2. Technology Description Relevant Prior art may be found in U.S. Patent Nos. 5,096,718 and 5,260,061 and the references cited therein. These patents disclose the use of certain propionic metabolites in certain foods to increase the shelf life of the resulting products.

JP 07-115950 suggests the combination of bacteriocins produced by lactic acid bacteria of propionibacterium in combination with either organic acids and their salts, fatty acid esters of polyhydric alcohols, amino acids, antibacterial peptides and proteins, polysaccharides comprising sugars, saccharic acids and amino sugars and their partial decomposition products, spices and their essential oils and plant components; and alcohols. The reference fails to suggest that the combination can be used to prevent the deterioration of foods as a result of mold and yeast (i.e., provide an antimycotic effect) by using the combination of bacteriocins with organic acids and their salts, fatty acid esters of polyhydric alcohols, amino acids, antibacterial peptides and proteins, polysaccharides comprising sugars, saccharic acids and amino sugars and their partial decomposition products, spices and their essential oils and plant components; and alcohols. The only mention in this reference in the treatment of foods having a pH of greater than 5.5, is in connection with a

hamburger composition, which, by definition would not suffer from mycotic bacteria (i.e., yeast and mold spoilage).

Despite the above teachings, there still exists a need in the art for expanding the use of propionibacteria metabolites to high pH foods, animal feeds and baked goods and to use such metabolites in combination with potentiators to provide a synergistic effect on antimycotic activity.

Brief Summary of the Invention It 5 now discovered, quite surprisingly, that the following types of foods which are ordinarily susceptible to spoilage by mold and/or yeast can utilize the compositions disclosed in U.S.

Patent No. 5,260,061 for their antimycotic properties: various baked goods including cakes, snack cakes, cookies, frostings, cheesecakes, pie crusts, pie fillings, confections, biscuits, batters, brownies, specialty breads, pastas, and any other food or beverage which has a pH of greater than 5.5, more preferably greater than 5.8 and most preferably greater than 6.0. The composition as disclosed in U.S. Patent No. 5,260,061 provides a mature propionibacterium growth medium that can provide inhibition of yeasts. This effect can occur without providing an undesirable flavor, odor, or appearance, even in "delicate" foods. The unexpected findings disclosed are especially dramatic in light of the breadth of activity and some of the low concentrations which provide yeast inhibition. An anti-yeast food additive can be obtained by growing propionibacteria, e.g.

Propionibacterium sherman ii, P. freudenreichii, P. pentosaceum, P. thoenii, P. arabinosum, P. rubrum, P. jensenii, P.

peterssonii, and related species (as identified in Malik et al., Can. J. Microbiol. 14:1185, 1968) in a milk, cheese whey, or broth medium, or other suitable nutrient mixtures. The resulting growth liquid can then be added to food and feed products to inhibit yeasts and molds. To facilitate storage and shipment, the

growth liquid may be dried to form a powder or frozen before use as an ant yeast food additive. The metabolites may be separated or purified or used as a mixture. Powdered or liquid natural metabolites of propionibacteria can be incorporated into various foods and feeds to render them less susceptible to spoilage by growth and/or enzymatic activity of yeasts. Anti-yeast activity may be obtained by incorporating viable propionibacteria directly into a food.

The growth medium for such Propionibacterium species may be formulated with milk or whey containing yeast extractives or fruit juices or any other broth media containing appropriate growth nutrients. The growth liquid, after development of the propionibacteria up to 106 to 1010 cells per ml, may be heat treated (pasteurized) to kill the inoculated and adventitious bacteria prior to use in liquid, condensed, dried, or frozen form. It is added in various concentrations (preferred between 0.01 and 10% of total weight) to food or feed where it functions to inhibit yeasts. This inhibition enables the shelf life and storage times of the food or feed to be increased.

In addition, the above materials may also be combined with certain potentiators such as EDTA, vanillin compounds (e.g., methyl vanillin or ethyl vanillin), citrate and/or its salts, sorbate and/or its salts or inositols to provide a synergistic antimycotic effect against a wider range of yeast and mold species over a broader range of effective pH levels.

It is an object of the present invention to provide a food product which includes a substance to inhibit the growth of yeasts and molds in high pH foods and feeds without harming the flavor, aroma, or other characteristics of the food product.

This, and other objects, will readily be apparent to those skilled in the art as reference is made to the detailed description of the preferred embodiment.

Detailed Description of the Preferred Embodiment In describing the preferred embodiment, certain terminology will be utilized for the sake of clarity. Such terminology is intended to encompass the recited embodiment, as well as all technical equivalents which operate in a similar manner for a similar purpose to achieve a similar result.

To the extent necessary for completion, the disclosures of U.S.

Patent Nos. 5,096,718 and 5,260,061 are hereby incorporated by reference.

For the purpose of this disclosure, "metabolite" is defined as a substance, other than water, produced by propionibacteria. An "active metabolite" or an "inhibitory metabolite" is a metabolite which inhibits the growth or reproduction of an undesired yeast or mold.

There are several aspects to the present invention as set forth below. It has been found possible to inhibit spoilage yeasts and molds and thereby extend the shelf life of many food products without adversely affecting flavor or aroma by adding a fermented growth mixture that may or may not contain viable organisms based on propionibacterium cultures with its metabolites or a fraction of such a growth mixture which fraction contains one or more inhibitory metabolites other than propionic acid. The mixture or fraction has a greater inhibitory effect than a weight of pure propionic acid which is equal to the pure propionic acid content of the mixture or fraction. Such substances are surprisingly excellent inhibitors of yeasts and molds.

Examples of the present invention are set forth hereinafter. It is intended that they be only illustrative. Propionibacterium strains identified by number are available from the American Type Culture Collection (ATCC) . The other cultures are widely

available or can be obtained from Oregon State University, Corvallis, Oreg., without cost. For example, Propionibacterium freudenreichii subsp. shermanii, ATCC strain #9616 can be used in accordance with the present invention.

It is discovered that Propionibacterium cultures can be used to produce a material, including one or more metabolites (other than propionic acid) that inhibit yeast and mold. The metabolites, which can be obtained as by-products of propionibacterial culture fermentation of skimmilk or other suitable medium can serve as flavor adjuncts and may also be inhibitory to a number of microorganisms, including themselves at the finish of fermentation. The shelf-life of a food product is extended by providing in or on the product one or more of such active metabolites. The degree of inhibition achieved is much greater than is due to propionic acid alone in the mixtures of metabolites studied. In some cases where excellent inhibition occurs, the amount of propionic acid is so low as to have no measurable effect at all. Also, end use levels of propionic acid less than about 0.1% are not accepted to be useful for inhibition of yeasts in general, or of molds in high pH (greater than about 5.5) systems. This indicates that some other unidentified inhibitory substance or combination of substances in propionibacteria fermented growth mixtures may be responsible for the excellent ability of such growth mixtures to extend the shelf life of food products against yeast and mold spoilage. While not wishing to be bound to any specific theory, it is believed that metabolites (other than propionic acid) may be responsible for such performance properties.

Small amounts of viable propionibacteria are used in the manufacture of Swiss cheese to form eyes by the production of CO and to impart the characteristic Swiss cheese flavor. In most food products, however, the presence of viable propionibacterial and Swiss cheese flavor would be unacceptable, eyes would not be

desired, and CO2 release may cause physical defects or bloat pacsing materials.

To facilitate storage and shipping, a propionibacteria growth mixture may be evaporated and frozen, or concentrated and dehydrated, e.g., by spray-drying, or freeze-drying, to form a powder.

A material according to the present invention is most readily used by mixing with and/or applying on a blendable food product, but should also be effective to treat the surface of solid food products, or the interior of such products, e.g. by injection.

The optimum amount to be used will depend on the composition of the particular food product to be treated, but can be determined by simple experimentation.

In most instances, substantial improvements in shelf life can be obtained by adding the material in and/or applying on an amount suffIciently small that it will have no deleterious effect on the flavor or aroma of the food product. This is possible because the material includes at least one propionibacteria metabolite which is active in inhibiting yeast and mold and does not impart a strong flavor such as that of propionic acid. More specifically, the liquid, condensed, or dried product, which typically comprises pasteurized cultured solids or liquids, containing the propionibacteria metabolites, is added to the food product in amounts between about 0.1 to about 2.0 percent by weight of the product, more preferably between about 0.25 to about 1.0 percent by weight of the product and most preferably between about 0.5 to about 0.75 percent by weight of the product. In the case where the agent is added to a dry mix to which is added liquid ingredients and thereafter cooked, such as a cake, the amount added is by weight of the rehydrated (wet) mix prior to cooing.

Commercially available materials, more specifically pasteurized cultured solids or liquids including propionibacteria metabolites

are sold by Rhone-Poulenc Inc. under the MICROGARDO trademark.

MICROGARD MG 100 is a pasteurized cultured skim milk that is standardized with skim milk solids and spray dried. MICPOGAPD MG 200 is a pasteurized cultured dextrose that has been standardized with maltodextrin and spray dried. MICROGARDO MG 250 is a condensed (frozen or liquid) version of the cultured dextrose product.

While the use of the propionibacterial metabolites, per se, can be used to inhibit yeast and mold on many foods, when combined with certain potentiating agents, a synergistic antimycotic effect is noticed. The potentiators which are preferred for use can be selected from the group consisting of chelators (e.g., EDTA (ethylenediamine tetraacetic acid) and inositol), the group consisting of essential oils and flavors (e.g., methyl vanillin, ethyl vanillin, allyl isothiocyanate, and guiacol), the group consisting of organic acids and salts of organic acids other than propionic acid, acetic acid and lactic acid and their respective salts (e.g., dimethyl fumarate, sodium citrate, potassium sorbate) . In practice, these potentiators are used in combination with the propionic bacteria metabolites in amounts ranging from about 50 to 2500 ppm by weight of the final food composition, more preferably between about 75 to about 800 ppm by weight of the final food composition.

The specific mechanism for why the combination yields synergistic results is not completely understood. One theory is that the "potentiator" functions to potentiate the metabolites. An alternative theory is that the metabolites are responsible for potentiating the "potentiator". Either theory is to be expressly covered by this invention.

The propionibacterial metabolite(s), optionally in combination with the potentiator, can be used in a number of foods which, prior to the present invention, were not believed possible. For example, prior teachings have suggested that propionibacterial

metabolites can effectively operate in foods having an acidic pH (i.e., pH less than about 5.5). Specific classes of foods suggested in accordance with U.S. Patent No. 5,260,061 include dairy foods, cultured foods, yogurt, Kissle-type products, fruit juice, salad dressings, pasta, sausages, and other meat products such as chicken, fish, crab and hamburger. Applicants have surprisingly discovered that propionibacterial metabolites, optionally in combination with the potentiator, can be used in connection with foods susceptible to spoilage by yeast and, more specifically, mold having a pH of greater than about 5.5, more preferably greater than about 5.8 and most preferably greater than about 6.0. In addition, the combination of propionibacterial metabolites with the potentiator can be used in connection with foods not suggested in the prior art.

Examples of foods which can be produced in accordance with the present invention include baked goods including cakes, snack cakes, cookies, frostings, cheesecakes, pie crusts, pie fillings, confections, biscuits, batters, brownies, specialty breads, and pastas. This above listing is not intended to be limiting as the invention specifically includes any food or beverage having a pH of greater than about 5.5 which is ordinarily susceptible to spoilage by yeast and/or mold.

It is also expected that the above metabolite(s) can be effective in extending the shelf-life of animal feeds with a pH greater than about 5.5.

The following non-limiting examples illustrate, generally, the effectiveness of propionibacterial metabolites plus potentiating agents, particularly in high pH foods, as materials that protect against spoilage yeasts and molds.

Example 1 - Coffee Cake Mold Testing To 100 parts of a dry coffee cake mix including the following ingredients in the following weight ratios (sugar: 11.4 parts, vegetable shortening: 11.4 parts, butter: 11.4 parts, bran flour: 6.0 parts, brown sugar: 5.7 parts, skim milk powder: 4.3 parts, honey: 2.8 parts; wheat germ: 2.0 parts, salt: 1.8 parts, soy flour (defatted): 1.0 parts, nutmeg (ground): 0.2 parts, egg yolks: 11.4 parts, egg white: 8.5 parts, vanilla: 0.2 parts, yeast: 5.7 parts, bread flour: 68.0 parts and cake flour: 23.0 parts) is added the following potential antimycotic agents to form four sample mixes: Sample A: Control (no agent added) Sample B: 0.2% (by weight of the final dry mixture) of Calcium Propionate Sample C: 0.75% (by weight of the final dry mixture) of MICROGARDX MG 200 Sample D: 0.75% (by weight of the final dry mixture) of MICROG RDO MG 200 and 500 ppm methyl vanillin (by weight of the final dry mixture) To each of the above mixtures is added 20.7 parts of water, the mixture is agitated with a hand mixer to uniformly disperse the dry mix into the water to form a cake batter and the batter is allowed to fully rise and is poured into a pan and baked at 360- 380 DF for about 20 minutes. The baked cakes are then allowed to cool to ambient temperatures and are wrapped with a clear and colorless polyethylene film. The cakes are maintained at room temperature for a period of time until mold forms on the cake.

The average days to molding of the cake samples is as follows: Sample A: Three Days Sample B: Seven to Ten Days Sample C: Four to Five Days

Sample D: Twenty-one to Twenty-eight Days Example 2 - Cheese Cake Yeast Testing To produce a cheesecake filling, the following is added to 3 cups of cottage cheese and 3 whole eggs and mixed until uniform: 3/4 cup of whipping cream, 3 tablespoons of melted butter, 5 tablespoons of sugar, 1/2 teaspoon lemon juice, 1/2 teaspoon grated lemon rind. Three fillings are produced, two as controls and one including 1.5 percent MICROGARDX MG 100 and 500 ppm methyl vanillin. The fillings are then poured into a crust and the cake is baked at 3500F for about 45 minutes or until the filling is firm.

The three cakes are stored at 400F for a period of six weeks after baking in order to determine the amount of yeast present in the cake. The results are as follows in Table 1. All data is presented in colonies per gram of yeast: Table 1 - One Week 35,000 185t000 500 Two Weeks 76,000 72,000 <100 Three Weeks 173,000 57,000 <100 I Four Weeks 1,140,000 630,000 200 Five Weeks 10,500,00 4,500,000 <100 0 Six Weeks 11,200,00 56,000,00 <100 0 0 Example 3 - In Vitro testing - MICROGARD + vanillin Combinations of MICROGARDX MG-250 (condensed pasteurized cultured dextrose) and methyl vanillin are tested in Mycophil broth. The

broth is adjusted to pH 6.5 and 5.3, and inoculated with either yeast (Rhodotorula mucilaginosa var. mucilaginosa) at about 5 log CFU/ml or mold spores (Aspergillus niger) at about 3 log spores/ml. The inoculated broth tubes are incubated at 300C for 24 hours for yeast and 48 hours for molds. Inhibition of yeast growth under different treatments is assayed by plate count and reported as CFU/ml. Inhibition of mold growth is recorded by visual observation and reported as no growth, slight growth (+) medium growth (++) or extended growth (+++). The results are shown in the following Tables 2 and 3.

Table 2 - Inhibition of MICROGARD MG-250 (MG) with methyl vanillin (V) against yeast at pH 5.3 and 6.5 Treatment CFU/m pH 5.3 pH 6.5 None 1.6 x 107 8.3 x 107 0.5% MG 8.5 x 105 -- 1% MG 6.3 x 104 2.3 x 107 500 ppm V 1.3 x 107 5.2 x 107 500 ppm V + 0.5% MG 5.1 x 104 -- 500 ppm V + 1% MG 1.3 x 102 7.2 x 10 Table 3 - Inhibition of MICROGARD MG-250 (MG) with methyl vanillin (V) against mold at pH 5.3 and 6.5 Treatment Growth * pH 5.3 pH 6.5 None f++ +++ 0.5% MG ++ not tested 1% MG + 500 ppm V +++

500 ppm V + 0.5% MG no growth not tested 500 ppm V + 1% MG no growth + Example 4 - In Vitro Testing - MICROGARD and Potassium Sorbate Combinations of MICROGARDS MG-200 with potassium sorbate are evaluated in Potato dextrose broth. The broth is adjusted to pH 6.0 or 4.0, and inoculated with mold spores (Penicillium spp.) at about 3 log spores/ml. The inoculated broth tubes are incubated at 250C for 5 days. Inhibition of mold growth is recorded by visual observation and reported as no growth, slight growth (+), medium growth (++) or extended growth (+++). The results are shown in Table 4.

Table 4 - Inhibition of MICROGARDS MG-200 (MG) with sorbate (S) against mold at pH 4.0 and 6.0 Treatment Mold Growth pH 4.0 pH 6.0 None +++ 0.5% MG ++ 500 ppm Sorbate ++ 1000 ppm Sorbate + 500 ppm S + 0.5% MG no growth no growth 1000 ppm S + 0.5 % MG ~ no growth no growth The above in vitro data demonstrates that MICROGARDS in combination with 500 ppm vanillin or sorbate greatly increases its inhibitory activity against yeast or/and mold at pH 5.3 and 6.5. The synergistic antimycotic effect generated by the combination makes the resulting food product containing the combination more inhibitory against mold and yeast, especially at

higher pH values where neither single component is especially inhibitory.

Example 5 - In Vitro Testing - MICROGARD and EDTA MICROGARDS MG-200 is used in combination with disodium-EDTA in Potato dextrose broth. The broth is adjusted to pH 6.0 or 5.3, and inoculated with mold spores (Penicillium spp. from moldy cheese) at about 3 log spores/ml. The inoculated broth tubes are incubated at 250C for 7 days. Inhibition of mold growth is recorded by visual observation and reported as no growth, slight growth (+), medium growth (++) or extended growth (+++). The results are shown in Table 5.

Table 5 - Inhibition of MICROGARDS MG-200 (MG) with disodium EDTA (EDTA) against mold at pH 5.3 and 6.0 Treatment Mold Growth pH 5.3 pH 6.0 None 1.0% MG ++ +++ ++ 500 ppm EDTA ++ ++ 1000 ppm EDTA ++ + 500 ppm EDTA+ 1.0% MG no growth no growth 1000 ppm EDTA + 1.0 % MG no growth no growth The above in vitro data demonstrates that MICROGARDX in combination with 500 or 1000 ppm EDTA greatly increases its inhibitory activity against mold at pH 5.3 and 6.0. The synergistic antimycotic effect generated by the combination makes the resulting food product containing the combination more inhibitory against mold, especially at higher pH values where neither single component is especially inhibitory.

Having described the invention in detail and by reference to the preferred embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the appended claims.