3,284,212 United States Patent Office Patented Nov. 8, 1966 1. 2 factors are naturally present, it is extremely difficult to 3,284,212 FOOD PRESERVATIVE COMPRISINGENZYMES preserve and store foods without taking abnormal pre AND ANTOXADANTS cautions to avoid these factors. For example, the sur Talmadge B. Tribble and Eugene L. Rondenet, Glenview, face exposure of foods to air greatly accelerates ran III., assignors to Flavor Corporation of America, Chi cidification of fatty components, which action weakens cago, Ill., a corporation of Delaware the natural antioxidants present in foods and thereby No Drawing. Filed Mar. 6, 1963, Ser. No. 263,134 helps to bring about complex adverse effects on enzyme 10 Claims. (C. 99-9) splitting systems which are interlocked in the protein, carbohydrate and fatty food constituents. In this way, This invention relates to improved means for preserv 0. food becomes susceptible to destructive attack by bac ing food. teria, molds and yeasts. More particularly, this invention concerns improved One familiar instance, exemplifying the relationship ingredients to be added to raw foods for the purpose of of air, moisture and heat to the chemical and biological preserving the same against deterioration, principally by changes which may take place in raw food are the ac controlling and regulating factors such as oxidation, 15 tivities of oxygen, microorganisms, enzymes and related microorganism activity and/or other chemical and bio phenomena believed to occur in ensiled foods. It is logical changes. generally believed that within the first 24 hours of stor One of the most important factors producing food ing a raw sillage crop within a silo, the atmospheric deterioration is oxidation. For oxidation occurs when oxygen is extremely active, causing high plant metabolism the oxygen present unites with other chemical elements or 20 and a resulting release of high heat energy accompanied groups of elements in foods to form oxides which cause by increased plant enzyme and microorganism activity. the breakdown of fatty or other carbohydrate food com Within the next 24-hour period, the oxygen demand ap ponents resulting in the ultimate deterioration of protein pears usually to exceed the available oxygen supply so matter. The reactions arising out of and accompanying that plant metabolism is greatly reduced and/or halted oxidation are believed to cause various chemical and bio 25 altogether, while enzyme activity continues with the logical changes resulting in such undesired results as aerobe microorganism activity being negligible, but with rancidification of fats, molding of carbohydrates and anaerobe microorganism activity increasing. Atmos putrefaction of proteins. Basically, the undesirable ef phenic oxidation activity under these latter conditions ap fects of food oxidation appear to stem primarily from pears to be negligible or non-existent, while enzymatic microorganism activity and chemical changes and/or re 30 oxidation activity greatly increases. Within the next 24 actions. hour interval, it is believed that atmospheric oxygen ac All organic foods also contain microscopic forms of tivity ceases altogether, along with plant metabolism, parasitic plant and vegetable life called microorganisms while the enzyme and anaerobe microorganism and bac which live on larger forms of life. Among the most com teria activities sharply increase, accompanied by high mon microorganisms in food, useful to man and animal, 35 enzyme oxidation activity. are bacteria, molds and yeasts. Not all such micro While the foregoing is very generalized, it nevertheless organisms, however, are necessarily harmful or undesir Serves to illustrate the complex and ever-changing nature able. For example, lactic acid bacteria are usefully em of the biological and chemical reactions and resulting ployed in making sauerkraut from fresh cabbage and problems which attend storing food and amply points up silage from fresh grass. In most foods, however, the 40 the necessity of means for controlling and regulating such excessive growth of yeast, bacteria or molds indicates and activities if successful food storage and preservation is to accompanies spoilage. be achieved. Such control and regulation usually re In addition to microorganisms, raw organic foods con quires minimal atmospheric air and moisture concentra tain certain substances called enzymes which appear to tions which, in many circumstances, may be most dif act somewhat as organic catalysts. A common example 45 ficult, if not impossible, to achieve. of such activity is demonstrated in the fruit ripening Aside from the physical difficulties encountered in con process. Even after the fruit is picked, enzyme activity trolling air and moisture, the problem of storing food is continues and if not halted, eventually will cause the further compounded by the reactions which occur within fruit to spoil and rot. Generally speaking, enzymes act 50 the food itself, as the same is transformed into a pre upon foods in two ways, either to add to or reduce water served state. In the case of an ensiled crop, for ex content or take up oxygen. There are three generally ample, such reactions stem from plant respiration, the recognized types of food enzyme activities, lipolytic, presence of enzymes within the plant cells, as well as amylolytic and proteolytic, and each type produces marked the presence of bacteria, yeasts and molds in the raw en effects which can contribute either to the spoilage or the 55 siled plant material. preservation of foods. For instance, lipoxidase, which is In brief, the present invention alleviates many of the a member of the lipolytic enzyme family, acts on fats difficulties and shortcomings heretofore experienced in and may cause butter, for example, to turn rancid. Cel food preservation, as above discussed, by providing im lulase and diastase, contrastingly assist in the preserva proved combinations of selected ingredients which are tion of foods by converting cellulose and starches into 60 added to food as a preserving means. The choice of sugars, alcohol and esters, the latter giving the character such ingredients, according to this invention, is designed to istic flavor to foods. Cellulase is especially effective in regulate and control oxidation processes and accompany hydrolyzing cellulose, while diastase, being high in alpha ing chemical and biological changes which take place in amylase with some beta amylase, is particularly active such a manner as to preserve the food for prolonged on starches. The proteolytic enzyme family include, 65 periods, while maintaining desirable nutritional ingre by way of example, papin, ficin, bromelin and others dients, appearance, color and general palatability values which generally act to hydrolyze and Teduce proteins. of the food. Fundamentally, the improved food preserva It is generally acknowledged that the processes and evo tive of this invention comprises predetermined combina lutions involved in food spoilage and deterioration are tions of selected antioxidants and enzymes which, when Anot fully understood, but it is known that the same are 70 added to foods in predetermined quantities, bring about greatly accelerated by the presence of air, moisture and the desired regulated control of occurring oxidation and heat, particularly in excessive amounts. Since these related chemical and biological activities. Thus, the 3,284,212 3 4. present invention provides improved means for prevent utilizing antioxidant preservatives or enzyme preserva ing food spoilage due to such factors as rancidification of tives or others of the presently recommended and fatty components, the molding of carbohydrates and the recognized preservative additives, as aforementioned, has putrefaction of proteins. In addition, this invention not proven completely effective for preserving ensiled produces noticeably improved results by way of retaining 5 foods and preventing spoilage. For instance, when such natural food coloring, taste, odor and general palatability known additive materials, according to present knowledge over prolonged time periods. and practice, are applied to ensiled forage crops, even The main object of this invention is to provide new and under ideal conditions of harvesting and storage, the same improved means for preserving food. have not produced materially better results by way of Another important object of this invention is to pro preserving nutrients, color, flavor and general palatability vide new and improved food preserving means comprising 10 than is obtainable by following recommended air and combined antioxidant and enzyme materials used as a food moisture control procedures without the benefit of such additive for preventing spoilage thereof. additives. Further, even in those instances where such An additional object of this invention is to provide new additives or preservatives have resulted in apparently and improved food preserving means which are effective better preserving effects, such effects have not proven to to avoid food spoilage over extended periods of time. be long-lasting so that the ultimate problem of spoilage A still further object of this invention is to provide a and waste has not been successfully met and wholly new and improved food preserving means, as set forth in avoided thereby. the preceding object, which, in addition to its marked Our present invention, whereby antioxidants are com ability to preserve food for extended periods, is also dis 20 bined with enzymes, by way of contrast, is highly effective tinguished by its capability of preserving desired nutri for preserving foods over extended periods. As a specific tional and palatable qualities therein. illustration of its merits and features, the same has proven Still another object of this invention is to provide a particularly effective in preserving ensiled foods, includ new and improved food preservative which, when applied ing but not necessarily limited to such silage foods and to raw, stored food preserves the same over extended 25 crops as grass, legume, corn and sorghum, immature periods of time while maintaining desirable nutritional grains converted to wet silage foods, mature grains, hay qualities therein without appreciable deterioration of the and other like feed and food materials and mixtures as foods' natural color, odor and taste. are normally prepared and stored by the farmer. This A still further important object of this invention is to will appear from the teachings and observed results provide a new and improved means for preserving food 30 related hereinafter. which is economical and simple to use and substantially In practicing the present invention on ensiled foods eliminates food spoilage and waste. and comparing the silage produced thereby with results Having thus described our invention, the best mode obtained under comparative conditions according to the presently contemplated for carrying out its teachings and best previously known ensiling and preservation practices, concepts shall now be set forth so as to enable those the following occurred: skilled in the art to which it pertains to make and use Silo I the same. Because of their markedly higher moisture content, An alfalfa hay crop was selected and an untreated por greater exposure to atmospheric oxygen and environmental tion thereof was ensiled in a first silo under the best known temperature fluctuations, raw silage foods present one of recommended procedures of air, moisture and storage con the most difficult food categories to preserve without ap 40 trol practice. After three weeks of ensiling, the silage preciable loss of available nutrients, flavor and general had the following content assay: palatability, particularly over extended periods. Previous to this invention, such silage foods were preserved, according to the best recognized principles and practices, either by carefully controlled harvesting, drying and storage procedures, directed principally toward the SILAGE I regulation and control of moisture and oxygen, or by adding to such natural or raw silage foods any of a variety Assay For- As Is IDry Wt of commercially available preservative materials. Such 50 Moisture, percent------78.12------materials include, by way of example, sodium metabi Ash, percent----- 1.96------8.95 sulphite, zinc bacitracin antibiotic, various antioxidants, Fat, percent.---- 0.62------2.83 Protein, percent. 6.68------30.53 enzymes, mineral acids and some natural carbohydrates Fibre, percent------4.95. 22.63 and starch materials, such as molasses and potato pulp. Carbohydrate, percent 7.67---- 35.05 pH (acid).------Among such known and recognized preservative ma f3 Caroteine--- 5.60 mg.fib-i- terials, the antioxidants have been considered most promis 55 Tocopherol.-- 1.00 mg./100gms. - Odor------AcidiciButyric------ing due to their recognized capacity to restrict oxidation Taste----- Sour Rancid and to retain natural carotenes and tocopherols present Color------Dark------in forage crops. Among the more common antioxidant materials utilized for this purpose, chiefly because they are presently considered non-toxic and safe for use in 60 foods, are butylated hydroxytoluene, butylated hydroxy anisole, propyl gallate, ethoxyquin, nordihydroguaiaretic acid, ascorbic acid and tocopherol. Such antioxidants are Silo II generally utilized either alone or with two or more there of in combination. It is also common practice to utilize 65 A Second portion of the same hay crop was also ensiled such antioxidant materials in combination with acids, such in a Second silo and treated with a recognized commercial as citric acid, to increase and promote their general ly available antioxidant preservative formulated as effectiveness. follows: Enzyme materials have also been resorted to because of Parts by weight their apparent ability to increase carbohydrate content re 70 Butylated hydroxytoluene (BHT) ------3. sulting in the conversion of complex starches into simple, Ethoxyquin ------3 digestible sugars. The enzymes are also believed to use Citric acid ------2 up available water or moisture so long as they remain Such antioxidant preservative was added to the hay by active. dusting eight grams thereof over each ton of hay accord For the most part, however, such past practice of 75 ing to recommended and recognized procedure for its uti 8,284,212 5 6 lization. After three weeks of ensiling, the following con In considering the above-indicated findings and observa tent assay of the resulting silage was observed: tions, it will be noted that the preservative formulation, SILAGE II according to this invention and utilized in sillage IV, is com posed of the same ingredients and proportionate amounts Assay For- As Is Dry Wt. 5 thereof employed in the antioxidant preservative for Silage II, in combination with the same ingredients and propor Moisture, percent------76.76------Ash, percent------2.11---- 9. 07 tionate amounts thereof found in the enzyme preservative Fat, percent------0.80 3.44 used in silage III. The resulting silages therefore afford a Protein, percent-- 6.87. 29.56 Fibre, percent.------5.8 22, 28 0 true comparison between the results obtained by using the Carbohydrate, percent 8.28 antioxidant and enzyme preservative ingredients alone and pH (acid)------3 Caroteine--- the results obtained by their combined effect according to Tocopherol.-- 4.20 mg./100gms------our present invention. Odor------AcidiciButyric------Comparing the above observed results, it will be readily Taste--- Souri Rancid.--. recognized that silage IV, produced according to the pres ent invention, is vastly superior and improved over any of the silages I-III, while silages II and III, utilizing antioxi Silo III dant preservatives and enzyme preservatives, respectively A third portion of the same hay crop was ensiled in a are not particularly superior to silage I, produced by air third silo utilizing a recognized commercial enzyme and moisture control practices. preservative added according to manufacturers' recom 20 More specifically, silage IV desirably contains signifi mended practice and procedures. Such enzyme preserva cantly less ash than any of the other three produced si tive was compounded as follows: lages. The fat content of silage IV, while somewhat less than the lowest corresponding factor observed in silages Parts by weight I-III, is nevertheless compartive in all four silages. The Diastase ------6 protein value of all four silages is substantially equivalent, Cellulase ------1. 25 while the fibre content of silage IV is substantially less This preservative was added to the hay by dusting the than the corresponding factor in any of the other three si same thereover in the ratio of seven grams of enzyme pre lages. This latter factor is most desirable since fibre is not servative per ton of hay. After three weeks of ensiling, particularly nutritional. the resulting silage had the following content assay: 30 In comparing carbohydrate content of the four silages, it will be observed that the carbohydrate contents of each SILAGE III of the several silages I-III are substantially equal, but that such factor in silage IV, produced according to the present Assay For- As Is invention, is vastly increased thereover. By comparing the Moisture, percent Wet or 'as is' percentages for the several silages, for ex Ash, percent. Fat, percent--- ample, it will be noted that silage IV has a carbohydrate Protein, percent-- - content which is substantially 169% of the carbohydrate Fibre, percent.------6.04---- 24.02 content for silage III, produced with the enzyme preserva Carbohydrate, percent---- 9.08---- 36.17 pH (acid).------5.65------40 tive and exhibiting the greatest carbohydrate percentage B caroteine--- 7.30 ling.ilb.------of any of silages I-III. As compared to silage I, produced Tocopherol 3.60 mg. 100gms Odor------Acidic, Butyric under the best known procedures of air and moisture con Taste Souri Rancid trol, Silage IV contains more than twice the carbohydrate Color.-- Dark------Content. In considering the important beta carotene content of the several silages, it will be recognized that silage IV, Silo IV produced in accordance with this invention, again exhibits A fourth portion of the same hay crop was treated ac vastly superior results in that factor. For example, the cording to the preservation practices of the present inven beta caroteine content of silage IV is in excess of 200% tion with a combined antioxidant and enzyme preserva of the corresponding factor in silage I; is some 146% of tive having the following formulation: 50 the beta caroteine in silage II; and some 1.78% of the Parts by weight beta carotene in silage III. Diastase ------6 It will also be observed that the tocopherol content in Ethoxyquin ------3 silage IV, while somewhat less than the corresponding Butylated hydroxytoluene (BHT) ------3 factor in silos II and III, is nevertheless of comparative Citric acid ------2 55 value thereto, while substantially exceeding the tocoph Cellulase ------1. erolsilage content I. of the air and moisture control produced This preservative was added to the hay by dusting sub Of further importance are the comparative pH values stantially 15 grams thereof over each ton of hay which was of the several produced silages. According to accepted 60 authority, a pH value of between 4.0 and 5.0 is considered then ensiled in a fourth silo. After three weeks of ensil most desirable and important for complete preservation ing, the resulting silage had the following content assay: of silage. Therefore, the pH value of 4.5 produced in SILAGE IV silage IV, according to this invention, is most significant and indicative of the achievement of the desired food Assay For- As Is Dry Wt. 65 preserving objective. By comparison with the other Moisture, percent------69.10------observed silages, the same demonstrates significant im Ash, percent------2.90.------7.30 provement over the indicated 5.7 pH value in the air and Fat, percent------0.69--- 2.23 Protein, percent--- 8.85. 28.64 moisture controlled silage I, the 5.2 pH value for the anti Fibre, percent.------3.80------12.29 Oxidant produced silage II and the 5.65 pH value for Carbohydrate, percent --- 15.37 ---. 49.74 70 the enzyme treated silage III. pH (acid)-- It is significant that of all four silages observed only Silage IV exhibited a retention of desirable natural odor, period.taste and color factors after the three-week ensiling While the foregoing exemplifies the working character 3,284,212 7 3. istics of this invention and establishes the same as pro bag and stored. A corresponding amount of untreated ductive of superior results when used for preserving en corn meal was likewise placed and stored in another air siled foods, as compared to the best known procedures of tight plastic bag. At the end of six months, the two air and moisture control, the use of antioxidant preserva plastic bags were opened and the corn meals compared. tives alone or the use of enzyme preservatives alone in 5 The corn meal treated with the preservative of our in silage production, experience has also shown that this vention had remained in a good state of preservation, invention is not restricted to that particular class of food with good fresh odor and taste, while the untreated corn preservation. Neither is the same restricted to the Specific meal had developed a definite stale odor and spoiled formulation of preservative ingredients set out herein taste. above, although based on experience to date, it is believed O To establish the effectiveness of this invention under that such indicated formulation of ingredients represents accelerated conditions of extreme moisture and heat, the a preferred embodiment of this invention for general following procedure was carried out: application. For instance, while the above-indicated Samples of the six-month old bagged hay and corn meal, preservative formula according to this invention specifies treated with the preservative of this invention described ethoxyquin and BHT antioxidants in equal proportionate immediately hereinabove, were diluted with an additional amounts, such proportions, while preferred, may be 50% amount of tap water and placed in separate open varied, and the amounts of the antioxidants also SucceSS flasks. The samples were then incubated at 39° C. for a fully may be varied from about one gram to about One period of two months. After two months in such an pound per ton of food. It has also been determined atmosphere designed to rapidly accelerate spoilage due that other equivalent antioxidants may be substituted 20 to extremely hot and humid climatic conditions, there were successfully for these particular ingredients, such as the no observed indications of rancidity, mold or decay pres antioxidants presently utilized in the food preserving art ent in any of the samples. and listed heretofore. From the foregoing, it is believed that those familiar In a like manner, the enzyme ingredients "diastase' with the art will readily recognize and appreciate the new and “cellulase' have been employed in amounts ranging 25 and improved food preserving means of this invention from about A00 gram to 50 grams per ton of food and which is effective over extended periods of time and which the specified proportions of such ingredients accordingly is demonstrative of marked improvements over previously varied. Also, equivalent enzymes, of the amylolytic, known preservative means for this purpose. While the lipolytic and proteolytic families, as previously men characteristics and features of the present invention have tioned, have been successfully substituted for the partic been described and related to particular observed results ular enzyme ingredients specified in our above preserva and related to an exemplary formula of ingredients, the tive formula. same is nevertheless susceptible to wide variation, modi In order to determine the lasting quality of the in fication and substitution of equivalents without departing proved preservative results obtained by the present inven from the spirit and scope of this invention. It is intended tion, particularly in silage foods, each of the above therefore that the present invention be unlimited by the specified exemplary silages I-IV was observed after six foregoing illustrative description thereof, except as may months of ensiling, with the following results: appear in the following appended claims. Silages I-III, treated according to recommended air We claim: and moisture control procedures, the antioxidant pre . A food preservative comprising in combination, servative procedure and the enzyme preservative proce 40 diastase, cellulase, ethoxyquin, butylated hydroxytoluene dure, respectively, each developed foul, putrid, moldy and citric acid; the antioxidants and enzymes respectively and rancid odors, significant loss of color and definite being combined substantially in the ratio of 1:1 by weight, Sour and rancid tastes. However, after six months, silage and the enzymes being combined substantially in the ratio IV, which was treated with the preservative according to of 6:1 by weight, diastase to cellulase. our invention, showed no signs of mold or putrefaction 2. A food preservative comprising non-toxic anti or any significant loss of color or deterioration of taste oxidants and enzymes combined in substantially equal from that observed at initial ensiling time. As a bene proportionate amounts by weight of antioxidants and ficial result of this improved durability of preservative enzymes; the antioxidants comprising ethoxyquin and effect, experience has shown that there is little or no feed butylated hydroxytoluene and the enzymes comprising a ing waste in silages preserved according to this invention. major proportion of diastase and a minor proportion of To determine the effectiveness of the present inven cellulase. tion to preserve and maintain fresh odors and tastes in 3. A food preservative comprising in combination: dry foods, as opposed to wet foods previously discussed, one ton of hay was treated with a liquid composition of Parts by weight 15 grams of the improved preservative of our invention, Diastase ------6 formulated as above indicated, one lb. of dextrose and Ethoxyquin ------3 enough water to make one gallon of liquid. The one Butylated hydroxytoluene ------3 ton of hay was then tumbled with the one gallon of such Citric acid ------2 liquid composition and a portion of the treated hay was Cellulase ------1. then encased within an air-tight plastic bag. At the 60 4. In a food preservative, the combination comprising Same time, an equal portion of untreated hay was like 3 parts by Weight ethoxyquin, 3 parts by weight butylated Wise encased in another air-tight plastic covering and hydroxytoluene and 7 parts by weight diastase and cellu Stored alongside the treated hay. After six months of lase, there being a substantially greater proportion by Storage, the plastic coverings were removed from the weight of diastase. treated and untreated hay. It was observed that the hay 5. In a food preservative, the combination comprising treated with the preservatives according to our invention 3 parts by Weight ethoxyquin, 3 parts by weight butylated had retained its original fresh odor and taste without any hydroxytoluene, 6 parts by weight diastase, and 1 part by indication of mold or other deterioration factors. Con Weight cellulase. trastingly, the untreated hay had developed a marked 6. In a food preservative, the combination comprising musty and stale odor and a moldy, rancid taste. 70 3 parts by weight ethoxyquin, 3 parts by weight butylated In still another practice of our invention, one ton of hydroxytoluene, 2 parts by weight citric acid, and sub ground corn meal was tumbled with one gallon of the stantially 6 parts by weight diastase and 1 part by weight liquid composition of water, dextrose and our preserva cellulase. tive, formulated as hereinabove set forth. The treated 7. In a food preservative, the combination comprising corn meal was then placed in a closed, air-tight plastic 3 parts by Weight ethoxyquin, 3 parts by weight butylated 3,284,212 9 10 hydroxytoluene, 2 parts by weight citric acid, and 7 parts diastase and 1 part by weight cellulase; 1 pound of dex by Weight enzymes consisting of diastase and cellulase. trose, and the remainder water. 8. In a food preservative, the combination comprising equal parts by weight of each ethoxyquin and butylated References Cited by the Examiner hydroxytoluene antioxidants, and enzymes consisting of cellulase diastase in amounts substantially equal by weight UNITED STATES PATENTS to the combined amounts of said antioxidants. Re. 23,523 7/1952. Baker ------99-150 9. A preserved ensiled food comprising ensiled food, 2,935,449 5/1960 Bavley. 3 parts by weight of each of the antioxidants ethoxyquin 2,988,449 6/1961 Hollenbeck. and butylated hydroxytoluene; 2 parts by weight citric 3,006,815 10/1961 Scott. acid, 6 parts by weight of the enzyme diastase, and 1 part O 3,051,572 8/1962 Tribble ------99-2 by weight of the enzyme cellulase; the amounts of said 3,063,912 11/1962. Otaka ------195-63 enzymes being from about A00 gram to about 50 grams 3,141,775 7/1964 Surgant ------999 per ton of ensiled food and the amounts of said anti 3,170,794 2/1965 Geffreys et al. ------99-9 oxidants being from about 1 gram to about 1 pound per 5 3,232,832 2/1966. Ono ------195-62 ton of ensiled food. 3,249,442 5/1966 Keyes et al. ------99-2 10. A liquid composition for use in preserving dry 3,250,622 5/1966 Brooks. foods comprising, per gallon of liquid, substantially 15 grams of a dry preservative compound comprising 3 parts A. LOUIS MONACELL, Primary Examiner. by weight of each ethoxyquin and butylated hydroxy 20 D. J. DONOVAN, Assistant Examiner. toluene, 2 parts by weight citric acid, 6 parts by weight