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WO2017079715A1 - Systèmes, procédés et compositions pour utiliser des préparations de protection de bactérie d'origine alimentaire pour favoriser la protection et la conservation d'aliments, de boissons et de surfaces non-comestibles - Google Patents

Systèmes, procédés et compositions pour utiliser des préparations de protection de bactérie d'origine alimentaire pour favoriser la protection et la conservation d'aliments, de boissons et de surfaces non-comestibles Download PDF

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Publication number
WO2017079715A1
WO2017079715A1 PCT/US2016/060760 US2016060760W WO2017079715A1 WO 2017079715 A1 WO2017079715 A1 WO 2017079715A1 US 2016060760 W US2016060760 W US 2016060760W WO 2017079715 A1 WO2017079715 A1 WO 2017079715A1
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Prior art keywords
food
safety
preparation
growth
precursor
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PCT/US2016/060760
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English (en)
Inventor
Richard RICHARD B. SMITTLE
John JOHN B. PHELPS
Gregory GREGORY D. SUNVOLD
John JOHN A. HOMMEYER
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Micro-Nature, Inc.
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Publication of WO2017079715A1 publication Critical patent/WO2017079715A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
    • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
    • A23B2/729—Organic compounds; Microorganisms; Enzymes
    • A23B2/783—Microorganisms; Enzymes

Definitions

  • the present teachings relate generally to systems, methods, and compositions that promote growth inhibition and/or death of certain unsafe and/or unsanitary microorganisms, including mold, yeast, pathogenic bacteria, and food-spoilage bacteria, on or in human or pet food, beverages, and/or non-edible surfaces. More particularly, the present teachings relate to systems, methods, and compositions that pertain to preparations of bacteria and their byproducts or metabolites, applied on or in human or pet food or their packaging, in beverages or their packaging, or on non-edible surfaces, to inhibit the growth of and/or kill mold, yeast, pathogenic bacteria, and food-spoilage bacteria.
  • High nutritional content, good quality ingredients, appearance, texture/mouth feel, and taste are the essential elements of a high-quality diet or food or beverage. Any responsible food or beverage product manufacturer must take great care to control the growth of microorganisms that compromise the sterility of food, including mold, to properly preserve the food or beverage to avoid spoilage and extend the shelf life of food. Numerous problems exist with conventional methods (e.g., retort, high-pressure processing (HPP), irradiation, freezing, chemical agents, organic/mineral acid direct addition) of controlling mold growth.
  • HPP high-pressure processing
  • irradiation irradiation
  • freezing freezing
  • chemical agents organic/mineral acid direct addition
  • Irradiation also has several drawbacks, such as: (1) consumer concerns about the safety of feeding irradiated foods and beverages, (2) capital investment costs, and (3) impacts on nutritional or palatability changes to the diet. Further, chemical agents used to control pathogens and spoilage also have significant drawbacks such as: (1) consumer-unfriendly label ingredients and perception of health concerns, (2) alteration of the nutrient profile of the diet that may have negative health consequences, and (3) reduced diet palatability. Further still, freezing is: (1) costly to perform and maintain, (2) requires a restrictive distribution model, and (3) is not preferred by consumers.
  • Conventional means of promoting food preservation and sanitation include the combining of live bacteria such as lactic acid bacteria, or more specifically, Pediococci bacterial species, with a carbohydrate energy source, into a nutrient-rich growth medium, to create a live bacterial cell-derived preparation.
  • live bacteria such as lactic acid bacteria, or more specifically, Pediococci bacterial species
  • a carbohydrate energy source such as a carbohydrate energy source
  • Such live-bacteria preparations may be used to kill or inhibit the growth of mold and other food-spoilage microorganisms.
  • the present teachings disclose a process for making a food-safety preparation.
  • the process includes: (i) obtaining a growth medium; (ii) introducing an additive to the growth medium to form a food-safety preparation precursor; (iii) inoculating the food-safety preparation precursor with one or more types of lactic acid bacteria to form an inoculated food-safety-preparation precursor; and (iv) incubating the inoculated food-safety-preparation precursor to produce a food-safety preparation.
  • An additive is at least one member chosen from a group comprising one or more supplemental amino acids, glycerol, and detergent.
  • a supplemental amino acid is at least one member chosen from a group comprising proline, serine, threonine, and cysteine.
  • Incubating may be carried out in a two step-process: (i) carrying out a first incubation of the inoculated growth medium at a temperature that ranges from about 33° C to about 38° C for a duration that is between about 16 hours and about 32 hours; and (ii) implementing a second incubation of the inoculated growth medium at a temperature that ranges from about 20° C to about 24° C for a duration that is between about 8 hours and about 24 hours.
  • the process for making a food-safety- preparation includes enhancing exposure of the inoculated food-safety-preparation precursor to air and/or oxygen during incubation. Enhancing exposure of the inoculated food-safety- preparation precursor during incubation may be carried out by at least one technique selected from a group comprising agitating the inoculated food-safety-preparation precursor in presence of air and/or oxygen, stirring the inoculated food-safety-preparation precursor in presence of the air and/or the oxygen, or pumping the air and/or the oxygen into the inoculated food-safety-preparation precursor.
  • Enhancing exposure of the inoculated food- safety-preparation precursor to air and/or oxygen during incubation preferably exposes the inoculated food-safety-preparation precursor, during incubation, to a volume of air and/or oxygen that is at least about two times the volume of the inoculated food-safety-preparation precursor.
  • enhancing exposure of the inoculated food-safety-preparation precursor to air and/or oxygen during incubation produces a food-safety-preparation that has a concentration value of dissolved oxygen in water that is between about 55% oxygen saturation (w/v)and about 95% oxygen saturation (w/v).
  • enhancing exposure of the food-safety-preparation precursor during incubation produces a food-safety-preparation that has a dissolved oxygen content that is between about 5.8 mg dissolved oxygen per liter of food-safety preparation and about 7.93 mg dissolved oxygen per liter of food-safety-preparation.
  • ascorbic acid and/or sodium erythorbate may be added to a growth medium, a food-safety-preparation precursor, an inoculated food- safety preparation precursor, or a food-safety preparation.
  • the present teachings disclose another process for making a food-safety preparation.
  • This process includes: (i) obtaining a growth medium; inoculating the growth medium with one or more types of lactic acid bacteria to form an inoculated growth medium; (iii) carrying out a first incubation of the inoculated growth medium at a temperature that ranges from about 33° C to about 38° C for a duration that is between about 8 hours and about 32 hours; and (iv) implementing a second incubation of the inoculated growth medium at a temperature that ranges from about 20° C to about 24° C for a duration that is between about 8 hours and about 36 hours, to produce the food-safety preparation.
  • the present teachings disclose a food-preservation and/or food-safety process.
  • the process includes: (i) obtaining a growth medium; (ii) introducing an additive to the growth medium to form a food-safety-preparation precursor; (iii) inoculating the food- safety-preparation precursor with one or more types of lactic acid bacteria to form an inoculated food-safety-preparation precursor; (iv) incubating the inoculated food-safety- preparation precursor to produce a food-safety preparation; and (v) associating the food- safety preparation with a food to substantially inhibit growth of mold and/or yeast on or in food during storage.
  • Substantially inhibiting growth of mold and/or yeast on or in food means that the growth of the mold and/or yeast on or in food in the presence of the food- safety-preparation is inhibited at least twice an amount of time that growth of mold and/or yeast on or in the food occurs in the absence of the food-safety preparation.
  • associating the food-safety preparation with a food may include (i) adding the food-safety preparation to an interior surface of a package used for storing the food, and (ii) packaging the food into the package to substantially inhibit growth of mold and/or yeast on or in the food.
  • associating may include (i) adding the food-safety preparation to a fat-containing coating ingredient to produce an activated fat-containing coating ingredient, and (ii) topically applying the activated fat-containing coating ingredient to a food to substantially inhibit growth of mold and/or yeast on or in the food.
  • the food-preservation and/or food- safety process further includes adding detergent to the food-safety preparation and/or to the food prior to associating the food-safety preparation with food, where the food contains fat, and associating is carried out at a temperature sufficient to melt the fat content in food.
  • a food-safety-preparation precursor may include: (i) growth medium; (ii) at least one additive; (iii) one or more supplemental amino acids; (iv) one or more types of lactic acid bacteria; and (v) a culture energy source.
  • An additive may be at least one member chosen from a group comprising glycerol, detergent, and one or more supplemental amino acids, such as proline, serine, threonine, and cysteine.
  • a food- safety-preparation precursor may also include sodium erythorbate and/or ascorbic acid.
  • a food-safety preparation may include: (i) growth medium; (ii) at least one type of lactic acid bacteria; and
  • lactic acid bacteria in a food-safety preparation is in a substantially non-fermenting state and/or in a substantially non-gas-producing state.
  • a food-safety preparation may also include at least one member selected from a group comprising dihydroxyacetone phosphate, hydrogen peroxide, sulfur dioxide, and sulfonic acid.
  • a food-safety preparation may include one or more volatile compounds, such as hydrogen peroxide, polyunstaturated fatty acid, and polyene.
  • a food-safety preparation may include sodium erythorbate and/or ascorbic acid.
  • Figure 1 is a flowchart for a food-safety process, according to one embodiment of the present teachings, and that includes certain steps involved inmaking and using food-safety preparations.
  • Figure 2 is an exemplarcontour plot correlatingvarious sodium erythorbate and sulfite concentrations added or present in food-safety preparations, as described in the flowchart of Figure 1 and applied to high-moisture meat infected with mold, to the number of days when mold is visible.
  • Figure 3 is an exemplar contour plot correlating various ascorbic acid and fermentate concentrations added or present in food-safety preparations, as described in the flowchart of Figure 1 and applied to pet food infected with mold, to the number of days when mold is visible.
  • Figure 4 is an exemplar bar graph showing optical density measured at a wavelength value of about 600 nm versus values of different incubation temperatures (in °C) used to preparefood safety preparations, as described in the flowchart of Figure 1 to show the amount of E. coli growth that occurs after infecting the food safety preparation with E. coli and incubating for about four hours.
  • the present teachings recognize that there exists a longstanding need for more effective means of processing food to: (1) to kill or inhibit the growth of undesirable microorganisms (including molds, yeast, pathogens, and food-spoilage microorganisms), (2) provide long- term (e.g., greater than 1 month to greater than 12 months) shelf-life of food product, (3) avoid costly manufacturing processes and equipment, (4) utilize widely accessible distribution models, (5) preserve the nutrient quality of ingredients closer to their raw state, and (6) provide alternative regulatory options.
  • undesirable microorganisms including molds, yeast, pathogens, and food-spoilage microorganisms
  • the disclosures of the present teachings enable the use of bacterial cultures to provide shelf-stable products for longer periods of time than current without the use of high amounts of heat, sealed packages, or costly capital equipment. Further, not only may the systems, methods, and compositions of the present teachings be used to achieve commercial sterility of edible surfaces (i.e., the outer surface of food) and the interior of foods or beverages, they may also be used to achieve sterility of non-edible surfaces, including and within hard-to-reach places, niches inside equipment, and inside and on various devices.
  • the present teachings provide systems, methods, and compositions that use lactic-acid-producing bacteria, or lactic acid bacteria, and their growth or fermentation byproducts and/or metabolites, to facilitate growth inhibition and/or killing of certain undesirable microorganisms on or in food and/or non-edible surfaces.
  • a food-safety preparation is applied on or in a food, or on or in a food packaging, to promote food safety and/or food preservation.
  • a food-safety preparation is anti- mycotic in nature or type.
  • this food-safety preparation may be used to inhibit growth of and/or kill mold or fungus on or in food.
  • Representative types of mold include Rhizopus spp., Rhizopus stolonifera, Rhizopusnigricans, Penicilliumspp., Aspergillus spp., Aspergillus niger, Cladosporum spp., or Botrytis spp.
  • a food-safety preparation is anti- yeast in nature or type.
  • this food-safety preparation may be used to inhibit growth of and/or kill yeast on or in food.
  • Representative types of yeast include Pichia spp., Pichia anomala, Saccharomyces spp., Zygosaccharomyces spp., Zygosaccharomyces bailii, or Rhodotorula spp.
  • a food-safety preparation is anti-pathogenic in nature or type.
  • this food-safety preparation may be used to inhibit growth of and/or kill pathogenic bacteria on or in food.
  • pathogenic bacteria include Salmonella, pathogenic Escherichia coli, Shigella spp., Listeria monocytogenes, Staphylococcus aureus, Campylobacter spp., Campylobacter jejuni,
  • Campylobacter coli Clostridia spp., Clostridium botulinum, Clostridium perfringens, Vibrio spp., Vibrio parahaemolyticus, or Vibrio cholera.
  • a food-safety preparation is anti food-spoilage in nature or type.
  • this food-safety preparation may be used to inhibit growth of and/or kill one or more spoilage microorganisms.
  • Representative types of food spoilage microorganism include Bacillus spp., Bacillus subtilis, Enterobacter spp., Enterobacter aerogenes, Proteus spp., Proteus vulgaris, Micrococcus spp., Micrococcus roseus, Erwinia spp., Alcaligenes spp., Clostridium spp., Pseudomonas spp., Pseudomonas fluorescens, Micrococcus spp., Enterococcus spp., Lactobacillus spp., Lactobacillus fructivorans, Leuconostoc spp., Lactococcus spp., Achromabacter spp., Alcaligenes spp., Flavobacterium spp., Acinetibacter spp., or Acetobacter spp.
  • a food-safety preparation may also target any combination of microorganisms and/or desired effects described herein.
  • the present systems, methods, and compositions are used tosterilize and/or promote safety of non-edible surfaces(e.g., work surfaces, cracks, or inside equipment and devices).
  • a food-safety preparation may be applied to various non-edible surfaces to promote the growth inhibition and/or killing of molds, yeasts, pathogenic bacteria, or food- spoilage microorganisms thereon.
  • the present teachings recognize that the manner in which lactic acid bacteria (e.g., Pediococci spp.) cultures are grown impacts the ability of such cultures to control growth of mold, yeast, pathogenic bacteria, and/or food-spoilage microorganisms when the food-safety preparation are applied to food or food packaging.
  • certain treatment steps and/or components or additives introduced during a process for making a food-safety preparation facilitate production of metabolites that kill and/or inhibit growth of molds, yeasts, pathogenic bacteria, and/or food-spoilage microorganisms.
  • the present teachings disclose a method of making and using a food-safety preparation precursor.
  • a food-safety preparation precursor may be thought of as combined and/or mixed components that, after being treated according to processes of the present teachings, produce a food-safety preparation.
  • a food-safety preparation precursor preferably includes a growth medium, at least one additive, a culture energy source, and one or more types of lactic acid bacteria.
  • a growth medium may be any nutrient-rich growth medium well known to those of skill in the art for growing and/or fermenting lactic acid bacteria.
  • a growth medium is at least one member chosen from a group comprising Trypto-Soy Broth, chicken broth, low sodium chicken broth, beef broth, yeast cell extract, Gold Cell yeast ethanol extract, Tech Grade yeast extract, chicken hydrolysate, hydrolyzed chicken protein, liver digest, hydrolyzed liver digest, dry palatant, petfood palatant, petfood dry digest, petfood dry palatant, dry flavoring, hydrolyzed collagen, hydrolyzed collagen from beef, hydrolyzed collagen from chicken, hydrolyzed collagen from fish, hydrolyzed collagen from pork, pea protein, vegetable protein, hydrolyzed vegetable protein, and hydrolyzed soy protein.
  • food-safety preparations that include chicken broth have been shown to produce enhanced killing power against Salmonella surrogates over those that include beef broth or commercially
  • a food-safety-preparation precursor includes a detergent.
  • a detergent in a food-safety-preparation precursor is at least one member selected from a group comprising Tween 20, polysorbate 20, polyoxyethylene (20) sorbitan monolaurate, poly oxy sorbitan monolaurate, lauric acid sources, Tween 60, lecithin, and palmitic acid.
  • a detergent is an emulsifier.
  • a detergent is present in a food-safety preparation at a concentration value that is between about 0.0001% (weight of detergent to volume of food-safety preparation (hereinafter, a weight to volume ratio is indicated by "w/v")) and about 5% (w/v), more preferably, between about 0.001% (w/v) and about 1.0% (w/v), more preferably, between about 0.01% (w/v) and about 0.5% (w/v), and more preferably, at a concentration that is about 0.1% (w/v).
  • the present teachings contemplate the use of any amount of detergent in a food-safety -preparation precursor, including using no amount of detergent.
  • a lactic acid bacteria in a food-safety preparation precursor may be any lactic acid bacteria well known to those of skill in the art that produces lactic acid and other metabolites during growth and/or fermentation.
  • a lactic acid bacteria is at least one member chosen from a group comprising Pediococcus acidilactici, Pediococcus pentosaceus, Lactococcus lactis, Lactococcus cremoris, Lactobacillus delbruckii var bulgaricus, Lactobacillus plantarum, Lactobacillus pentosum, Streptococcus thermophilus, Lactobacillus sakei, Lactobacillus curvatus, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus gasseri, Bifid
  • a lactic acid bacteria is a Pediococcispp., and more preferably, a Pediococcus pentosaceus or a Pediococcus acidilactici.
  • the present teachings also contemplate use of more than one type of lactic acid bacteria in a food-safety-preparation precursor ⁇ e.g., a combination of Pediococcus pentosaceus and Pediococcus acidilactici).
  • a food-safety-preparation precursor may also include one or more supplemental amino acids, which support growth and/or fermentation of lactic acid bacteria during incubation.
  • one or more supplemental amino acids in a food- safety-preparation precursor also promote enhanced anti-mycotic, anti-yeast, and/or anti- pathogen effects of a subsequent food-safety preparation produced according to the processes of the present teachings.
  • Theseamino acids are considered "supplemental," insofar as they are supplemental to amino acids that may be naturally occurring in certain growth media. In other words, these supplemental amino acids are added to a growth medium during a process for making a food-safety preparation.
  • one or more supplemental amino acids in a food-safety-preparation precursor include hydroxyl amino acids, such as threonine, proline, and serine.
  • a concentration value of proline or serinein a food-safety-preparation precursor may be between about 0.0001% (w/v) and about 1.0% (w/v), more preferably, between about 0.001%) (w/v) and about 0.5% (w/v), more preferably, between about 0.01% (w/v) and about
  • a concentration value of threonine in a food-safety-preparation precursor may be between about 0.0005%> (w/v) and about 5% (w/v), preferably, between about 0.0055%> (w/v) and about 1.0% (w/v), more preferably, between about 0.05% (w/v) and about 0.15% (w/v), and most preferably, is about 0.1% (w/v).
  • a supplemental amino acid is cysteine.
  • a concentration value of cysteine in a food-safety-preparation precursor is between about 0.001% (w/v) and about 0.5% (w/v), preferably, between about 0.01% (w/v) and about 1% (w/v), more preferably, between about
  • a culture energy source in a food-safety-preparation precursor may be any culture energy source well known to those of skill in the art for producing a source of energy to growing and/or fermenting bacteria.
  • a culture energy source is at least one member chosen from a group comprising apple juice, apple juice concentrate, dextrose, dextrose monohydrate, dextrose hydride, grape sugar, D-glucose, fructose, corn sugar, sucrose, lactose, maltose, corn syrup solids, hydrolyzed corn syrup, high fructose corn syrup, molasses, blackstrap molasses, levulose, glucose, galactose, xylose, ribose, mannose, cellobiose, arabinose, ribose, salicin, amygdalin, esculin, sorbose, amino acids, high fructose corn syrup, apple pulp, honey, sugar, maple syrup, pear juice, grape juice, orange
  • a culture energy source is dextrose, and present in a food-safety preparation at a concentration of up to about 5% (w/v), or more preferably, between about 1% (w/v) and about 3% (w/v).
  • concentrations of dextrose lower than those used in conventional techniques, in conjunction with the treatment conditions described herein allows Pediococci to grow into higher populations useful for subsequent enhanced metabolic production of antimicrobial agents, which include antifungal agents.
  • the culture energy sources of the present teachings provide effectivesources of carbohydrate in the form of simple sugars (e.g., monosaccharides, disaccharides, tri-saccharides, or saccharide chains up to but less than seven monomeric units of glucose, fructose, galactose, xylose, rhamnose, lactose, mannose, sorbitol, ribose, or arabinose linked together) that are readily metabolized by growing and/or fermenting bacteria during incubation.
  • simple sugars e.g., monosaccharides, disaccharides, tri-saccharides, or saccharide chains up to but less than seven monomeric units of glucose, fructose, galactose, xylose, rhamnose, lactose, mannose, sorbitol, ribose, or arabinose linked together
  • dextrose is an example of a substantially pure source of such simple sugars (i.e., glucose) for
  • carbohydrate equivalents where one carbohydrate equivalent is defined as the amount of carbohydrate provided by one unit of dextrose.
  • calculating a concentration value of carbohydrate equivalents in a culture energy source will help standardize the contribution of simple sugar carbohydrates in a culture energy source.
  • apple juice concentrate is predominantly water with about 41% simple sugars that are readily metabolized by bacteria. Accordingly, apple juice concentrate may be thought of as having about 41% carbohydrate equivalents (e.g., so 100 g of apple juice concentrate provides about 41 g of carbohydrate equivalents).
  • FOS short-chain fructooligosaccharides
  • short-chain FOS may be thought of as having about 100% carbohydrate equivalents.
  • concentration of carbohydrate equivalents in a food-safety-preparation precursor ranges from about 0.01% (w/v)to about 10% (w/v), preferably, ranges from about 0.1%) (w/v)to about 5% (w/v), more preferably ranges from about 0.5% (w/v)to about 1.5%
  • a food-safety-preparation precursor may also include glycerol.
  • glycerol is present in a food-safety-preparation precursor at a concentration value that is between about 0.01% (w/v) and about 5.0% (w/v), preferably, between about 0.1% (w/v) and about 3% (w/v), more preferably between about 0.5%) (w/v) and about 1.5% (w/v), and is, most preferably, about 1% (w/v).
  • the present teachings contemplate the use of any amount of glycerol in a food-safety- preparation precursor, including no glycerol.
  • a food-safety-preparation precursor includes ascorbic acid and/or sodium erythorbate.
  • the present teachings recognize that sodium erythorbate and/or ascorbic acid may be used in the systems, methods, and compositions of the present teachings as potent antioxidants to capture free oxygen to create oxygen-level adjusted food-safety preparations that more potently exhibit anti-mycotic, anti-yeast, anti- pathogen, and/or anti-spoilage microorganism effects.
  • sodium erythorbate and/or ascorbic acid is present in a food-safety-preparation precursor at a concentration at a concentration value that is between about 0.01% (w/v) and about 5.0% (w/v), preferably, between about
  • sodium erythorbate and/or ascorbic acid may be not be included in a food-safety- preparation precursor and still be added, to the same effect, in a later step (e.g., after incubation that results in a finished food-safety preparation, as explained below).
  • a food-safety-preparation precursor includes a growth medium, a culture energy source, and one or more supplemental amino acids.
  • a food-safety-preparation precursor includes a growth medium, a culture energy source, one or more supplemental amino acids, and glycerol.
  • a food-safety- preparation precursor includes a growth medium, a culture energy source, and glycerol.
  • a food-safety-preparation precursor includes a growth medium, a culture energy source, one or more supplemental amino acids, and ascorbic acid and/or sodium erythorbate.
  • a food-safety-preparation precursor includes a growth medium, a culture energy source, one or more supplemental amino acids, glycerol, and ascorbic acid and/or sodium erythorbate.
  • a food-safety-preparation precursor includes a growth medium, a culture energy source, glycerol, and ascorbic acid and/or sodium erythorbate.
  • a food-safety-preparation precursor includes a growth medium, a culture energy source, one or more supplemental amino acids, and a detergent.
  • a food-safety-preparation precursor includes a growth medium, a culture energy source, one or more supplemental amino acids, glycerol, and a detergent.
  • a food-safety-preparation precursor includes a growth medium, a culture energy source, glycerol, and a detergent.
  • a food-safety-preparation precursor includes a growth medium, a culture energy source, one or more supplemental amino acids, ascorbic acid and/or sodium erythorbate, and a detergent.
  • a food-safety- preparation precursor includes a growth medium, a culture energy source, one or more supplemental amino acids, glycerol, ascorbic acid and/or sodium erythorbate, and a detergent.
  • a food-safety- preparation precursor includes a growth medium, a culture energy source, glycerol, ascorbic acid and/or sodium erythorbate, and a detergent.
  • a food-safety preparation may be thought of as the composition that is produced following treatment of a food-safety-preparation precursor according to processes of the present teachings.
  • a food-safety preparation may be a bacterial growth culture, an incubate (i.e., a bacterial culture during growth phase of the bacteria), a fermentate (i.e., after fermentation such that bacteria are in a substantially non-growing and/or non gas-producing state), a non-live- bacteria sample (i.e., where living bacteria has been removed and/or bacteria is substantially dead), or a concentrate (i.e., a bacterial growth culture, an incubate, a fermentate, or a non- live-bacteria sample that has been concentrated (i.e., by removal of moisture)).
  • a food-safety preparation includes a growth medium, at least one type of lactic acid bacteria, and a sulfite.
  • a growth medium and at least one type of lactic acid bacteria are the same as or substantially similar to their counterparts described above with reference to food-safety-preparation precursors.
  • lactic acid bacteria in a food-safety preparation is in a substantially non- fermenting and/or non-gas-producing state.
  • lactic acid bacteria is substantially depleted of living bacteria.
  • lactic acid bacteria in a food-safety preparation is dead, or living bacteria has been removed.
  • a food-safety preparation includes a sulfite.
  • the present teachings recognize that the presence of sulfite, a known food preservative, is due, in part to the conversion of sulfhydryl groups on cysteine to sulfite during a process for making a food-safety preparation according to the present teachings.
  • sulfite is present in a food-safety preparation at a concentration value that is between about 50 ⁇ g per 100 g of food and about 3,000 ⁇ g per 100 g of food, preferably, between about 50 ⁇ g per 100 g of food and about 1,250 ⁇ g per 100 g of food, more preferably, between about 300 ⁇ g per 100 g of food and about 900 ⁇ g per 100 g of food, more preferably, between about 500 ⁇ g per 100 g of food and about 750 ⁇ g per 100 g of food,and is, most preferably, about 650 ⁇ g per 100 g of food.
  • a food-safety preparation is combined with food such that at least about 50 ⁇ g of sulfite per 100 g of food is used.
  • a food-safety preparation may also include at least one member selected from a group comprising dihydroxyacetone phosphate, hydrogen peroxide, sulfur dioxide, and sulfonic acid.
  • a group comprising dihydroxyacetone phosphate, hydrogen peroxide, sulfur dioxide, and sulfonic acid.
  • active components e.g., active ingredient that is anti-mycotic, anti-yeast, anti-spoilage or anti-pathogenic
  • active components e.g., active ingredient that is anti-mycotic, anti-yeast, anti-spoilage or anti-pathogenic
  • active components e.g., active ingredient that is anti-mycotic, anti-yeast, anti-spoilage or anti-pathogenic
  • the present teachings recognize that due to the volatile nature of such components, placing them generally on food or food packaging facilitates killing and/or growth inhibition of undesirable molds, yeasts, pathogens, and/or food-spoilage microorganisms.
  • the present teachings disclose a preserved and/or safe food.
  • a preserved and/or safe food may be thought of as a food that is preserved and/or made safe by the addition of a food-safety preparation on or in the food, and/or on or in packaging that contains the food.
  • preserved and/or safe food is rendered preserved and/or safe due to the presence of the food-safety preparations of the present teachings on, in, or around the food.
  • the present teachings disclose a process 100, according to one embodiment of the present teachings, for making and using a food-safety preparation.
  • the process may begin with a step 102, which includes obtaining a growth medium.
  • the growth medium is one or more of the same growth mediums or substantially similar to the growth mediums described above in connection with a food-safety-preparation precursor.
  • the obtained growth media contains certain additional components and/or additives, such as a detergent, one or more supplemental amino acids, a culture energy source, or any other component useful in promoting growth and/or fermentation of lactic acid bacteria in subsequent steps. In other embodiments of the present teachings, however, one or more of such components are added in subsequent steps.
  • a step 104 includes introducing an additive to said growth medium to form a food- safety preparation precursor.
  • an additive is at least one member selected from a group comprising one or more supplemental amino acids, glycerol, and detergent.
  • an "additive" may be thought of as any component that is added to or made a part of a food-safety-preparation precursor that promotes and/or facilitates anti-mycotic, anti-yeast, and/or anti-pathogen activity of a subsequently produced food-safety preparation.
  • the present teachings also recognize that any of these additives may be contained in a growth medium that is obtained in step 102.
  • the detergent contemplated in step 104 may be the same as or substantially similar to the detergent described above in connection with a food-safety-preparation precursor. Further, as explained above, the detergent may also be included as a component of a growth medium that is obtained according to step 102.
  • the detergent is a source of detoxified oleic acid that is important in lactic acid bacteria growth and cell membrane integrity. Accordingly, the addition of a detergent to a food-safety-preparation precursor ultimately provides a food- safety preparation, which effectively promotes growth inhibition or killing of pathogenic microorganisms contained within certain hydrophobic matrices, such as fat. These detergents may be used to detoxify fatty acids to produce detoxified fatty acids, which are incorporated in certain desirable cells (e.g., which promote growth inhibition or killing of pathogenic microorganism) inside a growth medium.
  • detergents or emulsifiers may also be added to a food or beverage, along with a food-safety preparation for, to render the food-safety preparation more capable of interacting with the bacteria, yeast, or mold, and thus killing or inhibiting growth of these microorganisms.
  • glycerol is added in step 104, it is preferably used in an effective amount to promote or facilitate anti-mycotic, anti-yeast, and/or anti-pathogen activity of an ultimately produced food-safety preparation.
  • the present teachings recognize that utilization of glycerol by lactic acid bacteria during growth and/or fermentation occurs as the primary culture energy source levels become depleted.
  • Certain metabolic pathways that utilize glycerol under the treatment conditions of the present teachings (e.g., a period of incubation at relatively lower temperatures and/or with introduction of additional oxygen and/or air during incubation, discussed in further detail below), promote the synthesis of small and intermediate chain unsaturated fatty acids, which are volatile and antifungal, and larger molecular weight compounds, such as polyenes, which are potent antifungal and antibiotic agents.
  • glycerol when grown in the presence of additional oxygen and/or air according to the present teachings, is also important in producing 3 -hydroxy -glyceraldehyde, a known antibacterial and antifungal agent.
  • the present teachings recognize that Pediococci grown in the presence of glycerol, under the treatment conditions of the present teachings, induces the production of glycerol 3-phosphate.
  • Glycerol-3-phosphate oxidase which is also produced by Pediococci (particularly when grown are relatively low temperature and in the presence of excess oxygen), converts glycerol 3-phosphate to dihydroxyacetone phosphate and hydrogen peroxide, both of which are potent anti-mycotic agents.
  • one or more supplemental amino acids are added in step 104, they are preferably of a type that is known to those of skill in the art to facilitate growth and/or fermentation of lactic acid bacteria in a subsequent step. Further, one or more supplemental amino acids added in step 104 may facilitate anti-mycotic, anti-yeast, and/or anti-pathogen activity of an ultimately produced food-safety preparation. According to one embodiment of the present teachings, one or more supplemental amino acids includes at least one hydroxyl amino acid chosen from a group comprising proline, serine, and threonine.
  • hydroxyl amino acids facilitate or promote the production of proteins found in acid mucins, These proteins are potent antibacterial agents that promote the growth of bacterial cells that provide a source of acid mucins, which inhibit the growth of, and/or kill, pathogenic bacteria. Hydroxylated amino acids also result in the formation of proteins that are enriched with hydroxyl sites that enable the attachment of sialic acid and N-acetyl glucosamine and sulfur, which are useful in inhibiting the growth of molds and yeast.
  • one more supplemental amino acids includes cysteine.
  • cysteine a sulfhydryl amino acid
  • sulfonated compounds e.g., sulfonic acid and sulfur dioxide
  • a culture energy source is also included in a food-safety -preparation precursors.
  • a culture energy source in step 102 of process 100 is same as or substantially similar to a culture energy source described above with reference to a food-safety-preparation precursor.
  • a culture energy source of the present teachings is included in a growth medium that is obtained in step 102, or a culture energy source may be added in a separate step, e.g., step 104.
  • an amount of culture energy source e.g., dextrose
  • an amount of one or more supplemental amino acids may be adjusted relative to each other.
  • the present teachings recognize that using relatively higher levels of a culture energy source during incubation may facilitate utilization by the bacteria of relatively higher amounts of one or more supplemental amino acids. This increased utilization of one or more supplemental amino acids by the bacteria during incubation promotes faster growth, division, and/or fermentation of the bacteria.
  • additional steps are carried out during step 104 and prior to advancing to step 106.
  • a detergent e.g., tween-80
  • glycerol e.g., tween-80
  • certain additional steps may be carried out to pasteurize the food- safety-preparation precursor prior to inoculating with lactic acid bacteria (i.e., in step 106, below).
  • the food-safety-preparation precursor may be heated up to a temperature that is between about 80° C and about 95° C, preferably between about 85° C and about 90° C, and more preferably, at about 88° C, to produce a pasteurized food-safety-preparation precursor.
  • a temperature that is between about 80° C and about 95° C, preferably between about 85° C and about 90° C, and more preferably, at about 88° C, to produce a pasteurized food-safety-preparation precursor.
  • the desired temperature e.g., about 88° C
  • the pasteurized food-safety-preparation precursor is cooled. If one or more supplemental amino acids are to be added, then they are added when the pasteurized food- safety-preparation precursor is cooled to a temperature that is between about 68° C and about 79° C, and preferably, at about 74° C. While wishing not to be bound to theory, this slight cooling step prior to introduction of one or more supplemental amino acids into a pasteurized food-safety-preparation precursor helps avoid Maillard reactions that are more likely to occur between a culture energy source (e.g., dextrose) and one or more supplemental amino acids at relatively higher temperatures (e.g., about 88° C).
  • a culture energy source e.g., dextrose
  • the pasteurized food-safety-preparation precursor is cooled to a temperature that is between about 22° C and about 57° C, and more preferably, between about 35° C and about 40° C.
  • a temperature that is between about 22° C and about 57° C, and more preferably, between about 35° C and about 40° C.
  • a step 106 includes inoculating the food-safety-preparation precursor with one or more types of lactic acid bacteria to form an inoculated food-safety-preparation precursor.
  • Lactic acid producing bacteria in step 106 are substantially similar to those described above in connection with a food-safety-preparation precursor and food-safety preparation described above.
  • step 106 includes inoculating with sufficiently effective amounts of lactic acid bacteria into a food-safety-preparation precursor to promote growth and/or fermentation of the lactic acid bacteria.
  • a food-safety-preparation precursor is inoculated with at least about 1 x 10 4 colony forming units of lactic acid bacteria per gram of growth media or per gram of food-safety-preparation precursor (expressed herein as units of "cfu/gram").
  • step 106 inoculating in step 106 is carried out prior to or at the same time as step 104 is carried out. In other embodiments of the present teachings, inoculating in step 106 is carried out after step 104.
  • a step 108 includes incubating the inoculated food-safety -preparation precursor to produce a food-safety preparation. Incubating, in step 108, may be carried out under a treatment condition that promotes or facilitates growth and/or fermentation of lactic acid bacteria.
  • incubating is carried out at a temperature that is between about 20° C and about 55° C, preferably between about 30° C and about 45° C, more preferably between about 33° C and about 38° C, and most preferably, at about 35° C. Incubating may be carried out at a duration that is between about 8 hours and about 36 hours, preferably between about 16 hours and about 32 hours, more preferably, between about 20 hours and about 28 hours, and most preferably, for about 24 hours.
  • incubating is carried out under two separate treatment conditions: (l)a first incubation stepis carried out at a relatively higher temperature, e.g., a temperature value that ranges from between about 20° C and about 55° C, preferably, between about 30° C and about 45° C, more preferably, between about 33° C and about 38° C, and most preferably, at about 35° C; and (2) a second incubation stepis carried out at a relatively lower temperature, e.g., a temperature value that ranges from between about 8° C and about 35° C, preferably, between about 15° C and about 28° C, more preferably, between about 20° C and about 26° C, and most preferably, at about 22° C.
  • a relatively higher temperature e.g., a temperature value that ranges from between about 20° C and about 55° C, preferably, between about 30° C and about 45° C, more preferably, between about 33° C and about 38° C, and most preferably, at about 35° C
  • the first incubation step carried out at a relatively higher temperature facilitates an increase in the population of lactic acid bacteria that preferentially utilizes the available culture energy source (e.g., glucose or dextrose); then, a second incubation step carried out at a relatively lower temperature ensures that the remaining energy source is glycerol, which facilitates, according to the present teachings, production of certain food-safety-preparation metabolites that facilitate anti-mycotic, anti-yeast, and/or anti pathogen effects of a food-safety preparation prepared.
  • the available culture energy source e.g., glucose or dextrose
  • each incubation step may be carried for any effective incubation time sufficient to promote the anti-mycotic, anti-yeast, and/or anti-pathogen effects of the food-safety preparations of the present teachings.
  • each of a first incubation step and a second incubation step is separately carried out for a duration that is between about 8 hours and about 36 hours, more preferably, between about 16 hours and about 32 hours, more preferably, between about 20 hours and 28 hours, and most preferably, for about 24 hours.
  • incubating in step 108 includes exposing the food-safety-preparation precursor to additional air and/or oxygen exposure during incubation than those found under ambient or normal conditions.
  • additional air exposure or “additional air and/or oxygen exposure” refers to the situation when more of the growth culture and/or food-safety-preparation precursor is exposed to additional air and/or oxygen during incubation.
  • additional air and/or oxygen exposure during incubation provides additional oxygen to bacterial cells while they are growing, dividing, and/or fermenting, which induces metabolic stress on the bacteria.
  • metabolic stress is inducement of certain self-defense mechanisms, causing the bacteria to secrete protective compounds, which may be anti-mycotic, antifungal, anti-pathogenic, and/or anti-spoilage in nature or type.
  • Providing additional air and/or oxygen exposure during step 108 may be carried a number of different ways.
  • additional air and/or oxygen exposure may be provided by agitating the food-safety-preparation precursor in the presence of air and/or oxygen, stirring the inoculated food-safety-preparation precursor in the presence of air and/or oxygen, pumping or bubbling air and/or oxygen into the food-safety-preparation precursor during incubation, or using containers that provide additional surface area exposure of the food- safety-preparation precursor to air and/or oxygen.
  • the volume of air and/or oxygen that is exposed to a food-safety-preparation precursor is at least about two times the volume of the food-safety-preparation precursor.
  • exposing a food-safety-preparation precursor to additional air and/or oxygen produces an enhanced oxygen content in the water component of the food-safety preparation.
  • exposure of a food-safety-preparation precursor to additional air and/or oxygen in such manner during incubation produces a dissolved oxygen content in water that is a value that ranges from between about 45%oxygen saturation (w/v)and about 95%oxygen saturation
  • enhancing exposure of the food-safety-preparation precursor during incubation produces a food-safety-preparation that has a dissolved oxygen content of between about 4.0 mg dissolved oxygen per liter of food-safety-preparation and about 8.5 mg dissolved oxygen per liter of food-safety-preparation, preferably between about 4.9 mg dissolved oxygen per liter of food-safety-preparation and about 8.0 mg dissolved oxygen per liter of food-safety- preparation, 5.8 mg dissolved oxygen per liter of food-safety preparation and about 7.93 mg dissolved oxygen per liter of food-safety-preparation.
  • the present teachings recognize that the combination of incubation using relatively lower temperatures, in conjunction with exposure to additional air and/or oxygen (which is more than the amount present in ambient conditions), produces food-safety preparations with potent anti-mycotic, anti -yeast, and/or anti-pathogen activity.
  • relatively lower temperatures allow for greater amounts of additional oxygen to become soluble in the water component of the food-safety-preparation precursor and available for oxidation, providing, in part, the advantage of increased production of sulfonic acid and sulfuric acid under the present treatment conditions.
  • lower temperature incubation with an increase in available oxygen increases the formation of volatile compounds, especially when glycerol is available as an energy source.
  • the present teachings believe that once the primary culture energy source (e.g., dextrose), is diminished or depleted, alternative adaptive enzyme systems are induced for utilization of glycerol. These alternative adaptive enzyme systems are associated with production of factors (e.g., hydrogen peroxide) that inhibit growth of molds, yeasts, pathogens, and food-spoilage microorganisms. Further still, lower temperatures result in an increase in dissolved oxygen and subsequently an increase in the synthesis of unsaturated fatty acids. Unsaturated fatty acids are volatile and toxic to yeasts and molds. By way of example, sorbic acid is a volatile 6-carbon double unsaturated fatty acid that has desirable anti-mycotic properties.
  • factors e.g., hydrogen peroxide
  • sodium erythorbate and/or ascorbic acid may be added at any time during or prior to step 108 to promote or facilitate anti- mycotic, anti-yeast, anti-pathogen, and/or anti-spoilage effects of a food-safety preparation.
  • Sodium erythorbate and ascorbic acid are described above in connection with a food-safety- preparation precursor.
  • a food-safety preparation produced according to steps 102-108 may be further modified by concentrating the food-safety preparation, or any precursor thereof.
  • concentrating includes removing moisture.
  • Concentrating may be carried out using a technique that concentrates a bacterial culture.
  • concentrating includes at least one technique selected from a group comprising removing supernatant, centrifuging, vacuum drying, filtrating, flocculating, freeze drying, rotary evaporation, reverse osmosis, and ion exchange.
  • living bacteria in a food-safety preparation may be killed by a number of different ways, including but not limited to use of chemical agents (e.g., ethanol) and/or heating.
  • a food-safety preparation may also be depleted of live bacteria by a number of different ways, including but not limited to centrifugation, filtration, or flocculation.
  • a food-safety preparation has been prepared.
  • a step 110 is preferably carried out.
  • the food-safety preparation is associated with a food or a food ingredient to substantially inhibit growth of mold and/or yeast on or in the food or food ingredient during storage.
  • associating includes applying the food-safety preparation to an interior surface of a food packaging.
  • a step of packaging the food into the food package (applied with a food-safety preparation) to substantially inhibit growth of mold and/or yeast on or in the food is carried out.
  • a food- safety preparation is present in or on a food product or in or on a food packaging that is associated with the food product at a ratio that is between about 0.1 ml of food-safety preparation per 100 g of food product and about 4 ml of food-safety preparation per 100 g of food product, preferably, between about .5 ml of food-safety-preparation per 100 g of food product and about 2 ml of food-safety preparation per 100 g of food product, and more preferably, between about 1 ml of food-safety preparation per 100 g of food product and about 1.5 ml of food-safety preparation per 100 g of food product.
  • the above-described associating step includes: a first step of adding the food-safety preparation to a fat-containing coating ingredient to produce an activated fat-containing coating ingredient; and then, a second step of topically applying the activated fat-containing coating ingredient to a food is carried out to substantially inhibit growth of mold and/or yeast on or in the food.
  • a food-safety preparation is added directly to food to inhibit growth of mold, yeast, and/or pathogens on or in the food.
  • the present teachings recognize that food-safety preparations having bacteria in various states may be introduced to food or beverages.
  • a food-safety preparation may include live bacteria in a substantially non-fermenting state when added to a food or beverage.
  • a food-safety preparation that includes no live bacteria may be added to a food or beverage such that the food-safety preparation will not cause the food or beverage to undergo fermentation or culturing.
  • a detergent prior to associating a food-safety preparation with a food that contains fat, a detergent may be added to the food-safety preparation, or added directly to the food at or near the same time as the food-safety preparation is added, to inhibit the growth of mold, yeast, pathogens, and/or spoilage microorganisms in the fat of food.
  • an amount of detergent added to the food- safety preparation is a value that ranges from between about 0.1% (w/v) to about 2% (w/v), and preferably, between about 0.5% (w/v) to about 1% (w/v).
  • the food-safety preparation with detergent is added to food heated to a degree sufficient to melt the fat content in food.
  • the fat is poultry fat.
  • the addition of bacterial cultures and associated metabolites, in or from the food-safety preparations of the present teachings, to food to prevent or inhibit the growth of certain microorganisms may be carried out by topically adding the food-safety preparation to the surface of edible products such as food, confectionary, and pet food products.
  • the food-safety preparations of the present teachings may be added after heat-processing steps have been carried out to avoid heat volatilizing the active components in the food- safety preparation.
  • the active components contained within the incubate, fermentate, concentrate, or culture may be topically added directly to the food product.
  • the incubate, fermentate, concentrate, or culture may be added into another coating that is topically applied to the food product.
  • One preferred embodiment includes topically adding the incubate, fermentate, concentrate, or culture to the food product by mixing the incubate, fermentate, concentrate, or culture into a palatant or flavor coating that is topically applied to the food product.
  • Another preferred embodiment includes topically adding the incubate, fermentate, concentrate, or culture to the food product by mixing the incubate, fermentate, concentrate, or culture into a fat coating that is topically applied to the food product.
  • the food-safety preparations of the present teachings may also be added prior to food processing, where the food processing does not involve fermentation of food.
  • a food-safety preparation may be added to a dry ingredient mixture prior to extruding the mixture for the purpose of making an extruded, pathogen killed, food or pet food product.
  • the addition of the food-safety preparation to address molds, yeasts, pathogens, and food spoilage microorganisms in the dry mixture prior to extruding the mixture provides the advantage of convenience to the food or pet food manufacturer and assures a homogeneous mixture of the bacterial cultures to address growth of molds, yeasts, pathogens, and food-spoilage microorganisms in the food or pet food product.
  • incorporating the food-safety preparation in the extruded food or pet food product provides the advantage of killing undesirable microorganisms that may infest the food or pet food product after extrusion but prior to consumption of the food or pet food product.
  • a food-safety preparation In addition to killing and/or inhibiting mold, yeast, pathogen, and food-spoilage microorganisms growth on food, additional uses of a food-safety preparation include impregnating packaging materials for use with edible (such as food wrap, food packaging, beverage packaging, or packets inserted amongst the food) or non-edible applications (such as leather goods, shoes, paint and so forth), particularly where the packaged products tend to accumulate mold.
  • edible such as food wrap, food packaging, beverage packaging, or packets inserted amongst the food
  • non-edible applications such as leather goods, shoes, paint and so forth
  • non-food uses include non-food uses.
  • preparations may be applied to: bath towels to prevent mildew/mold smell; wigs or fake hair; head gear such as hats, goggles, ear warmers and the like; carpets, carpet under-lays, basement molding, flooring, window seals, calking, paints in order to inhibit mold growing on surfaces such as those in basements; outdoor decking, stadium seating, signage, any outdoor materials susceptible to mold growth; in a coating for preserving documents and books (e.g., comics, manuscripts) and historical artifact preservation; cleaning devices such as Swiff er® cloths, brooms, dry mops, and the like; impregnating into "active" clothing including outdoor wear, Under Armour®, exercise gear, sports gear, anything that can get wet from sweat or weather; kitchen items that are prone to mold and microbial contamination such as cutting boards, wood counters, plastic dish drying racks, vending machines (
  • using a food-safety preparation inhibits visible mold growth at least twice as long as not using the preparation.
  • visible mold inhibition occurs for a time period at least 20% longer than when not using the food-safety preparation.
  • Example 1 Development of a live bacteria concentrate culture containing Pediococci
  • percentage values of components ⁇ e.g., dextrose, Tween 80, proline, serine, threonine, or cysteine) added to or included in a composition refer to an approximate value of percentage, by weight, of that component relative to the volume of the composition, which is expressed herein as "w/v.”
  • values of components added into a composition referring to a volume of a component ⁇ e.g., water or broth) relative to the volume of the composition are herein expressed as "v/v.”
  • supplemental amino acids were added to the pasteurized broth mixture to make an amino-acid-enriched broth mixture with about 0.1% threonine (w/v), about 0.05% serine (w/v), about 0.05% proline (w/v), and about 0.1%) cysteine (w/v).
  • the amino-acid-enriched broth mixture was further cooled to about 52° C and then swirled to create a homogenous broth suspension. The homogenous broth suspension was then inoculated with Pediococcus acidilactici and P.
  • pentosaceus at the level of about 1 x 10 7 colony forming units of bacteria per gram of culture medium mixture (hereinafter referred to as "cfu/g") to produce an inoculated chicken broth.
  • the inoculated chicken broth was placed in a sealed container and then incubated at about 35° C for about 48 hours (hereinafter referred to as "h") to allow it to ferment to produce a fermented growth culture. After about 48 h, the fermented growth culture was removed from the 35° C environment. Approximately 400 mL of fermented growth culture were centrifuged at about 2,600 g for about 15 minutes (hereinafter referred to as "min").
  • the supernatant was discarded from this, and the recovered bacterial cells were resuspended in about 40 mL of Butterfield's Phosphate Buffered Diluent ("BPBD") at about 1 x 10 10 cfu/g to create a live- bacteria concentrate culture.
  • BPBD Butterfield's Phosphate Buffered Diluent
  • the resuspended bacterial cells were stored in refrigeration (where the temperature was approximately 4° C).
  • Example 2 Development of a live Pediococci culture grown in Trypto Soy Broth with glycerol and in the presence of additional air exposure.
  • development of a live Pediococci culture grown in Trypto-Soy Broth with glycerol and incubated in the presence of additional air exposure is shown, according to one embodiment of the present teachings.
  • glycerol is added as a substrate to an incubation growth media containing Pediococci to induce the production of glycerol 3 -phosphate.
  • Pediococci grown at low temperatures and in the presence of excess air or oxygen produces glycerol 3 -phosphate oxidase that then converts glycerol 3 -phosphate to dihydroxy acetone phosphate and hydrogen peroxide creating a potent antimicrobial mixture.
  • a bacterial incubation broth was made up according the formula found in Table 1.
  • *Trypto Soy Broth consists of about 56.7% casein digest peptone, about 10% papain digest of soy bean meal; about 16.7% sodium chloride; about dextrose; and about 8.3% dipotassium phosphate.
  • **Tween 80 is also referred to as polysorbate 80.
  • the mixture was heated to about 121° C at about 15 psi for about 15 min to create an incubation broth.
  • the incubation broth was cooled to about 35° C and then inoculated with about 1 x 10 7 cfu/g of Pediococcus acidilactici and P. pentosaceus to create an inoculated broth.
  • About 500 mL aliquots of the inoculated broth were poured into 22.9 cm x 22.9 cm TeflonTM coated pans. This resulted in the inoculated broth being about 1 cm deep.
  • pans containing the inoculated broth were loosely covered with aluminum foil and incubated at about 35° C for about 24 h.
  • the pans containing inoculated broth were then transferred to an ambient temperature environment (approximately 22° C) and incubated for about an additional 96 h to create the culture of live Pediococci grown in Trypto Soy Broth with glycerol and in the presence of additional air exposure. This culture was then placed in sealed glass containers that were stored at approximately 4° C until further use.
  • Example 3 Development of a live bacteria grown in chicken broth with supplemental amino acids and in the presence of additional air exposure
  • a bacterial incubation broth was made up according the formula found in Table 2.
  • Tween 80 is also referred to as polysorbate 80.
  • a chicken broth mixture with about 1% dextrose (w/v), about 1% glycerol (w/v), and about 0.1% Tween 80 (w/v) was prepared, and this mixture was then pasteurized to about 88° C to create a pasteurized broth mixture.
  • supplemental amino acids were added to make an amino-acid-enriched broth mixture with about 0.1% threonine (w/v), about 0.05% serine (w/v), about 0.05% proline (w/v), and about 1.0% cysteine (w/v).
  • the amino-acid-enriched broth mixture was further cooled to about 52° C and then swirled to create a homogenous broth suspension.
  • the homogenous broth suspension was then inoculated with Pediococcus acidilactici and P. pentosaceus at the level of about 1 x 10 7 cfu/g. Approximately 500 mL aliquots of the inoculated broth were poured into 22.9 cm x 22.9 cm TeflonTM coated pans. This resulted in the inoculated broth being about 1 cm deep.
  • the pans containing the inoculated broth were loosely covered with aluminum foil and incubated at about 35° C for about 24 h. Incubation was then continued at about 22° C for about 96 h to create a live bacteria grown in chicken broth with supplemental amino acids and in the presence of additional air exposure. The live bacteria culture grown in chicken broth with supplemental amino acids and in the presence of additional air exposure was then placed in sealed glass containers that were stored at about 4° C until further use.
  • Example 4 Comparison of live bacteria cultures
  • Example 1 live bacteria concentrate
  • Example 2 culture of live bacteria grown in Trypto Soy Broth with glycerol and in the presence of additional air exposure
  • Example 3 culture of live bacteria grown in chicken broth with glycerol and supplemental amino acids and in the presence of additional air exposure. Table 3 summarizes the attributes and differences between these culture sources.
  • Example 5 Comparison of live bacteria cultures derived from various formulas and processes for anti-mold benefits
  • live Pediococci cultures grown using different formulas and processes are evaluated for their effects on mold inhibition.
  • the sources of the live Pediococci cultures were as described in Example 1 (live bacteria concentrate), Example 2 (culture of live bacteria grown in Trypto Soy Broth with glycerol and in the presence of additional air exposure), and Example 3 (culture of live bacteria grown in chicken broth with glycerol and supplemental amino acids and in the presence of additional air exposure).
  • Mold organisms used in this experiment were Aspergillis spp., Aspergillis niger, Cladosporum spp. isolated from dog kibbles, Rhizopium stolonifera, and Penicillum rodquerfortii .
  • One of four approximate moisture levels (5 mL, 10 mL, 16 mL, or 23 mL) was added to about 100 g of kibble to create kibbles susceptible to mold growth.
  • the approximate water activity (hereinafter "Aw") corresponding to each of the various moisture levels of treated high moisture kibbles was 0.64 Aw for 5 mL/100 g kibble, 0.77 Aw for 10 mL/100 g kibble, 0.88 Aw for 16 mL/100 g kibble, and 0.91 Aw for 23 mL/100 g kibble.
  • the moisture added to the kibbles was obtained from: 1) about 1 mL of a water solution containing individual mold organisms (about 1,000 fragments/mL of water), 2) about 1 mL of one of the three live Pediococci cultures; and 3) the balance of moisture coming from the addition of water to kibbles.
  • a fragment sometimes referred to as a propagule, is a component of a mold that is sufficient to start the growth of a mold on an edible or inedible surface.
  • a corresponding water-based control for each moisture level was made by substituting water for the added live Pediococci cultures. The moisture sources and kibbles were combined into Ziploc® tubs or bags that were sealed, shaken to insure proper mixing, and then stored at about 22° C. Tubs and bags were inspected daily to determine if mold growth had occurred. A hand lens was used to confirm suspect visual eye observations of mold growth on the samples.
  • Results are noted in Tables 4, 5, 6, and 7 and indicate that all 3 sources of live Pediococci culture inhibit mold growth (Tables 4, 5, 6, and 7). Mold growth for the live bacteria grown in chicken broth with supplemental amino acids and in the presence of additional air exposure, and for the live Pediococci grown in Trypto Soy Broth with glycerol and in the presence of additional air exposure, was only evident on the higher amounts of added moisture (Table 6 - 16 mL culture/100 g kibble and Table 7 - 23 mL culture/100 g kibble). Further, live Pediococci bacteria grown in the presence of additional air exposure resulted in greater mold inhibition properties than those grown in the absence of additional air exposure (Table 6). Greatest mold inhibition occurs with live bacteria cultures grown in chicken broth with supplemental amino acids and in the presence of additional air exposure (Table 6). Cladosporum spp. and Rhizopium stolonifera were the mold species most sensitive to inhibition (Table 7).
  • Example 6 Comparison of live bacteria cultures derived from various formulas and processes for anti-yeast benefits
  • Example 1 culture of live bacteria concentrate
  • Example 2 culture of live Pediococci grown in Trypto Soy Broth with glycerol and in the presence of additional air exposure
  • Example 3 culture of live bacteria grown in chicken broth with supplemental amino acids and in the presence of additional air exposure.
  • yeast organisms used in this experiment were Zygosaccharomyces bailii, Pichia anomala, Saccharomyces cerevisiae, and wild-type (unidentified) yeast spp. isolated from green juice.
  • Yeast organisms were grown on a plate containing potato dextrose agar at about 22° C for approximately 5 days.
  • a loop about 10 ⁇ . from each plate was transferred into about 100 ⁇ _, BPBD to create a solution containing about 1,000,000 yeast organisms per gram.
  • yeast spoilage sources each containing one of the following yeasts: Zygosaccharomyces bailii; Pichia anomala; Saccharomyces cerevisiae; and wild-type yeast spp. isolated from green juice.
  • a microtiter well experiment was conducted to evaluate the efficacy of live Pediococci bacteria added into chicken broth and yeast spoilage microorganisms.
  • About 100 ⁇ of chicken broth was added into microtiter wells 2 through 12.
  • about 100 ⁇ of the live- bacteria concentrate described in Example 1 was added to wells 1 and 2.
  • approximately 1 :2 serial dilutions were made for every subsequent well. Unless otherwise indicated, each serial dilution referred to in these Examples refers to a dilution of a volume into another volume, or v/v.
  • wells 1-12 had the following approximate serial dilutions (v/v) of inoculum: 1 : 1, 1 :2, 1 :4, 1 :8, 1 : 16, 1 :32, 1 :64, 1 : 128, 1 :256, 1 :512, 1 : 1024, and 1 :2048.
  • about 100 ⁇ . of one of the four single yeast spoilage microorganism sources were added to wells 1-12.
  • Four control wells were made that contained about 100 ⁇ . chicken broth and about 100 ⁇ . of each source of yeast spoilage microorganism.
  • Microtiter plates were then incubated at about 22° C for about 4 days. Upon completion of incubation, about 100 ⁇ .
  • iodonitrotetrazolium color reagent were added to each well.
  • Microtiter plates were then placed into an environment having a temperature of approximately 35° C for about 2-4 hours to enable a color change to occur.
  • a yellow or amber color indicated no growth
  • a pink color indicated inhibition of growth
  • a red color indicated definite growth of the yeast organism.
  • Results are shown in Table 8, 9, 10, and 11, and they indicate that all sources of live Pediococci bacteria effectively inhibited growth of the various yeast sources. Results further indicate that adding air and growing at lower temperatures improved the ability of the live Pediococci bacteria sources to inhibit the growth of various yeast sources. Alternatively, the live-bacteria concentrate grown without additional air and at elevated temperatures was less effective than other live Pediococci bacteria sources. Pichia anomala was the most sensitive to the impact of a source of live Pediococci bacteria.
  • Example 7 Impact of live bacteria cultures derived from various formulas and processes on listeria growth
  • Results are shown in Table 8, 9, 10, and 11, and they indicate that all sources of live Pediococci bacteria effectively inhibited growth of the various yeast sources. Results further indicate that adding air and growing at lower temperatures improved the ability of the live Pediococci bacteria sources to inhibit the growth of various yeast sources. Alternatively, the live-bacteria concentrate grown without additional air and at elevated temperatures was less effective than other live Pediococci bacteria sources. Pichia anomala was the most sensitive to the impact of a source of live Pediococci bacteria.
  • Example 1 live bacteria concentrate
  • Example 2 live Pediococci culture grown in Trypto Soy Broth with glycerol and in the presence of additional air exposure
  • Example 3 live bacteria culture grown in chicken broth with glycerol and supplemental amino acids and in the presence of additional air exposure.
  • the Listeria organisms used in this experiment were sourced from Listeria innocuous ATCC 33090. To avoid laboratory health risks, Listeria innocua served as a surrogate organism indicative of the effects of Listeria monocytogenes. Listeria innocua was added into the Trypto Soy Broth (TSB; as described in Example 2) that resulted in about 1,000,000 cfu/g to create Listeria innocua TSB.
  • TTB Trypto Soy Broth
  • a microtiter well experiment was conducted to evaluate the efficacy of live Pediococci bacteria added into chicken broth and against Listeria innocua. About 100 ⁇ of chicken broth were added into microtiter wells 2 through 15. Next, about 100 ⁇ of one of the live Pediococci cultures were added to wells 1 and 2. Starting with well 2, approximately 1 :2 serial dilutions were made for every subsequent well.
  • wells 1-15 had the following approximate serial dilutions (v/v) of inoculum: 1 : 1, 1 :2, 1 :4, 1 :8, 1 : 16, 1 :32, 1 :64, 1 : 128, 1 :256, 1 :512, 1 : 1024, 1 :2048, 1 :4096, 1 :8192, and 1 : 16384. Then about 20 ⁇ of the Listeria innocua TSB were then added to wells 1-15. This process was repeated for each source of the live Pediococci cultures. A control well was made that contained about 100 ⁇ chicken broth and about 20 ⁇ . of Listeria innocua TSB.
  • Microtiter plates were then incubated at about 22° C for about 3 days. Upon completion of incubation, about 100 ⁇ . of iodonitrotetrazolium reagent were added to each well. Microtiter plates were then placed into an environment having a temperature of approximately 35° C for about 2-4 hours to enable the color change to occur. In each microtiter well, a yellow or amber color indicated no growth, a pink color indicated inhibition of growth, and a red color indicated definite growth of the Listeria organism.
  • Results are shown in Table 12. Results indicated that all live bacteria cultures killed and inhibited the growth of Listeria innocua. Results further indicated that live Pediococci grown in chicken broth with supplemental amino acids and in the presence of additional air exposure had the greatest impact in killing and inhibiting the growth of Listeria innocua. Live Pediococci grown in Trypto Soy Broth with glycerol and in the presence of additional air exposure were much more effective than live bacteria concentrate in killing and inhibiting the growth of Listeria innocua.
  • Example 8 Impact of live bacteria cultures derived from various formulas and processes on Salmonella surrogates growth
  • Pediococci cultures grown using different formulas and different processes evaluated for their effects on growth of Salmonella surrogates.
  • Example 1 live bacteria concentrate
  • Example 2 culture of live Pediococci grown in Trypto Soy Broth with glycerol and in the presence of additional air exposure
  • Example 3 culture of live bacteria grown in chicken broth with glycerol and supplemental amino acids and in the presence of additional air exposure.
  • E. coli strains ATCC BAA 1427, 1428, 1429, 1430 and 1431 served as the Salmonella surrogate organisms for this experiment.
  • E. coli were added into a solution of BPBD that resulted in about 100,000 cfu/g to create the Salmonella surrogates source.
  • a microtiter well experiment was conducted to evaluate the efficacy of live Pediococci bacteria added into chicken broth and against the Salmonella surrogates. About 100 ⁇ chicken broth were added into microtiter wells 2-15. Next, about 100 ⁇ of one of the live Pediococci cultures were added to wells 1 and 2. Starting with well 2, approximately 1 :2 serial dilutions were made for every subsequent well.
  • wells 1-15 had the following approximate serial dilutions (v/v) of inoculum: 1 : 1, 1 :2, 1 :4, 1 :8, 1 : 16, 1 :32, 1 :64, 1 : 128, 1 :256, 1 :512, 1 : 1024, 1 :2048, 1 :4096, 1 :8192, and 1 : 16384. Then about 100 ⁇ of the Salmonella surrogates were added to wells 1-15. This process was repeated for each source of the live Pediococci cultures. A control well was made that contained about 100 ⁇ _, chicken broth and about 100 ⁇ _, of Salmonella surrogates.
  • Microtiter plates were then incubated at about 22° C for about 2 days. Upon completion of incubation, about 100 ⁇ _, of iodonitrotetrazolium reagent were added to each well. Microtiter plates were then placed into an environment having a temperature of approximately 35° C for a duration that ranges from about 2 h to about 4 h to enable the color change to occur. In each microtiter well, a yellow or amber color indicated no growth, a pink color indicated inhibition of growth, and a red color indicated definite growth of the Listeria organism.
  • Results are shown in Table 13 and indicated that all live bacteria cultures killed Salmonella surrogates. Results further indicated that both live Pediococci grown in chicken broth with glycerol and supplemental amino acids and in the presence of additional air exposure, and live Pediococci grown in Trypto Soy Broth with glycerol and in the presence of additional air exposure, were similar in their effects to each other and had a greater impact in killing Salmonella surrogates than live-bacteria concentrate.
  • Example 9 Inhibition of indigenous mold associated with pet food kibbles by a live-bacteria culture
  • the source of the live Pediococci culture was prepared as described in Example 3.
  • Mold organisms assessed were those indigenous (or naturally associated with) to the dog food kibbles used in this experiment.
  • the first kibble source consisted of dry dog food kibble coated only with fat. Under typical commercial circumstances dog food kibble is coated with fat and palatant. The reason this kibble was used in this particular experiment was to remove any mold inhibitors that might be added to the kibble based on the addition of palatant.
  • the second kibble source was lams® Mini Chunks, a premium pet food marketed for a wide range of adult dogs.
  • the third kibble source was Purina® Dog Chow Light and Healthy, a reduced fat kibble to minimize impact of adding a externally to the kibble and interfering with laboratory assays to determine mold growth.
  • Additional moisture (about 12 g water / 50 g of kibble) was added to the kibbles to enhance their susceptibility to mold growth.
  • the water activity corresponding to the enhanced moisture levels of each of the treated high moisture kibbles was about 0.9200 Aw for the dry dog food with no added fat, about 0.9100 Aw for the lams Mini Chunks, and about 0.8858 Aw for the reduced fat kibble.
  • the moisture added to 50 g kibbles was obtained from: 1) about 0.5 mL of the live Pediococci culture (about log 8.8 cfu/mL); and 2) about 11.5 mL of moisture coming from the addition of water to kibbles.
  • a corresponding water-based control using the no added fat kibbles was made by substituting water for the added live Pediococci cultures.
  • the moisture sources and kibbles were combined into Ziploc® tubs that were sealed, shook for about 30 sec to insure proper mixing, and then stored at about 22° C. Tubs were inspected daily to determine if mold growth had occurred. Results are noted in Table 14 and indicate that mold growth was inhibited on all three sources of kibbles. Of the three sources of kibbles, mold growth was delayed the most in the commercial low fat dog kibbles.
  • Examples 10, 11, and 12 disclose the use of non-live Pediococci cultures that have been further concentrated and their effects on inhibiting mold and yeast growth in a green juice or dog food kibbles that have been considerably hydrated.
  • Example 10 Development of a live pediococci culture grown in Trypto Soy Broth
  • a bacterial incubation broth was made up according to the formula found in Table 15.
  • *Trypto Soy Broth consists of about 56.7% casein digest peptone, about 10% papain digest of soy bean meal; about 16.7% sodium chloride; about dextrose; and about 8.3% dipotassium phosphate. **Tween 80 is also referred to as polysorbate 80.
  • the mixture was heated to about 121° C at about 15 psi for about 15 min to create an incubation broth.
  • the incubation broth was cooled to about 35° C and then inoculated with about 1 x 10 7 cfu/g of a blend of Pediococcus pentosaceus and P. acidilactici to create an inoculated broth.
  • the inoculated broth was incubated at about 35° C for about 48 h to create a live Pediococci culture.
  • the live Pediococci culture was concentrated by about 4X ⁇ i.e., about 400 mL concentrated to about 100 mL) by heating to about 80° C for about 10 min, which also killed the bacteria, to create a 4X concentrated non-live culture.
  • the 4X concentrated non-live culture was then placed in sealed glass containers that were stored at about 4° C until further use.
  • Example 1 1 Impact of the 4X concentrated non-live culture on inhibiting mold and yeast growth in a green juice
  • a non-live culture is evaluated for its effects on mold and yeast growth inhibition and also describes the source of 4X concentrated non-live cultures. Mold organisms used in this experiment were Aspergillis spp., Aspergillis niger,
  • Yeast organisms used in this experiment were Rhodotorula spp., Pichia anomala,Pichia membranefaciens, Zygosaccharomyces bailii and Saccharomyces cereviseae.
  • Green juice was made by grinding up and filtering out (Hamilton Beach) the juice from about 500 g of kale, about 500 g of spinach, and about 5 g of ginger.
  • Microtissue wells were used to evaluate mold growth in the presence or absence of the 4X concentrated non-live culture. About 3 mL of the previously described green juice were placed into each well. Next, about 100 ⁇ _, of the 4X concentrated non-live culture was rapidly mixed into each well containing green juice. After adding the 4X concentrated non- live culture, about 1 ⁇ , of each mold and yeast organism previously described was individually streaked into a well containing the green juice and 4X concentrated non-live culture. Corresponding control wells were made that contained no 4X concentrated non-live culture. Then, the microtissue wells were covered and incubated at about 22° C. Wells were observed daily for mold or yeast growth.
  • Example 12 Impact of 4X concentrated non-live culture on inhibiting mold and yeast growth in high-moisture dog food kibbles
  • the effects of a non-live concentrate on mold and yeast inhibition in high- moisture dog food kibbles are shown.
  • the source of the 4X concentrated non-live culture was prepared as described in Example 10.
  • Mold organisms used in this experiment were Aspergillis niger, Cladosporum spp. isolated from dog kibbles, and Penicillum rodquerfortii .
  • Yeast organisms used in this experiment were Rhodotorula spp.
  • Microtissue wells were used to evaluate mold growth in the presence or absence of the 4X concentrated non-live culture.
  • dog food kibbles that contained less than about 10% moisture two hydrated kibble mixtures were prepared. The first hydrated kibble mixture, containing about 33.33% ground kibbles, about 33.33%) of a sterile bacteriological agar growth medium, and about 33.33% water, was mixed together to prepare a hydrated mixture with an approximate Aw of 0.99.
  • a second hydrated kibble mixture containing about
  • Results are shown in Tables 18 and 19 and indicate that control samples grew mold or yeast after 2 or 3 days. Results further indicate that most mold species did not grow on hydrated kibble at about 0.99 Aw in the presence of the 4X concentrated non-live cultures for at least about 6 days. Mold growth was delayed until at least about 13 days when hydrated kibbles at approximately 0.88 Aw were treated with 4X concentrated non-live cultures.
  • Examples 13-16 Improved efficacy of live bacteria fermentate grown with glycerol and incubated at a relatively lower temperature in the presence of additional air exposure is shown by Examples 13-16.
  • two different sources of live bacteria fermentate were developed then evaluated in their efficacies against yeast spoilage microorganisms.
  • adding glycerol to the growth medium and incubating the growth medium at lower temperatures in the presence of additional air exposure improved the ability of live bacteria fermentate to inhibit yeast spoilage
  • Example 13 Development of a live bacteria IPX concentrate fermentate grown in chicken broth with supplemental amino acids for 48 h
  • Tween 80 is also referred to as polysorbate 80.
  • a chicken broth mixture with about 2% dextrose (w/v) and about 0.1% Tween 80 (w/v) was prepared and then pasteurized to about 88° C to create a pasteurized broth mixture.
  • the pasteurized broth mixture When the pasteurized broth mixture had cooled to about 74° C, it was used to prepare an amino-acid-enriched broth mixture with about 0.1% threonine (w/v), about 0.05% serine (w/v), about 0.05% proline (w/v), and about 1.0% cysteine (w/v).
  • the amino-acid-enriched broth mixture was further cooled to about 38° C and then swirled to create a homogenous broth suspension.
  • the homogenous broth suspension was then inoculated with P. acidilactici and P. pentosaceus at the level of about 1 x 10 7 cfu/g.
  • the inoculated broth was incubated at about 35° C for about 48 h to create an incubated broth.
  • the bacterial cells were recovered from the incubated broth by centrifuging at about 2,600 g for about 15 min. Recovered bacterial cells from about 10 volumes of incubated broth were then resuspended in about 1 volume of BPBD to create a live-bacteria 10X concentrate fermentate.
  • Example 14 Inhibition of various yeast species by live-bacteria IPX concentrate fermentate grown in chicken broth and supplemental amino acids for 48 h
  • yeast organisms used in this experiment were Zygosaccharomyces bailii, Pichia anomala, Saccharomyces cerevisiae, and wild-type (unidentified) yeast spp. isolated from green juice.
  • Yeast organisms were grown on a plate containing potato dextrose agar at about 22° C for about 5 days.
  • a loop (about 10 ⁇ ) from each plate was transferred into about 100 mL BPBD to create a solution containing about 1,000,000 yeast organisms per approximately 1 mL of BPBD.
  • yeast spoilage microorganisms sources each containing one of the following yeasts: Zygosaccharomyces bailii, Pichia anomala, Saccharomyces cerevisiae, and wild-type yeast spp. isolated from green juice.
  • a microtiter well experiment was conducted to evaluate the efficacy of the live bacteria 10X concentrate fermentate on inhibiting the growth of the yeast spoilage microorganism sources.
  • microtiter plates were then incubated at about 22° C for about 4 days. Upon completion of incubation, about 100 ⁇ ⁇ of iodonitrotetrazolium color reagent was added to each well. Microtiter plates were then placed into an environment having a temperature of about approximately 35° C environment for a duration that ranges from about 2 h to about 4 h to enable the color change to occur. In each microtiter well a yellow or amber color indicated no growth, a pink color indicated inhibition of growth, and a red color indicated definite growth of the yeast organism.
  • Results are shown in Table 21 and indicated that all sources of yeast were inhibited by the live-bacteria 10X concentrate. Results further indicate that the greatest inhibition occurred with the S. cerevisiae yeast source whereas Z. bailii and wild-type yeast from green juice were more resistant to inhibition.
  • Microtiter experiment determines a minimal inhibitory concentration (MIC) for each organism evaluated.
  • Example 15 Development of a live-bacteria fermentate grown in chicken broth with supplemental amino acids and glycerol and in the presence of additional air exposure at low temperatures
  • a bacterial incubation broth was made up according the formula found in Table 22.
  • Tween 80 is also referred to as polysorbate 80.
  • supplemental amino acids were added to prepare an amino-acid-enriched broth mixture with about 0.1% threonine (w/v), about 0.05% serine (w/v), about 0.05% proline (w/v), and about 1.0% cysteine (w/v) .
  • the amino-acid-enriched broth mixture was further cooled to about 38° C and then swirled to create a homogenous broth suspension.
  • the homogenous broth suspension was then inoculated with Pediococcus acidilactici and P. pentosaceus at the level of about 1 x 10 7 cfu/g.
  • Example 16 Growth inhibition of various yeast species by live-bacteria fermentate grown in chicken broth with amino acids and glycerol and in the presence of additional air exposure at low temperatures
  • yeast organisms used in this experiment were Zygosaccharomyces bailii, Pichia anomala, Saccharomyces cerevisiae, and wild-type (unidentified) yeast spp. isolated from green juice.
  • Yeast organisms were grown on a plate containing potato dextrose agar at about 22° C for about 5 days.
  • a loop (about 10 ⁇ ) from each plate was transferred into about 100 mL BPBD diluent to create a solution containing about 1,000,000 yeast organisms per about 1 mL of BPBD.
  • yeast spoilage microorganism sources each containing one of the following yeasts: Zygosaccharomyces bailii, Pichia anomala, Saccharomyces cerevisiae, and wild-type yeast spp. isolated from green juice.
  • a microtiter well experiment was conducted to evaluate the efficacy of the live bacteria fermentate to inhibit the growth of the yeast spoilage microorganism sources.
  • About 100 ⁇ chicken broth were added into microtiter wells 2-12.
  • about 100 ⁇ of the live-bacteria fermentate described in Example 16 were added to wells 1 and 2.
  • approximately 1 :2 serial dilutions (v/v) were made for every subsequent well.
  • wells 1-12 had the following approximate serial dilutions (v/v) of inoculum: 1 : 1, 1 :2, 1 :4, 1 :8, 1 : 16, 1 :32, 1 :64, 1 : 128, 1 :256, 1 :512, 1 : 1024, and 1 :2048.
  • about 100 ⁇ . of one of the four single microorganism sources were added to wells 1-12.
  • Four control wells were made that contained about 100 ⁇ chicken broth and about 100 ⁇ of each source of yeast spoilage microorganism. Microtiter plates were then incubated at about 22° C for about five days.
  • iodonitrotetrazolium color reagent Upon completion of incubation, about 100 ⁇ of iodonitrotetrazolium color reagent were added to each well. Microtiter plates were then placed into an environment having a temperature of approximately 35° C for about two to four hours to enable the color change to occur. In each microtiter well a yellow or amber color indicated no growth, a pink color indicated inhibition of growth, and a red color indicated definite growth of the yeast organism.
  • Results are shown in Table 23. Results indicated that all sources of yeast were inhibited by the live-bacteria fermentate grown in chicken broth with amino acids and glycerol and in the presence of additional air exposure at low temperatures. Results further indicate that the greatest inhibition occurred with the S. cerevisiae and wild-type yeast sources whereas Z bailii was more resistant to inhibition.
  • Example 16 When results in this Example 16 are compared to those observed in Example 14, it is evident that adding glycerol, increasing air exposure during incubation, and reducing incubation temperature, improve the ability of the resulting live bacteria fermentate to inhibit yeast growth.
  • Microtiter experiment determines a minimal inhibitory concentration (MIC) for each organism evaluated.
  • Examples 17 and 18 demonstrate the impact of a live bacteria fermentate grown with glycerol and incubated in the presence of additional air exposure at low temperatures on inhibiting mold growth in high moisture pet food.
  • Example 17 Development of a live bacteria fermentate
  • development of live bacteria fermentate grown in chicken broth with amino acids and glycerol and in the presence of additional air exposure at low temperatures is shown.
  • a bacterial incubation broth was made up according the formula found in Table 24.
  • Tween 80 is also referred to as polysorbate 80.
  • an amino-acid-enriched broth mixture with about 0.1% threonine (w/v), about 0.05% serine (w/v), about 0.05% proline (w/v), and about 1.0% cysteine (w/v) was prepared.
  • the amino-acid-enriched broth mixture was further cooled to about 38° C and then swirled to create a homogenous broth suspension.
  • the homogenous broth suspension was then inoculated with Pediococcus acidilactici and P. pentosaceus at the level of about 1 x 10 7 cfu/g to form an inoculated broth.
  • the inoculated broth was then incubated at about 35° C for about 24 h. Incubation was then continued at about 22° C for about 96 h with agitation by continuously stirring the inoculated broth mixture with a magnetic stir bar to create a live bacteria fermentate grown in chicken broth, amino acids, and glycerol, and incubated with agitation ⁇ i.e., with additional air exposure) at low temperature.
  • the live bacteria grown in chicken broth, amino acids, and glycerol, and incubated with agitation at low temperatures was then placed in sealed brown polypropylene screw capped bottles that were stored at about 4° C until further use.
  • Example 18 Impact of live bacteria fermentate on inhibiting mold growth in high-moisture pet food This shows the impact of a live bacteria fermentate on mold growth in a high moisture pet food product.
  • the source of the live bacteria fermentate was made in the manner described in Example 17.
  • Live bacteria fermentate from Example 17 was applied at the rate of about 1 mL live bacteria fermentate per about 100 g of two different high-moisture pet food products, "A" and “B.”
  • the high moisture pet food product “A” contained pieces sized about 2 cm by about 1 cm and weighed on average about 1.5 g.
  • the high moisture pet food product “A” further contained no more than about 64% moisture with an Aw of about 0.9949 and contained at least about 13% protein and at least about 11% fat.
  • the high moisture pet food product “B” contained pieces sized about 2 cm by about 3.5 cm and weighed about 2 g.
  • the high moisture pet food product “B” further contained no more than about 66% moisture with an Aw of about 0.9900 and contained at least about 19% protein and at least about 6.5% fat.
  • Results are reported in Table 25. Results indicated that the addition of live bacteria fermentate resulted in at least 25 days of extended shelf-life in products stored at about 4° C.
  • Example 19 Live bacteria fermentate and additional Tween 80 increased kill of Salmonella surrogates in fat This example shows the use of live bacteria fermentates with various levels of Tween 80 in killing Salmonella in fat.
  • the sources of live bacteria fermentate used the same formula and incubation process as that used to obtain the live bacteria fermentate in Example 15.
  • the procedure used to evaluate the anti-Salmonella efficacy of the live bacteria fermentate sources grown on various levels of Tween 80 was as follows. Chicken fat was melted at about 45° C and about 100 g of melted fat was placed in Ziploc® tubs. E. coli strains ATCC BAA 1427, 1428, 1429, 1430 and 1431 served as the Salmonella surrogate organisms for this experiment. E. coli were added into a solution of BPBD that resulted in about 1,000,000 cfu/mL of BPBD to create the Salmonella surrogates source.
  • Tween 80 and water was added to the chicken fat, Salmonella surrogates, and live bacteria fermentate.
  • the mixture of Tween 80 and water consisted of adding about 1 mL of mixture with the mixture being either about 100%, about 50%, about 10%), or about 5% Tween 80 with the balance being water (v/v).
  • E. coli levels were determined by the pour plate technique using violet red bile glucose agar plates incubated for 24 h at 35° C. Results in Table 26 are expressed as logio amount of E. coli reduction from control. E. coli levels after about 4 h or about 24 h incubation with live bacteria fermentate obtained from various levels of Tween 80 were subtracted from control samples. Results indicate that the maximum reduction of E.
  • Tween 80 occurred after about 24 h exposure to about 0.5% (w/v) or more Tween 80.
  • Example 20 Creation of samples with various levels of sulfite
  • This example shows the development of a live bacteria fermentate made with glycerol and incubated in the presence of additional air exposure at low temperatures.
  • a bacterial incubation broth was made up according to the following process. First, a chicken broth mixture with about 1% dextrose (w/v), about 0.1% Tween 80 (w/v), and about 1% glycerol (w/v), and the remainder as chicken broth (v/v), was pasteurized to about 88° C to create a pasteurized broth mixture. When the pasteurized broth mixture had cooled to about
  • supplemental amino acids were added to produce an amino-acid-enriched broth mixtures with about 0.1 % threonine (w/v), about 0.05% serine (w/v), about 0.05% proline (w/v), and one of three approximate levels of cysteine (0%, 0.1%, or 0.2%) (w/v).
  • the amino-acid-enriched broth mixtures were further cooled to about 38° C and then swirled to create homogenous broth suspensions.
  • the homogenous broth suspensions were then inoculated with Pediococcus acidilactici and Pediococcus pentosaceus at the level of about 1 x 10 7 cfu/g to create inoculated broth suspensions.
  • the inoculated broth suspensions were incubated at about 35° C for about 24 h without agitation to create initial incubated broths. Incubation of the initial incubated broths was then continued at about 22° C for about 24 h either with or without agitation to create six fully incubated broths.
  • the initial incubated broths were agitated in order to incorporate additional oxygen at the level of about 60% oxygen saturation to about 89% oxygen saturation, (or about 5.8 mg dissolved oxygen per liter of incubated broth to about 7.93 mg dissolved oxygen per liter of incubated broth).
  • Sodium erythorbate was then added into the fully incubated broths at a level that produced about 1.0% sodium erythorbate (w/v), such that the sodium erythorbate would act as an antioxidant to capture free oxygen to create oxygen-level adjusted fermentates.
  • Bacterial cells in the oxygen-level adjusted fermentates were removed by centrifuging at about 2,600 g for about 10 min and then decanting and recovering the supernatant of each sample to create finished fermentates, i.e., food-safety preparations.
  • Table 27 represents the various combinations of formulas and processes used to create the finished fermentate samples represented in Table 28. After being created, the levels of sulfite, sulfide, and sulfate in each sample were determined.
  • mg of sulfide, sulfite, sulfide, ascorbic acid, or sodium erythorbate per dL or L of fermentate is expressed as "mg/dL” or "mg/L,” respectively.
  • Combinations of the finished fermentate applied at various approximate levels (0.1 ml, 1 ml, 1.5 ml, 1.75 ml, and 2.5 ml) per 100 g of food made using 0.1% cysteine (w/v) and incubated about 24 h quiescently followed by about 24 h agitation along with various approximate levels of sodium erythorbate in fermentate (about 0 mg/dL, about 10 mg/dL, about 15 mg/dL, about 30 mg/dL, and about 35 mg/dL) were evaluated for their abilities to inhibit mold according to the following procedure.
  • About 1 mL of the finished fermentate containing either about 0 mg/dL, about 10 mg/dL, about 15 mg/dL, about 30 mg/dL, or about 35 mg/dL of sodium erythorbate in fermentate was added to about 100 g of a high moisture pet food contained within a transparent rigid plastic tub (Ziploc®) or sealed plastic bag.
  • the high moisture pet food contained pieces of about 2 cm by about 1 cm and weighed on average about 1.5 g.
  • the high moisture pet food product further contained no more than about 64% moisture with an Aw of about 0.9949 and contained at least about 13% protein and at least about 11%) fat.
  • a lid was then placed onto the rigid plastic tub to create a sealed container that was stored at ambient (about 22° C) conditions. Each day thereafter, the contents were visually inspected without opening the containers to determine if visual mold growth was detected.
  • This methodological approach serves as a rapid screening assay of mold inhibition on the surface of food. Days until mold inhibition are reported in Table 28.
  • Example 21 Impact of sulfites and sodium erythorbate on mold inhibition.
  • This example shows the impact of sulfite and sodium erythorbate levels in fermentates on mold inhibition.
  • the impact of sulfites and sodium erythorbate from the fermentates produced in Example 20 on mold inhibition is demonstrated using a central composition and response surface statistical analysis.
  • the statistical analysis involved a 2x2 factorial central composite design.
  • Central composite designs enable estimation of the quadratic terms. Further, these designs enable obtaining the desirable design properties for the orthogonal blocking and rotatability (Minitab® 17, 17.3.1, 2016, Minitab® Inc., State College, PA). Results are shown in Figure 2.
  • Figure 2 is a contour plot showing the impact of sulfite and sodium erythorbate levels on the number of days until relatively high-moisture food treated with the food-safety preparations and infected with mold visually shows mold.
  • Figure 2 includes an x-axis 202 showing ⁇ g of sulfite per 100 g of food product and a y-axis 204 showing mg of sodium erythorbate per 100 g of food product.
  • Figure 2 also shows a region 206, which indicates the levels of sulfite and sodium erythorbate that results in visible mold growth on the food produce in less than four days, a region 208, which indicates the levels of sulfite and sodium erythorbate that results in visible mold growth on the food product between 4 days and less than 6 days, a region 210, which indicates the levels of sulfite and sodium erythorbate that results in visible mold growth on the food product between 6 days and less than 8 days, a region 212, which indicates the levels of sulfite and sodium erythorbate that results in visible mold growth on the food product between 8 days and less than 10 days, a region 214, which indicates the levels of sulfite and sodium erythorbate that results in visible mold growth on the food product between 10 days and less than 12 days, and a region 216, which indicates the levels of sulfite and sodium erythorbate that results in visible mold growth on the food product at 12 days
  • Fermentates comprising both sulfite and sodium erythorbate effectively retarded mold growth more than fermentate containing sulfite or sodium erythorbate alone. There was a significant (P ⁇ 0.05) interaction between the concentration of sodium erythorbate and sulfite. This indicates a synergistic effect of sodium erythorbate and sulfite.
  • mold growth was inhibited for about 6 days or more on ambient (about 22° C) stored high moisture meat (Aw of about 0.995) that was heavily contaminated with mold (about 1,000 propagules of fungi per piece of meat).
  • Example 22 Creation of a cell-free fermentate with sodium erythorbate
  • This example shows the development of a cell-free fermentate made with glycerol and sodium erythorbate and incubated for about 48 h.
  • the fermentate was made up according to the formula in Table 29 and the following process.
  • a chicken broth mixture with about 1% dextrose (w/v), about 0.1% Tween 80 (w/v), and about 1% glycerol (w/v) was prepared and then pasteurized to about 88° C to create a pasteurized broth mixture.
  • the pasteurized broth mixture had cooled to about 74° C, this was used to prepare an amino-acid-enriched broth mixture with about 0.1 % threonine (w/v), about 0.05% serine (w/v), about 0.05% proline (w/v), and about 0.1% cysteine (w/v).
  • the amino-acid-enriched broth mixture was further cooled to about 38° C and then swirled to create a homogenous broth suspension.
  • the homogenous broth suspension was then inoculated with Pediococcus acidilactici and P. pentosaceus at the level of about 1 x 10 7 cfu/g.
  • the inoculated broth was incubated at about 35° C for about 24 h without agitation to create an initial incubated broth. Incubation of the initial incubated broth was then continued at about 22° C for about 24 h with agitation (using a magnetic stir bar set at about 160 rpm to about 180 rpm) to create a fully incubated broth.
  • the initial incubated broth was agitated in order to incorporate additional oxygen at the level of between about 60% to about 89% oxygen saturation, or about 5.8 mg dissolved oxygen/L fully incubated broth to about 7.93 mg dissolved oxygen/L fully incubated broth.
  • sodium erythorbate was at the level about 1% (w/v) to act as an antioxidant to capture free oxygen to create an oxygen-level adjusted fermentate.
  • Bacterial cells in the oxygen-level adjusted fermentate were removed by centrifuging at about 2,600 g for about 10 minutes and decanting and recovering the supernatant to create a finished fermentate.
  • Example 23 The effect of a fermentate with mold inhibition properties on Salmonella and
  • the impact of the finished fermentate from Example 22 is shown to have effective antimicrobial activity against Salmonella and Listeria moncytogenes surrogates.
  • a microtiter well experiment was conducted to evaluate the efficacy of live Pediococci bacteria added into chicken broth and against either Salmonella surrogates, E. coli Types 1427, 1428, 1429, 1430 and 1431, or Listeria surrogate, Listeria innocua ATCC 33090.
  • About 100 ⁇ chicken broth were added into microtiter wells 2 through 12.
  • about 100 ⁇ of the finished fermentate from Example 22 were added to wells 1 and 2.
  • 1 :2 serial dilutions (v/v) were made for every subsequent well.
  • wells 1-12 had the following serial dilutions (v/v) of inoculum: 1 : 1, 1 :2, 1 :4, 1 :8, 1 : 16, 1 :32, 1 :64, 1 : 128, 1 :256, 1 :512, 1 : 1024, and 1 :2048. Then about 100 ⁇ of either the Salmonella surrogates or Listeria innocua were added to wells 1-12. A control well was made that contained about 100 ⁇ . chicken broth and about 100 iLoi Salmonella surrogates. Microtiter plates were then incubated at about 22° C for about two days.
  • iodonitrotetrazolium reagent Upon completion of incubation, about 100 ⁇ L of iodonitrotetrazolium reagent were added to each well. Microtiter plates were then placed into an approximately 35° C environment for two to four hours to enable the color change to occur. In each microtiter well a yellow or amber color indicated no growth, a pink color indicated inhibition of growth and a red color indicated definite growth of the pathogenic organisms, Salmonella or Listeria surrogates.
  • Table 30 shows the microtiter analysis killing concentration of the fermentate obtained from Example 22 compared to Salmonellaand Listeria surrogates.
  • Results indicate growth inhibition of Salmonella and Listeria surrogates up to at least a 1 :8 (v/v) dilution of the fermentate obtained from Example 22.
  • Example 24 Creation of a fermentate made with air incorporation This example shows development of a fermentate made with glycerol and incubated with additional air exposure at low temperatures.
  • a bacterial incubation broth was made up according to the formula represented in Table 31 and according to the following process. First, a chicken broth mixture with about 1% dextrose (w/v), about 0.1% Tween 80 (w/v), and about 1% glycerol (w/v), was prepared and then pasteurized to about 88° C to create a pasteurized broth mixture.
  • the amino-acid-enriched broth mixture was further cooled to about 38° C and then swirled to create a homogenous broth suspension.
  • the homogenous broth suspension was then inoculated with Pediococcus acidilactici and P. pentosaceus at the level of about 1 x 10 7 cfu/g.
  • the inoculated broth was incubated at about 35° C for about 24 h without agitation to create an initial incubated broth.
  • Incubation of the initial incubated broth was then continued at about 22° C for about 24 h either with agitation using a magnetic stir bar rotating at about 160 rpm to about 180 rpm in a beaker to create a fully incubated broth that had about 60% oxygen saturation to about 89% oxygen saturation ⁇ i.e., about 5.8 mg dissolved oxygen/L fully incubated broth to about 7.93 mg dissolved oxygen/L fully incubated broth).
  • Bacterial cells in the fully incubated broth were removed by centrifuging at about 2,600 g for about 10 min and decanting and recovering the supernatant to create a fermentate made with air incorporation.
  • Example 25 Evaluation of a fermentate and ascorbic acid on mold inhibition
  • This example shows the impact, on mold inhibition, of various levels of a fermentate and ascorbic acid added to high moisture pet food.
  • the fermentate from Example 24 and ascorbic acid at various levels were added to about 100 g of high moisture pet food product to evaluate the impact on mold inhibition.
  • the high moisture pet food contained pieces of about 2 cm by about 1 cm and weighed on average 1.5 g.
  • the high moisture pet food product further contained no more than about 64% moisture with an Aw of about 0.9949 and contained at least about 13% protein and at least about 11 % fat.
  • About 100 g of high moisture pet food was placed into transparent Ziploc tubs.
  • various levels of ascorbic acid and the fermentate were placed onto the high moisture pet food and stored at ambient (about 22° C) conditions. Table 32 also indicates the day on which visual mold was observed on the high moisture pet food.
  • Figure 3 is a contour plot graph showing the impact different amounts of fermentate and ascorbic acid in food-safety preparation on days until visible mold growth on food infected with mold and treated with the food-safety preparations.
  • Figure 3 includes an x-axis 302, showing ml of food-safety preparation in the form of a fermentate per 100 g of food, and a y-axis 304, showing mg of ascorbic acid in the food-safety preparation per 100 g of food.
  • Figure 3 also shows a region 306, which shows the amounts of fermentate and amounts of ascorbic acid that results in visible mold growth on food in less 4 days, regions 308a and
  • region 308b which show the amounts of fermentate and amounts of ascorbic acid that results in visible mold growth on food between 4 days and less than 5 days
  • region 310a and 310b which show the amounts of fermentate and amounts of ascorbic acid that results in visible mold growth on food between 5 days and less than 6 days
  • a region 312 which shows the amounts of fermentate and amounts of ascorbic acid that results in visible mold growth on food between 6 days and less than 7 days
  • region 314a and 314b which show the amounts of fermentate and amounts of ascorbic acid that results in visible mold growth on food at 7 days
  • a region 316 which shows the amounts of fermentate and amounts of ascorbic acid that results in visible mold growth on food at 8 days
  • a region 318 which shows the amounts of fermentate and amounts of ascorbic acid that results in visible mold growth on food in 8-9 days
  • a region 320 which shows the amounts of fermentate and amounts of ascorbic acid that results in visible mold growth on food in greater than 9 days.
  • Results indicate a significant (P ⁇ 0.05) effect of ascorbic acid on lengthening the time until the product molded. Further, the fermentate in this application resulted in a non-significant impact on time to mold. The fermentate demonstrated moderate efficacy when present at up to about 1.3 ml per 100 g of product with less than about 8 mg per 100 g of ascorbic acid. Further, central composite results suggest that a higher level of fermentate, about 2.4 ml per 100 g of product with less than 15 mg of ascorbic acid per 100 g of product, provides moderate anti-mold efficacy.
  • Example 26 Creation of a Lactobacillus plantarum fermentate
  • This example shows preparation of a fermentate made with a Lactobacillus plantarum.
  • a bacterial incubation broth was made up according to the formula represented in Table 33 and the following process.
  • chicken broth mixture with about 1% dextrose, about 0.1% Tween 80, and about 1% glycerol was prepared and then pasteurized to about 88° C to create a pasteurized broth mixture.
  • the pasteurized broth mixture had cooled to about 74° C, it was used to prepare an amino-acid enriched broth mixture with about 0.1 % threonine (w/v), about 0.05%) serine (w/v), about 0.05%> proline (w/v), and about 0.1%> cysteine (w/v).
  • the amino-acid-enriched broth mixture was further cooled to about 38° C and then swirled to create a homogenous broth suspension.
  • the homogenous broth suspension was then inoculated with Lactobacillus plantarum NRRL 6055 at the level of about 1 x 10 7 cfu/g.
  • the inoculated broth was incubated at about 35° C for about 24 h without agitation. After this incubation, the cells were separated from the supernatant by centrifuging at about 2,600 g for about 10 minutes and decanting off the supernatant. Collected cells were resuspended into about 25 ml of BPBD to create a 40X concentrated cell preparation.
  • Example 27 Lactobacillus plantarum reduces mold growth on cut strawberries.
  • Example 28 Impact of temperature on the efficacy of fermentate to inhibit E. coli growth. This examples shows the impact of incubating fermentates at various temperature on the growth inhibition of Salmonella surrogates.
  • the source of fermentate used in this experiment was prepared as described in Example 22.
  • One centrifuge tube containing about 45 mL of fermentate was kept at room temperature (about 22° C).
  • Eight centrifuge tubes containing about 45 mL of fermentate were placed into a water bath adjusted to about 93° C. The temperature in the tubes placed in the water bath was monitored such that a tube was removed once the fermentate reached the following approximate temperatures: 38°, 43°, 49°, 54°, 60°, 66°, 71°, and 77° C. Once removed from the water bath, the tubes were stored at refrigeration (about 4° C) until analysis.
  • fermentate efficacy involved determining how much inhibition of ari E. coli organism occurred in the presence of fermentates heated to various temperatures.
  • the procedure for analyzing fermentate efficacy utilized the following process. Add about 400 ⁇ . of fermentate to about 5 mL of chicken broth to create a fermentate broth mixture. Into the fermentate broth mixture, about 100 ⁇ . of antibiotic (kanamycin) resistant E. coli was added to create an inoculated broth. The inoculated broth was incubated for about 12 h at about 35° C to create an incubated broth. About 250 ⁇ .
  • antibiotic kanamycin
  • BD® Sparks, MD trypticase soy broth
  • kanamycin Gold Biotechnologies, St. Louis, MO
  • Tubes containing antibiotic treated broth were incubated at about 35° C for about 8 h and monitored every hour for changes in optical density (OD).
  • OD was assessed at a wavelength of 600 nm (“OD 6 oo") using an Unico 1100RS spectrophotometer.
  • Figure 4 is a bar graph 400 showing growth of E. coli in food-safety preparations that were prepared using various incubation temperatures.
  • Figure 4 includes an x-axis 402, which shows incubation temperature, in degrees C, of various food-safety preparations, and a y-axis 404, which shows growth of E. coli, as measured by optical density (OD 6 oo)-
  • Figure 4 also includes a bar 406, showing an OD 6 oo of
  • a bar 408 showing an OD 6 oo of 0.033 for samples incubated at about 38° C
  • a bar 410 showing an OD 6 oo of 0.038 for samples incubated at about 43° C
  • a bar 412 showing an OD 6 oo of 0.035 for samples incubated at about 49° C
  • a bar 414 showing an OD 6 oo of 0.062 for samples incubated at about 54° C
  • a bar 416 showing an OD 6 oo of 0.065 for samples incubated at about 60° C
  • a bar 418 showing an OD 6 oo of 0.071 for samples incubated at about 66° C
  • a bar 420 showing an OD 6 oo of 0.104 for samples incubated at about 71° C, and a bar 422, showing an OD 60 o of 0.205 for samples incubated at about 77° C.
  • Results at the four-hour assessment point are generally representative of treatment differences over the hourly assessments. Data indicate the amount of antibiotic-resistant E. coli growth that occurred after four hours of incubation at 35° C. The more E. coli growth, the lower the activity of the fermentate. As such, results indicate that up to about 49° C, there is little change in E. coli growth indicating the fermentate efficacy is unaffected by heating up to about 49° C. However, intermediate heating levels, about 54° to about 66° C, result in a moderate increase in OD, indicating that some efficacy of the fermentate has been lost. The greatest loss of fermentate efficacy occurs when heating at about 71° C and above.

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Abstract

La présente invention concerne un procédé de conservation d'aliment et/ou de protection d'aliment. Le procédé consiste (i) à obtenir un milieu de croissance ; (ii) à introduire un additif dans le milieu de croissance pour former un précurseur de préparation de protection d'aliment ; (iii) à inoculer un ou plusieurs types de bactéries d'acide lactique dans le précurseur de préparation de protection d'aliment pour former un précurseur de préparation de protection d'aliment inoculé ; (iv) à incuber le précurseur de préparation de protection d'aliment inoculé pour produire une préparation de protection d'aliment ; (v) à associer la préparation de protection d'aliment à un aliment pour sensiblement inhiber la croissance de moisissures et/ou de levures sur ou dans l'aliment pendant le stockage.
PCT/US2016/060760 2015-11-06 2016-11-07 Systèmes, procédés et compositions pour utiliser des préparations de protection de bactérie d'origine alimentaire pour favoriser la protection et la conservation d'aliments, de boissons et de surfaces non-comestibles WO2017079715A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11058131B2 (en) 2015-04-16 2021-07-13 Kennesaw State University Research And Service Foundation, Inc. Escherichia coli O157:H7 bacteriophage Φ241
CN114847413A (zh) * 2022-05-31 2022-08-05 西南民族大学 一种牦牛肉源诱食剂、制备方法及其在宠物食品添加剂中的应用

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Publication number Priority date Publication date Assignee Title
WO1991013556A1 (fr) * 1990-03-12 1991-09-19 Yhtyneet Paperitehtaat Oy Matiere de conditionnement supprimant l'oxygene d'un emballage et procede de production
EP1250050B1 (fr) * 2000-01-11 2004-10-27 Intralytix Inc. Procedes et dispositifs pour l'assainissement par bacteriophages
US20090246336A1 (en) * 2008-03-25 2009-10-01 Ecolab Inc. Bacteriophage treatment for reducing and preventing bacterial contamination
EP2769630A1 (fr) * 2013-02-26 2014-08-27 Purac Biochem N.V. Procédé de production de nisine amélioré
WO2014145369A1 (fr) * 2013-03-15 2014-09-18 Micro-Nature Llc Procédés de production de bouillie de viande améliorés et compositions

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991013556A1 (fr) * 1990-03-12 1991-09-19 Yhtyneet Paperitehtaat Oy Matiere de conditionnement supprimant l'oxygene d'un emballage et procede de production
EP1250050B1 (fr) * 2000-01-11 2004-10-27 Intralytix Inc. Procedes et dispositifs pour l'assainissement par bacteriophages
US20090246336A1 (en) * 2008-03-25 2009-10-01 Ecolab Inc. Bacteriophage treatment for reducing and preventing bacterial contamination
EP2769630A1 (fr) * 2013-02-26 2014-08-27 Purac Biochem N.V. Procédé de production de nisine amélioré
WO2014145369A1 (fr) * 2013-03-15 2014-09-18 Micro-Nature Llc Procédés de production de bouillie de viande améliorés et compositions

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11058131B2 (en) 2015-04-16 2021-07-13 Kennesaw State University Research And Service Foundation, Inc. Escherichia coli O157:H7 bacteriophage Φ241
CN114847413A (zh) * 2022-05-31 2022-08-05 西南民族大学 一种牦牛肉源诱食剂、制备方法及其在宠物食品添加剂中的应用

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