WO1999044444A1 - Reduction of bacterial pathogens on fresh food items - Google Patents

Reduction of bacterial pathogens on fresh food items Download PDF

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Publication number
WO1999044444A1
WO1999044444A1 PCT/US1999/004586 US9904586W WO9944444A1 WO 1999044444 A1 WO1999044444 A1 WO 1999044444A1 US 9904586 W US9904586 W US 9904586W WO 9944444 A1 WO9944444 A1 WO 9944444A1
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Prior art keywords
percent
concentration
active ingredient
lactic acid
coli
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PCT/US1999/004586
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French (fr)
Inventor
Michael P. Doyle
Kumar S. Venkitanarayanan
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The University Of Georgia Research Foundation, Inc.
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Priority to CA002322301A priority Critical patent/CA2322301A1/en
Priority to AU30661/99A priority patent/AU3066199A/en
Publication of WO1999044444A1 publication Critical patent/WO1999044444A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/358—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B7/00—Preservation or chemical ripening of fruit or vegetables
    • A23B7/02—Dehydrating; Subsequent reconstitution
    • A23B7/022—Dehydrating; Subsequent reconstitution with addition of chemicals before or during drying, e.g. semi-moist products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B7/00—Preservation or chemical ripening of fruit or vegetables
    • A23B7/10—Preserving with acids; Acid fermentation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B7/00—Preservation or chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
    • A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
    • A23B7/154—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B7/00—Preservation or chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
    • A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
    • A23B7/157—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3481—Organic compounds containing oxygen
    • A23L3/3508—Organic compounds containing oxygen containing carboxyl groups

Definitions

  • the field of this invention is the area of food safety; in particular, the invention relates to compositions and methods for treatment of produce or meat products to reduce or inactivate bacterial pathogens on that produce or meat.
  • Escherichia coli O157:H7 which causes hemorrhagic colitis and hemolytic uremic syndrome, has emerged as a food-borne pathogen of major public health concern [Padhye and 2
  • the USDA-FSIS Pathogen Reduction final rule recommends antimicrobial treatments as a method for reducing or inactivating pathogenic bacteria in foods [USDA-FSIS (1995) Proposal Billing Code 3410-DM-P. Docket No. 93-016P.RIN 0583.AB69. USDA, Beltsville, MD]. Effective methods to reduce or eliminate pathogens in foods are important to the successful implementation of Hazard Analysis Critical Control Point (HACCP) programs by the food industry. Lactic acid is a multipurpose GRAS (Generally Regarded As Safe) chemical that has an antimicrobial effect on a variety of bacterial pathogens [Anderson and Marshall (1990) J. FoodSci. 55:903-905; Greer and Dilts (1992) Food Res. Int.
  • E. coli O157:H7 has unusual tolerance to acidic conditions [Zhao et al. (1993) Appl. Environ. Microbiol. 59:2526-2530; Brackett et al. (1994) J. Food Prot. 57:198-203; Conner and Kotrola (1995) Appl. Environ. Microbiol. 61 :382-385].
  • strains of E. coli O157:H7 are sensitive to heat, with D values similar to those of Salmonella spp. [Doyle et al. (1997) In Food Microbiology: Fundamentals and Frontiers (Eds. Doyle, M.P., Beuchat, L.R. and Montville,
  • compositions and methods of the present invention reduce the numbers of pathogenic bacteria on the surfaces of fresh fruits and vegetables, especially by inactivating enteric pathogens including, but not limited to, Escherichia coli strains such as those of O157:H7, Salmonella spp, Listeria monocytogenes, Campylobacter, Yersinia, Vibrio species, including without limitation, cholerae, parahaemolyticus and vulnificus.
  • enteric pathogens including, but not limited to, Escherichia coli strains such as those of O157:H7, Salmonella spp, Listeria monocytogenes, Campylobacter, Yersinia, Vibrio species, including without limitation, cholerae, parahaemolyticus and vulnificus.
  • the method of the present invention is to wash or soak the fresh food items in a solution comprising lactic acid and either hydrogen peroxide, sodium benzoate or glycerol monolaurate, under conditions of time (30 sec to 30 min) and temperature (5°C to 70 °C) such that numbers of at least one bacterial pathogen are reduced. Optimization of conditions allows for maximum impact on bacterial number and minimal impact on food taste, texture and visual qualities. Desirably the pH of the solution is below 3.5, preferably from about 0.5 to about 3.5.
  • a suitable composition comprises 1% to 3% acid, desirably 1.5%, lactic acid plus 0.1% to 3% hydrogen peroxide or 0.05% to 1.0%, desirably 0.1%, sodium benzoate, or 0.001% to 0.05%, desirably 0.005% glycerol monolaurate.
  • the lactic acid in the wash or soak solution can be substituted by one or more of acetic, malic, propionic, tartaric, mandelic and phosphoric acid, at similar concentration and pH.
  • Pathogens which are inactivated or whose numbers are reduced include, without limitation, species of Salmonella, Yersinia, Listeria, Campylobacter and Escherichia, especially E. coli O157:H7, and Vibrio species, including without limitation, cholerae, parahemolyticus and vulnificus.
  • a solution comprising an acid, especially lactic acid, and either hydrogen peroxide, sodium benzoate or glycerol monolaurate, under conditions of time (30 sec to 30 min) and temperature (4°C to 70 °C) such that numbers of at least one bacterial pathogen are reduced. Optimization of conditions allows for maximum impact on bacterial number and minimal impact on food taste, texture and visual qualities.
  • the pH of the solution is below 3.5, preferably from about 0.5 to about 3.5.
  • a suitable composition comprises 1% to 1.5% lactic acid plus a second component: 0.1% to 2% hydrogen peroxide, 0.1% sodium benzoate or 0.005% glycerol monolaurate.
  • the lactic acid in the wash or soak solution can be substituted by one or more of acetic, malic, propionic, tartaric, mandelic and phosphoric acid, at similar concentration and pH.
  • Figure 1 illustrates the inactivation of E. coli O157:H7 in 0.1% peptone containing 1.0 or 1.5% lactic acid plus 0.1% hydrogen peroxide at 8°C, 22°C and 40°C. Arrow indicates that E. coli O157:H7 was not detected at the minimum level of sensitivity ( ⁇ 10 CFU/ml).
  • Figure 2 shows the inactivation of E. coli O157:H7 in 0.1% peptone containing 1.0 or 1.5% lactic acid plus 0.1% sodium benzoate at 8° C, 22° C and 40 °C. Arrow indicates that E. coli O157:H7 was not detected at the minimum level of sensitivity ( ⁇ 10 CFU/ml).
  • Figure 3 depicts the inactivation of E. coli O157:H7 in 0.1% peptone containing 1.0 or 1.5% lactic acid plus 0.005% glycerol monolaurate at 8 °C, 22 °C and 40°C. Arrow indicates that E. coli O157:H7 was not detected at the minimum level of sensitivity ( ⁇ 10 CFU/ml). 5
  • Figures 4A-4C demonstrate the inactivation of E. coli O157:H7 in 0.1% peptone containing 1.5% lactic acid, 0.1% hydrogen peroxide, 0.1% sodium benzoate or 0.005% glycerol monolaurate at 40°C (Fig. 4A), 22°C (Fig. 4B) and 8°C (Fig. 4C).
  • Figure 5 is a flowchart for the fresh fruit model experiment in which known populations of E coli Ol 57:H7 were applied to apples and oranges and treated with certain washing steps, followed by determination of the bacterial cell counts.
  • Figure 6 illustrates where apples were inoculated with E coli O157:H7 in the experiment outlined in Fig. 5.
  • the pH of the different combinations of treatments ranged from 2.75-3.04, with the control (0.1% peptone) of pH 7.05 (Table 1).
  • Treatments containing lactic acid and hydrogen peroxide had the lowest pH (2.75-2.88), followed by those containing lactic acid and glycerol monolaurate (pH 2.81-2.91), and lactic acid and sodium benzoate (pH 2.93-3.04).
  • E. coli O157:H7 in 0.1% peptone containing 1.0 or 1.5% lactic acid plus 0.1% sodium benzoate at 8°, 22° and 40°C is depicted in Figure 2.
  • E. coli O157:H7 populations were reduced to undetectable levels (as determined by both plating and enrichment procedures) after 15 min at 40°C, whereas a reduction of only 2.0 to 2.5 log E coli O157:H7/ml was observed at 30 min at 8°C, and at 20 min at 22 °C, respectively.
  • E. coli O157:H7 populations were reduced by 2.0 log CFU/ml in 30 min, > 5.0 log CFU/ml in 20 min, and to undetectable levels in 15 min, at 8°, 22° and 40°C, respectively (Figure 3).
  • Lactic acid is biocidal to a wide range of microorganisms; however, E coli O157:H7 is unusually tolerant to its antimicrobial properties.
  • Conner and Kotrola [(1995) Appl. Environ. Microbiol. 61 :382-385] determined that E. coli O157:H7 was able to survive for up to 56 days in TSB acidified to pH 4.7 with lactic acid.
  • Abdul-Raouf et al. (1993) Appl. Environ. Microbiol. 59:2364-2368] determined that E coli O157:H7 survived well in beef slurries with lactic acid.
  • lactic acid plus hydrogen peroxide was most effective in reducing E coli O157:H7 populations.
  • the maximum reduction in E. coli O157:H7 populations was about 2.5 log CFU/ml, achieved by treatment with lactic acid plus hydrogen peroxide.
  • the combination of 1.5% lactic acid and 0.1 % hydrogen peroxide had the greatest antimicrobial activity, with > 6.0 log E coli O157:H7/ml reduction occurring within the shortest exposure time, at both 22° and 40 °C.
  • Hydrogen peroxide is a GRAS approved chemical level for use as an antimicrobial agent in cheese making and whey processing at the 0.05% level [Davidson et al.
  • E. coli O157:H7 In a model fruit experiment, known numbers ( ⁇ 5 x 10 7 ) of E. coli O157:H7 were applied to the stem ends of oranges and apples, and the fruits were allowed to dry. The average population of E. coli O157:H7 recovered from apples after inoculation was 7.0 log CFU/apple. Immersion of apples in water containing 1.5% lactic acid plus 1.5% hydrogen peroxide at 40°C for 5, 10 or 15 min. resulted in complete inactivation of E. coli O157:H7 ( ⁇ 5 log CFU/apple). However, control apples immersed in sterile deionized water at 40 °C for the same durations yielded a mean E. coli O157:H7 population of 5.4 log CFU/apple. Further, an average count of 4.5 log CFU/ml of the pathogen was recovered from the deionized water in which the inoculated apples were submerged, whereas, no E. coli
  • O157:H7 could be detected in the treatment water containing lactic acid and hydrogen peroxide (as determined by both plating and enrichment procedures).
  • the exemplified wash solution (1.5% lactic acid, 1.5% hydrogen peroxide similarly reduces numbers of bacterial pathogens on fresh food items (e.g., raw fruits and vegetables) including, but not limited to, Escherichia coli including O157:H7, Salmonella spp.. Vibrio spp., Shigella spp. and Listeria spp.
  • Glycerol monolaurate (monolaurin) is a food-grade chemical approved by the Food and Drug Administration as an emulsifier (21 CFR GRAS 182.4505). Besides its emulsification property, glycerol monolaurate exhibits antimicrobial activity against many spoilage and pathogenic microorganisms [Kabara (1979) J. Am. Oil Chem. Soc. 56:760A- 767A; Beuchat (1980) Appl. Environ. Microbiol. 39:1178-1182; Kato, N. (1981) J. Food Safety 3:12-15]. Gram-positive bacteria are more sensitive to the antimicrobial effects of monolaurin than gram-negative bacteria [Monk et al. (1996) J. Appl.
  • the antimicrobial activity of glycerol monolaurate is pH dependent and can be increased by decreasing the pH of the medium [Kato and Shibasaki (1976) J. Antibacterial. Antifung. Agents 4:254-261 ; Oh and Marshall (1992) J. Food Prot. 55:449-450].
  • the above combination treatments used in media tests or as produce washes or soaks desirably at a solution temperature between about 8 and about 70 °C, preferably between about 20 and about 45 °C, and for a time from about 2 to about 30 minutes are effective in reducing or inactivating Salmonella, Yersinia, Campylobacter, Listeria, Vibrio species, as well as E coli O157:H7 or other species or strains of Escherichia.
  • lactic acid at a concentration of 1 to 2% is the preferred acid component of the produce washing or soaking solution
  • other acids can be substituted therefore including, but not limited to, acetic acid, malic, propionic, tartaric, mandelic and phosphoric acid, at similar concentrations and pHs as the lactic acid.
  • the term produce means fresh fruits and vegetables.
  • Carcasses include skinned or de-haired or de-feathered carcasses of beef, hog, buffalo, game or poultry, among others.
  • Cut meat products are cuts of meat or poultry, e.g., chicken breasts or wings or roasts or steaks, but ground meat products are not included within this definition.
  • a produce wash is a solution used to bathe the surface of produce, and typically is in 11 contact with the produce from about 30 sec to about 5 min.
  • a produce soak is a solution in which produce items are immersed for a time from about 30 sec to about 30 min.
  • the terms and solutions can be used interchangeably unless otherwise distinguished. It is understood that the temperature at which produce is washed or soaked will influence the length of time necessary to reduce or inactivate bacteria thereon, with warmer temperatures leading to a shorter time necessary for treatment.
  • the wash or soak solutions of the present invention can be used to reduce bacterial number, especially bacterial pathogen number, on the surfaces of fruits, vegetables, raw cut meat products, fish, shellfish, at the consumer level (in the household), in commercial food preparation environments, on fruits and/or vegetables prior to juicing, by wholesale or retail operators, and/or at the level of the harvest, meat packing plant or slaughterhouse, fishing boat, and so on, without limitation.
  • the present methods are particularly useful for inactivating E. coli. O157:H7 on the surfaces of fresh fruits and vegetables.
  • E. coli O157:H7 Five strains of E. coli O157:H7 [E06 (milk isolate), E08 (meat isolate), E10 (meat isolate), El 6 (meat isolate), and E22 (calf feces isolate)] were used for the study.
  • the strains of E. coli O157:H7 were each cultured in 100 ml of sterile tryptic soy broth (TSB) (Difco Laboratories, Detroit, MI) in 250 ml Erlenmeyer flasks at 37°C for 24 hr with agitation (150 rpm).
  • TLB sterile tryptic soy broth
  • the viable bacterial population in each culture was confirmed by plating 0.1 ml portions of appropriately diluted culture on tryptic soy agar (TSA) (Difco Laboratories, Detroit, MI) plates and incubating the plates at 37° C for 24 hr. An approximately equal population of each of the five strains was combined and 0.1 ml of the suspension was used as the inoculum.
  • TSA tryptic soy agar
  • E. coli O157:H7 (10 7 CFU) was added, and the treatments were incubated in a water bath (Pharmacia LKB, Piscataway, NJ) at 8°C for 0, 20 and 30 min; at 22 °C for 0, 10 and 20 min; and at 40 °C for 0, 5 and 10 min. Following incubation, a 1 ml portion of the inoculated treatment was serially diluted (1:10) in 9 ml of sterile 0.1% peptone water and 0.1 ml portions from appropriate dilutions were surface plated in duplicate on TSA plates.
  • a water bath Pharmacia LKB, Piscataway, NJ
  • a volume of 1 ml of the inoculated solution after exposure to the treatment for 0, 5 and 10 min also was transferred to separate 250 ml Erlenmeyer flasks containing 100 ml of sterile TSB and incubated at 37°C for 24 hr. Colonies of E. coli O157:H7 were enumerated on TSA plates after incubation at 37°C for 24 hr. Following enrichment in TSB, the culture was streaked on Sorbitol MacConkey agar No.3 (Oxoid Division, Unipath Co., Ogdensburg, NY) and the 13 plates were incubated at 37 °C for 24 hr.
  • coli O157:H7 (10 7 CFU) was added and the solutions were incubated in a water bath at 8°C for 0, 20 and 30 min; at 22°C for 0, 10 and 20 min; and at 40°C for 0, 10 and 15 min. Following incubation, the populations of E. coli O157:H7 that survived in each treatment were determined according to the procedure described herein above.
  • pH of 0.1% peptone solution before and after addition of each combination of chemicals was determined (pH meter model 350, Corning Inc., Corning, NY). At least duplicate samples were assayed for each treatment and the entire study was replicated four times.
  • E. coli O157:H7 Five strains of E. coli O157:H7 ( ⁇ -06, E-08, E-10, E-16, and E-22) were used for the study. Each strain was grown individually in 100 ml of tryptic soy broth (TSB, Difco, Detroit, MI) at 37°C with agitation (150 rpm) for 20 h. Following incubation, the bacteria (100 ml) were sedimented by centrifugation (4,000 x g for 30 min) in separate tubes and washed and resuspended in 10 ml of sterile 0.1% peptone water. The five strains of the pathogen (10 ml each) were combined and used as the inoculum.
  • TAB tryptic soy broth
  • the bacterial population in the suspension was determined by plating 0.1 ml of appropriate dilutions of the suspension on duplicate Sorbitol MacConkey agar plates (SMA, Oxoid Division, Unipath Co., NY) and incubating the plates at 37 °C for 24 h.
  • Apples and oranges were inoculated and treated according to the flowchart in Fig. 5.
  • Red Delicious apples and navel oranges were purchased from a local grocery store.
  • the populations of E. coli O157:H7 on apples and oranges were determined as follows. To each bag containing 50 ml of sterile 0.1% peptone water, an apple or orange was 15 transferred in such a way that the inoculated stem end of the fruit was submerged in the diluent. The bag containing the fruit was sealed and placed on a bench-top orbitary shaker and subjected to shaking at 400 rpm for 2 min. A 1.0 ml portion of solution from each "Whirl-pak" bag was serially diluted with 9.0 ml of sterile 0.1% peptone water and 0.1 ml portions from appropriate dilutions were plated on duplicate tryptic soy agar (TSA, Difco,
  • E. coli O157:H7 by E. coli O157:H7 latex agglutination assay (Remel Microbiology Products, Lenexa, KS). The entire experiment with each fruit was replicated three times. In the case of oranges, exposure times of only 10 and 15 min with the treatment solution or water (control) were evaluated.

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Abstract

Methods and compositions for treating uncooked food items, such as produce, so as to reduce viable bacteria, especially enteropathogenic bacteria, on the surfaces of produce and other uncooked food products. Use of a solution containing lactic acid and at least one of hydrogen peroxide, sodium benzoate or glycerol monolaurate, in contact with the food item for 5 sec to 30 min. effectively reduces certain foodborne pathogens, especially Escherichia coli O157:H7. The figure is a flowchart for the fresh fruit model experiment in which known populations of E. coli O157:H7 were applied to apples and oranges and treated with certain washing steps, followed by determination of the bacterial cell counts.

Description

REDUCTION OF BACTERIAL PATHOGENS ON FRESH FOOD ITEMS
CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from United States Provisional Application No. 60/076,487, filed March 2, 1998.
ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT This invention was made, at least in part, with funding from the United States Food and Drug Administration. Accordingly, the United States Government has certain rights in this invention.
BACKGROUND OF THE INVENTION
The field of this invention is the area of food safety; in particular, the invention relates to compositions and methods for treatment of produce or meat products to reduce or inactivate bacterial pathogens on that produce or meat.
Reported outbreaks of food-borne illness involving fruits and vegetables have increased during the last decade. However, there are no effective methods currently available to reduce or inactivate pathogenic bacteria on produce. There is a need in the art for methods and compositions to be used as surface treatments to reduce or inactivate bacterial pathogens including, but not limited to, Esche chia coli O157:H7, Listeria monocytogenes, and Salmonella enteritidis on fruits and vegetables.
Escherichia coli O157:H7, which causes hemorrhagic colitis and hemolytic uremic syndrome, has emerged as a food-borne pathogen of major public health concern [Padhye and 2
Doyle (1991) Appl. Environ. Microbiol. 57:2693-2698]. A massive outbreak of E. coli O157:H7 infection involving more than 9,500 cases and nine deaths occurred in Japan in 1996. A recent outbreak in the United States was associated with consumption of unpasteurized apple juice [Centers for Disease Control and Prevention (1997) Morbid. Mortal. Weekly Rep. 46:4-8]. A wide variety of foods including meat, milk, fruits and vegetables have been implicated as vehicles of E. coli O157:H7 infection.
The USDA-FSIS Pathogen Reduction final rule recommends antimicrobial treatments as a method for reducing or inactivating pathogenic bacteria in foods [USDA-FSIS (1995) Proposal Billing Code 3410-DM-P. Docket No. 93-016P.RIN 0583.AB69. USDA, Beltsville, MD]. Effective methods to reduce or eliminate pathogens in foods are important to the successful implementation of Hazard Analysis Critical Control Point (HACCP) programs by the food industry. Lactic acid is a multipurpose GRAS (Generally Regarded As Safe) chemical that has an antimicrobial effect on a variety of bacterial pathogens [Anderson and Marshall (1990) J. FoodSci. 55:903-905; Greer and Dilts (1992) Food Res. Int. 25:355- 364]. However, several researchers have determined that E. coli O157:H7 has unusual tolerance to acidic conditions [Zhao et al. (1993) Appl. Environ. Microbiol. 59:2526-2530; Brackett et al. (1994) J. Food Prot. 57:198-203; Conner and Kotrola (1995) Appl. Environ. Microbiol. 61 :382-385]. Despite their acid tolerance, strains of E. coli O157:H7 are sensitive to heat, with D values similar to those of Salmonella spp. [Doyle et al. (1997) In Food Microbiology: Fundamentals and Frontiers (Eds. Doyle, M.P., Beuchat, L.R. and Montville,
T.J. pp. 171-191. American Society for Microbiology, Washington, D.C.].
The antimicrobial activity of organic acids can be increased or potentiated when combined with other food preservatives [Bøgh-Sørensen (1994) In Food Preservation by Combined Processes (Eds. Leistner, L. and Gorris, L.G.M.) pp. 17-18. Food Linked Agro- Industrial Research Concerted Action No.7, Subgroup B. The Netherlands; Marshall and
Kim (1996) J. Food Quality 19:317-329] or heat [Greer and Dilts (1992) Food Res. Int. 25:355-364; Smulders et al. (1986) J. Food Technol. 21 :419-436]. Many GRAS chemicals, including sodium benzoate [Zhao et al. (1993) Appl. Environ. Microbiol. 59:2526-2530], sodium nitrite and potassium sorbate [Tsai and Chou (1996) J. Sci. FoodAgric. 71 :10-12], 3 and trisodium phosphate [Kim and Slavik (1994) J. FoodSci. 59:20-22], have been reported to possess antibacterial activity against E coli O157:H7. However, these chemicals are not entirely effective in eliminating or inactivating the pathogen.
There is a longfelt need in the art for improved food safety, especially as related to surface contamination of fresh food items including, but not limited to. fresh fruits and vegetables. Surface contamination of such foods eaten raw has resulted in life-threatening illnesses, for example, when contaminated with viable E coli O157:H7 cells.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide methods and compositions effective for inactivating or reducing the number of bacteria on the surfaces of produce and other uncooked food items. In particular, the compositions and methods of the present invention reduce the numbers of pathogenic bacteria on the surfaces of fresh fruits and vegetables, especially by inactivating enteric pathogens including, but not limited to, Escherichia coli strains such as those of O157:H7, Salmonella spp, Listeria monocytogenes, Campylobacter, Yersinia, Vibrio species, including without limitation, cholerae, parahaemolyticus and vulnificus. The method of the present invention is to wash or soak the fresh food items in a solution comprising lactic acid and either hydrogen peroxide, sodium benzoate or glycerol monolaurate, under conditions of time (30 sec to 30 min) and temperature (5°C to 70 °C) such that numbers of at least one bacterial pathogen are reduced. Optimization of conditions allows for maximum impact on bacterial number and minimal impact on food taste, texture and visual qualities. Desirably the pH of the solution is below 3.5, preferably from about 0.5 to about 3.5. A suitable composition comprises 1% to 3% acid, desirably 1.5%, lactic acid plus 0.1% to 3% hydrogen peroxide or 0.05% to 1.0%, desirably 0.1%, sodium benzoate, or 0.001% to 0.05%, desirably 0.005% glycerol monolaurate. The lactic acid in the wash or soak solution can be substituted by one or more of acetic, malic, propionic, tartaric, mandelic and phosphoric acid, at similar concentration and pH.
It is a further object of this invention to provide methods and compositions for 4 reducing numbers of or inactivating bacteria, especially pathogens, on the surfaces of fish, shellfish, raw cut meat products and carcasses. Pathogens which are inactivated or whose numbers are reduced include, without limitation, species of Salmonella, Yersinia, Listeria, Campylobacter and Escherichia, especially E. coli O157:H7, and Vibrio species, including without limitation, cholerae, parahemolyticus and vulnificus. Fish, shellfish, meat carcasses
(hog, beef, lamb, buffalo and the like) are washed with or immersed in a solution comprising an acid, especially lactic acid, and either hydrogen peroxide, sodium benzoate or glycerol monolaurate, under conditions of time (30 sec to 30 min) and temperature (4°C to 70 °C) such that numbers of at least one bacterial pathogen are reduced. Optimization of conditions allows for maximum impact on bacterial number and minimal impact on food taste, texture and visual qualities. Desirably the pH of the solution is below 3.5, preferably from about 0.5 to about 3.5. A suitable composition comprises 1% to 1.5% lactic acid plus a second component: 0.1% to 2% hydrogen peroxide, 0.1% sodium benzoate or 0.005% glycerol monolaurate. The lactic acid in the wash or soak solution can be substituted by one or more of acetic, malic, propionic, tartaric, mandelic and phosphoric acid, at similar concentration and pH.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the inactivation of E. coli O157:H7 in 0.1% peptone containing 1.0 or 1.5% lactic acid plus 0.1% hydrogen peroxide at 8°C, 22°C and 40°C. Arrow indicates that E. coli O157:H7 was not detected at the minimum level of sensitivity (< 10 CFU/ml).
Figure 2 shows the inactivation of E. coli O157:H7 in 0.1% peptone containing 1.0 or 1.5% lactic acid plus 0.1% sodium benzoate at 8° C, 22° C and 40 °C. Arrow indicates that E. coli O157:H7 was not detected at the minimum level of sensitivity (< 10 CFU/ml).
Figure 3 depicts the inactivation of E. coli O157:H7 in 0.1% peptone containing 1.0 or 1.5% lactic acid plus 0.005% glycerol monolaurate at 8 °C, 22 °C and 40°C. Arrow indicates that E. coli O157:H7 was not detected at the minimum level of sensitivity (< 10 CFU/ml). 5
Figures 4A-4C demonstrate the inactivation of E. coli O157:H7 in 0.1% peptone containing 1.5% lactic acid, 0.1% hydrogen peroxide, 0.1% sodium benzoate or 0.005% glycerol monolaurate at 40°C (Fig. 4A), 22°C (Fig. 4B) and 8°C (Fig. 4C).
Figure 5 is a flowchart for the fresh fruit model experiment in which known populations of E coli Ol 57:H7 were applied to apples and oranges and treated with certain washing steps, followed by determination of the bacterial cell counts.
Figure 6 illustrates where apples were inoculated with E coli O157:H7 in the experiment outlined in Fig. 5.
DETAILED DESCRIPTION OF THE INVENTION
Reported outbreaks of food-borne illness involving Escherichia coli O157:H7 have increased in the United States during the last decade, with a variety of food products being implicated as vehicles of infection. Meats, including beef, hamburger and chicken have been shown to contain bacterial pathogens including, without limitation, E. coli O157:H7, Salmonella spp., Listeria monocytogenes and Campylobacter spp. Fish and shellfish may be contaminated with Vibrio species including, but not limited to , cholerae, parahaemolyticus and vulnificus. Additionally, fresh fruits and vegetables and unpasteurized fruit juice products have also been shown to contain dangerous bacterial pathogens and have been linked to enteric disease outbreaks. The young, the elderly and those with compromised immune systems are especially vulnerable to infection. Unless effective means are developed to reduce bacterial numbers on food products, food-borne infections are likely to become an increasing problem as the population of this country ages.
With the goal of effective treatments of contaminated produce to reduce numbers of pathogenic bacteria, the present inventors carried out studies to determine the efficacy of combinations of various GRAS chemicals and moderate temperatures to kill E. coli Ol 57:H7, chosen as a model for enteric bacterial pathogens. A five-strain mixture of E. coli O157:H7 of approximately 106 CFU/ml was inoculated into 0.1% peptone solutions containing 1.0 or 6
1.5% lactic acid plus 0.1% to 2% hydrogen peroxide, 0.1% sodium benzoate or 0.005% glycerol monolaurate. The solutions were incubated at 8°C for 0, 15 and 30 min; at 22 °C for 0, 10 and 20 min; or at 40°C for 0, 10 and 15 min, and populations of E coli O157:H7 were determined at each sampling time.
The pH of the different combinations of treatments ranged from 2.75-3.04, with the control (0.1% peptone) of pH 7.05 (Table 1). Treatments containing lactic acid and hydrogen peroxide had the lowest pH (2.75-2.88), followed by those containing lactic acid and glycerol monolaurate (pH 2.81-2.91), and lactic acid and sodium benzoate (pH 2.93-3.04).
The effect of 1.0 or 1.5% lactic acid plus 0.1% hydrogen peroxide at 8°, 22° and 40°C on the five-strain mixture of E coli O157:H7 is illustrated in Figure 1. E coli
O157:H7 populations declined to undetectable levels (as determined by both plating and enrichment procedures) after 10 min of exposure to 1.0 and 1.5% lactic acid plus 0.1% hydrogen peroxide at 40 °C, and after 20 min of exposure to 1.5% lactic acid plus 0.1% hydrogen peroxide at 22°C. However only a 2.5 log E. coli O157:H7/ml reduction occurred in 1.5% lactic acid plus 0.1% hydrogen peroxide held for 30 min at 8 °C.
The inactivation of E. coli O157:H7 in 0.1% peptone containing 1.0 or 1.5% lactic acid plus 0.1% sodium benzoate at 8°, 22° and 40°C is depicted in Figure 2. In the presence of 1.5% lactic acid plus 0.1% sodium benzoate, E. coli O157:H7 populations were reduced to undetectable levels (as determined by both plating and enrichment procedures) after 15 min at 40°C, whereas a reduction of only 2.0 to 2.5 log E coli O157:H7/ml was observed at 30 min at 8°C, and at 20 min at 22 °C, respectively.
In the treatment containing 1.5% lactic acid plus 0.005% glycerol monolaurate, E. coli O157:H7 populations were reduced by 2.0 log CFU/ml in 30 min, > 5.0 log CFU/ml in 20 min, and to undetectable levels in 15 min, at 8°, 22° and 40°C, respectively (Figure 3).
Selective culture media can inhibit the recovery of heat- and acid-stressed cells of E coli O157:H7 [Abdul-Raouf et al. (1993) Appl. Environ. Microbiol. 59:2364-2368; Clavero 7 and Beuchat (1996) Appl. Environ. Microbiol. 62:2735-2740]. For example, Sorbitol MacConkey agar (SMA), when used as an enumeration medium, failed to recover E. coli O157:H7 cells after they were subjected to stress by heat and acids. Hence, in this study, TSA and TSB were used as the media for enumeration and enrichment, respectively.
Lactic acid is biocidal to a wide range of microorganisms; however, E coli O157:H7 is unusually tolerant to its antimicrobial properties. Conner and Kotrola [(1995) Appl. Environ. Microbiol. 61 :382-385] determined that E. coli O157:H7 was able to survive for up to 56 days in TSB acidified to pH 4.7 with lactic acid. Similarly, Abdul-Raouf et al. [(1993) Appl. Environ. Microbiol. 59:2364-2368] determined that E coli O157:H7 survived well in beef slurries with lactic acid. Our studies with lactic acid (1.5%), hydrogen peroxide (0.1 %), sodium benzoate (0.1%), and glycerol monolaurate (0.005%) as sole treatments on E. coli O157:H7 in 0.1% peptone water revealed that none of the chemicals could reduce E. coli O157:H7 populations by 5.0 log CFU/ml (Figure 4A, 4B, 4C). Of the four chemicals evaluated, lactic acid had the greatest biocidal effect, reducing E. coli O157:H7 counts by approximately 1.3 log CFU/ml, 2.0 log CFU/ml, and 4.0 log CFU/ml after 30 min of exposure at 8°, 20 min at 22° and 10 min at 40 °C, respectively. No substantial reduction (< ca. 1.0 log CFU/ml) of E. coli O157:H7 occurred in treatments of 0.1% hydrogen peroxide, 0.1% sodium benzoate, or 0.005% glycerol monolaurate.
Of the combinations of treatments used in this study, lactic acid plus hydrogen peroxide was most effective in reducing E coli O157:H7 populations. At 8°C, the maximum reduction in E. coli O157:H7 populations was about 2.5 log CFU/ml, achieved by treatment with lactic acid plus hydrogen peroxide. The combination of 1.5% lactic acid and 0.1 % hydrogen peroxide had the greatest antimicrobial activity, with > 6.0 log E coli O157:H7/ml reduction occurring within the shortest exposure time, at both 22° and 40 °C. Hydrogen peroxide is a GRAS approved chemical level for use as an antimicrobial agent in cheese making and whey processing at the 0.05% level [Davidson et al. (1983) In Naturally Occurring and Miscellaneous Food Antimicrobials. (Eds. Brannen, A.L. and Davidson, M.P.) pp. 385-391. Marcel Dekker, Inc., New York]. A treatment of milk with 0.06% hydrogen peroxide at 130°F for 1 min was reported to be biocidal to Clostridium spp. and Bacillus spp. 8
[Nielsen (1987) Am. Dairy Ref 39(6):32-34]. E. coli populations on chicken carcasses were reduced significantly by 5,300 ppm of hydrogen peroxide in chiller water [Lillard and Thomson (1983) J Food Sci. 48:125-126]. However, the efficacy of 0.1% hydrogen peroxide in inactivating E. coli O157:H7 within 30 min at 8°, 20 min at 22° or 10 min at 40 °C is minimal. The addition of 1.0% lactic acid in combination with 0.1% hydrogen peroxide greatly enhances the antimicrobial activity of the two chemicals.
In a model fruit experiment, known numbers (~5 x 107) of E. coli O157:H7 were applied to the stem ends of oranges and apples, and the fruits were allowed to dry. The average population of E. coli O157:H7 recovered from apples after inoculation was 7.0 log CFU/apple. Immersion of apples in water containing 1.5% lactic acid plus 1.5% hydrogen peroxide at 40°C for 5, 10 or 15 min. resulted in complete inactivation of E. coli O157:H7 (<5 log CFU/apple). However, control apples immersed in sterile deionized water at 40 °C for the same durations yielded a mean E. coli O157:H7 population of 5.4 log CFU/apple. Further, an average count of 4.5 log CFU/ml of the pathogen was recovered from the deionized water in which the inoculated apples were submerged, whereas, no E. coli
O157:H7 could be detected in the treatment water containing lactic acid and hydrogen peroxide (as determined by both plating and enrichment procedures).
In the case of oranges, the mean inoculation level was 7.3 log CFU/orange. Oranges immersed in water containing 1.5% lactic acid plus 1.5% hydrogen peroxide at 40 °C for 10 or 15 min. yielded a mean E. coli Ol 57:H7 population of 1.5 log CFU and < 1.0 log
CFU/apple (detected by enrichment only), respectively. However, control apples subjected to immersion in deionized water yielded a bacterial count of > 5.5 log CFU/orange after 10 and 15 min. exposure times. As observed in the studies with apples, no E. coli O157:H7 survived in the treatment solution after immersion of oranges, whereas, a mean E coli O157:H7 count of 4.5 log CFU/ml was recovered from the deionized water. See Tables 1 and 2 for a summary of these results.
Immersion of apples and oranges in water containing 1.5% lactic acid plus 1.5% hydrogen peroxide at 40 °C was very effective in reducing E. coli O157:H7 on the fruits. 9
Although immersion of fruits in water itself reduced the population of E. coli O157:H7 by 1 to 1.5 log CFU, a significant population of the pathogen survived in the water, representing a potential source of recontamination or cross contamination, in case the same water is used for soaking or washing fruits.
The exemplified wash solution (1.5% lactic acid, 1.5% hydrogen peroxide similarly reduces numbers of bacterial pathogens on fresh food items (e.g., raw fruits and vegetables) including, but not limited to, Escherichia coli including O157:H7, Salmonella spp.. Vibrio spp., Shigella spp. and Listeria spp.
Glycerol monolaurate (monolaurin) is a food-grade chemical approved by the Food and Drug Administration as an emulsifier (21 CFR GRAS 182.4505). Besides its emulsification property, glycerol monolaurate exhibits antimicrobial activity against many spoilage and pathogenic microorganisms [Kabara (1979) J. Am. Oil Chem. Soc. 56:760A- 767A; Beuchat (1980) Appl. Environ. Microbiol. 39:1178-1182; Kato, N. (1981) J. Food Safety 3:12-15]. Gram-positive bacteria are more sensitive to the antimicrobial effects of monolaurin than gram-negative bacteria [Monk et al. (1996) J. Appl. Bacteriol. 81 :7-18]. The antimicrobial activity of glycerol monolaurate is pH dependent and can be increased by decreasing the pH of the medium [Kato and Shibasaki (1976) J. Antibacterial. Antifung. Agents 4:254-261 ; Oh and Marshall (1992) J. Food Prot. 55:449-450]. The combination of lactic acid (1.0 and 1.5%) and monolaurin (0.005%) inactivated > 6.0 log E coli O157:H7/ml at 40°C in 15 min. At 22°C, this treatment reduced E. coli O157:H7 by approximately 5 log
CFU/ml in 20 minutes. The antimicrobial activity of monolaurin is attributed to its effect on bacterial cell membrane permeability resulting in inhibition of synthesis of macromolecules [Galibraith and Miller (1973) J. Appl. Bacteriol. 36:659-675; Vadehra et al. (1985) In The Pharmacological Effect ofLipids II (Ed. Kabara, J.j.) pp. 89-95. American Oil Chemists Society, Champaign, IL]. Reducing the pH of the suspending medium increases the uptake of monolaurin by bacterial cells [Oh and Marshall (1992) J. Food Prot. 55:449-450].
Although treatment containing lactic acid and sodium benzoate was less effective against E. coli O157:H7 than the other two treatments studied, > 6.0 log CFU/ml inactivation 10 of the pathogen was observed in 1.5% lactic acid plus 0.1% sodium benzoate held at 40°C for 15 min. E. coli O157:H7 populations were reduced by only 2.0 to 2.5 log CFU/ml after 20 min at 22°C and 30 min at 8°C, respectively.
Temperature was a critical factor influencing antimicrobial activity of the different treatments on E coli O157:H7. All three combinations of the GRAS chemicals evaluated were effective in inactivating > 6.0 log E. coli O157:H7/ml at 40°C for 15 min. At 8°C, treatments for up to 30 min could only reduce < 3.0 log E. coli O157:H7/ml. This study also indicated that a combination of lactic acid with hydrogen peroxide, sodium benzoate or glycerol monolaurate was more inhibitory to E. coli O157:H7 than when they were present individually. Based on the retail price of the chemicals determined to be effective, the combinations and concentrations of chemicals reported herein are practical and can be applied as surface treatments to inactivate E. coli O157:H7 on suitable raw agricultural commodities.
The above combination treatments used in media tests or as produce washes or soaks, desirably at a solution temperature between about 8 and about 70 °C, preferably between about 20 and about 45 °C, and for a time from about 2 to about 30 minutes are effective in reducing or inactivating Salmonella, Yersinia, Campylobacter, Listeria, Vibrio species, as well as E coli O157:H7 or other species or strains of Escherichia.
While lactic acid at a concentration of 1 to 2% is the preferred acid component of the produce washing or soaking solution, other acids can be substituted therefore including, but not limited to, acetic acid, malic, propionic, tartaric, mandelic and phosphoric acid, at similar concentrations and pHs as the lactic acid.
Within the scope of this invention the term produce means fresh fruits and vegetables. Carcasses include skinned or de-haired or de-feathered carcasses of beef, hog, buffalo, game or poultry, among others. Cut meat products are cuts of meat or poultry, e.g., chicken breasts or wings or roasts or steaks, but ground meat products are not included within this definition.
A produce wash is a solution used to bathe the surface of produce, and typically is in 11 contact with the produce from about 30 sec to about 5 min. A produce soak is a solution in which produce items are immersed for a time from about 30 sec to about 30 min. However, the terms and solutions can be used interchangeably unless otherwise distinguished. It is understood that the temperature at which produce is washed or soaked will influence the length of time necessary to reduce or inactivate bacteria thereon, with warmer temperatures leading to a shorter time necessary for treatment.
The wash or soak solutions of the present invention can be used to reduce bacterial number, especially bacterial pathogen number, on the surfaces of fruits, vegetables, raw cut meat products, fish, shellfish, at the consumer level (in the household), in commercial food preparation environments, on fruits and/or vegetables prior to juicing, by wholesale or retail operators, and/or at the level of the harvest, meat packing plant or slaughterhouse, fishing boat, and so on, without limitation. The present methods are particularly useful for inactivating E. coli. O157:H7 on the surfaces of fresh fruits and vegetables.
All references cited in the present application are incorporated by reference herein to the extent that they are not inconsistent with the present disclosure.
The following examples are provided for illustrative purposes, and are not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified sequences and methods which occur to the skilled artisan are intended to fall within the scope of the present invention.
EXAMPLES
Example 1. Test Chemicals
Stock solutions of 10 and 15% lactic acid, 10% hydrogen peroxide, and 10% sodium benzoate (Sigma Chemical Co., St. Louis, MO) were prepared in sterile distilled water and filter sterilized (0.22 μm nylon filter, Nalgene, Rochester, NY). A stock solution of 0.5% glycerol monolaurate was prepared in 95% ethanol. 12
Example 2. Bacterial Cultures and Media
Five strains of E. coli O157:H7 [E06 (milk isolate), E08 (meat isolate), E10 (meat isolate), El 6 (meat isolate), and E22 (calf feces isolate)] were used for the study. The strains of E. coli O157:H7 were each cultured in 100 ml of sterile tryptic soy broth (TSB) (Difco Laboratories, Detroit, MI) in 250 ml Erlenmeyer flasks at 37°C for 24 hr with agitation (150 rpm). Following incubation, the bacteria were sedimented by centrifugation (4000 x g for 20 min), and washed and resuspended in 10 mM phosphate buffered saline (PBS, pH 7.2). The optical density of the solution was determined (OD of 0.5 at 640 nm represents approximately 109 CFU/ml, CFU are colony forming units). Each culture was diluted with 9 ml of sterile 0.1% peptone to yield an approximate bacterial population of 108 CFU/ml. The viable bacterial population in each culture was confirmed by plating 0.1 ml portions of appropriately diluted culture on tryptic soy agar (TSA) (Difco Laboratories, Detroit, MI) plates and incubating the plates at 37° C for 24 hr. An approximately equal population of each of the five strains was combined and 0.1 ml of the suspension was used as the inoculum.
Example 3. Sample Inoculation and Treatments
One ml of 10 or 15% lactic acid solution and 100 μl of 10% hydrogen peroxide solution were added to individual tubes containing 8.9 ml of 0.1% peptone water to yield solutions containing 1.5% lactic acid plus 0.1% hydrogen peroxide and 1% lactic acid plus 0.1% hydrogen peroxide, respectively. Controls were prepared by adding 1.1 ml of sterile distilled water to 8.9 ml of 0.1% peptone. To each tube, 100 μl of the five-strain mixture of
E. coli O157:H7 (107 CFU) was added, and the treatments were incubated in a water bath (Pharmacia LKB, Piscataway, NJ) at 8°C for 0, 20 and 30 min; at 22 °C for 0, 10 and 20 min; and at 40 °C for 0, 5 and 10 min. Following incubation, a 1 ml portion of the inoculated treatment was serially diluted (1:10) in 9 ml of sterile 0.1% peptone water and 0.1 ml portions from appropriate dilutions were surface plated in duplicate on TSA plates. A volume of 1 ml of the inoculated solution after exposure to the treatment for 0, 5 and 10 min also was transferred to separate 250 ml Erlenmeyer flasks containing 100 ml of sterile TSB and incubated at 37°C for 24 hr. Colonies of E. coli O157:H7 were enumerated on TSA plates after incubation at 37°C for 24 hr. Following enrichment in TSB, the culture was streaked on Sorbitol MacConkey agar No.3 (Oxoid Division, Unipath Co., Ogdensburg, NY) and the 13 plates were incubated at 37 °C for 24 hr. Representative sorbitol-negative colonies from the plates were then confirmed as E. coli O157:H7 by the E. coli O157:H7 latex agglutination assay (Remel Microbiology Products, Lenexa, KS) and the API-20Ε diagnostic test kit (Biomerieux, Hazelwood, MO).
Similarly, 1 ml of 10 or 15% lactic acid solution and 100 μl of 10% sodium benzoate or 0.5% glycerol monolaurate (monolaurin) solution were added to individual tubes containing 8.9 ml of 0.1% peptone water to yield solutions containing 1.5% lactic acid plus 0.1% sodium benzoate or 0.005% glycerol monolaurate and 1% lactic acid plus 0.1% sodium benzoate or 0.005% glycerol monolaurate, respectively. To each of the tubes, 100 μl of the five-strain mixture of E. coli O157:H7 (107 CFU) was added and the solutions were incubated in a water bath at 8°C for 0, 20 and 30 min; at 22°C for 0, 10 and 20 min; and at 40°C for 0, 10 and 15 min. Following incubation, the populations of E. coli O157:H7 that survived in each treatment were determined according to the procedure described herein above.
At all the three incubation temperatures, the populations of E. coli O157:H7 that survived in 0.1 % peptone solution containing solely 1.5% lactic acid or 0.1 % hydrogen peroxide, or 0.1% sodium benzoate or 0.005% glycerol monolaurate also were determined.
The pH of 0.1% peptone solution before and after addition of each combination of chemicals was determined (pH meter model 350, Corning Inc., Corning, NY). At least duplicate samples were assayed for each treatment and the entire study was replicated four times.
Example 4. Statistical Analysis
For each treatment, the data from the independent replicate trials were pooled and the mean value with standard deviation was determined [Steele and Torrie (1980) Principles and Procedures of Statistics. McGraw Hill, New York].
Example 5. Fresh Fruit Model Testing
The efficacy of the combination of lactic acid with hydrogen peroxide for inactivating Escherichia coli O157:H7 on apples and oranges was tested. 14
Five strains of E. coli O157:H7 (Ε-06, E-08, E-10, E-16, and E-22) were used for the study. Each strain was grown individually in 100 ml of tryptic soy broth (TSB, Difco, Detroit, MI) at 37°C with agitation (150 rpm) for 20 h. Following incubation, the bacteria (100 ml) were sedimented by centrifugation (4,000 x g for 30 min) in separate tubes and washed and resuspended in 10 ml of sterile 0.1% peptone water. The five strains of the pathogen (10 ml each) were combined and used as the inoculum. The bacterial population in the suspension was determined by plating 0.1 ml of appropriate dilutions of the suspension on duplicate Sorbitol MacConkey agar plates (SMA, Oxoid Division, Unipath Co., NY) and incubating the plates at 37 °C for 24 h.
Apples and oranges were inoculated and treated according to the flowchart in Fig. 5.
Red Delicious apples and navel oranges were purchased from a local grocery store.
For each experiment, a total of nine apples or oranges were washed in water containing 0.15% crystal clear residue remover (Bonagra Technologies and Services, CA), scrubbed and rinsed with deionized water to remove the external wax coating on their surfaces. After washing, the apples and oranges were dried with a paper towel. The fruits were placed on a sterile plastic tray and 100 μl of E. coli O157:H7 culture (108 CFU) was applied near the stem end of the fruit as shown in Figure 6. The inoculated fruits were dried in a laminar flow hood for 1 h at room temperature (23 °C). After drying, the populations of E. coli O157:H7 on three apples or oranges were determined (baseline). From the remaining six fruits, three of them (inoculated stem end) were held immersed in 50 ml of sterile deionized water containing 1.5% lactic acid plus 1.5% hydrogen peroxide at 40°C for 5, 10 or 15 min. (treatment). Simultaneously, the remaining three apples or oranges were immersed in sterile deionized water at 40 °C for the same durations specified above (control). Immediately after treatments, each apple or orange was transferred to a separate, sterile, 500 ml "Whirl- pak" plastic bag (Nasco, Fort Atkinson, Wisconsin) containing 50 ml of sterile 0.1% peptone water and the population of E. coli O157:H7 was determined on fruits as described above.
The populations of E. coli O157:H7 on apples and oranges were determined as follows. To each bag containing 50 ml of sterile 0.1% peptone water, an apple or orange was 15 transferred in such a way that the inoculated stem end of the fruit was submerged in the diluent. The bag containing the fruit was sealed and placed on a bench-top orbitary shaker and subjected to shaking at 400 rpm for 2 min. A 1.0 ml portion of solution from each "Whirl-pak" bag was serially diluted with 9.0 ml of sterile 0.1% peptone water and 0.1 ml portions from appropriate dilutions were plated on duplicate tryptic soy agar (TSA, Difco,
Detroit, MI) plates. The colonies of bacteria were enumerated after incubating the plates at 37°C for 24 h. The population of E coli O157:H7 in the 50 ml of peptone water in each bag was determined and was expressed per apple or orange. A volume of 1 ml of 0.1% peptone water from each bag also was transferred to separate 250 ml Erlenmeyer flasks containing 100 ml of sterile TSB and incubated at 37 °C for 24 hr. Following enrichment in TSB, the culture was streaked on SMA and the plates were incubated at 37°C for 24 h. Representative sorbitol-negative colonies from
SMA plates and colonies from TSA plates were confirmed as E. coli O157:H7 by E. coli O157:H7 latex agglutination assay (Remel Microbiology Products, Lenexa, KS). The entire experiment with each fruit was replicated three times. In the case of oranges, exposure times of only 10 and 15 min with the treatment solution or water (control) were evaluated.
16
Table 1. Inactivation of E. coli O157:H7 on apples by combination of 1.5% lactic acid + 1.5% hydrogen peroxide at 40 °C.
Samples Surviving bacterial population (mean log cfu/apple) after
5 min. exposure 10 min. exposure 15 min. exposure
Baseline 7.0 ±0.05 7.0 ±0.05 6.95 ± 0.08
Treatment 0a 0a 0a
Control 5.80±0.16 5.30±0.14 5.20 ±0.25
Treatment solution 0a 0a 0a (cfuml)
Control solution 4.56 ±0.16 4.53 ±0.13 4.40 ±0.37 (cfu/ml)
Figure imgf000018_0001
"negative by enrichment.
Table 2. Inactivation of E. coli O157:H7 on oranges by combination of 1.5% lactic acid + 1.5% hydrogen peroxide at 40°C.
Samples Surviving bacterial population (mean log cfu/orange) after
10 min. exposure 15 min. exposure
Baseline 7.27 ±0.15 7.30 ±0.13
Treatment 1.5 ±1.00 0.90 ±0.33
Control 5.58 ±0.13 5.70 ±0.11
Treatment solution 0a 0a (cfu/ml)
Control solution 4.51 ±0.17 4.53 ±0.15 (cfu/ml)
Figure imgf000018_0002
"negative by enrichment.

Claims

17I CLAIM:
1. A method for reducing bacterial numbers on a fresh food item suspected of being contaminated with at least one foodborne pathogen, said method comprising the step of contacting the fresh food item in a decontaminating solution comprising a first active ingredient, wherein said first active ingredient is an acid in a concentration from about 0.3 to about 5 percent (volume/volume) selected from the group consisting of lactic, acetic, malic, propionic, tartaric, mandelic, and phosphoric acid, and a second active ingredient selected from the group consisting of hydrogen peroxide at a concentration of from about 0.1% to 5% percent, glycerol monolaurate at a concentration from about 0.0001% to about 0.1 percent (vol/vol) and sodium benzoate at a concentration of from about 0.005 to about 0.5 percent,
wherein the fresh food item is in contact with the decontaminating solution for a time and at a temperative sufficient to reduce the numbers of at least one foodborne pathogen at least 1000-fold.
2. The method of claim 1 wherein said first active ingredient is lactic acid at a concentration of from 1 to about 2 percent lactic acid and wherein said second active ingredient is hydrogen peroxide at a concentration of from about 0.1 to about 2 percent.
3. The method of claim 1 wherein said first active ingredient is lactic acid at a concentration of about 1.5 percent and wherein said second active ingredient is hydrogen peroxide at a concentration of about 1.5 percent.
4. The method of claim 1 wherein said first active ingredient is lactic acid at a concentration of from about 1 to about 2 percent and wherein said second active ingredient is sodium benzoate at a concentration from about 0.05 to about 0.5 percent.
5. The method of claim 4 wherein said first active ingredient is lactic acid at a 18 concentration between 1 and 2 percent and wherein said second active ingredient is sodium benzoate at a concentration of 0.1 percent.
6. The method of claim 1 wherein said first active ingredient is lactic acid at a concentration from about 1 to about 2 percent and wherein said second active ingredient is glycerol monolaurate at a concentration of 0.001 to 0.05 percent.
7. The method of claim 1 wherein the foodborne pathogen suspected of contaminating the fresh food item is Escherichia coli O157:H7.
8. The method of claim 1 wherein the contacting of the decontaminating solution and the fresh food item is for about 30 seconds to about 30 minutes.
9. The method of claim 1 wherein the contacting of the decontaminating solution and the fresh food item is at a temperature from about 4 to about 50° C.
10. The method of claim 1 wherein the fresh food item to be contacted with the decontaminating solution is a fruit or a vegetable.
19
AMENDED CLAIMS
[received by the International Bureau on 28 June 1999 (28.06.99) ; original claim 1 amended ; remaining claims unchanged ( 1 page) ]
1. A method for reducing bacterial numbers on a fresh food item suspected of being contaminated with at least one foodborne pathogen, said method comprising the step of contacting the fresh food item in a decontaminating solution comprising a first active ingredient, wherein said first active ingredient is an acid in a concentration from about 0.3 to about 5 percent (volume/volume) selected from the group consisting of lactic, acetic, malic, propionic. tartaric, mandelic. and a second active ingredient selected from the group consisting of hydrogen peroxide at a concentration of from about 0.1% to 5% percent, glycerol monolaurate at a concentration from about 0.0001% to about 0.1 percent (vol/vol) and sodium benzoate at a concentration of from about 0.005 to about 0.5 percent,
wherein the fresh food item is in contact with the decontaminating solution for a time and at a temperative sufficient to reduce the numbers of at least one foodborne pathogen at least 1000-fold.
2. The method of claim 1 wherein said first active ingredient is lactic acid at a concentration of from 1 to about 2 percent lactic acid and wherein said second active ingredient is hydrogen peroxide at a concentration of from about 0.1 to about 2 percent.
3. The method of claim 1 wherein said first active ingredient is lactic acid at a concentration of about 1.5 percent and wherein said second active ingredient is hydrogen peroxide at a concentration of about 1.5 percent.
4. The method of claim 1 wherein said first active ingredient is lactic acid at a concentration of from about 1 to about 2 percent and wherein said second active ingredient is sodium benzoate at a concentration from about 0.05 to about 0.5 percent.
5. The method of claim 4 wherein said first active ingredient is lactic acid at a
PCT/US1999/004586 1998-03-02 1999-03-02 Reduction of bacterial pathogens on fresh food items WO1999044444A1 (en)

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