative inhibitory effect of several important acidulants against L. monocytogenes was acetic > lactic > citric > malic _> hydrochloric acid (Sorrells et al . , J. Food Prot . , 52, 571-573 (1989) ; Ahamad and Marth, J. Food Prot . , 52, 688- 695 (1989) ) . In previously reported studies, growth of L. monocytogenes was prevented only when acidification was sufficient to reduce the pH to approximately 4.4 to 5.0. The dissociation constant (pK _a ) of most organic acids lies between pH 3 and 5. Since the undissociated form of the acid is believed to be responsible for the antimicrobial effect, organic acids themselves generally show antibacterial activity only in foods with pH values

below 5.0 (Doores, Organic Acids. pp. 95-136. In Davidson, P.M. and Branen, A.L. (eds.) Antimicrobials in Foods, 2nd ed. , Marcel Dekker, Inc., New York (19^3) .

As discussed in detail below, NISAPLIN (TM) (Aplin and Barrett) proved to be effective in combatting Listeria monocytogenes in ultrapasteurized liquid whole egg products. The present method uses nisin in a range of from 10 - 10,000 IU/g of liquid whole egg product, and more preferably in the range of from 100-1,000 IU/g of liquid whole egg product, to combat survival and growth of L. monocytogenes in ultrapasteurized liquid whole egg product stored at temperatures over 40°F. The method can also be used in pasteurized liquid whole egg products.

Most preferably, when the present invention is used to combat L. monocytogenes , the pH of the liquid whole egg product is decreased to below 7.5; preferably the pH is between 6 and 7. This decrease in pH acts synergistically with lanthionin bacteriocins to increase the anti-listerial effects of the bacteriocins. The alteration in pH may be accomplished using any suitable means, including inorganic acids (for example, phosphoric acid) or organic acids. Organic acids are preferred including, but not limited to, citric acid, acetic acid, lactic acid and malic acid. Citric acid is presently most preferred.

While the invention has been described in detail, above, for the control of B. cereus-induced coagulation, it will be appreciated that the same information applies with respect to the control of Streptococcus faecalis .

As used herein, the term "combat" or "combatting" in reference to microorganisms in egg products may refer to either a decrease in absolute numbers of a microorganism over time, or a decrease in the rate of growth of a microorganism (over that rate which would otherwise be experienced) .

The present invention is explained in greater detail in the following Examples, where °C means degrees Centigrade, °F means degrees Fahrenheit, ml means milliliter, g means gram, mm means millimeter, M means molar, μl means microliter, w/v means weight per volume, v/v means volume per volume, h means hour, LWE means liquid whole egg, BHI means brain heart infusion, PW means peptone water, MIC means minimum inhibitory concentration, IU means international units, SPC means standard plate count, and CFU means colony forming units.

EXAMPLE 1

Coagulation in Ultrapasteurized Li uid Whole Egg Products

This example illustrates that coagulation in ultrapasteurized liquid whole egg products subjected to abuse temperatures is due to the growth of Bacillus species.

I. Identification of Bacillus species.

Samples of unspoiled and spoiled ultrapasteurized liquid whole egg product and reduced cholesterol ultrapasteurized liquid whole egg product, as well as raw (non-ultrapasteurized) reduced cholesterol liquid whole egg product and raw whole eggs were plated on Brain Heart Infusion Agar, Trypticase Peptone-Glucose- Yeast Extract Agar (TPGY) , YM agar (for yeast/molds) , Plate Count Agar (PCA) , Mannitol-Egg Yolk-Polymyxin agar (MYP) and MRS agar + CaCo ₃ (for lactobacilli) .

Samples were plated both without heat shocking and after heat shocking for 10 minutes at 80°C or 20 minutes at 60°C. Heat shocking helps select for spore- forming microorganisms . MRS plates were incubated microaerophilically at room temperature, TPGY plates were incubated anaerobically at 30°C, and the rest were incubated aerobically at 30°C. The resulting microbiological growth was examined and characterized.

Samples of spoiled reduced cholesterol ultrapasteurized liquid whole egg product (pH 5.65 with a firm, yogurt-like consistency and a slightly lighter color, resulting from temperature abuse) grew 3 x 10 ⁷ - 3 x 10 ⁸ /ml of two very similar colony types. These both were identified as large, gram-positive, facultatively anaerobic, catalase-positive rods, some of which were in chains, which had oval, mostly central endospores that did not swell the rod (indicating that these were Group I Bacillus species) .

Samples of unspoiled reduced cholesterol ultrapasteurized liquid whole egg product (pH 7.0-7.05) grew 7 x 10 ³ /ml gram-positive, catalase-negative short rods/cocci, possibly lactobacilli. Samples of spoiled ultrapasteurized liquid whole egg product (pH 5.6 with a similar consistency as spoiled reduced cholesterol ultrapasteurized liquid whole egg product) grew 2 x 10 ⁶ /ml Group I Bacillus, and 2 x 10 ⁵ /ml gram-positive, catalase-negative short rods/cocci. Samples of unspoiled ultrapasteurized liquid whole egg product (pH 7.1) had no detectable colonies; if any bacteria were present their numbers were below 10 ¹ /ml.

Samples of raw (unpasteurized) reduced cholesterol liquid whole egg product (pH 6.7) contained 10 ^s /ml similar short rods/cocci, as well as 10 ⁴ /ml very motile short gram-negative rods. No Bacillus were detected, although they may have been present at less than lOVml.

Raw whole eggs (pH 7.1) had a total aerobic plate count of 2 x 10 ³ /ml, which included at least 10 colony types, and a wide variety of rods and cocci. No Bacillus were detected, although they may have been present at less than 10 ¹ /ml.

No yeasts or molds were detected. The Bacillus and the short rods/cocci survived heat shocking in the egg products, but their numbers were reduced 2-3 logs.

The following tests focus on the Bacillus and the short rod/cocci isolates obtained from the samples.

II . Bacillus Tests

A total of five Bacillus isolates (four from reduced cholesterol ultrapasteurized liquid whole egg product and one from ultrapasteurized liquid whole egg product) were further tested to try to identify the species. An API (TM) Rapid CH (TM) kit (Analytab Products, Division of Sherwood Medical, Plainview, NY) was used to identify biochemical and nutritional characteristics of the isolates. All tests were run in duplicate.

Results for all tests were identical : -gram positive -catalase positive

-spores oval, mostly central, and do not swell the rod -motile (looks like flagellar motion) -casein hydrolysis positive -ferment glucose, but not arabinose, xylose, or mannitol -facultative anaerobe

-grow in up to 7% NaCl, but not 10% NaCl -grow in 0.001% lysozyme -grow in pH 7.5, 6.8, 5,7, but not 5.0

-grow at 12°C-42°C, but not 4°C or 50°C -Voges-Proskauer positive -starch hydrolysis positive

-grew faster in nutrient broth supplemented with 1% glucose, 1% maltose, 1% sucrose, or 0.1-1.0% beta-cyclodextrin (BCD) than in nutrient broth alone (which has no glucose)

-lecithinase positive Based on these results, four of the five

Bacillus isolates were identified as Bacillus cereus, and

one was identified as a closely related species, Bacillus thuringi ens is .

III . Microbial Growth in Reduced Cholesterol Ultrapasteurized Liquid Whole Egg Product Six isolates (four Bacillus and two short rods/cocci from both regular and reduced cholesterol ultrapasteurized liquid whole egg product) were added at an initial concentration of 10 ³ -10 ⁴ /ml to unspoiled reduced cholesterol ultrapasteurized liquid whole egg product, and also to unspoiled reduced cholesterol ultrapasteurized liquid whole egg product containing 0.1% glucose, 0.1% sucrose, 0.1% maltose, 0.1% beta- cyclodextrin (BCD) , or 0.01% amylase. Samples of unspoiled reduced cholesterol ultrapasteurized liquid whole egg product were used as controls.

After approximately 20 hours at room temperature (~21°C) , all samples with added Bacillus had coagulated and the pH had dropped to 5.5-5.7. Numbers of Bacillus had risen to ~3 x 10 ⁸ /ml. After 20 hours in samples with added short rods/cocci, the pH had dropped to 5.6-6.15, and numbers of short rods/cocci had risen to ~10 ⁸ /ml, but the samples had not coagulated. After 3 days at room temperature, these samples still had not coagulated. Control samples of unspoiled reduced cholesterol ultrapasteurized liquid whole egg product with no added bacteria coagulated after two days at room temperature.

No difference was noted in the results from the samples with sugars or amylase added.

IV. Survival at Pasteurization Temperatures

Tubes of phosphate buffer and tubes of unspoiled reduced cholesterol ultrapasteurized liquid whole egg product were prewarmed in a 70°C water bath. Isolates (both Bacillus and short rods/cocci) were added

at an initial concentration of ~10 ⁴ /ml . After four minutes, the tubes were removed, cooled quickly in an ice water bath, diluted, and plated.

Both types of organisms survived. Numbers of Bacillus dropped - 2 logs in both buffer and egg product. Numbers of the short rods/cocci stayed relatively the same in both buffer and egg product.

V. Reduction of pH

Lowering the pH of reduced cholesterol ultrapasteurized liquid whole egg product to 5.6 with HCl did not cause coagulation of the product when stored 48 hours at room temperature.

VI . Conclusions

This example indicates that ultrapasteurized liquid whole egg product and reduced cholesterol ultrapasteurized liquid whole egg product contain at least one strain of Bacillus cereus and also gram- positive short rods/cocci (possibly a lactobacillus) , both which are able to survive pasteurization at 70°C for four minutes. With temperature abuse of the product, it appears that both are able to grow to high numbers . Both are capable of lowering the pH of the product. However, Bacillus outgrows the short rods/cocci in the product and causes coagulation of the product, while the short rods/cocci do not. Coagulation of the product is not caused merely by a drop in pH.

EXAMPLE 2

Identification of Enterococci in Ultrapasteurized Liguid Whole Egg Product This example demonstrates that enterococci can survive ultrapastuerization techniques utilizing temperatures of up to 158°F for 3.5 minutes, and are present in ultrapasteurized liquid whole egg product.

I . Identification of Enterococci

Samples of reduced cholesterol liquid whole egg product were plated on three petri plates containing Standard Methods Agar (SMA) . Following bacterial growth, eight isolates were sampled from each of the three SMA petri plates, for a total of 24 isolates.

The 24 isolates were transferred into Brain Heart Infusion (BHI) ^' broth. Of the eight isolates from each single plate, six were incubated at 37°C and two were incubated at room temperature (22°C) for 18-20 hours. Following incubation, gram stains were made of the organisms in each tube. The isolates were then transferred to BHI broth and incubated at 45 and 50°C; transfers were also made into BHI broth containing 6.5% salt and to BHI broth adjusted to pH 9.6; these were incubated at 37°C. The isolates were checked for catalase activity by streaking onto Tryptic Soy Agar with Yeast Extract (TSAYE) . Catalase activity was determined by using 2 or 3 drops of hydrogen peroxide on each colony and checking for gas production.

Good growth was observed in all cultures (at temperatures of 22, 37, 45 and 50 °C) . Gram stains showed all isolates to be gram positive ovoid cocci in chains consisting of two to six units. Some clumping was observed. All isolates were catalase negative and all colonies were similar in appearance on the TSAYE streak plates. Growth occurred in all 6.5% salt BHI cultures and in pH 9.6 BHI broth. These growth characteristics are typical of Enterococci. This group of microbes is noted for its wide range of growth characteristics and its ability to survive milk pasteurization temperatures.

II. Thermal Inactivation Studies:

Isolates 1-24, above, were transferred and incubated at 37°C for 18-20 hours in BHI broth. A second transfer and incubation at 37°C for 18 -20 hours in BHI broth was performed. Two ml of each of the 24 isolates

was transferred to test tubes for the heat treatment . The initial population of the cultures heat treated was approximately 7.8 x 10 ⁸ . Heating was done in a water bath at 70°C (158°F) . The tubes were held at this temperature for 3.5 minutes and then immediately transferred to an ice bath for immediate cooling. Survival was determined by serial dilution of the heated isolates in 0.1% peptone water followed by pour plating with TSAYE. The plates were incubated at 37°C for 24 hours. After that time the number of survivors was determined by counting the colonies on the plates. All cultures survived the heat treatment of 158°F for 3.5 minutes. The average population of the survivors was 1.1 x 10 ⁵ . This gave an approximate reduction of 3 to 4 logs, insufficient reduction to conclude that this heat treatment controls these organisms.

This example illustrates that Enterococci can survive ultrapasteurization treatments and can contaminate ultrapasteurized liquid whole egg products.

EXAMPLE 3

Combatting Coagulation in Heat Abused Product

This example demonstrates the effectiveness of nisin preparation in combatting coagulation in packages of reduced cholesterol ultrapasteurized liquid whole egg stored at abuse temperatures.

I . Inoculated packages.

Six packages of reduced cholesterol ultrapasteurized liquid whole egg product were inoculated with coagulated product in an amount sufficient to provide approximately 10 ⁶ cfu/g of spoilage causing bacteria. Each package received one of six doses of nisin preparation: 0, 500, 1,000, 2,000, 5,000, or 10,000 IU/g of liquid egg product. Packages were stored at 70°F to simulate extreme temperature abuse conditions.

The results, shown in Table 1, indicate that nisin extends the time to coagulation in inoculated packages kept at abuse temperatures in a dose dependent fashion.

TABLE 1

Time-to-coagulation at 70°F for Inoculated

Packages of Reduced Cholesterol Ultrapasteurized Liquid

Whole Egg Product Containing Nisin Preparation

Nisin Hours at 70°F

Test No. Level IU/g Until

Coagulation

1 0 16

2 500 20

3 1,000 25

4 2,000 25

5 5,000 44

6 10,000 48

II . Non-inoculated packages

Nisin preparation was added to six non- inoculated 1/2 pint packages of reduced cholesterol ultrapasteurized liquid whole egg product at the same dosages as above (0, 500, 1,000, 2,000, 5,000, or 10,000 IU/g) . Packages were stored at 70°F. Coagulation in the control package containing no nisin occurred between 36 and 48 hours. In packages containing nisin, coagulation did not occur until day 7 or 8. (Data not shown) .

III. Effect of Nisin at Seven Days

This experiment demonstrated the effectiveness of nisin in combatting coagulation of ultrapasteurized liquid whole egg product stored at 70°F. Seven 1/2 pint packages of fresh reduced cholesterol ultrapasteurized

liquid whole egg product were used. Nisin in the form of commercially available NISAPLIN (TM) was placed in five packages at doses of 500, 1,000, 2,000, 5,000, and 10,000 IU/g of product. One package was opened but received no nisin (open control) ; one package was kept closed and received no nisin (closed control) . Packages were stored at 70°F starting on 8/26/92 at 4:00 p.m. Samples were monitored daily for signs of coagulation and spoilage.

For both closed and open control packages, coagulation of the product occurred within 48 hours. Packages with nisin from 500 - 10,000 IU/g product spoiled but did not coagulate at day seven. Table 2.

TABLE 2

Reduced Cholesterol Ultrapasteurized Liquid Whole Egg

Product Stored at 70°F

Sample NISAPLIN (TM) Days to IU/g Coagulate

Closed 0 2 Package

Open Control 0 2

Level 1 500 7+ days Spoiled ¹

Level 2 1,000 7+ days Spoiled ¹

Level 3 2,000 7+ days Spoiled ¹

Level 4 5,000 7+ days Spoiled ¹

Level 5 10,000 7+ days Spoiled ¹

Spoiled but did not coagulate.

EXAMPLE 4 Effect of Nisin (250 IU/g) on Microbial Growth

This example demonstrates the effect of nisin, 250 IU/g of liquid whole egg product, on microbiological

growth in ultrapasteurized liquid whole egg product stored at 70°F.

Nisin (250 IU/g) was added to ultrapasteurized reduced-cholesterol liquid whole egg product. The product was then stored at 70°F for one week. Samples were taken daily and examined microbiologically. Results are shown in Table 3 , and indicate that while the total plate count rose over time, as did the pH, the number of B. cereus organisms remained constant. At the end of one week, no coagulation was noted except at the surface of the product carton.

TABLE 3 Nisin at 250 IU/g.

Time Age Temp TPC S. Cereus L. Bacillus PH Spoilage or (days) (°F) Coagulation

Time Zero 19:00 0 — 70 <10 <1 7.04 none

Day 1 11:00 1 70 80 <10 <1 7.04 none

Day 2 10:40 2 70 800 <10 <1 7.07 none

I

Day 3 13:00 3 70 1000 <10 <1 7.09 none I

Day 4 7:00 4 70 21000 <10 <1 7.14 none

Day 5 13:00 5 70 161000 <10 <10 7.22 none

10 Day 6 14:00 6 70 408000 <10 <10 7.22 none

Week 1 10:00 7 70 5.80E+07 <10 <10 7.23 No coagulation except Surface of Carton (odor)

EXAMPLE 5

Effect of Nisin (100 IU/gram) on Microbial Growth

This example demonstrates the effect of nisin, 100 IU/g of liquid whole egg product, on microbiological growth in ultrapasteurized liquid whole egg product stored at various temperatures .

Nisin (100 IU/g product) was added to raw

(unpasteurized) reduced cholesterol liquid whole egg product. After ultrapasteurization, twelve 1/2-pint packages of the ultrapasteurized reduced cholesterol liquid whole egg product with added nisin were studied: four packages were stored at 40°F, four were stored at

50°F, and four at 70°F. Standard Plate Counts (SPC) were performed on samples from the packages at various times to determine the colony forming units per gram of product

(cfu/g) ; results are shown in Table 4. Anaerobic counts per gram of product were also performed at various times during incubation; results are shown in Table 5.

TABLE 4 Effect of Nisin (100 IU/g) on Bacterial Growth

CFU/gm Product

40°F 50°F 70°F

0 week <10, <10, (not done) (not done) <10, <10

1 week (not done) (not done) <100, <100, <100, <100

4 week <10, <10, <10, <10, (not done) <10, <10 <10, <10

6 week <10, <10, <10, <10, (not done) <10, <10 <10, <10

8 week <10, <10, (not done) (not done) <10, <10

TABLE 5 Effect of Nisin (100 IU/gm) on Anaerobe Growth

Anaerobic Count/gm

40°F 50°F 70°F

1 week (not done) (not done) 1, 1, 1, 1

4 weeks <1, <1, <1, <ι, (not done)

<1, <1 <1, <1

6 weeks <10, <10, <10, <10, (not done) <10, <10 <10, <10

8 weeks <1, <1, (not done) (not done)

<1, <1

III. Storage at 40°F:

At 0 weeks of storage time, all packages stored at 40°F showed <10 cfu/g. Similarly, at 4 weeks and at 6 weeks of storage time all packages showed <10 cfu/g. At 8 weeks of storage time, two packages showed <10 cfu/g, while two showed 10 cfu/g. For anaerobic organisms, all four packages stored at 40°F showed <1 anaerobic count/g at 4 weeks storage time. At 6 weeks time, all four packages showed <10 anaerobic count/g. At 8 weeks time, all four packages showed <1 anaerobic count/g. III. Storage at 50°F: All four packages stored at 50°F showed <10 cfu/g at 4 weeks storage time and <10 cfu/g at 6 weeks storage time. For anaerobic organisms, all packages showed _< 1 anaerobic count/g at 4 weeks and <10 anaerobic count/g at 6 weeks. III. Storage at 70°F:

Packages stored at 70°F were sampled only at the end of one week. All packages stored at 70°F showed _< 100 cfu/g at 1 week storage time. All packages showed 1 anaerobic count/g at 1 week storage time.

EXAMPLE 6 Experimental Product

The product evaluated in the following Examples

(Examples 7-14) consisted of commercially broken and ultrapasteurized reduced-cholesterol liquid whole egg

(LWE) (SIMPLY EGGS™, M.G. Waldbaum Co., Gaylord,

Minnesota) , from a single production lot aseptically packaged in 227 gram aliquots and frozen within 4 hours of processing. Frozen LWE was thawed at 4°C for 18 hours before use. Pasteurized LWE was selected for use in this study so that the fate of L. monocytogenes could be monitored with minimal interference from the diverse competing microflora generally present in commercially- broken raw LWE. See, e . g. , ICMSF, Eggs and egg products, pp. 521-566. In Microbial Ecology of Foods, Vol. II, Academic Press, New York (1980) . Thawed LWE from replicate cartons was pooled to provide multiple 700 mL aliquots in sterile Erlenmeyer flasks. Flasks were stored at 4°C for up to 2 hours before pH adjustment, nisin supplementation, and inoculation.

EXAMPLE 7 pH Adjustment and Nisin Addition

Bulk LWE (700 ml) in sterile Erlenmeyer flasks was continuously mixed using a magnetic stirring plate during pH monitoring and adjustment. At intervals, 5 mL subsamples were aseptically removed and the pH was determined using an ACCUMET ^® model 10 pH meter equipped with a silver/silver chloride combination electrode

(Fisher Scientific, Fair Lawn, NJ) . One half of the total volume of LWE used was pH-adjusted from an initial pH of 6.9 to a final pH of 7.5 by the addition of 0.07 percent w/v of sodium hydroxide, added as a 50 percent w/v stock solution (Fisher Scientific) . The remaining LWE was acidified to a final equilibrium pH of 6.6 by the addition of 0.07 percent w/v citric acid (Sigma Chemical Co., St. Louis, MO) added as a sterile

25 percent w/v stock solution. NISAPLIN™, a dried commercial nisin preparation with a potency of 1 x 10 ⁶ international units (IU)/g, was supplied by Applied Microbiology Inc., New York. pH-adjusted LWE was further subdivided into triplicate sterile flasks and supplemented with 0 percent, 0.01 percent or 0.1 percent (w/v) NISAPLIN™ to provide final nisin concentrations of 0, 100, and 1000 IU/ml of LWE, respectively.

EXAMPLE 8 Bacterial and Culture Conditions

L. monocytogenes strains Scott A (clinical isolate, serotype 4b) , F5069 (raw milk isolate, serotype 4b) , 675-3 (raw milk isolate) and raw egg isolates NCF- U2K3 and NCF-FIKK4 were used. Strain 675-3 was obtained from Dr. T. R. Klaenhammer (NC State University) ; the remaining four strains were obtained from Dr. P. M. Foegeding (NC State University) . Parent cultures were maintained in brain heart infusion (BHI) broth (Difco, Detroit, MI) supplemented with 40 percent glycerol and stored at -20°C. Working stock cultures were prepared by transferring 0.1 mL of frozen stock culture to 10 mL of BHI broth incubated at 37°C for 24 hours. By transferring 0.1 mL of the working stock culture to 10 mL of fresh BHI (37°C for 24 hours) , stationary phase cultures containing 1.4 x 10 ⁹ to 2.9 x 10 ⁹ CFU/mL were obtained. Two milliliter aliquots of each of the 5 strains were pooled, centrifuged for 10 minutes at 9000 x g, resuspended in 10 L of 0.1 percent peptone water (PW) , and diluted 1:1000 in sterile PW to produce the final inoculum suspension. LWE used in the Listeria challenge study was inoculated at 0.2 percent (v/v) to provide a target inoculum level of 3 x 10 ³ CFU/mL. Lactococcus lactis subsp. cremoris ATCC 14365, a nisin sensitive indicator organism used in nisin bioassays, was obtained from Dr. F. Breidt (NC State University) and was

transferred twice in MRS broth (Difco) (30°C, 24 hours) before use.

EXAMPLE 9 Storage Studies Individual samples for storage and evaluation consisted of 9 mL of LWE in sterile capped 16 mm diameter test tubes stored under static conditions at 4°C (39°F) or 10°C (50°F) in thermostatically controlled incubators. Unlike a previous study by Foegeding and Leasor (1990) , a mineral oil overlay (to minimize free oxygen transfer) was not added to the LWE samples due to concerns that the hydrophobic nisin molecules might partition within the oil layer. At selected intervals, duplicate Listeria- inoculated samples were removed from storage, vortexed and surface-plated onto tryptose phosphate agar (Difco) with 0.05 percent each of esculin and ferric ammonium citrate (Sigma) (TPA-FE; Donnelly and Briggs, J ^" . Food Prot . , 49, 994-998 (1986)) and incubated at 37°C for 48 hours. Direct surface plating of 0.5 mL of LWE onto duplicate plates permitted a detection limit of 1 CFU per mL. Round, regular-edged blue-gray colonies, often with indented centers, which caused a blackening of the medium were enumerated as L. monocytogenes . Although the isolation of microorganisms other than listeria on TPA-FE rarely occurred, confirmation of presumptive Listeria isolates was carried out by conducting catalase tests and preparing wet mounts . Uninoculated control samples were also periodically plated onto TPA-FE to confirm the absence of naturally-occurring Listeria spp. During the Listeria growth studies, duplicate samples from each test variable were analyzed for L. monocytogenes after storage periods of 0, 3 , ' and 7 days, and after 2, 4, 6, 8, and 12 weeks at 4°C or 10°C. Before microbiological testing, each sample was evaluated for changes in sensory quality (i.e., color, aroma and consistency) by an experienced panelist. At each of the

above sampling intervals, a 2.5 mL aliquot of each LWE sample was transferred to a fresh test tube to determine product pH as previously described.

EXAMPLE 10 Residual Nisin Assays

The residual activity (IU/mL) of added nisin in LWE was determined by extraction and bioassay using a horizontal agar well diffusion assay developed by Aplin & Barret, Ltd. (Anonymous, Laboratory method: Nisin assay (egg) , Laboratory procedure 25.1.93, pp. 1-10, Aplin S Barrett Ltd., Dorset, England, 1993) . The procedures used in the present study differed only in the indicator microorganism (Lactococcus lactis subsp. cremoris ATCC 14365) and the agar medium (MRS agar, Difco) used for well diffusion testing. The use of this L. lactis subsp. cremoris strain in nisin bioassays has been previously described (Harris et al . , Appl . Environ . Microbiol . 58, 1477-1483 (1992)) . After 24 h at 30°C, mean inhibition zone diameters around LWE extract wells were measured and recorded. Residual nisin concentrations were estimated by comparing these zone widths with linear regression lines (mean r ² =0.84) obtained by plotting mean inhibition zone width (mm) versus the log concentration of purified nisin tested in duplicate standard curve bioassay plates. Purified nisin with a potency of 3.7 x 10 ⁷ IU/g was obtained from Aplin & Barrett, Ltd. (Dorset, England) . Stock solutions of purified nisin containing 8 x 10 ⁴ IU/mL in 0.02 HCl (pH 2.0) were prepared, autoclaved (121°C for 15 min) , and stored at -20°C before use. Nisin bioassays were conducted on duplicate samples of LWE representing each test variable (following sensory, microbiological, and pH testing) after storage periods of 0, 2, 8, and 12 weeks.

EXAMPLE 11 Minimum Inhibitory Concentration (MIC) Determination

Appropriate MIC values for each of the 5 L . monocytogenes strains used in the liquid egg challenge study were determined in pH-adjusted BHI broth supplemented with nisin levels ranging from 0 to 1,000 IU/mL. BHI broth (initial pH 7.5) was prepared and dissolved at room ^' temperature according to the manufacturer's instructions with the exception that only 90% of the recommended volume of distilled water was added. Aliquots of this solution were adjusted to pH 7.6 using 0.008% w/v NaOH (added as a 1M solution) , or to pH 6.6 using 0.40% v/v of 5M HCl, or to pH 6.6 using 0.13% w/v citric acid (added as a 25% w/v solution) before making up the final volume using distilled water. The pH-adjusted BHI broth was dispensed into 10 mL aliquots in test tubes autoclaved (121°C for 15 min) . Final equilibrium pH values in the sterilized BHI broth tubes were pH 7.6 (NaOH) , pH 6.7 (HCl) , and pH 6.7 (citric acid) . Final nisin concentrations of 0 to 1000 IU/mL (in 10 increments) were obtained by the addition of small volumes (≤ 12 μl) of sterile purified nisin stock solution (8 x 10 ⁴ IU/mL) to each lOmL tube of BHI broth. The addition of the nisin stock solution did not alter the equilibrium pH of the media. A series of triplicate tubes of BHI broth encompassing 10 nisin levels was prepared for each of the three pH variables .

BHI broth cultures (37°C, 24h) of each of the five L. monocytogenes test strains were diluted in PW to provide a final inoculum level (1% v/v) of approximately 2 x 10 ³ CFU/mL of pH adjusted, nisin-supplemented BHI broth. Tubes without nisin served as positive controls and uninoculated tubes containing nisin were used to verify the sterility of the test media. Growth of L. monocytogenes was monitored by visual observation for turbidity after incubation at 37°C for 7 days (no changes in growth patterns were observed after additional

incubation for a total of 10 days) . The presence of L. monocytogenes in turbid tubes was confirmed by isolation onto TPA-FE plates (37°C, 48 hours) , and the sterility of the BHI series containing the lowest concentration of nisin yielding no turbidity was tested in the same manner. The MIC for each strain was defined as the lowest concentration of nisin which completely inhibited growth in all three tubes of the triplicate series.

EXAMPLE 12 Results of L. monocytocrenes Challenge Study

The results of the above Examples demonstrate that the bactericidal effect of nisin against L. monocytogenes in ultrapasteurized LWE was enhanced in product acidified to pH 6.6 using citric acid as compared to product at pH 7.5. LWE at pH 7.5 was selected as a control product representative of the average pH of citric acid-free commercially pasteurized LWE (Ball et al . , J. Food Sci . , 52, 1212 (1987)) . The survival and growth characteristics of L. monocytogenes in nisin- supplemented LWE at pH 7.5 are presented in Figure 1. A consistent L. monocytogenes initial inoculum level of approximately 3.3 log CFU/mL (to simulate postpasteurization contamination) was used for all LWE variables evaluated. On the day of product inoculation, a period of approximately 6 hours at 4°C elapsed between the time of inoculation and the plating of samples onto TPA-Fe to enumerate viable L. monocytogenes populations. As shown in Figure 1, the presence of nisin at 100 IU/mL and 1000 IU/mL reduced the initial Listeria population by 0.5 and 1.6 log cycles, respectively. The initial decimal reductions observed in nisin-supplemental LWE are deemed to reflect the varying nisin sensitivities of the 5 strains used in the L. monocytogenes pooled inoculum (see MIC determination data, Example 6) . Within 7 days at 4°C, logarithmic growth of L . monocytogenes in LWE containing 0 or 100 IU/mL nisin had

begun, and maximum populations exceeding log 6.0 CFU/mL were achieved within 4 weeks (Figure 1) . Previous research has shown that L. monocytogenes strains F5069, NCF-U2k3, and NCF-F1KK4 may attain maximum populations of log 6.0 to 7.0 CFU/mL within approximately 3 weeks in ultrapasteurized LWE at 4°C (Foegeding and Leasor, 1990) . In the current study, the presence of 1000 IU/mL of nisin permitted survival and limited growth of L. monocytogenes in LWE at pH 7.5. After an initial 1.6-log cycle reduction in the viable population, surviving listeriae grew to levels equal to, yet not exceeding the initial inoculum level (log 3.4 CFU/mL) within 4 weeks at 4°C with no further growth through the end of the 12-week storage study. Similar results were obtained in pH 7.5 LWE stored at 10°C (representative of moderate refrigerated temperature abuse) except that the growth rates (the apparent slope of the growth curves) and the maximum Listeria populations achieved in the presence of 0 or 100 IU/mL of nisin increased (Figure 1) . After a lag period of approximately 2 weeks, recovery and eventual growth of L. monocytogenes (either nisin-resistant cells or cells which evaded the effect of nisin) was evident in pH 7.5 LWE containing nisin at 1000 IU/mL. The inhibitory effect of nisin at 1000 IU/mL against L. monocytogenes during modest periods of simulated temperature abuse

(i.e., ≤ 2 weeks at 10°C) was also considered noteworthy.

Table 6 summarizes the effects of the presence of various L. monocytogenes populations on the pH and shelf life (i.e., elapsed time to detectable spoilage) of inoculated ultrapasteurized LWE. Relatively large populations of L. monocytogenes (≥ 6.4 log CFU/mL) achieved at 4°C in pH 7.5 LWE resulted in only minor changes in product pH and no detectable changes in product sensory quality. At 10°C, overt spoilage of LWE occurred within 8 to 12 weeks due to the growth of competing psychrotrophic spoilage microorganisms

identified as Xanthomonas spp (non-pathogenic) . However, the growth of the spoilage strain lagged behind that of L. monocytogenes, which achieved populations exceeding log 6.0 CFU/mL within 1 week at 10°C in LWE containing 0 or 100 IU/mL of nisin (Figure 1) . Xanthomonas spp. are not capable of growth at temperatures below 5°C and were not detected in LWE samples incubated at 4°C.

Table 6

10

15

20 d - Competitive growth of gram-negative rods (Xanthomonas spp ) at 6 - 7 log CFU/mL was detected on TPA-FE agar

As presented in Figure 2, the sensitivity of L. monocytogenes to nisin at 1000 IU/mL was enhanced in LWE acidified to pH 6.6. Within only 6h post-inoculation, viable Listeria populations, initially at log 3.3 CFU/mL, were reduced by 1.0 log cycle (100 IU/mL of nisin) and 3.3 log cycles (1000 IU/mL of nisin) in comparison to the nisin-free controls. In the presence of 1000 IU/mL of nisin, L. monocytogenes populations remained below detection (i.e., < 1 CFU/mL) in 29 of 30 samples analyzed over the 12-week storage periods at 4 or 10°C (Figure 2) . Acidification of ultrapasteurized LWE to pH 6.6 did not in itself have an inhibitory effect on L. monocytogenes ; the growth curves for the pathogen at pH 6.6 (0 IU/mL and 100 IU/mL of nisin) did not differ substantially from the corresponding growth curves obtained in pH 7.5 LWE at both 4 and 10°C (Figure 1) . Indeed, L. monocytogenes has been reported to survive or grow in laboratory media acidified to pH values as low as 4.3 to 5.2 (Ahamad and Marth, J ^" . Food Prot . , 52, 688 (1989) ; Farber et al . , Lett. Appl . Microbiol . 9, 181-183 (1989) ; McClure et al . , Lett . Appl . Microbiol . , 9 , 95-99 (1989) ; Parish and Higgins, J ^" . Food Prot . , 52, 144-147 (1989)) .

As presented in Table 6, the presence of 1000 IU/mL of nisin extended the shelf-stability of the Listeria-inoculated LWE (pH 6.6) to greater than 12 weeks at both 4 and 10°C. Product containing nisin at 0 or 100 IU/mL was visibly spoiled (loss of color) within 8 weeks at 4°C. At 10°C, pH 6.6 LWE supplemented with 0 or 100 IU/mL of nisin underwent an acidic spoilage within 4 to 6 weeks and contained large populations of both L. monocytogenes and the psychrotrophic spoilage organism Xanthomonas (Table 6) .

The residual activity of nisin in ultrapasteurized LWE was monitored using a bioassay procedure at intervals throughout the Listeria challenge study. The nisin concentrations reported are based on the construction of independent standard curves. The

activity of nisin in LWE was generally more stable during storage at 4°C than at 10°C at both pH values tested

(Table 7) . Inactivation of nisin during storage at 10°C for 12 weeks may reflect the impact of metabolic by¬ products from large populations of L. monocytogenes and/or Xanthomonas on the structural stability and activity of nisin molecules. In pH 6.6 LWE, residual nisin activity was detected through 12 weeks at 10°C, while bioactive nisin was not detected beyond 8 weeks at 10°C in LWE at pH 7.5. At 4°C, residual nisin levels were in general comparable at both pH values evaluated

(Table 7) .

Table 7

Residual Nisin Concentrations as Measured by Bioassay in Listeria-inoculated LWE (n=2) stored

Under Refrigeration.

Nisin Storage addition temp. Initial Residual nisin level (IU/mL):

(IU/mL) (°C) PH Wk 0 Wk 2 Wk 8 Wk 12

0 4 7.5 <0.2 ^a <0.01 ^a <23 ^a <1.0 ^a

100 4 7.5 62 140 100 50

1000 4 7.6 1500 2600 1000 680

0 10 7.5 ND ^b <0.01 <0.23 <1.0

100 10 7.5 ND 93 22 <1.0

1000 10 7.6 ND 2100 280 <10

0 4 6.5 <0.2 <0.01 <0.23 <1.0

100 4 6.6 78 110 75 54

1000 4 6.6 1100 3300 740 770

0 10 6.5 ND <0.01 <0.23 <1.0

100 10 6.6 ND 92 54 33

1000 10 6.6 ND 2900 550 340

Nisin detection limits vary by sampling date due to differences between linear regression eαuations generated for each purified msin standard curve

" ND Not determined

EXAMPLE 13 MIC values of nisin against L. monocvtogenes

Because nisin was shown to be more bactericidal to L. monocytogenes in LWE at pH 6.6 than at pH 7.5, the effects of pH (7.6 vs. 6.7) and acidulant (citric acid vs . hydrochloric acid) on the minimum inhibitory concentration (MIC) of purified nisin against L. monocytogenes was investigated. A secondary objective was to determine whether nisin levels below 1000 IU/mL would inactivate L. monocytogenes populations (at log 3.0 CFU/mL) in a model broth system (BHI broth) . Agar media were not tested because nisin is generally more inhibitory to sensitive bacterial strains in liquid systems than in solid or semisolid systems (Ray, Nisin of Lactococcus lactis ssp lactis as a food biopreservative, pp. 207-264, In Ray B. and Daeschel, M. Food Biopreservatives of Microbial Origin, CRC Press, Boca Raton, FL (1992) ) .

As listed in Table 8, the MIC values for five L. monocytogenes strains in BHI broth (pH 7.6) ranged from 200 to 600 IU/mL, with a mean MIC of 380 IU/mL. The increased sensitivity of L. monocytogenes to nisin at reduced pH was again demonstrated in BHI broth at 37°C. The mean MIC value for the five L. monocytogenes strains in BHI adjusted to pH 6.7 using the inorganic acid HCl was 220 IU/mL (a mean MIC reduction of 42% relative to the pH 7.6 controls) . Strain 675-3 was an exception to this trend, with an MIC of 200 IU/mL at both pH 7.6 and 6.7 (HCl) . In BHI adjusted to pH 6.7 using citric acid the effectiveness of nisin against the L. monocytogenes increased further for 4 of 5 strains, with a mean MIC value of 105 IU/mL (an MIC reduction of 72% relative to the values observed at pH 7.6) . As a nutritionally- complex liquid medium, BHI broth acted as a model system for MIC determinations in LWE.

Table 8

Effect of pH and Acidulant on MIC of Purified Nisin Against L. monocytogenes in BHI ^a Broth

MIC (IU/ml) in BHI Broth ³

L. pH 7.6 pH 6.7 pH 6.7 monocytogenes (HCl) (citrate) strain ^b

Scott A 300 200 50

F5069 600 175 175

675-3 200 200 100

NCF-F1KK4 300 300 100

NCF-U2K3 500 200 100

Brain heart infusion broth. ^b Initial inoculum level = 10 ³ CFU/mL.

An anti-listerial effect of citric acid plus nisin in LWE and BHI broth was observed at pH 6.6 to 6.7.

The results of the Examples presented herein indicate that nisin limits the survival and growth of L. monocytogenes in LWE acidified with citric acid to pH 6.7. The use of citric acid as an acidulant is desirable from the standpoint that citrate is a GRAS (Generally Recognized as Safe) additive currently used in a variety of liquid egg products to bind iron, thereby preventing color loss (greening) during cooking.

EXAMPLE 14

Effect of pH and Nisin Concentration on the Shelf-life of Uninoculated Ultrapasteurized Liguid Whole Egg

An uninoculated liquid whole egg (LWE) shelf- stability study was conducted. Tests were conducted to assess the effects of product pH (7.5 vs. 6.7) and nisin concentration (0, 100, or 1000 IU/mL of nisin, added as

NISAPLIN™) on the organoleptic shelf-life and equilibrium

pH of the product (Table 9) and on residual nisin concentrations (Table 10) during storage at 4°C and 10°C. In addition, the effects of the above variables on naturally-occurring microbial populations were evaluated by determining mesophilic aerobic plate counts (surface plating onto PCA, 37°C, 48h) and mesophilic aerobic spore counts (heat-shocked diluted samples surface plated onto TGE agar, 37°C, 48h) .

As presented in Figure 3 (pH 7.5 LWE) and Figure 4 (pH 6.6 LWE), the presence of 1000 IU/mL of nisin extended the microbiological shelf-life of LWE at

4°C at both pH values, yielding aerobic plate counts of less than log 2.0 CFU/mL after 12 weeks of storage

(essentially unchanged from day zero) . When incubated under conditions simulating temperature abuse (i.e. 10°C) , spoilage populations (predominantly Xanthomonas spp.) exceeded the USDA-defined shelf-life upper limits (i.e., aerobic plate count log 4.0 CFU/mL) within 4 weeks in the presence of 0 or 100 IU/mL of nisin. Acidification to pH 6.6 (1000 IU/mL of nisin) extended the interval to spoilage at 10°C to a total of approximately 6 weeks (Figure 4) . Mesophilic aerobic spore populations (initially at ≤2.00 log CFU/mL) remained virtually unchanged throughout the 12-week storage study (data not presented) . The only exception to this trend was the pH 7.5 nisin-free LWE stored at 10°C, which yielded a mesophilic aerobic spore count of log 4.0 CFU/mL after 12 weeks.

Table 9

Shelf-life of Uninoculated Ultrapasteurized LWE (n=2) under Refrigeration

Nisin Storage Time to Initial Final Sensory Mesophilic (IU/ml) (°C) spoi1age* pH pH ^a Comments APC ^b week 12 (weeks) (log CFU/mL)

0 4 >12 7.5 8.0 Normal 6.4 ^C

100 4 >12 7.5 8.2 Normal 3.3

1000 4 >12 7.6 8.3 Normal 1.5

10 7.5 6.8 Loss of color; high 8.4 viscosity

100 10 >12 7.5 8.1 Normal 7.9 ^d I

4 <_

10 1000 10 >12 7.6 8.1 Normal 7.4 ^d I

0 4 12 6.5 7.1 Slightly viscous; slight 5.4 ^C fermented off-odor

100 4 >12 6.6 7.1 Normal 4.2 ^C

1000 4 >12 6.6 7.1 Normal 1.0

10 12 6.5 6.4 Loss of color; 7.9 ^d sl ightly viscous; fermented off- odor

15 100 10 12 6.5 7.1 Sl ight fermented 6.9 ^d off-odor sl ightly viscous

1000 10 >12 6.6 7.0 Normal 6.9 ^d

Spoilage was defined as the first sampling period at which changes in the color, consistency, or aroma of the liquid egg were detected. pH at detectable spoilage or at the end of the 12-week storage study.

Aerobic plate count.

20 Mixed cultures including Bacillus spp. and Nocardia spp.

Gram-negative rods (Xanthomonas spp.).

Table 10

Residual Nisin Concentration Measured by Bioassay in Uninoculated Ultrapasteurized LWE Under Refrigeration

Nisin Storage Initial Residual nisin level (IU/mL) at: 5 (IU/mL) temp. pH Week 0 Week 2 Week 8 Week 12

0 4 7.5 <0.2 ^a <0.003 ^a <1.2 ^a <0.004 ^a

100 4 7.5 62 77 67 4

1000 4 7.6 1500 1000 760 56 j i

0 10 7.5 ND ^b <0.003 <1.2 <0.004 '

10 100 10 7.5 ND 57 <1.2 <0.004

1000 10 7_ _L 6 ND 8_00 2_50 11

0 4 6.5 <0.2 <0.003 <1.2 <0.004

100 4 6.6 78 64 86 9

1000 4 6^ 1100 76_0 S30 34_0

15 0 10 6.5 ND <0.003 <1.2 <0.004

100 10 6.6 ND 76 25 0.6

1000 10 6.6 ND 640 1000 15 a - Nism detection limits vary by sampling date due to differences between linear regression equations generated for each purified nisin standard curve b - ND, Not determined

20

The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.