Classifications

• • 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
  • A23B4/00—Preservation of meat, sausages, fish or fish products
  • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
  • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
  • A23B4/24—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/12—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group, wherein Cn means a carbon skeleton not containing a ring; Thio analogues thereof
• • 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
  • A23B2/742—Organic compounds containing oxygen
  • A23B2/754—Organic compounds containing oxygen containing carboxyl groups
  • A23B2/758—Carboxylic acid esters
• • 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
  • A23B2/762—Organic compounds containing nitrogen
• • 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
  • A23B4/00—Preservation of meat, sausages, fish or fish products
  • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
  • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
  • A23B4/20—Organic compounds; Microorganisms; Enzymes
• • 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
  • A23B70/00—Preservation of non-alcoholic beverages
  • A23B70/10—Preservation of non-alcoholic beverages by addition of preservatives

Definitions

• the present invention relates to a method for reduction and/or prevention of the activity of gram-negative bacteria in or on products by using a composition based on fatty acid esters of glycerol.
• the present invention further is directed to the products resulting from using this method.
• Fatty acid monoesters of glycerol are known for their antibacterial activity against yeasts, fungi and food-spoiiage bacteria. They a!so have antibacteriai activity against certain gram-positive food pathogenic bacteria such as for example Listeria and Clostridium. They are on themselves however not or hardly effective against gram-
• the enhancer may be: an ⁇ -hydroxy acid, a ⁇ -hydroxy acid, other carboxylic acids, a chelating agent other than a carboxyiic acid, a phenolic compound (e.g. certain antioxidants and parabens) or a C1-C10 - monohydroxy alcohol.
• a phenolic compound e.g. certain antioxidants and parabens
• a C1-C10 - monohydroxy alcohol e.g. certain antioxidants and parabens
• Further suitable enhancers are compounds highly specific for binding ferrous and/or ferric ion such as siderophores (e.g.
• enterobactin enterobactin, pyochelin
• iron binding proteins e.g lactoferrin, transferrin
• chelators such as bacteriocins, antibacteriai enzymes, sugars, sugar alcohol and combinations thereof.
• Above-mentioned chelating agent is described to be an organic compound capable of multiple coordination sites with a meta! ion in solution. Typically these chelating agents are polyanionic compounds and coordinate best with polyvalent metal ions.
• Examples are ethylene diamine tetraacetic acid (EDTA) and salts thereof, various phosphate-based and/or phosphonic acid-based compounds, adipic acid, succinic acid, diethylenetriaminepenta-acetic acid, 1-hydroxyethy!ene and certain carboxylic acids such as ⁇ - and ⁇ -hydroxy acids, malic acid and tartaric acid.
• EDTA ethylene diamine tetraacetic acid
• salts thereof various phosphate-based and/or phosphonic acid-based compounds
• adipic acid succinic acid
• diethylenetriaminepenta-acetic acid 1-hydroxyethy!ene
• carboxylic acids such as ⁇ - and ⁇ -hydroxy acids, malic acid and tartaric acid.
• the present invention provides a totally different means to enhance the antibacteriai activity of fatty acid esters of glycerol against gram-negative bacteria.
• the present invention results in a very effective means against gram-negative bacteria, which may be applied in a wide-variety of products and applications ranging from technical products and applications to products for consumption and/or persona! care.
• the present invention is directed to a method for reduction and/or prevention of the activity, growth and/or presence of gram-negative bacteria in a product or on a surface comprising contacting said product or surface with a composition comprising a combination of a) fatty acid ester of glycerol and b) polylysine and/or a salt of poiyiysine, wherein said fatty acid ester of glycerol is applied as antibacterial or antimicrobial agent and not as emulsifier.
• Fatty acid esters of glycerol are usually applied as emulsifiers but it has now been surprisingly found that they may be applied as antibacterial agent against specifically gram-negative bacteria as wel! by using them in a composition comprising poiyiysine and/or a salt hereof.
• the combination of poiyiysine and/or salt with fatty acid esters of glycerol is capable of not simply enhancing the activity of both components whereby said enhancing effect is the sum of the individual activities of both the poiyiysine and the glycerol fatty acid ester, but it unexpectedly demonstrates a synergistic effect on the antibacterial activity as both components works in synergy resulting in an antibacterial activity which is significantly higher than the sum of the activities of the individuai
• enhancers are themselves not very effective against gram- negative bacteria and thus have to be appiied in high quantities to show any enhancing antibacterial effect, in the quantities needed to achieve this antibacterial effect, said enhancers negatively affect the quality of the food product in which they are appiied in terms of taste, color, odor and/or texture.
• Organic acids for example are not that effective by themselves against gram- negative bacteria and in order to have an enhancing effect, a considerable amount of the organic acid is needed.
• the acid however negatively affects taste, texture and other properties of many products as they lower the phi of said food product in the quantity necessary to have any antibacteriai effect.
• Acids are for example known to negatively affect the texture of protein-rich products (e.g. meat products) because they lead to for example the denaturation of the proteins.
• the fatty acid esters of glycerol used in the method according to the present invention are also not very effective on their own against gram-negative bacteria, but in combination with poiylysine and/or a salt hereof, a synergisticaliy increased antibacterial activity is obtained at concentrations of the glycerol fatty acid esters that are acceptable in many food and drink products.
• Polylysine is known to exert an antibacterial activity against gram-negative bacteria. Both ⁇ -po!y!ysine and ⁇ - ⁇ o!ySysine have antibacteria! activity although the latter one in significant greater extent as described by Shima et al. (Nov. 1984). As described in the article, ⁇ -polyiysine can effectively be used against gram-positive and -negative bacteria
• Hiraki et al. (2000) describe that when ⁇ -poiylysine is used together with antibacterial agents such as glycine, acetic acid (in the form of vinegar), ethanol or thiamine iauryisulfonate, its antibacterial efficiency is greatly enhanced. No mention is however made of a method wherein fatty acid esters of glycerol are combined with ⁇ -polylysine in
• compositions b comprising ⁇ -polylysine in combination with mono-and di-glycerol esters and other antibacterial components such as protamines, ethano!, glycine, lysozyme etceteras.
• compositions are described to be effective against spore-forming and iactic acid-producing bacteria, against putrefactive bacteria such as Leuconostoc (Gram- positive) and against yeasts and fungi such as Candida.
• prior art is directed to the use of fatty acid esters of glycerol as emulsifier or -j surfactant such as e.g. in JP 2002-274742 in blends that may comprise antibacteria! agents such as poSylysine and others.
• fatty acid esters of glycerol have been used as antibacteria! agent against gram-negative bacteria in a composition comprising polyiysine and/or a salt hereof.
• Prior art does not describe that fatty acid esters of glycerol may be used to synergistica ⁇ y increase the antimicrobial effect of polylysine and/or any salt hereof.
• the present invention thus comprises a method for reduction or prevention of the presence, growth and/or activity of gram-negative bacteria comprising contacting said bacteria with a composition comprising a combination of a) fatty acid ester of fatty acid and glycerol and b) po!y!ysine and/or a sait hereof, wherein said fatty acid ester of fatty acid and glycerol is used as antibacterial agent.
• Polyiysine may be present as ⁇ -poiylysine, as ⁇ -poiylysine or as a mixture hereof.
• ⁇ -Polyiysine is preferred as it has a higher antibacterial activity against gram-negative bacteria compared to the other forms of poiylysine and thus lesser amounts of this antibacteria! agent are needed to achieve a satisfactory synergy in the antibacterial
• ⁇ -poly!ysine preferably comprises 30 to 50 L-lysine monomers linked by the peptide bonds between the free carboxyi groups and the e-amino groups.
• the fatty acid esters of the present invention may also be combined with one or more saits of poiylysine.
• the glycerol fatty acid ester of the present invention aiso referred to as glyceride or glycerol or giycerol-based fatty acid ester, may comprise a monoester, a di-ester or a tri-ester of glycerol or mixtures hereof.
• esters are produced often lead to mixture of the various mono-, di and/or tri-esters possible as commonly known.
• the esters can be separated from these mixtures by different techniques known by the 5 person skilled in the art.
• mono-esters these mono- esters of glycerol comprise the pure components as well as mixtures which mainly comprise mono-esters but also comprise di- and tri-esters as further components of said
• the method according to the present invention is even more effective if the bacteria or the product or surface containing the bacteria is contacted with a composition comprising glycerol fatty acid ester, polylysine and/or a salt thereof and further one or more lactylates.
• Lactylates are fatty acid esters of Sactic acid (and/or the sait of iactic acid) and are well-known to the person skilled in the art. These components are
• lactylate components are often obtained as mixtures of for example a mixture of predominantly monolactylates and further comprising dilactyiates due to the way in which they are prepared. St may be very well possible that also higher polymerized lactylates are present
• lactylates may be obtained in their pure form (e.g. only the mono-form) by means of for example chromatographic separation or by any other means known to the person skilled in the art.
• the antibacterial composition used in the method according to the present invention further may comprise one or more organic acids and/or their salts or esters as
• these components further enhance the antibacterial activity.
• one or more organic acids and/or their salts or esters selected from iactic acid, acetic acid, citric acid, malic acid, fumaric acid, tartaric acid, gluconic acid, propionic acid and caproic acid are used because these acids do not have a negative impact on the product quality with respect to for example taste, odor and color of the product.
• the antibacterial composition used in the method according to the present invention further comprises one or more metai-chelating agents.
• the chelating agent may be selected from for example ethylene diamine tetraacetic acid (EDTA) and salts thereof, diethySenetriaminepenta-acetic acid and salts therof, various phosphate- based compounds such as sodium hexametaphosphate, sodium acid pyrophosphate, and
• polyphosphoric acid organophosphonate chelating compounds such as: phytic acid, 1 ,1- diphosphonic acid, siderophores and iron binding proteins such as enterobacterin and lactoferrin, and carboxylic acids and hydroxy carboxylic acids and/or salts thereof such as for example and not iimited to succinic acid, ascorbic acid, glycoiic acid, benzoic acid, octanoic acid and adipic acid.
• the method according to the present invention is very effective against gram-negative bacteria of the famiiy of Escherichia (e.g. Escherichia coil), Salmone ⁇ a (e.g. Salmonella spp), Campylobacter (e.g.
• Campylobacter spp Campylobacter spp
• Pseudomonas e.g. Pseudomonas spp
• the polylysine-components and glyceride- components in the composition act synergistica ⁇ y upon these specific target organisms whereby an antibacteria! activity is achieved that is sufficient to prevent and/or reduce the presence, growth and/or activity of these gram-negative bacteria.
• the method according to the present invention is applicable in a great variety of products and applications, ranging from for example products of Sow and high pH-values, highly concentrated and diluted products, products usable in the technical field (e.g. in detergents for bossi or house-hold use), in the pharmaceutical field (e.g. for cleaning/disinfection of equipment or in the preparation of pharmaceutical compositions or their packaging), in personal care (e.g. in manufacture of cosmetics, shampoos, creams and lotions), in the feed industry (e.g. for cleaning of equipment, in the manufacture, storage, handling and preparation of anima! feed and drink products) and in the food and drink industry.
• products usable in the technical field e.g. in detergents for panoramic or house-hold use
• the pharmaceutical field e.g. for cleaning/disinfection of equipment or in the preparation of pharmaceutical compositions or their packaging
• personal care e.g. in manufacture of cosmetics, shampoos, creams and lotions
• the feed industry e.g. for cleaning of equipment, in the
• the present invention therefore relates to the use of a glycerol fatty acid ester as antibacteria! agent against gram-negative bacteria in a composition comprising poSylysine and/or a salt hereof for the reduction and/or prevention of the presence, growth or activity of gram-negative bacteria, and in particular the bacteria mentioned earlier, in the manufacture, handling, storage and preparation of detergents, of cosmetic products and of personal-care products.
• the method according to the present invention for reduction or prevention of the presence, growth or activity of gram-negative bacteria in detergent-products, cosmetic products and personal-care products comprises contacting said products with a composition comprising glycerol fatty acid ester and poiylysine and/or a salt hereof according to the various embodiments of the present invention during one or more stages of manufacture, handling, storage or preparation of said products, wherein said glycerol fatty acid ester is used as antibacterial agent.
• a composition comprising glycerol fatty acid ester and poiylysine and/or a salt is further found to be very usable for cleaning surfaces.
• the method according to the present invention therefore also is directed to reduction or prevention of the presence, growth or activity of gram-negative bacteria on a surface, and in particular the bacteria mentioned eariier, comprising contacting said surface with an antibacterial composition comprising glycerol fatty acid ester and poiylysine and/or a salt hereof, wherein said glycerol fatty acid ester is used as antibacterial agent.
• the present invention further is directed to the use of glycerol fatty acid ester as antibacterial agent against gram-negative bacteria in a composition comprising polylysine and/or a salt hereof in the manufacture, handling, storage and preparation of food and drink products for the feed industry and of food and drink products for human
• the method according to the present invention for reduction or prevention of the presence, growth or activity of gram-negative bacteria in food and drink products for animais or human consumption comprises contacting said products with a composition comprising glycerol fatty acid ester and poiyiysine and/or a salt hereof during one or more stages of the food processing process such as in the manufacture, handiing, storage or preparation of said products, wherein said glycerol fatty acid ester is being applied as antibacterial agent.
• Examples of food and drink products are beverages such as for example carbonated and non-carbonated beverages and fruit or vegetable-based juices, protein- rich products such as for example various meat and fish products, dressings, sauces and toppings, ready-to-eat and ready-to-drink products, refrigerated and high temperature- treated products etceteras. These products can be very well manufactured or treated with the method according to the present invention. The resulting products are not negatively affected in organoleptic quality in terms of e.g. taste, texture and color while the products are being protected against food spoilage and/or food poisoning by the presence and activity of gram-negative bacteria.
• Glycerol fatty acid esters will normally be present in a food or drink product in an amount of up to 5 % by weight of the product, preferably from 0.0001 % to 5 %, preferably from 0.0001 % to 2 %, preferably from 0.0001 % to 1 %.
• PoSylysine will normally be present in a food or drink product in an amount of up to 1 % by weight of the product, preferably from 0.0001 % to 1 %, preferably from 0.0001 % to 0.1 %, preferably from 0.0001 % to 0.01 %, preferably from 0.0001 % to 0.001%.
• EDTA, organophosphates and polyphosphates will normally be present in a food or drink product in an amount of up to 1 % by weight of the product, preferably from 0.0001 % to 1 %.
• Lactylate will normally be present in a food or drink product in an amount of up to 1 % by weight of the product, preferably from 0.0001 % to 1 %, or even from 0.0001 % to 0.1 % and most preferably from 0.0001 % to 0.01 %.
• Organic acids such as for example iactic acid, fumaric acid, succinic acid, tartaric acid, ascorbic acid, glycolic acid, benzoic acid, acetic acid, propionic acid, octanoic acid, malic acid and adip ⁇ c may be present in a food or drink product in an amount of up to 10 % by weight of the product, preferably from 0.0001 % to 10 %, preferably from 0.0001 % to 5 %.
• the above-mentioned food and 5 drink products are contacted with the composition of the present invention comprising glycero! fatty acid ester and poSylysine and/or a sait hereof, in a preferred embodiment of the method according to the present invention, the food and drink products are injected the above-mentioned composition.
• the composition is then present in the interior part
• the method comprises surface -treating the products with the composition comprising gSycerol fatty acid ester and poiyiysine and/or a sait hereof. This may be done not oniy in the finai product stage but also during or in for example the disinfection of carcasses in the manufacture of meat products or in the washing step applied for fruit and vegetables.
• the antibacterial composition comprising gSycerol fatty acid ester and poiyiysine and/or a sait hereof.
• Ib composition may be brought in contact with or introduced into the product to be treated by various means such as for exampie as a spray, a rinse or a wash solution or as solution wherein the various food products are dipped.
• composition of the 20 present invention is used as active ingredient in the final product or as component of for example a wash solution or spray
• the components of the composition will vary in concentration and in internal ratio as will be obvious to the person skilled in the art.
• composition comprising glycerol fatty acid and poiyiysine and./or a salt hereof may be avaiiable in solid or liquid form.
• the composition is in liquid form, it generally is in 5 the form of an aqueous composition, which may be a solution or a dispersion.
• Such an aqueous composition generally comprises, based on total weight of the solution, from 0.0001 wt% to up to 40 wt%, more preferably from 0.1 wt% to 35 wt%, and most preferably from 1 to 25 wt% of poiyiysine and from 0.0001 wt% up to 45wt.%, more preferably from 1 to 40 wt%, and most preferably from 5 to 35 wt% of a glycerol fatty acid 0 ester according to the present invention.
• the composition may further comprise a lactyiate in an amount of 0 to 45 wt% and more preferably from 5 to 35 wt% and further an organic acid in the range of 0 to 45 wt% and more preferably from 0 to 30 wt%.
• the glycerol fatty acid ester and the poiyiysine or the salt thereof may be introduced in the liquid composition by means of carriers.
• carriers The person skilied in the art 5 knows what type of carriers can be used. Among various we ⁇ -known carriers, it was found that polyethylene glycol and/or lactate function very well as carrier.
• the carrier may be present in concentrations of about 50 to 98 wt%.
• various emuisifiers known to the person skilled in the art may be added. Preferably emulsifiers such as polysorbates (e.g.
• poiysorbate 60 or 80 and lecithine are applied in concentrations of for example 0.1 to 25%, more preferably 1-10% and most preferably 2 to 4% based on 100% fatty acid derivative, such as glycerol fatty acid ester and/or lactySate, if the latter component is used in the composition in addition to glycerol fatty acid ester and polylysine or a salt thereof.
• fatty acid derivative such as glycerol fatty acid ester and/or lactySate
• composition comprising glycerol fatty acid ester and polylysine or the salt thereof is in solid form, it will generally be in the form of a powder comprising particles of the relevant components.
• the composition in solid form generally comprises, based on total weight of the powder, from 0.0001 wt% to up to 40 wt%, more preferably from 0,1 wt% to 35 wt%, and most preferably from 1 to 25 wt% of polyiysine and from 0.0001 wt% up to 45wt.%, more preferably from 1 to 40 wt%, and most preferably from 5 to 35 wt% of of a glycerol fatty acid ester according to the present invention.
• composition may further comprise a Sactyiate in an amount of 0 to 45 wt% and more preferably from 0 to 35 wt% and further an organic acid in the range of 0 to 45 wt% and more preferably from 0 to 30 wt%.
• the foliowing non-limiting examples further iiiustrate the present invention.
• the following cultures were used in a study: Escherichia coli (ATCC 8739), Escherichia co// serotype O157:H7 (ATCC 700728), Salmonella typhimu ⁇ um (ATCC 13311 ) and Salmonella enti ⁇ tidis (ATCC 13076). All cultures were transferred daily in screw-capped tubes containing 10 ml brain heart infusion broth. Cultures were incubated at 30° C without agitation. Brain heart infusion broth was prepared with increasing amounts of the mono/di glyceride and polylysine.
• the concentration range for the capryiic (C8) mono/di glyceride was as from 0 to 0.18 % in 10 0.02 % steps, for the capric (C10) mono/di glyceride was as from 0 to 0.09 % in 10 0.01 % steps, for the iauric (C12)mono/di glyceride was as from 0 to 0.009 % in 10 0.001 % steps.
• Mono/di glycerides were combined with polyiysine.
• the concentration range for the poiylysine was as from 0 to 0.0675 % in 10 0.0075 % steps, resulting in 100 different media.
• the pH of the media was adjusted to 6.1 - 6,2 with 1 N HC!
• the Bioscreen® calculates maximum specific growth rates.
• the purpose of further data processing is to ascertain whether two amino acids act independently of each other or whether they stimulate each other in their inhibitory action (synergy) or cancel out each other inhibitory effect (antagonism).
• ⁇ ⁇ max . s /(K 5 + s)
• ⁇ max represents the maximum specific growth rate
• s standing concentration of the growth limiting substrate in the medium
• ⁇ f(s,p)
• p the concentration of inhibitor P.
• the logistic dose response function a robust fitting function for transition phenomena in life sciences. J. Clinical Ligand Assay 24: 270 - 274).
• O x .O y The experimentally observed effect of combinations of X and Y on the relative growth rate is defined as O x y .
• Synergy, independent effect, and antagonism can be visualized in a plot of O ⁇ versus O x .O y .
• This is exemplified in Fig. 1 - 4, wherein different plots are given of O Cx ⁇ p L y s (experimentally observed relative growth rate in the presence of mixtures of a monogSyceride and poiylysine) versus Qc xG -Qp L y s (predicted relative growth rate in the presence of mixtures of a lactylate and poiylysine) for Salmonella typhimurium (ATCC 13311) and Salmonella entiritidis (ATCC 13076) showing the synergy in inhibition between Sactyiates and poSyiysine.
• the solid line in this graph represents the line where the experimentally observed relative growth rate (Oc xL p L y s ) equals the predicted relative growth rate (O CXL .Op Ly s) and where the lactyiate and polylysine act as independent inhibitors.
• Figure 1 represents a plot of experimentally observed relative growth rate of Salmonella typhimurium in the presence of mixtures of C8-glyceride and poSyiysine (O CSG p L y s ) versus predicted relative growth rate in the presence of mixtures of C8-g!yceride and poiylysine
• Figure 2 represents a piot of experimentally observed relative growth rate of Salmonella entiritidis in the presence of mixtures of a C8-gSyceride and poSyiysine (O CSG. piy s ) versus predicted relative growth rate in the presence of mixtures of C8 ⁇ glyceride and poiylysine
• Figure 3 represents a piot of experimentally observed relative growth rate of Salmonella typhimurium in the presence of mixtures of a C10-glyceride and poSyiysine (O CI Q G PL y s ) versus predicted relative growth rate in the presence of mixtures of C10-giyceride and poiylysine (OcioG-OpLy ⁇ ).
• Figure 4 represents a plot of experimentaiSy observed relative growth rate of Salmonella entiritidis in the presence of mixtures of a C10-giyceride and poiylysine (0 CI OG. p t y s ) versus predicted relative growth rate in the presence of mixtures of C10-glyceride and polySysine
• Figures 1-4 demonstrate that poiyiysine and giycerides in the various combinations tested act synergistically upon the target organism as O xy / O x .O y ⁇ 1 and > 0 (represented by the dots below the soiid line).
• synergy examples are given in Table 1 such as for example the synergy between 0.0225 % (w/w) poiyiysine and 0.12 % (w/w) C8-glyceride or 0.0225 % (w/w) poiyiysine and 0.09 %(w/w) C10-g!yceride.
• the relative growth rate of Escherichia coii (ATCC 8739), Escherichia coli serotype O157:H7 (ATCC 700728), Salmonella typhimurium (ATCC 13311) or Salmonella enti ⁇ tidis (ATCC 13076) in a broth containing 0.0225 % (w/w) poiyiysine and 0.12 % (w/w) C8-g!yceride or 0.0225 % (w/w) polySysine and 0.09 %(w/w) C10-g!yceride is in all cases lower than can be expected on the basis of the relative growth rate of these organisms in media containing either polySysine or one of the giyceride esters.
• Example 2 antimicrobial effect in contaminated [ . chicken . fjlft and milk
• Salmonella Typhimurium ATCC 13311 and Escherichia coli O157:H7 ATCC 700728 were grown in sterile screw capped tubes containing Brain heart infusion broth for 18 - 24 hours at 30 0 C.
• Fiiets were subsequently sterilized by gamma-irradiation (average radiation dose: 12 kiioGray).
• Chicken fiiets were briefly dipped and completeiy submersed in 1 I of a solution containing the appropriate formulation and then transferred to 400 mi Bagfilter® lateral filter bags (interscience, St Nom, France) containing 5 mi of the appropriate formulation. Bags were vacuum-sealed and incubated at 12 0 C for up to 7 days untii further analysis. Time zero samples were plated within 30 min after dipping.
• Sterile iow fat milk was purchased fro a iocaS supermarket and 100 mi quantities were transferred to a series of sterile screw topped bottles.
• ⁇ -Poiylysine The sodium salt of glyceryl mono/di octanoate (C8 mono/di giyceride) and glyceryl mono/di decanoate (C10 mono/di giyceride) was added to a concentration as shown in Tabie 2.
• the different milk preparations were inoculated with an overnight culture of Escherichia coii O157:H7.
• the starting ceil density was iog 10 2.5 - 3.0.
• compositions of the formulations that were studied are shown in Table 2.
• ⁇ - Polylysine and mono/di glycerides were dissolved in demineralised water and sterilized for 20 min at 12O 0 C.
• ⁇ -Polylyssne was purchased from Chisso America lnc (New York, USA), The sodium salt of glyceryl mono/di octanoate (C8 mono/di giyceride) and glyceryl mono/di decanoate (C10 mono/di giyceride) were purchased from Caravan Ingredients (Lenexa, Kansas, USA).
• Table 3 Effect of combinations of ⁇ -poiyiysine ( ⁇ - PL) with mono/di glycerides on Salmonella Typhimurium on chicken filets at 12 0 C; expressed in iog 10 colony forming units (CFU) / m!

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