BACKGROUND OF THE INVENTION [0002] Control of bacteria in animal feed is an ongoing challenge for the industry, even more so as consumers focus on the health and safety of the meat they feed their families. Control of bacteria is desirable to protect the health of the animal for which the feed is intended. Gut bacteria in the animal compete for nutrients and can be detrimental to the health and performance of the animal. Second, and of growing importance, the reduction of bacteria in feed is an important part in the reduction and control of food borne diseases.

[0003] The use of antibiotics in animal feed has recently come under scrutiny, in part due to concerns that overuse of antibiotics may result in resistance to the antibiotics in the animals treated. Additionally, upon consumption of animals treated with antibiotics, humans may

experience problems due to allergies to the antibiotics, or in becoming resistant to the antibiotics as well. Thus, a need exists for alternative means of inhibiting bacteria in animal feed.

[0004] Two main alternatives are currently available for control of bacteria in feed: thermal and chemical methods. Heat treatment of feed is costly, but efficient.

However, heat treatment alone does not avoid re- contamination of the feed between the time the feed is treated and consumed by the animal.

[0005] Chemical treatments primarily involve the use of formaldehyde or an organic acid to the feed.

Formaldehyde has been used extensively in the UK, although its use remains under scrutiny by the EU Commission.

[0006] Organic acids, on the other hand, have many applications in the animal feed industry worldwide. For many years, nutritionists have used organic acids in piglet diets for their positive effect on health and growth. For instance, formic acid is used to decontaminate raw materials, and propionic acid is used to control mold.

Other organic acids commonly used include fumaric, citric and lactic acids. Organic acids are also known to modify the gastrointestinal flora.

[0007] Approvals for the use of non-therapeutic antibiotics in animal feed have been withdrawn in several countries and the practice of feeding low-dose growth promoters is fast disappearing worldwide. Already, producers who want to sell in the drug-free market are searching for non-pharmaceutical replacements for feed antibiotics. Most data suggest that the growth-promoting effect of antibiotics can be entirely ascribed to their antimicrobial activity and the physiological repercussions from that. Thus, the search for replacements has focused on naturally occurring molecules with known anti-microbial activity.

[0008] The primary effect of antibiotics is antimicrobial; all of the digestibility and performance

effects can by explained by their impact on the gastrointestinal microflora and the resulting reduction in immune stimulation. Organic acids have antimicrobial activity; however, there appear to be effects of organic acids beyond those attributed to antimicrobial activity.

Reductions in certain species of bacteria are associated with feeding organic acids, which are particularly effective against acid intolerant species like E. coli, Salmonella and Campylobacter. Both antibiotics and organic acids improve protein and energy digestibility by lowering the incidence of background immune stimulation and the resulting synthesis and secretion of immune mediators, by reducing production of ammonia and other growth-depressing microbial metabolites and perhaps by reducing the overall microbial load. Unlike antibiotics, the antimicrobial activity of organic acids is pH dependent. Organic acids have a clear and significant benefit in weanling piglets, and have been observed to benefit poultry performance.

Organic acids have several additional effects that go beyond those of antibiotics. These include reduction in digesta pH and increased pancreatic secretion.

[0009] Pigs are susceptible to weaning stresses (separation from sows, environmental changes, and physical effects of solid feed) and a variety of pathogens such as E. coli and rotavirus. These pathogens are reduced in adult animals by the reduction of pH in the stomach, but young pigs have lower hydrochloric acid secretion from the stomach. In addition, the failure to acidify gastric contents coupled with low pancreatic enzyme secretion can lead to insufficient nutrient digestion and also increase the susceptibility of weaning pigs to enteric diseases.

[0010] A number of studies have documented the effects of organic acids on performance in young swine, particularly early weaned piglets. A recent publication by Partanen ("Organic acids: their efficacy and modes of action in pigs, "in Gut Environment of Pigs, p. 201, Piva, A. et al. , eds, Nottingham University Press, Nottingham, UK

(2001) ) reviews the literature in this area and provides the results of a meta-analysis of existing data. Only studies using individual acids in the absence of antibiotics and copper are considered. In the analysis of 46 weaned piglet and 23 fattening pig trials, significant feed-to-gain improvements were seen with formic, fumaric, and citric acids and also with calcium diformate. Weight gain and feed intake effects were significant for formic acid and diformate. The author concludes that dietary acids have a beneficial effect, especially on weaned piglets, that is primarily associated with changes in the gastrointestinal microflora. See Eidelsburger et al. , J.

Anim. Physiol. Anim. Nutr. 68: 82-92 (1992).

[0011] Without being limited to a particular theory, it is believed that one mechanism for the action of organic acids as antimicrobials is as follows. The acid changes the microbial populations in accordance with its antimicrobial spectrum of activity. Once inside the cell, the change in pH causes the dissociation of these weak acids. The multiple effects of organic acids are due to this intracellular dissociation and the cellular response to it.

The antimicrobial activity is a result of the deleterious effects of the free proton and, perhaps, the free anion on the bacterial or fungal cell. In the gut enterocytes, the dissociation is thought to result in the synthesis of secretin, a hormone that stimulates pancreatic secretion.

Thus, organic acids have benefits that go beyond antimicrobial activity. For feeds, the activity to control fungal growth dominates, while in the gut, the populations being affected are primarily the bacteria whose growth is most affected by acidic conditions. It should be emphasized, however, that the mechanism of action of organic acids is quite different from, and in addition to, that of inorganic acids such as HC1. See, e. g., Eidelsburger et al., supra. The importance of low pH on the antimicrobial activity of organic acids can be explained by its effect on the dissociation of the acid. At low pH, more

of the organic acid will be in the undissociated form.

Undissociated organic acids are lipophilic and can diffuse across cell membranes, including those of bacteria and molds. See Huyghebaert, Report: CLO-DW (1999) and Eidelsburger et al. , supra. Once in the bacterial cell, the higher pH of its cytoplasm causes dissociation of the acid, and the resulting reduction in pH of the cell contents will have the effects of disruption of enzymatic reactions and nutrient transport systems. See Cherrington et al., Adv.

Microb. Physio. , 32: 87-108 (1991). In addition, the process of transporting the free proton out of the cell is energy requiring and this contributes to reduced energy availability for proliferation, resulting in some degree of bacteriostasis. This direct antimicrobial activity is believed to be responsible for feed and food sanitation effects that contribute to the use of organic acids as preservatives.

[0012] Most food production animals, especially pigs, poultry and cattle, require supplemental methionine in their diets for proper growth and reproduction. 2-hydroxy- 4- (methylthio) butanoic acid (HMBA, sold under the trade name Alimet by Novus International, Inc. (St. Louis, Missouri) ) is a popular source of supplemental methionine for animal diets. Alimets has become a preferred source over powder d, l-methionine (DLM), for feed mills wanting to solve the common problems associated with increased production capacity because of the efficiency advantages of the physical form of liquids. Easier bulk handling, accurate dosage, elimination of packaging with its disposal and the inventory shrink issues, as well as dust reduction, are popular features. Alimet contains 88% methionine activity, while liquid DLM contains only 40% methionine activity. This low level of relative activity in DLM means water takes the place of valuable energy and protein components in the feed, reducing nutrient density.

[0013] Preventing or delaying the growth of mold in animal feed compositions is beneficial, in that less feed

is lost to spoilage, and illnesses associated with the molds or toxins they produce can be avoided.

[0014] Mold in feed rations can render the feed unfit for consumption. Moldy feed may decrease the digestibility and/or palatability of the feed, both of which can adversely affect production and health of the animal.

Additionally, many molds produce mycotoxins which affect the nutrient value of feed, or which may be hazardous to the health of animals, including livestock and humans.

Aspergillus aflatoxin B1 mycotoxin is a potent liver carcinogen; certain Penicillium mycotoxins affect liver or kidney function; and Fusarium mold species are associated with pulmonary edema in swine, liver cancer in rats, and abnormal bone development in chicks and pigs. United States Department of Agriculture, "Grain Fungal Diseases & Mycotoxin Reference, "available at http://www. usda. gov/gipsa/pubs/pubs. htm. Pigs are particularly sensitive to the presence of Fusarium mycotoxins, especially deoxynivalenol (DON), also known as vomitoxin.

[0015] Detoxifying feed which has been contaminated with mycotoxins can be quite difficult, and often is accomplished only by subjecting the feed to extreme processing conditions. For example, corn contaminated with aflatoxin, a mycotoxin produced by the Aspergillus species of mold, can be detoxified by treating the corn under pressure with hot, moist ammonia. Thus, the need exists for a way to prevent formation of mold and the mycotoxins they may produce.

[0016] Mold growth in untreated, stored feeds is especially prevalent in hot, humid conditions. Higher temperatures increase the chance for mold growth, particularly when coupled with high moisture levels in the feed. In chicken feed, mold growth rarely occurs in grains containing less than 14-15% moisture. However, even feeds with a low average moisture level may have pockets with high moisture levels, producing microclimates ideal for

mold growth. This often happens in warmer climates, especially where cool night time temperatures cause condensation inside silos or bins where grains or feed is stored. Even these relatively small pockets of mold growth can be problematic; mycotoxins can be problematic to birds at parts-per-million levels (e. g. , tricothecenes, produced by the Fusarium species, and aflatoxins, produced by the Aspergillus species).

[0017] It is known that certain feed additives prevent mold growth (i. e. , act as antifungals) or bind existing mycotoxins. Such additives are often used where moisture control in grain or mixed feed is logistically difficult. The use of certain organic acids, including propionic and acetic acid, as mold inhibitors is known.

Adsorbents to bind mycotoxins include aluminosilicates and clay loam products, whose chemical structure allow the capture of aflatoxin, and perhaps other mycotoxins.

Mycotoxin adsorbents have variable efficacy, due in part to their variable binding characteristics. Use of a separate mold inhibitor or mold adsorbent increases the costs associated with animal feed.

[0018] A previous study (Doerr et al., Poultry Science, 74 (1), 23 (1995) ) suggested that Alimets may reduce the growth of mold in samples of Sabouraud's dextrose broth, potato dextrose agar, or ground corn (19% moisture) treated with either Aspergillus parasiticus, Aspergillus ochraceus, or Fusarium moniliforme, and in ground corn (17. 5% moisture) with its naturally occurring fungal flora.

[0019] Enhancing the palatability of animal food is an endless endeavor by food manufacturers. Addition of palatants to the food is desirable as a means to increase acceptance by the animals, resulting in improved health of the animal, increased weight gain, etc. Palatants are frequently used in foods for canines, felines, and aquaculture.

SUMMARY OF THE INVENTION [0020] Accordingly, the present invention provides methods for inhibiting bacteria in animal feed.

[0021] In another aspect, the present invention provides methods for inhibiting mold in food, food ingredients, and animal feed compositions.

[0022] In another aspect, the present invention provides an anti-bacterial composition comprising a compound of Formula I, as defined herein, for use in inhibiting bacteria in animal feed.

[0023] In yet another aspect, the present invention provides an anti-bacterial composition comprising a compound of Formula I and one or more organic acids for use in inhibiting bacteria in animal feed.

[0024] Briefly, therefore, the present invention is directed to an anti-bacterial composition comprising a compound of Formula I, as defined herein.

[0025] The present invention is also directed to an anti-bacterial composition comprising a compound of Formula I and one or more organic acids.

[0026] The present invention is also directed to a method for inhibiting bacteria in animal feed, said method comprising treating said feed with an anti-bacterial composition comprising a compound of Formula I.

[0027] The present invention is also directed to a method for inhibiting bacteria in animal feed, said method comprising treating said feed with an anti-bacterial composition comprising a compound of Formula I and one or more organic acids.

[0028] The present invention is also directed to a method for inhibiting bacteria in silage, said method comprising treating said silage with an anti-bacterial composition comprising a compound of Formula I.

[0029] The present invention is also directed to a method for inhibiting bacteria in silage, said method comprising treating said silage with an anti-bacterial

composition comprising a compound of Formula I and one or more organic acids.

[0030] The present invention is also directed to a method for inhibiting bacteria in animal feed comprising computing the concentration in said feed of a compound of Formula I necessary to inhibit bacteria present in said animal feed, and applying said compound of Formula I to said feed in a proportion sufficient to achieve said concentration.

[0031] The present invention is also directed to a method for inhibiting bacteria in animal feed comprising heat-treating said animal feed, computing the concentration in said feed of a compound of Formula I necessary to inhibit bacteria present in said animal feed, and applying said compound of Formula I to said feed in a proportion sufficient to achieve said concentration.

[0032] The present invention is also directed to a method for inhibiting bacteria in animal feed comprising computing the concentration in said feed of said compound of Formula I and another organic acid or mixture of organic acids necessary to inhibit bacteria present in said animal feed, and applying said compound of Formula I and said organic acid or mixture of organic acids to said feed in a proportion sufficient to achieve said concentration.

[0033] The present invention is also directed to a method for inhibiting bacteria in animal feed comprising heat-treating said animal feed, computing the concentration in said feed of a compound of Formula I and said organic acid or mixture of organic acids necessary to inhibit bacteria present in said animal feed, and applying said compound of Formula I and said organic acid or mixture of organic acids to said feed in a proportion sufficient to achieve said concentration.

[0034] The present invention is also directed to a method of method for inhibiting mold in animal feed comprising directly or indirectly making information available for computing the concentration in said feed of a

compound of Formula I necessary to inhibit bacteria present in said animal feed which has been heat-treated, and directly or indirectly making information available for applying said compound of Formula I to said feed in a proportion sufficient to achieve said concentration.

[0035] The present invention is also directed to a method of method for inhibiting mold in animal feed comprising directly or indirectly making information available for computing the concentration in said feed of a compound of Formula I and another organic acid or combination of organic acids necessary to inhibit bacteria present in said animal feed, and directly or indirectly making information available for applying said compound of Formula I and said organic acid or combination of organic acids to said feed in a proportion sufficient to achieve said concentration.

[0036] The present invention is also directed to a method for inhibiting bacteria in animal feed, said method comprising heat-treating the feed in combination with treating said feed with an anti-bacterial composition comprising a compound of Formula I and comprising one or more organic acids.

[0037] The present invention is directed to a method of inhibiting mold in an animal feed composition, the method comprising applying a compound of Formula I to said feed composition, wherein said feed composition comprises corn and soy.

[0038] The present invention is also directed to a method for delaying the formation of mold in an animal feed composition, the method comprising applying a compound of Formula I to said feed composition, wherein said feed composition comprises corn and soy.

[0039] The present invention is also directed to a method of inhibiting the formation of mold in an animal feed composition, the method comprising applying a compound of Formula I to said feed composition, wherein said feed composition has a moisture content of about 17% or less.

[0040] The present invention is also directed to a method for inhibiting mold in silage, said method comprising treating said silage with an anti-fungal composition comprising a compound of Formula I.

[0041] The present invention is also directed to a method for inhibiting mold in silage, said method comprising treating said feed with an anti-fungal composition comprising a compound of Formula I and one or more organic acids.

[0042] The present invention is also directed to a method for inhibiting the growth of mold in an animal feed composition, the method comprising computing the concentration in said feed composition of a compound of Formula I necessary to inhibit the growth of mold in said feed composition; and applying said compound of Formula I to said feed composition in an amount sufficient to achieve said concentration.

[0043] The present invention is also directed to a method for inhibiting the growth of mold in animal feed comprising computing the concentration in said feed of a compound of Formula I necessary to inhibit the growth of mold in said animal feed; and applying said compound of Formula I to said feed in said concentration.

[0044] The present invention is also directed to a method for improving the mold resistance of an animal feed composition, the method comprising discontinuing the use of DL-methionine as a feed supplement; computing the concentration in said feed of a compound of Formula I necessary to inhibit the growth of mold in said animal feed; and applying said compound of Formula I to said feed in an amount sufficient to achieve said concentration.

[0045] The present invention is also directed to a method for inhibiting mold in animal feed comprising directly or indirectly making information available for computing the concentration in said feed of a compound of Formula I necessary to inhibit mold present in said animal feed; and directly or indirectly making information

available for applying said compound of Formula I to said feed in an amount sufficient to achieve said concentration.

[0046] The present invention is also directed to method for inhibiting mold in animal feed comprising directly or indirectly making information available for computing the concentration in said feed of a compound of Formula I and another organic acid or mixture of organic acids necessary to inhibit mold present in said animal feed; and directly or indirectly making information available for applying said compound of Formula I and said organic acid or mixture of organic acids to said feed in an amount sufficient to achieve said concentration.

[0047] The present invention is also directed to the use of a compound of Formula I in the manufacture of a nutrient composition for inhibiting mold in animal feed by treating said feed with said nutrient composition.

[0048] The present invention is also directed to the use of a compound of Formula I and one or more organic acids in the manufacture of a nutrient composition for inhibiting mold in animal feed by treating said feed with said nutrient composition.

[0049] The present invention is also directed to a method of inhibiting mold in an animal feed composition, the method comprising monitoring the concentration of methionine supplement in said feed composition, adding additional amounts of said methionine supplement as needed to achieve an anti-mold effective concentration of methionine supplement in said feed composition.

[0050] The present invention is also directed to a method of enhancing the palatability of animal food, particularly dog and cat food, and food for aquaculture.

[0051] Other objects and features will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS [0052] Figure 1A is a graph illustrating the effect of varying doses (0.108, 0.3, and 0.83 g/L) of formic acid

and Alimets at pH 4.5 and 6.75 on the number of colony forming units of S. enteritidis after 4 hours.

[0053] Figure 1B is a graph illustrating the effect of varying doses (0.108, 0.3, and 0.83 g/L) of formic acid and Alimet at pH 4.5 and 6.75 on the number of colony forming units of E. coli after 4 hours.

[0054] Figure 1C is a graph illustrating the effect of varying doses (0.108, 0.3, and 0.83 g/L) of formic acid and Alimets at pH 4.5 and 6.75 on the number of colony forming units of L. plantarum after 6 hours.

[0055] Figure 1D is a graph illustrating the effect of varying doses (0.108, 0.3, and 0.83 g/L) of formic acid and Alimets at pH 4.5 and 6.75 on the number of colony forming units of C. jejuni after 6 hours.

[0056] Figure 2A is a graph illustrating the pH- dependent antibacterial effect of formic acid and Alimet on the number of colony forming units of S. enteritidis.

[0057] Figure 2B is a graph illustrating the pH- dependent antibacterial effect of formic acid and Alimets on the number of colony forming units of E. coli.

[0058] Figure 2C is a graph illustrating the pH- dependent antibacterial effect of formic acid and Alimets on the number of colony forming units of L. plantarum.

[0059] Figure 2D is a graph illustrating the pH- dependent antibacterial effect of formic acid and Alimet on the number of colony forming units of C. jejuni.

[0060] Figure 3 is a graph illustrating the effect of varying doses (1,3 and 5 g/L) formic acid and Alimets on the number of colony forming units of S. enteritidis after 4 hours at pH 4.5 and 6.75.

[0061] Figures 4A and 4B are graphs illustrating the effect of varying doses of a combination of formic acid and Alimets on the number of colony forming units of S. enteritidis after 4 hours at pH 4.5 and 6.75.

[0062] Figure 5 is a graph comparing the effects of hydrochloric acid, formic acid, lactic acid, and Alimets on

the number of colony forming units of E. coli over time, at pH 4 and 7.3.

[0063] Figure 6 is a graph showing the effect of moisture level on the number of colony forming units of Salmonella in meat meal premix.

[0064] Figure 7 is a graph showing the percent recovery of Salmonella for different levels of Alimets in meat meal premix containing 20% moisture.

[0065] Figure 8 is a graph showing the effect of Alimets on the number of colony forming units of Salmonella in meat meal premix containing 20% moisture.

[0066] Figure 9 is a graph illustrating the % C02 in the headspace for a starter mash with a moisture level of 16.8% having no DLM or Alimet@ ; with 0. 2% DLM; and with 0.2% Alimet@.

[0067] Figure 10 is a graph illustrating the % C02 in the headspace for a starter mash with a moisture level of 14. 8% having no DLM or Alimet@ ; with 0.2% DLM; and with 0.2% Alimet@.

[0068] Figure 11 is a graph illustrating the % C02 in the headspace for a starter mash with a moisture level of 12. 8% having no DLM or Alimet@ ; with 0.2% DLM; and with 0. 2% Alimet@.

[0069] Figure 12 is a graph illustrating the % C02 in the headspace for a starter mash with a moisture level of 14.8% having no DLM or Alimet@ ; with 0.2% Alimet@ ; with 2 lb/ton 65% propionic acid plus 0.2% DLM; with 1.5 lb/ton 65% propionic acid plus 0.2% DLM; and with 1.0 lb/ton 65% propionic acid plus 0.2% DLM.

[0070] Figure 13 is a graph illustrating the % CO2 in the headspace for a starter mash with a moisture level of 14.8% with 2 lb/ton 65% propionic acid; with 1.0 lb/ton 65% propionic acid; and with 1.0 lb/ton 65% propionic acid plus 0. 2% Alimet@.

[0071] Figure 14 is a graph illustrating the % C02 in the headspace for a starter mash with a moisture level of 14.8% with 2 lb/ton 65% propionic acid; with 1.5 lb/ton 65%

propionic acid; and with 1.5 lb/ton 65% propionic acid plus 0.2% Alimet@.

[0072] Figure 15 is a graph illustrating the % CO2 in the headspace for a starter mash treated with propionic acid and propionic acid plus 0.2% Alimets with a moisture level of 16.8%, 14. 8%, 12.8%.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0073] A method has been discovered for inhibiting microbes in animal feed, said method comprising treating said feed with a compound of Formula I.

[0074] Compounds of Formula I have the following structure: [0075] wherein Ru ils an alkyl group having from one to four carbon atoms; [0076] n is an integer from 0 to 2; [0077] R2 is selected from the group consisting of hydroxy, amino,-OCOR3, or-NHCOR3 ; [0078] and wherein R3 is an organic acid derivative; [0079] or a salt thereof.

[0080] The term"organic acid derivative"means a derivative of any suitable organic acid resulting from removal of the carboxyl function from the acid. Preferably, the organic acid has from one to eight carbon atoms.

Suitable organic acid derivatives include, but are not limited to, derivatives of formic acid, acetic acid, propionic acid, butyric acid, benzoic acid, lactic acid, malic acid, tartaric acid, mandelic acid, citric acid, fumaric acid, sorbic acid, boric acid, succinic acid, adipic acid, glycolic acid, and glutaric acid.

[0081] Preferably, RI is methyl, ethyl, propyl (including n-propyl and isopropyl), or butyl (including n- butyl, sec-butyl, and t-butyl).

[0082] In a preferred embodiment, the compound of Formula I is selected from the following list of compounds: [0083] l-hydroxy-1-(methylthio) acetic acid; [0084] 1-hydroxy-1-(ethylthio) acetic acid; [0085] 1-hydroxy-1-(propylthio) acetic acid; [0086] 1-hydroxy-1-(butylthio) acetic acid; [0087] 1-amino-1-(methylthio) acetic acid; [0088] 1-amino-1-(ethylthio) acetic acid; [0089] 1-amino-1-(propylthio) acetic acid; [0090] 1-amino-1-(butylthio) acetic acid; [0091] 1-carboxy-1-(methylthio) acetic acid; [0092] 1-acetyloxy-1-(methylthio) acetic acid; [0093] 1-propionyloxy-1-(methylthio) acetic acid; [0094] 1-butyryloxy-1-(methylthio) acetic acid; [0095] 1-benzoyloxy-1-(methylthio) acetic acid; [0096] 1-lactoyloxy-1-(methylthio) acetic acid; [0097] 1-[2-carboxy-2-(hydroxy)propionyloxy]-1- (methylthio) acetic acid; [0098] 1-[2-carboxy-1-(hydroxy)propionyloxy]-1- (methylthio) acetic acid; [0099] 1-[2-carboxy-1-,2-(dihydroxy)propionyloxy]-1- (methylthio) acetic acid; [0100] 1- [hydroxy (phenyl) acetyl] oxy-1- (methylthio) acetic acid; [0101] 1-[2,3-dicarboxy-2-(hydroxy)butyryloxy]-1- (methylthio) acetic acid; [0102] 1-[2-carboxy-1-carboxymethyl-1- (hydroxy) propionyloxy]-1-(methylthio) acetic acid; [0103] 1- (3-carboxyacryloyl) oxy-l- (methylthio) acetic acid; [0104] 1-(2,4-pentadienovloxv)-1-(methylthio) acetic acid; [0105] 1-(2-carboxypropionyloxy)-1-(methylthio) acetic acid; [0106] 1- [ (4-carboxy) amyloxy]-l- (methylthio) acetic acid; [0107] 1-glycoloyloxy-1-(methylthio) acetic acid;

[0108] 1-glutaroyloxy-1-(methylthio) acetic acid ; [0109] 1-formylamino-1-(methylthio) acetic acid; [0110] 1-acetylamino-1-(methylthio) acetic acid; [0111] 1-propionylamino-1-(methylthio) acetic acid; [0112] 1-butyrylamino-1-(methylthio) acetic acid; [0113] 1-benzoylamino-1-(methylthio) acetic acid; [0114] 1-lactoylamino-1-(methylthio) acetic acid; [0115] 1-[2-carboxy-2-(hydroxy)propionylamino]-1- (methylthio) acetic acid; [0116] 1-[2-carboxy-1-(hydroxy)propionylamino]-1- (methylthio) acetic acid; [0117] 1-[2-carboxy-1,2-(dihydroxy)propionylamino]-1- (methylthio) acetic acid; [0118] 1- [hydroxy (phenyl) acetyl] amino-1- (methylthio) acetic acid; [0119] 1-[2,3-dicarboxy-2-(hydroxy)butyrylamino]-1- (methylthio) acetic acid; [0120] 1-[2-carboxy-1-carboxymethyl-1- (hydroxy) propionylamino]-1-(methylthio) acetic acid; [0121] 1-(3-carboxyacryloyl)amino-1- (methylthio) acetic acid; [0122] 1- (2, 4-pentadienoylamino)-l- (methylthio) acetic acid; [0123] 1-[2-carboxypropionylamino)-1- (methylthio) acetic acid; [0124] 1- [ (4-carboxy) amylamino]-1- (methylthio) acetic acid; [0125] 1-glycoloylamino-1-(methylthio) acetic acid; [0126] 1-glutaroylamino-1- (methylthio) acetic acid; [0127] 1-carboxy- (ethylthio) acetic acid; [0128] 1-acetyloxy-(ethylthio) acetic acid; [0129] 1-propionyloxy- (ethylthio) acetic acid; [0130] 1-butyryloxy- (ethylthio) acetic acid; [0131] 1-benzoyloxy- (ethylthio) acetic acid; [0132] 1-lactoyloxy-(ethylthio) acetic acid; [0133] 1-[2-carboxy-2-(hydroxy)propionyloxy]- (ethylthio) acetic acid;

[0134] 1-[2-carboxy-1-(hydroxy)propionyloxy]- (ethylthio) acetic acid; [0135] 1-[2-carboxy-1, 2-(dihydroxy) propionyloxy]- (ethylthio) acetic acid; [0136] 1- [hydroxy (phenyl) acetyl] oxy- (ethylthio) acetic acid; [0137] 1-[2,3-dicarboxy-2-(hydroxy)butyryloxy]- (ethylthio) acetic acid; [0138] 1-[2-carboxy-1-carboxymethyl-1- (hydroxy) propionyloxy]- (ethylthio) acetic acid; [0139] 1- (3-carboxyacryloyl) oxy- (ethylthio) acetic acid; [0140] 1- (2, 4-pentadienoyloxy)- (ethylthio) acetic acid; [0141] 1- (2-carboxypropionyloxy)- (ethylthio) acetic acid; [0142] 1-[(4-carboxy)amyloxy]-(ethylthio) acetic acid; [0143] 1-glycoloyloxy- (ethylthio) acetic acid; [0144] 1-glutaroyloxy- (ethylthio) acetic acid; [0145] 1-formylamino-(ethylthio) acetic acid; [0146] 1-acetylamino-(ethylthio) acetic acid; [0147] 1-propionylamino-(ethylthio) acetic acid; [0148] 1-butyrylamino-(ethylthio) acetic acid; [0149] 1-benzoylamino-(ethylthio) acetic acid; [0150] 1-lactoylamino-(ethylthio) acetic acid; [0151] 1-[2-carboxy-2-(hydroxy)propionylamino]- (ethylthio) acetic acid; [0152] 1-[2-carboxy-1-(hydroxy)propionylamino]- (ethylthio) acetic acid; [0153] 1-[2-carboxy-1,2-(dihydroxy)propionylamino]- (ethylthio) acetic acid; [0154] 1- [hydroxy (phenyl) acetyl] amino- (ethylthio) acetic acid; [0155] 1-[2,3-dicarboxy-2-(hydroxy)butyrylamino]- (ethylthio) acetic acid; [0156] 1-[2-carboxy-1-carboxymethyl-1- (hydroxy) propionylamino]- (ethylthio) acetic acid;

[0157] 1- (3-carboxyacryloyl) amino- (ethylthio) acetic acid; [0158] 1- (2, 4-pentadienoylamino)- (ethylthio) acetic acid; [0159] 1- (2-carboxypropionylamino)- (ethylthio) acetic acid; [0160] 1- [ (4-carboxy) amylamino]- (ethylthio) acetic acid; [0161] 1-glycoloylamino-(ehtylthio) acetic acid; [0162] 1-glutaroylamino-(ethylthio) acetic acid; [0163] 1-carboxy- (propylthio) acetic acid; [0164] 1-acetyloxy- (propylthio) acetic acid; [0165] 1-propionyloxy- (propylthio) acetic acid; [0166] 1-butyryloxy- (propylthio) acetic acid; [0167] 1-benzoyloxy- (propylthio) acetic acid; [0168] 1-lactoyloxy-(propylthio) acetic acid; [0169] 1-[2-carboxy-2-(hydroxy)propionyloxy]- (propylthio) acetic acid; [0170] 1-[2-carboxy-1-(hydroxy)propionyloxy]- (propylthio) acetic acid; [0171] 1-[2-carboxy-1,2-(dihydroxy)propionyloxy]- (propylthio) acetic acid; [0172] 1- [hydroxy (phenyl) acetyl] oxy- (propylthio) acetic acid; [0173] 1-[2,3-dicarboxy-2-(hydroxy)butyryloxy]- (propylthio) acetic acid; [0174] 1-[2-carboxy-1-carboxymethyl-1- (hydroxy) propionyloxy]-(propylthio) acetic acid; [0175] 1- (3-carboxyacryloyl) oxy- (propylthio) acetic acid; [0176] 1- (2, 4-pentadienoyloxy)- (propylthio) acetic acid; [0177] 1- (2-carboxypropionyloxy)- (propylthio) acetic acid; [0178] 1-[(4-carboxy)amyloxy]-(propylthio) acetic acid; [0179] 1-glycoloyloxy- (propylthio) acetic acid;

[0180] 1-glutaroyloxy- (propylthio) acetic acid; [0181] 1-formylamino-(propylthio) acetic acid; [0182] 1-acetylamino-(propylthio) acetic acid; [0183] 1-propionylamino-(propylthio) acetic acid; [0184] 1-butyrylamino- (propylthio) acetic acid; [0185] 1-benzoylamino-(propylthio) acetic acid; [0186] 1-lactoylamino-(propylthio) acetic acid; [0187] 1-[2-carboxy-2-(hydroxy) propionylamino]- (propylthio) acetic acid; [0188] 1-[2-carboxy-1-(hydroxy)propionylamino]- (propylthio) acetic acid; [0189] 1-[2-carboxy-1,2-(dihydroxy)propionylamino]- (propylthio) acetic acid; [0190] 1- [hydroxy (phenyl) acetyl] amino- (propylthio) acetic acid; [0191] 1-[2,3-dicarboxy-2-(hydroxy)butyrylamino]- (propylthio) acetic acid; [0192] 1-[2-carboxy-1-carboxymethyl-1- (hydroxy) propionylamino]- (propylthio) acetic acid; [0193] 1- (3-carboxyacryloyl) amino- (propylthio) acetic acid; [0194] 1- (2, 4-pentadienoylamino)- (propylthio) acetic acid; [0195] 1- (2-carboxypropionylamino)- (propylthio) acetic acid; [0196] 1- [ (4-carboxy) amylamino]- (propylthio) acetic acid ; [0197] 1-glycoloylamino-(propylthio) acetic acid; [0198] 1-glutaroylamino-(propylthio) acetic acid; [0199] 1-carboxy- (butylthio) acetic acid; [0200] 1-acetyloxy-(buthlthio) acetic acid; [0201] 1-propionyloxy- (butylthio) acetic acid; [0202] 1-butyryloxy- (butylthio) acetic acid; [0203] 1-benzoyloxy- (butylthio) acetic acid; [0204] 1-lactoyloxy- (butylthio) acetic acid; [0205] 1-[2-carboxy-2-(hydroxy)propionyloxy]- (butylthio) acetic acid;

[0206] 1-[2-carboxy-1-(hydroxy)propionyloxy]- (butylthio) acetic acid; [0207] 1-[2-carboxy-1,2-(dihydroxy)propionyloxy]- (butylthio) acetic acid; [0208] 1- [hydroxy (phenyl) acetyl]oxy-(butylthio) acetic acid; [0209] 1-[2,3-dicarboxy-2-(hydroxy)butyryloxy]- (butylthio) acetic acid; [0210] 1-[2-carboxy-1-carboxymethyl-1- (hydroxy) propionyloxy]-(butylthio) acetic acid; [0211] 1- (3-carboxyacryloyl) oxy- (butylthio) acetic acid; [0212] 1- (2, 4-pentadienoyloxy)- (butylthio) acetic acid ; [0213] 1- (2-carboxypropionyloxy)- (butylthio) acetic acid; [0214] 1- [ (4-carboxy) amyloxy]- (butylthio) acetic acid; [0215] 1-glycoloyloxy- (butylthio) acetic acid; [0216] 1-glutaroyloxy- (butylthio) acetic acid; [0217] 1-formylamino- (butylthio) acetic acid; [0218] 1-acetylamino- (butylthio) acetic acid; [0219] 1-propionylamino- (butylthio) acetic acid; [0220] 1-butyrylamino- (butylthio) acetic acid; [0221] 1-benzoylamino-(butylthio) acetic acid; [0222] 1-lactoylamino- (butylthio) acetic acid; [0223] 1-[2-carboxy-2-(hydroxy)propionylamino]- (butylthio) acetic acid; [0224] 1-[2-carboxy-1-(hydroxy)propionylamino]- (butylthio) acetic acid; [0225] 1-[2-carboxy-1,2-(dihydroxy)propionylamino]- (butylthio) acetic acid; [0226] 1- [hydroxy (phenyl) acetyl] amino- (butylthio) acetic acid; [0227] 1- [2, 3-dicarboxy-2- (hydroxy) butyrylamino] - (butylthio) acetic acid; [0228] 1-[2-carboxy-1-carboxymethyl-1- (hydroxy) propionylamino]-(buthlthio) acetic acid;

[0229] 1- (3-carboxyacryloyl) amino- (butylthio) acetic acid; [0230] 1- (2, 4-pentadienoylamino)- (butylthio) acetic acid; [0231] 1- (2-carboxypropionylamino)- (butylthio) acetic acid; [0232] 1- [ (4-carboxy) amylamino]- (butylthio) acetic acid; [0233] 1-glycoloylamino- (butylthio) acetic acid; [0234] 1-glutaroylamino- (butylthio) acetic acid; [0235] 2-hydroxy-3- (methylthio) propanoic acid; [0236] 2-hydroxy-3- (ethylthio) propanoic acid; [0237] 2-hydroxy-3- (propylthio) propanoic acid; [0238] 2-hydroxy-3- (butylthio) propanoic acid; [0239] 2-amino-3- (methylthio) propanoic acid; [0240] 2-amino-3- (ethylthio) propanoic acid; [0241] 2-amino-3- (propylthio) propanoic acid; [0242] 2-amino-3- (butylthio) propanoic acid; [0243] 2-carboxy-3- (methylthio) propanoic acid; [0244] 2-acetyloxy-3- (methylthio) propanoic acid; [0245] 2-propionyloxy-3- (methylthio) propanoic acid; [0246] 2-butyryloxy-3- (methylthio) propanoic acid; [0247] 2-benzoyloxy-3- (methylthio) propanoic acid; [0248] 2-lactoyloxy-3- (methylthio) propanoic acid; [0249] 2- [2-carboxy-2- (hydroxy) propionyloxy]-3- (methylthio) propanoic acid; [0250] 2-[2-carboxy-1-(hydroxy) propionyloxy]-3- (methylthio) propanoic acid; [0251] 2-[2-carboxy-1, 2-(dihydroxy) propionyloxy]-3- (methylthio) propanoic acid; [0252] 2- [hydroxy (phenyl) acetyl] oxy-3- (methylthio) propanoic acid; [0253] 2- [2, 3-dicarboxy-2- (hydroxy) butyryloxy]-3- (methylthio) propanoic acid; [0254] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionyloxy]-3- (methylthio) propanoic acid;

[0255] 2- (3-carboxyacryloyl) oxy-3- (methylthio) propanoic acid; [0256] 2- (2, 4-pentadienoyloxy) -3- (methylthio) propanoic acid; [0257] 2- (2-carboxypropionyloxy)-3- (methylthio) propanoic acid; [0258] 2- [ (4-carboxy) amyloxy]-3- (methylthio) propanoic acid; [0259] 2-glycoloyloxy-3- (methylthio) propanoic acid; [0260] 2-glutaroyloxy-3- (methylthio) propanoic acid; [0261] 2-formylamino-3- (methylthio) propanoic acid; [0262] 2-acetylamino-3- (methylthio) propanoic acid; [0263] 2-propionylamino-3- (methylthio) propanoic acid; [0264] 2-butyrylamino-3- (methylthio) propanoic acid; [0265] 2-benzoylamino-3- (methylthio) propanoic acid; [0266] 2-lactoylamino-3- (methylthio) propanoic acid; [0267] 2- [2-carboxy-2- (hydroxy) propionylamino]-3- (methylthio) propanoic acid; [0268] 2-[2-carboxy-1-(hydroxy) propionylamino]-3- (methylthio) propanoic acid; [0269] 2- [2-carboxy-l, 2- (dihydroxy) propionylamino]-3- (methylthio) propanoic acid; [0270] 2- [hydroxy (phenyl) acetyl] amino-3- (methylthio) propanoic acid; [0271] 2- [2, 3-dicarboxy-2- (hydroxy) butyrylamino]-3- (methylthio) propanoic acid; [0272] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionylamino]-3- (methylthio) propanoic acid; [0273] 2- (3-carboxyacryloyl) amino-3- (methylthio) propanoic acid; [0274] 2- (2, 4-pentadienoylamino) -3- (methylthio) propanoic acid; [0275] 2- (2-carboxypropionylamino)-3- (methylthio) propanoic acid; [0276] 2- [ (4-carboxy) amylamino]-3- (methylthio) propanoic acid; [0277] 2-glycoloylamino-3- (methylthio) propanoic acid;

[0278] 2-glutaroylamino-3- (methylthio) propanoic acid; [0279] 2-carboxy-3- (ethylthio) propanoic acid; [0280] 2-acetyloxy-3- (ethylthio) propanoic acid; [0281] 2-propionyloxy-3- (ethylthio) propanoic acid; [0282] 2-butyryloxy-3- (ethylthio) propanoic acid; [0283] 2-benzoyloxy-3- (ethylthio) propanoic acid; [0284] 2-lactoyloxy-3- (ethylthio) propanoic acid; [0285] 2- [2-carboxy-2- (hydroxy) propionyloxy]-3- (ethylthio) propanoic acid; [0286] 2-[2-carboxy-1-(hydroxy) propionyloxy]-3- (ethylthio) propanoic acid; [0287] 2-[2-carboxy-1, 2-(dihydroxy) propionyloxy]-3- (ethylthio) propanoic acid; [0288] 2- [hydroxy (phenyl) acetyl] oxy-3- (ethylthio) propanoic acid; [0289] 2- [2, 3-dicarboxy-2- (hydroxy) butyryloxy]-3- (ethylthio) propanoic acid; [0290] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionyloxy]-3- (ethylthio) propanoic acid; [0291] 2- (3-carboxyacryloyl) oxy-3- (ethylthio) propanoic acid; [0292] 2- (2, 4-pentadienoyloxy)-3- (ethylthio) propanoic acid; [0293] 2- (2-carboxypropionyloxy)-3- (ethylthio) propanoic acid; [0294] 2- [ (4-carboxy) amyloxy]-3- (ethylthio) propanoic acid; [0295] 2-glycoloyloxy-3- (ethylthio) propanoic acid; [0296] 2-glutaroyloxy-3- (ethylthio) propanoic acid; [0297] 2-formylamino-3- (ethylthio) propanoic acid; [0298] 2-acetylamino-3- (ethylthio) propanoic acid; [0299] 2-propionylamino-3- (ethylthio) propanoic acid; [0300] 2-butyrylamino-3- (ethylthio) propanoic acid; [0301] 2-benzoylamino-3- (ethylthio) propanoic acid; [0302] 2-lactoylamino-3- (ethylthio) propanoic acid; [0303] 2- [2-carboxy-2- (hydroxy) propionylamino]-3- (ethylthio) propanoic acid;

[0304] 2-[2-carboxy-1-(hydroxy) propionylamino]-3- (ethylthio) propanoic acid; [0305] 2-[2-carboxy-1, 2-(dihydroxy) propionylamino]-3- (ethylthio) propanoic acid; [0306] 2- [hydroxy (phenyl) acetyl] amino-3- (ethylthio) propanoic acid; [0307] 2- [2, 3-dicarboxy-2- (hydroxy) butyrylamino]-3- (ethylthio) propanoic acid; [0308] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionylamino]-3- (ethylthio) propanoic acid; [0309] 2- (3-carboxyacryloyl) amino-3- (ethylthio) propanoic acid; [0310] 2- (2, 4-pentadienoylamino) -3- (ethylthio) propanoic acid; [0311] 2- (2-carboxypropionylamino)-3- (ethylthio) propanoic acid; [0312] 2- [ (4-carboxy) amylamino]-3- (ethylthio) propanoic acid; [0313] 2-glycoloylamino-3- (ethylthio) propanoic acid; [0314] 2-glutaroylamino-3- (ethylthio) propanoic acid; [0315] 2-carboxy-3- (propylthio) propanoic acid; [0316] 2-acetyloxy-3- (propylthio) propanoic acid; [0317] 2-propionyloxy-3- (propylthio) propanoic acid; [0318] 2-butyryloxy-3- (propylthio) propanoic acid; [0319] 2-benzoyloxy-3- (propylthio) propanoic acid; [0320] 2-lactoyloxy-3- (propylthio) propanoic acid; [0321] 2- [2-carboxy-2- (hydroxy) propionyloxy]-3- (propylthio) propanoic acid; [0322] 2-[2-carboxy-1-(hydroxy) propionyloxy]-3- (propylthio) propanoic acid; [0323] 2-[2-carboxy-1, 2-(dihydroxy) propionyloxy]-3- (propylthio) propanoic acid; [0324] 2- [hydroxy (phenyl) acetyl] oxy-3- (propylthio) propanoic acid; [0325] 2- [2, 3-dicarboxy-2- (hydroxy) butyryloxy] -3- (propylthio) propanoic acid;

[0326] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionyloxy]-3- (propylthio) propanoic acid; [0327] 2- (3-carboxyacryloyl) oxy-3- (propylthio) propanoic acid; [0328] 2- (2, 4-pentadienoyloxy) -3- (propylthio) propanoic acid; [0329] 2- (2-carboxypropionyloxy)-3- (propylthio) propanoic acid; [0330] 2- [ (4-carboxy) amyloxy]-3- (propylthio) propanoic acid; [0331] 2-glycoloyloxy-3- (propylthio) propanoic acid; [0332] 2-glutaroyloxy-3- (propylthio) propanoic acid; [0333] 2-formylamino-3- (propylthio) propanoic acid; [0334] 2-acetylamino-3- (propylthio) propanoic acid; [0335] 2-propionylamino-3- (propylthio) propanoic acid; [0336] 2-butyrylamino-3- (propylthio) propanoic acid; [0337] 2-benzoylamino-3- (propylthio) propanoic acid; [0338] 2-lactoylamino-3- (propylthio) propanoic acid; [0339] 2- [2-carboxy-2- (hydroxy) propionylamino]-3- (propylthio) propanoic acid; [0340] 2-[2-carboxy-1-(hydroxy) propionylamino]-3- (propylthio) propanoic acid; [0341] 2-[2-carboxy-1, 2-(dihydroxy) propionylamino]-3- (propylthio) propanoic acid; [0342] 2-hydroxy (phenyl) acetyl] amino-3- (propylthio) propanoic acid; [0343] 2- [2, 3-dicarboxy-2- (hydroxy) butyrylamino]-3- (propylthio) propanoic acid; [0344] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionylamino]-3- (propylthio) propanoic acid; [0345] 2- (3-carboxyacryloyl) amino-3- (propylthio) propanoic acid; [0346] 2- (2, 4-pentadienoylamino) -3- (propylthio) propanoic acid; [0347] 2- (2-carboxypropionylamino)-3- (propylthio) propanoic acid;

[0348] 2- [ (4-carboxy) amylamino] -3- (propylthio) propanoic acid; [0349] 2-glycoloylamino-3- (propylthio) propanoic acid; [0350] 2-glutaroylamino-3- (propylthio) propanoic acid; [0351] 2-carboxy-3- (butylthio) propanoic acid; [0352] 2-acetyloxy-3- (butylthio) propanoic acid; [0353] 2-propionyloxy-3- (butylthio) propanoic acid; [0354] 2-butyryloxy-3- (butylthio) propanoic acid; [0355] 2-benzoyloxy-3- (butylthio) propanoic acid; [0356] 2-lactoyloxy-3- (butylthio) propanoic acid; [0357] 2- [2-carboxy-2- (hydroxy) propionyloxy]-3- (butylthio) propanoic acid; [0358] 2-[2-carboxy-1-(hydroxy) propionyloxy]-3- (butylthio) propanoic acid; [0359] 2-[2-carboxy-1, 2-(dihydroxy) propionyloxy]-3- (butylthio) propanoic acid; [0360] 2- [hydroxy (phenyl) acetyl] oxy-3- (butylthio) propanoic acid; [0361] 2- [2, 3-dicarboxy-2- (hydroxy) butyryloxy]-3- (butylthio) propanoic acid; [0362] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionyloxy]-3- (butylthio) propanoic acid; [0363] 2- (3-carboxyacryloyl) oxy-3- (butylthio) propanoic acid; [0364] 2- (2, 4-pentadienoyloxy)-3- (butylthio) propanoic acid; [0365] 2- (2-carboxypropionyloxy)-3- (butylthio) propanoic acid; [0366] 2- [ (4-carboxy) amyloxy]-3- (butylthio) propanoic acid; [0367] 2-glycoloyloxy-3- (butylthio) propanoic acid; [0368] 2-glutaroyloxy-3- (butylthio) propanoic acid; [0369] 2-formylamino-3- (butylthio) propanoic acid; [0370] 2-acetylamino-3- (butylthio) propanoic acid; [0371] 2-propionylamino-3- (butylthio) propanoic acid; [0372] 2-butyrylamino-3- (butylthio) propanoic acid; [0373] 2-benzoylamino-3- (butylthio) propanoic acid;

[0374] 2-lactoylamino-3- (butylthio) propanoic acid; [0375] 2- [2-carboxy-2- (hydroxy) propionylamino]-3- (butylthio) propanoic acid; [0376] 2-[2-carboxy-1-(hydroxy) propionylamino]-3- (butylthio) propanoic acid; [0377] 2-[2-carboxy-1, 2-(dihydroxy) propionylamino]-3- (butylthio) propanoic acid; [0378] 2- [hydroxy (phenyl) acetyl] amino-3- (butylthio) propanoic acid; [0379] 2- [2, 3-dicarboxy-2- (hydroxy) butyrylamino]-3- (butylthio) propanoic acid; [0380] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionylamino]-3- (butylthio) propanoic acid; [0381] 2- (3-carboxyacryloyl) amino-3- (butylthio) propanoic acid; [0382] 2- (2, 4-pentadienoylamino) -3- (butylthio) propanoic acid; [0383] 2- (2-carboxypropionylamino)-3- (butylthio) propanoic acid; [0384] 2- [ (4-carboxy) amylamino]-3- (butylthio) propanoic acid; [0385] 2-glycoloylamino-3- (butylthio) propanoic acid; [0386] 2-glutaroylamino-3- (butylthio) propanoic acid; [0387] 2-hydroxy-4- (methylthio) butanoic acid; [0388] 2-hydroxy-4- (ethylthio) butanoic acid; [0389] 2-hydroxy-4- (propylthio) butanoic acid; [0390] 2-hydroxy-4- (butylthio) butanoic acid; [0391] 2-amino-4- (methylthio) butanoic acid; [0392] 2-amino-4- (ethylthio) butanoic acid ; [0393] 2-amino-4- (propylthio) butanoic acid; [0394] 2-amino-4- (butylthio) butanoic acid; [0395] 2-carboxy-4- (methylthio) butanoic acid; [0396] 2-acetyloxy-4- (methylthio) butanoic acid; [0397] 2-propionyloxy-4- (methylthio) butanoic acid; [0398] 2-butyryloxy-4- (methylthio) butanoic acid; [0399] 2-benzoyloxy-4- (methylthio) butanoic acid; [0400] 2-lactoyloxy-4- (methylthio) butanoic acid;

[0401] 2- [2-carboxy-2- (hydroxy) propionyloxy]-4- (methylthio) butanoic acid; [0402] 2-[2-carboxy-1-(hydroxy) propionyloxy]-4- (methylthio) butanoic acid; [0403] 2-[2-carboxy-1, 2-(dihydroxy) propionyloxy]-4- (methylthio) butanoic acid; [0404] 2- [hydroxy (phenyl) acetyl] oxy-4- (methylthio) butanoic acid; [0405] 2- [2, 3-dicarboxy-2- (hydroxy) butyryloxy]-4- (methylthio) butanoic acid; [0406] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionyloxy]-4- (methylthio) butanoic acid; [0407] 2- (3-carboxyacryloyl) oxy-4- (methylthio) butanoic acid; [0408] 2- (2, 4-pentadienoyloxy)-4- (methylthio) butanoic acid; [0409] 2- (2-carboxypropionyloxy)-4- (methylthio) butanoic acid; [0410] 2- [ (4-carboxy) amyloxy]-4- (methylthio) butanoic acid; [0411] 2-glycoloyloxy-4- (methylthio) butanoic acid; [0412] 2-glutaroyloxy-4- (methylthio) butanoic acid; [0413] 2-formylamino-4- (methylthio) butanoic acid; [0414] 2-acetylamino-4- (methylthio) butanoic acid; [0415] 2-propionylamino-4- (methylthio) butanoic acid; [0416] 2-butyrylamino-4- (methylthio) butanoic acid; [0417] 2-benzoylamino-4- (methylthio) butanoic acid; [0418] 2-lactoylamino-4- (methylthio) butanoic acid; [0419] 2- [2-carboxy-2- (hydroxy) propionylamino]-4- (methylthio) butanoic acid; [0420] 2-[2-carboxy-1-(hydroxy) propionylamino]-4- (methylthio) butanoic acid; [0421] 2-[2-carboxy-1, 2-(dihydroxy) propionylamino]-4- (methylthio) butanoic acid; [0422] 2- [hydroxy (phenyl) acetyl] amino-4- (methylthio) butanoic acid;

[0423] 2- [2, 3-dicarboxy-2- (hydroxy) butyrylamino]-4- (methylthio) butanoic acid; [0424] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionylamino]-4- (methylthio) butanoic acid; [0425] 2- (3-carboxyacryloyl) amino-4- (methylthio) butanoic acid; [0426] 2- (2, 4-pentadienoylamino) -4- (methylthio) butanoic acid; [0427] 2- (2-carboxypropionylamino)-4- (methylthio) butanoic acid; [0428] 2- [ (4-carboxy) amylamino]-4- (methylthio) butanoic acid; [0429] 2-glycoloylamino-4- (methylthio) butanoic acid; [0430] 2-glutaroylamino-4- (methylthio) butanoic acid; [0431] 2-carboxy-4- (ethylthio) butanoic acid; [0432] 2-acetyloxy-4- (ethylthio) butanoic acid; [0433] 2-propionyloxy-4- (ethylthio) butanoic acid; [0434] 2-butyryloxy-4- (ethylthio) butanoic acid; [0435] 2-benzoyloxy-4- (ethylthio) butanoic acid; [0436] 2-lactoyloxy-4- (ethylthio) butanoic acid; [0437] 2- [2-carboxy-2- (hydroxy) propionyloxy]-4- (ethylthio) butanoic acid; [0438] 2-[2-carboxy-1-(hydroxy) propionyloxy]-4- (ethylthio) butanoic acid; [0439] 2-[2-carboxy-1, 2-(dihydroxy) propionyloxy]-4- (ethylthio) butanoic acid; [0440] 2- [hydroxy (phenyl) acetyl] oxy-4- (ethylthio) butanoic acid; [0441] 2- [2, 3-dicarboxy-2- (hydroxy) butyryloxy]-4- (ethylthio) butanoic acid; [0442] 2-[2-carboxy-1-carboxymethyl-1- (hydroxy) propionyloxy]-4- (ethylthio) butanoic acid; [0443] 2- (3-carboxyacryloyl) oxy-4- (ethylthio) butanoic acid; [0444] 2- (2, 4-pentadienoyloxy)-4- (ethylthio) butanoic acid;

[0445] 2- (2-carboxypropionyloxy)-4- (ethylthio) butanoic acid; [0446] 2- [ (4-carboxy) amyloxy]-4- (ethylthio) butanoic acid; [0447] 2-glycoloyloxy-4- (ethylthio) butanoic acid; [0448] 2-glutaroyloxy-4- (ethylthio) butanoic acid; [0449] 2-formylamino-4- (ethylthio) butanoic acid; [0450] 2-acetylamino-4- (ethylthio) butanoic acid; [0451] 2-propionylamino-4- (ethylthio) butanoic acid; [0452] 2-butyrylamino-4- (ethylthio) butanoic acid; [0453] 2-benzoylamino-4- (ethylthio) butanoic acid; [0454] 2-lactoylamino-4- (ethylthio) butanoic acid; [0455] 2- [2-carboxy-2- (hydroxy) propionylamino]-4- (ethylthio) butanoic acid; [0456] 2-[2-carboxy-1-(hydroxy) propionylamino]-4- (ethylthio) butanoic acid; [0457] 2-[2-carboxy-1, 2-(dihydroxy) propionylamino]-4- (ethylthio) butanoic acid; [0458] 2- [hydroxy (phenyl) acetyl] amino-4- (ethylthio) butanoic acid; [0459] 2- [2, 3-dicarboxy-2- (hydroxy) butyrylamino]-4- (ethylthio) butanoic acid; [0460] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionylamino]-4- (ethylthio) butanoic acid; [0461] 2- (3-carboxyacryloyl) amino-4- (ethylthio) butanoic acid; [0462] 2- (2, 4-pentadienoylamino) -4- (ethylthio) butanoic acid; [0463] 2- (2-carboxypropionylamino)-4- (ethylthio) butanoic acid; [0464] 2- [ (4-carboxy) amylamino]-4- (ethylthio) butanoic acid; [0465] 2-glycoloylamino-4- (ethylthio) butanoic acid; [0466] 2-glutaroylamino-4- (ethylthio) butanoic acid; [0467] 2-carboxy-4- (propylthio) butanoic acid; [0468] 2-acetyloxy-4- (propylthio) butanoic acid; [0469] 2-propionyloxy-4- (propylthio) butanoic acid;

[0470] 2-butyryloxy-4- (propylthio) butanoic acid; [0471] 2-benzoyloxy-4- (propylthio) butanoic acid; [0472] 2-lactoyloxy-4- (propylthio) butanoic acid; [0473] 2- [2-carboxy-2- (hydroxy) propionyloxy]-4- (propylthio) butanoic acid; [0474] 2-[2-carboxy-1-(hydroxy) propionyloxy]-4- (propylthio) butanoic acid; [0475] 2- [2-carboxy-l, 2- (dihydroxy) propionyloxy]-4- (propylthio) butanoic acid; [0476] 2- [hydroxy (phenyl) acetyl] oxy-4- (propylthio) butanoic acid; [0477] 2- [2, 3-dicarboxy-2- (hydroxy) butyryloxy]-4- (propylthio) butanoic acid; [0478] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionyloxy]-4- (propylthio) butanoic acid; [0479] 2- (3-carboxyacryloyl) oxy-4- (propylthio) butanoic acid; [0480] 2- (2, 4-pentadienoyloxy)-4- (propylthio) butanoic acid; [0481] 2- (2-carboxypropionyloxy)-4- (propylthio) butanoic acid; [0482] 2- [ (4-carboxy) amyloxy]-4- (propylthio) butanoic acid; [0483] 2-glycoloyloxy-4- (propylthio) butanoic acid; [0484] 2-glutaroyloxy-4- (propylthio) butanoic acid; [0485] 2-formylamino-4- (propylthio) butanoic acid; [0486] 2-acetylamino-4- (propylthio) butanoic acid; [0487] 2-propionylamino-4- (propylthio) butanoic acid; [0488] 2-butyrylamino-4- (propylthio) butanoic acid; [0489] 2-benzoylamino-4- (propylthio) butanoic acid; [0490] 2-lactoylamino-4- (propylthio) butanoic acid; [0491] 2- [2-carboxy-2- (hydroxy) propionylamino]-4- (propylthio) butanoic acid; [0492] 2-[2-carboxy-1-(hydroxy) propionylamino]-4- (propylthio) butanoic acid; [0493] 2-[2-carboxy-1, 2-(dihydroxy) propionylamino]-4- (propylthio) butanoic acid;

[0494] 2- [hydroxy (phenyl) acetyl] amino-4- (propylthio) butanoic acid; [0495] 2- [2, 3-dicarboxy-2- (hydroxy) butyrylamino]-4- (propylthio) butanoic acid; [0496] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionylamino]-4- (propylthio) butanoic acid; [0497] 2- (3-carboxyacryloyl) amino-4- (propylthio) butanoic acid; [0498] 2- (2, 4-pentadienoylamino) -4- (propylthio) butanoic acid; [0499] 2- (2-carboxypropionylamino)-4- (propylthio) butanoic acid; [0500] 2- [ (4-carboxy) amylamino] -4- (propylthio) butanoic acid; [0501] 2-glycoloylamino-4- (propylthio) butanoic acid; [0502] 2-glutaroylamino-4- (propylthio) butanoic acid; [0503] [0504] 2-carboxy-4- (butylthio) butanoic acid; [0505] 2-acetyloxy-4- (butylthio) butanoic acid; [0506] 2-propionyloxy-4- (butylthio) butanoic acid; [0507] 2-butyryloxy-4- (butylthio) butanoic acid; [0508] 2-benzoyloxy-4- (butylthio) butanoic acid; [0509] 2-lactoyloxy-4- (butylthio) butanoic acid; [0510] 2- [2-carboxy-2- (hydroxy) propionyloxy]-4- (butylthio) butanoic acid; [0511] 2-[2-carboxy-1-(hydroxy) propionyloxy]-4- (butylthio) butanoic acid; [0512] 2-[2-carboxy-1, 2-(dihydroxy) propionyloxy]-4- (butylthio) butanoic acid; [0513] 2- [hydroxy (phenyl) acetyl] oxy-4- (butylthio) butanoic acid; [0514] 2- [2, 3-dicarboxy-2- (hydroxy) butyryloxy]-4- (butylthio) butanoic acid; [0515] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionyloxy]-4- (butylthio) butanoic acid; [0516] 2- (3-carboxyacryloyl) oxy-4- (butylthio) butanoic acid;

[0517] 2- (2, 4-pentadienoyloxy)-4- (butylthio) butanoic acid; [0518] 2- (2-carboxypropionyloxy)-4- (butylthio) butanoic acid; [0519] 2- [ (4-carboxy) amyloxy]-4- (butylthio) butanoic acid; [0520] 2-glycoloyloxy-4- (butylthio) butanoic acid; [0521] 2-glutaroyloxy-4- (butylthio) butanoic acid; [0522] 2-formylamino-4- (butylthio) butanoic acid; [0523] 2-acetylamino-4- (butylthio) butanoic acid; [0524] 2-propionylamino-4- (butylthio) butanoic acid; [0525] 2-butyrylamino-4- (butylthio) butanoic acid; [0526] 2-benzoylamino-4- (butylthio) butanoic acid; [0527] 2-lactoylamino-4- (butylthio) butanoic acid; [0528] 2-[2-carboxy-2-(hydroxy) propionylamino]-4- (butylthio) butanoic acid; [0529] 2-[2-carboxy-1-(hydroxy) propionylamino]-4- (butylthio) butanoic acid; [0530] 2-[2-carboxy-1, 2-(dihydroxy) propionylamino]-4- (butylthio) butanoic acid; [0531] 2- [hydroxy (phenyl) acetyl] amino-4- (butylthio) butanoic acid; [0532] 2- [2, 3-dicarboxy-2- (hydroxy) butyrylamino]-4- (butylthio) butanoic acid; [0533] 2- [2-carboxy-l-carboxymethyl-l- (hydroxy) propionylamino]-4- (butylthio) butanoic acid; [0534] 2- (3-carboxyacryloyl) amino-4- (butylthio) butanoic acid; [0535] 2- (2, 4-pentadienoylamino) -4- (butylthio) butanoic acid; [0536] 2- (2-carboxypropionylamino)-4- (butylthio) butanoic acid; [0537] 2- [ (4-carboxy) amylamino]-4- (butylthio) butanoic acid; [0538] 2-glycoloylamino-4- (butylthio) butanoic acid; and [0539] 2-glutaroylamino-4- (butylthio) butanoic acid.

[0540] In a more preferred embodiment, the compound of Formula I is selected from the group of compounds wherein Ru ils methyl; n is 2; Ra is hydroxy or-OCOR3 ; and R3 is a derivative of formic acid, acetic acid, propionic acid, butyric acid, benzoic acid, lactic acid, malic acid, tartaric acid, mandelic acid, citric acid, fumaric acid, sorbic acid, boric acid, succinic acid, adipic acid, glycolic acid, or glutaric acid. In an even more preferred embodiment, the compound of Formula I is selected from the group of compounds wherein R1 is methyl; n is 2; R2 is hydroxy or-OCOR3 ; and R3 is a derivative of formic acid, propionic acid, butyric acid, lactic acid, citric acid, or fumaric acid.

[0541] Representative salts of the compound of Formula I include the ammonium, magnesium, calcium, lithium, sodium, potassium, selenium, iron, copper, and zinc salts. In a preferred embodiment, the compound of Formula I is in the form of the calcium salt.

Representative amides include methylamide, dimethylamide, ethylmethylamide, butylamide, dibutylamide, butylmethylamide, alkyl ester of N-acyl methionates (e. g., alkyl N-acetyl methionates. Representative esters include the methyl, ethyl, n-propyl, isopropyl, butyl esters, namely n-butyl, sec-butyl, isobutyl, and t-butyl esters, pentyl esters and hexyl esters, especially n-pentyl, isopentyl, n-hexyl and isohexyl esters.

[0542] In various preferred embodiment, the compound of Formula I is 2-hydroxy-4- (methylthio) butanoic acid (HMBA) or a salt, amide or ester thereof. In still more preferred embodiments, the compound of Formula I is HMBA.

[0543] Preferably, the concentration of the compound of Formula I in the feed compositions described herein is between about 0. 01% and about 5%. In various preferred embodiments, the concentration is between 0. 01% and about 4%; between 0.02% and about 3%; between 0.03% and about 2% ; between 0.04% and about 1% ; between about 0. 05% and about 0.6% ; and between about 0.06% and about 0.525%. In various

particularly preferred embodiments, the concentration is about 0. 075% ; about 0. 125% ; about 0.15% ; about 0. 225% ; about 0.25% ; about 0. 3% ; about 0.375% ; and about 0. 5%.

[0544] In another embodiment of the present invention, the methods of inhibiting microbes in animal feed comprises treating said feed with a compound of Formula I and one or more organic acids. Preferably, the organic acid has a pKa < 5.5. In one embodiment, the organic acid is a carboxyl-substituted hydrocarbon moiety. The hydrocarbon moiety may be further substituted by one or more substituents such as halogen; oxygen-containing groups such as alkoxy, aryloxy, hydroxy, protected hydroxy, keto, acyl, acyloxy; nitrogen-containing groups such as nitro, amino, amido, cyano; and sulfur-containing groups such as thiol, thioalkyl, and sulfonyl. In a preferred embodiment, said organic acids are selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, benzoic acid, lactic acid, malic acid, tartaric acid, mandelic acid, citric acid, fumaric acid, sorbic acid, boric acid, succinic acid, adipic acid, glycolic acid, and glutaric acid, or combinations thereof. In one embodiment, the organic acid is formic acid, propionic acid, butyric acid, lactic acid, or combinations thereof.

[0545] Preferably, the combined concentration of the compound of Formula I and the organic acid or mixture of organic acids in the food compositions described herein is between about 0. 01% and about 5%. In various preferred embodiments, the combined concentration is between about 0. 015% and about 4%; between about 0. 02% and about 3% ; between about 0. 05% and about 2.5% ; between about 0.075% and about 2% ; between about 0.1% and about 1.5% ; between about 0.15% and about 1% ; between about 0.4% and about 0.9% ; between about 0.5% and about 0. 8% ; between about 0.01% and about 5% ; between about 0.01% and about 4. 5% ; between about 0. 05% and about 4%; between about 0.08% and about 3%; between about 0.1% and about 2.5% ; between about 0.01% and about 0.8% ; between about 0. 01% and about 0.5% ;

between about 0.05% and about 0.6% ; and between about 0.06% and about 0.525%.

In various other preferred embodiments, the concentration of said compound of Formula I and said organic acid in the food compositions described herein is as follows: Concentration of the Concentration of the compound of Formula I organic acid between about 0. 01% and between about 0. 01% and about 0. 5% about 0. 5% between about 0. 1% and between about 0. 1% and about 0.4% about 0.5% about 0. 125% about 0. 375% about 0.225% about 0.225% about 0.25% about 0.25% about 0. 375% about 0. 125% about 0.3% about 0. 5% [0546] In a preferred embodiment of the present invention, the antimicrobial compositions comprises a compound of Formula I and one or more other acidulants.

Such acidulants are typically strong acids, and are preferably mineral acids. Examples of such acidulants include phosphoric acid, phosphorous acid, sulfuric acid, hydrochloric acid, hydrobromic acid, and nitric acid. In one embodiment, the acidulant is phosphoric acid.

[0547] In a more preferred embodiment, the antimicrobial compositions comprise a compound of Formula I, one or more organic acids, as defined above, and one or more other acidulants as defined above.

[0548] Preferably, the pH of the feed is between about 3 and about 8. Even more preferably, the pH is between about 4 and about 7. Still more preferably, the pH is between about 4.5 and about 6.75. The pH may be measured by placing a known quantity of the feed and placing it in a known quantity of distilled water. The pH of the resulting

solution, after sitting, may be measured by any standard means for measuring pH.

[0549] The following embodiments are particularly preferred for the addition of combinations of Alimet and formic acid to feed (concentrations expressed in wt% of feed composition): [0550] about 0. 125% Alimet and about 0.375% formic acid at pH from about 4.5 to about 6.75 ; [0551] about 0.25% Alimets and about 0. 25% formic acid at pH from about 4.5 to about 6.75 ; [0552] about 0.375% Alimets and about 0. 125% formic acid at pH from about 4.5 to about 6.75 ; [0553] about 0. 5% Alimet at pH from about 4.5 to about 6.75 ; [0554] about 0. 3% Alimets and about 0.5% formic acid at pH from about 4.5 to about 6.75.

[0555] In another preferred embodiment, the above- mentioned organic acid is a mixture of formic acid and propionic acid, wherein the formic acid comprises from about 95% to about 5% of the organic acid mixture and the propionic acid comprises from about 5% to about 95% of the organic mixture. Preferably, formic acid comprises from about 85% to about 15% of the organic acid mixture, and propionic acid comprises from about 15% to about 85% of the organic acid mixture. In another preferred embodiment, formic acid comprises from about 85% to about 65% of the organic acid mixture, and propionic acid comprises from about 15% to 35% of the organic acid mixture. In another preferred embodiment, formic acid comprises about 75% of the organic acid mixture, and propionic acid comprises about 25% of the organic acid mixture. This formic/propionic acid mixture can then be combined with the compound of Formula I according to the ratios described above.

[0556] In a preferred embodiment, the antimicrobial compositions comprise a compound of Formula I, preferably HMBA or a salt thereof, and a first organic acid, as

defined herein. Preferably, the first organic acid is selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, benzoic acid, lactic acid, malic acid, tartaric acid, mandelic acid, citric acid, fumaric acid, sorbic acid, boric acid, succinic acid, adipic acid, glycolic acid, and glutaric acid. In another preferred embodiment, the antimicrobial compositions may further comprise one or more components selected from: a second organic acid, a third organic acid, and an acidulant. Preferably, the second organic acid and third organic acid are independently selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, benzoic acid, lactic acid, malic acid, tartaric acid, mandelic acid, citric acid, fumaric acid, sorbic acid, boric acid, succinic acid, adipic acid, glycolic acid, and glutaric acid. Preferably, the acidulant is selected from the group consisting of phosphoric acid, sulfuric acid, phosphorous acid, hydrochloric acid, hydrobromic acid, and nitric acid.

[0557] It has been discovered that certain antimicrobial compositions of the invention have an improved odor, when the compound of Formula I is Alimet@, compared to similar compositions without Alimet@. For example, blends comprising formic acid have a pungent odor.

In such blends without Alimet@, this odor is more readily detectable than in the same blends containing Alimet@.

Without being limited to a particular theory, it is believed that the Alimets in the blends may lower the vapor pressure of the other organic acids in the blends.

Alternatively, the Alimets may mask the disagreeable odors.

[0558] In another preferred embodiment is provided a method for inhibiting bacteria in silage, said method comprising treating said silage with an anti-bacterial composition comprising a compound of Formula I. Preferably, the compound of Formula I is added to the silage at about 1 lb/ton to 80 lb/ton of fresh forage, more preferably at about 2 lb/ton to 50 lb/ton of fresh forage, more

preferably about 3 lb/ton to 45 lb/ton of fresh forage, more preferably about 4 lb/ton to 40 lb/ton of fresh forage, more preferably about 5 lb/ton to 35 lb/ton of fresh forage, more preferably about 7 lb/ton to 30 lb/ton of fresh forage, more preferably about 9 lb/ton to 25 lb/ton of fresh forage, more preferably about 10 lb/ton to 20 lb/ton of fresh forage. Optionally, the compositions may further comprise an acidulant, as described herein.

[0559] In another preferred embodiment is provided a method for inhibiting bacteria in silage, said method comprising treating said silage with an anti-bacterial composition comprising a compound of Formula I and one or more organic acids as described above. Preferably, the compound of Formula I and other organic acid (s) are added to the silage at about 2 lb/ton to 125 lb/ton of fresh forage combined. In one embodiment, the compound of Formula I and other organic acid (s) are added to the silage at about 4 lb/ton to 100 lb/ton of fresh forage combined. In another embodiment, the compound of Formula I and other organic acid (s) are added to the silage at about 5 lb/ton to 90 lb/ton of fresh forage combined, more preferably about 7 lb/ton to 80 lb/ton of fresh forage combined, more preferably about 8 lb/ton to 70 lb/ton of fresh forage combined, more preferably about 9 lb/ton to 60 lb/ton of fresh forage combined, more preferably about 10 lb/ton to 55 lb/ton of fresh forage combined, more preferably about 12 lb/ton to 50 lb/ton of fresh forage combined, more preferably about 15 lb/ton to 30 lb/ton of fresh forage combined. Optionally, the compositions may further comprise an acidulant, as described herein.

[0560] In a preferred embodiment of the invention, the bacteria inhibited according to the methods of the present invention is from the family Enterobacteriaceae, Campylobacter or Lactobacillaceae. In another preferred embodiment, the bacteria is from the family Campylobacter or Lactobacillaceae. In another preferred embodiment, the bacteria is from the genus Lactobacillus or Campylobacter.

In another preferred embodiment, the bacteria is L. plantarum or C. jejuni. In a particularly preferred embodiment, the bacteria is from the family Enterobacteriaceae. In an even more preferred embodiment, the bacteria is from the genus Salmonella or Escherichia.

In a still more preferred embodiment, the bacteria is S. enteritidis or E. coli.

[0561] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I may be used in the manufacture of a nutrient composition for inhibiting bacteria in animal feed. These nutrient compositions may further comprise one or more organic acids, as described above. Optionally, the compositions may further comprise an acidulant, as described herein.

[0562] In still yet another embodiment of the present invention, the above-mentioned compounds of Formula I may be used in a method for inhibiting bacteria in animal feed comprising computing the concentration in said feed of said compound of Formula I necessary to inhibit bacteria present in said animal feed, and applying said compound of Formula I to said feed in an amount sufficient to achieve said concentration.

[0563] In another embodiment of the present invention, the above-mentioned compounds of Formula I and above-mentioned organic acids may be used in a method of inhibiting bacteria in animal feed comprising computing the concentration in said feed of a compound of Formula I and another organic acid or mixture of organic acids necessary to inhibit bacteria present in said animal feed, and applying said compound of Formula I and said organic acid or mixture of organic acids to said feed in an amount sufficient to achieve said concentration.

[0564] In still yet another embodiment of the present invention, the above-mentioned compounds of Formula I may be used in a method of method for inhibiting mold in animal feed comprising directly or indirectly making information available for computing the concentration in said feed of a

compound of Formula I necessary to inhibit bacteria present in said animal feed, and directly or indirectly making information available for applying said compound of Formula I to said feed in an amount sufficient to achieve said concentration.

[0565] In another embodiment of the present invention, the above-mentioned compounds of Formula I and above-mentioned organic acids may be used in a method of method for inhibiting mold in animal feed comprising directly or indirectly making information available for computing the concentration in said feed of said compound of Formula I and said organic acid or mixture of organic acids necessary to inhibit bacteria present in said animal feed, and directly or indirectly making information available for applying said compound of Formula I and said organic acid or mixture of organic acids to said feed in an amount sufficient to achieve said concentration.

[0566] In another embodiment of the present invention, the above-mentioned animal feeds may be heat- treated, either before or after administration of the above-mentioned compounds of Formula I and/or organic acids.

[0567] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I may be used in a method of inhibiting bacteria in animal feed, the method comprising monitoring the concentration of methionine supplement in said feed composition, adding additional amounts of said methionine supplement as needed to achieve an anti-bacterially effective concentration of methionine supplement in said feed composition.

[0568] Treatment of the animal feed compositions with the compounds of Formula I and with the other organic acids disclosed herein may be done by mixing the compound of Formula I (and other organic acid, if present) with the other ingredients in the feed, such as the corn, soybean meal, meat meal premix, other feed supplements, etc. , as the feed is being formulated. Alternatively, the compound

of Formula I and optional other organic acid (s) may be applied to a pre-mixed or pre-pelleted feed. In either case, the compound of Formula I and optional organic acid (s) are preferably added as liquids, and uniformly disperse throughout the bulk of the feed composition when applied. When the compound of Formula I and another organic acid are both used in the methods of the present invention, preferably said compound of Formula I and said other organic acid or acids are mixed together before application to the animal feeds. This pre-mixed compound of Formula I/organic acid (s) blend can be applied to the animal feed ingredients during formulation of the feed compositions, or can be applied to pre-mixed or pre-pelleted feed.

[0569] In one embodiment of the present invention is presented a method of inhibiting mold in an animal feed composition, the method comprising applying a compound of Formula I to said feed composition, wherein said feed composition comprises corn and soy.

[0570] In another embodiment of the present invention, the methods of inhibiting mold in animal feed comprises treating said feed with an antifungally-effective amount of a compound of Formula I and one or more organic acids as described above.

[0571] In another preferred embodiment, the above- mentioned organic acid is a mixture of formic acid and propionic acid, wherein the formic acid comprises from about 95% to about 5% of the organic acid mixture and the propionic acid comprises from about 5% to about 95% of the organic mixture. Preferably, formic acid comprises from <BR> <BR> po<BR> about 85% to about 15% of the organic acid mixture, and propionic acid comprises from about 15% to about 85% of the organic acid mixture. In another preferred embodiment, formic acid comprises from about 85% to about 65% of the organic acid mixture, and propionic acid comprises from about 15% to 35% of the organic acid mixture. This organic acid mixture can then be combined with the compound of Formula I according to the ratios described above.

[0572] In another embodiment of the present invention is presented a method of inhibiting mold in an animal feed composition, the method comprising applying a compound of Formula I and one or more organic acids to said feed composition, wherein said feed composition comprises corn and soy. In one embodiment, said organic acid is selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, benzoic acid, lactic acid, malic acid, tartaric acid, mandelic acid, citric acid, fumaric acid, sorbic acid, boric acid, succinic acid, adipic acid, glycolic acid, and glutaric acid, or combinations thereof. In another embodiment, the organic acid is formic acid, propionic acid, or combinations thereof.

[0573] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I may be used in a method for delaying the formation of mold in an animal feed composition, the method comprising applying a compound of Formula I to said feed composition, wherein said feed composition comprises corn and soy.

[0574] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I and above-mentioned organic acids may be used in a method for delaying the formation of mold in an animal feed composition, the method comprising applying a compound of Formula I and one or more organic acids to said feed composition, wherein said feed composition comprises corn and soy.

[0575] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I may be used in a method of inhibiting the formation of mold in an animal feed composition, the method comprising applying a compound of Formula I to said feed composition, wherein said feed composition has a moisture content of abut 17% or less. Preferably, the moisture content is at least 0. 01%.

In another embodiment, the moisture content is at least 1%.

In another embodiment, the moisture content is at least 5%.

In another embodiment, the moisture content is at least 10%.

[0576] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I and above-mentioned organic acids may be used in a method of inhibiting the formation of mold in an animal feed composition, the method comprising applying a compound of Formula I and one or more organic acids to said feed composition, wherein said feed composition has a moisture content of about 17% or less. Preferably, the moisture content is at least 0. 01%. In another embodiment, the moisture content is at least 1%. In another embodiment, the moisture content is at least 5%. In another embodiment, the moisture content is at least 10%.

[0577] In yet another embodiment of the present invention is provided a method for inhibiting mold in silage, said method comprising treating said silage with an anti-fungal composition comprising a compound of Formula I.

Preferably, the compound of Formula I is added to the silage at about 1 lb/ton to 40 lb/ton of fresh forage, more preferably about 5 lb/ton to 30 lb/ton of fresh forage, more preferably about 7 lb/ton to 25 lb/ton of fresh forage, more preferably about 10 lb/ton to 20 lb/ton of fresh forage.

[0578] In yet another embodiment of the present invention is provided a method for inhibiting mold in silage, said method comprising treating said silage with an anti-fungal composition comprising a compound of Formula I and one or more organic acids. Preferably, the compound of Formula I and other organic acid(s) are added to the silage at about 5 lb/ton to 50 lb/ton of fresh forage combined, more preferably about 8 lb/ton to 40 lb/ton of fresh forage combined, more preferably about 10 lb/ton to 30 lb/ton of fresh forage combined, more preferably about 15 lb/ton to 25 lb/ton of fresh forage combined.

[0579] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I may

be used in a method for inhibiting the growth of mold in an animal feed composition, the method comprising computing the concentration in said feed of a compound of Formula I necessary to inhibit the growth of mold in said feed composition; and applying said compound of Formula I to said feed composition in said concentration.

[0580] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I and organic acids may be used in a method for inhibiting the growth of mold in an animal feed composition, the method comprising computing the concentration in said feed of a compound of Formula I and another organic acid or mixture of organic acids necessary to inhibit the growth of mold in said feed composition; and applying said compound of Formula I and said organic acid or mixture of organic acids to said feed composition in said concentration.

[0581] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I may be used in a method for inhibiting the growth of mold in animal feed comprising computing the concentration in said feed of a compound of Formula I necessary to inhibit the growth of mold in said animal feed; and applying said compound of Formula I to said feed in said concentration.

[0582] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I and organic acids may be used in a method for inhibiting the growth of mold in animal feed comprising computing the concentration in said feed of a compound of Formula I and another organic acid or mixture of organic acids necessary to inhibit the growth of muld in said animal feed ; and applying said compound of Formula I and said organic acid or mixture of organic acids to said feed in said concentration.

[0583] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I may be used in a method for improving the mold resistance of an animal feed composition, the method comprising

discontinuing the use of DL-methionine as a feed supplement; computing the concentration in said feed of a compound of Formula I necessary to inhibit the growth of mold in said animal feed; and applying said compound of Formula I to said feed in said concentration.

[0584] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I and said organic acids may be used in a method for improving the mold resistance of an animal feed composition, the method comprising discontinuing the use of DL-methionine as a feed supplement; computing the concentration in said feed of a compound of Formula I and another organic acid or mixture of organic acids necessary to inhibit the growth of mold in said animal feed; and applying said compound of Formula I and said organic acid or mixture of organic acids to said feed in said concentration.

[0585] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I may be used in a method for inhibiting mold in animal feed directly or indirectly making information available for computing the concentration in said feed of said compound of Formula I necessary to inhibit mold present in said animal feed; and directly or indirectly making information available for applying said compound of Formula I to said feed in said concentration.

[0586] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I and organic acids may be used in a method for inhibiting mold in animal feed directly or indirectly making information available for computing the concentration in said feed of said compound of Formula I and the concentration of said organic acid or mixture of organic acids necessary to inhibit mold present in said animal feed; and directly or indirectly making information available for applying said compound of Formula I and said organic acid or mixture of organic acids to said feed in said concentration.

[0587] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I may be used in the manufacture of a nutrient composition for inhibiting mold in animal feed by treating said feed with said nutrient composition in an anti-mold effective amount.

In another embodiment, the nutrient composition may also comprise one or more of the above-mentioned organic acids, or a mixture thereof.

[0588] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I may be used in a method of inhibiting mold in an animal feed composition, the method comprising monitoring the concentration of methionine supplement in said feed composition, adding additional amounts of said methionine supplement as needed to achieve an anti-mold effective concentration of methionine supplement in said feed composition.

[0589] In yet another embodiment of the present invention, the above-mentioned compounds of Formula I may be used in a method of enhancing the palatability of animal food, the method comprising treating the food with a compound of Formula I in an amount sufficient to give a concentration of the compound of Formula I in the food of between about 0.01 wt. % and about 0.5 wt. %. Preferably, the food is food for canines, felines, or aquaculture. For dogs, the concentration of the compound of Formula I in the food is preferably between about 0.05 % and about 0.15% ; for cats, it is preferably between about 0. 20% and 0. 30%.

For both dogs and cats, the compound of Formula I is preferably HMBA or DLM.

[0590] In a preferred embodiment, the compositions or combinations described herein comprise HMBA, or a salt, ester or amide thereof; and a first organic acid selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, benzoic acid, lactic acid, malic acid, tartaric acid, mandelic acid, citric acid, fumaric acid, sorbic acid, boric acid, succinic acid,

adipic acid, glycolic acid, and glutaric acid. Even more preferably, the first organic acid is selected from the group consisting of formic acid, propionic acid, butyric acid, and lactic acid.

[0591] In various preferred embodiments, the compositions or combinations further comprise an acidulant selected from the group consisting of mineral acids, preferably selected from the group consisting of phosphoric acid, sulfuric acid, phosphorous acid, hydrochloric acid, hydrobromic acid, and nitric acid; a second organic acid selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, benzoic acid, lactic acid, malic acid, tartaric acid, mandelic acid, citric acid, fumaric acid, sorbic acid, boric acid, succinic acid, adipic acid, glycolic acid, and glutaric acid. In even more preferred embodiments, the first organic acid and second organic acid are independently selected from the group consisting of formic acid, propionic acid, butyric acid, and lactic acid; and/or a third organic acid selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, benzoic acid, lactic acid, malic acid, tartaric acid, mandelic acid, citric acid, fumaric acid, sorbic acid, boric acid, succinic acid, adipic acid, glycolic acid, and glutaric acid.

[0592] In a still more preferred embodiment, the compositions and combinations described herein comprise HMBA, formic acid, propionic acid, and phosphoric acid.

Preferably, the content of HMBA is from about 5% to about 20% of the sum of the HMBA, formic acid, propionic acid, and phosphoric acid content; the content of the formic acid is from about 65% to about 85% of said sum; the content of the propionic acid is from about 1% to about 15% of said sum; and the content of the phosphoric acid is from about 5% to about 20% of said sum. Even more preferably, the content of HMBA is about 10% of said sum; the content of the formic acid is about 75% of said sum ; the content of the propionic acid is about 5% of said sum; and the content

of the phosphoric acid is about 10% of said sum.

Alternatively, the content of HMBA is from about 20% to about 40% of said sum, the content of the formic acid is from about 45% to about 65% of said sum, the content of the propionic acid is from about 1% to about 20% of said sum, and the content of the phosphoric acid is from about 1% to about 15% of said sum; even more preferably, the content of HMBA is about 30% of said sum, the content of the formic acid is about 55% of said sum, the content of the propionic acid is about 10% of said sum, and the content of the phosphoric acid is about 5% of said sum.

[0593] In another more preferred embodiment, the compositions and combinations described herein comprise HMBA, butyric acid, lactic acid, and phosphoric acid.

Preferably, the content of HMBA is from about 20% to about 40% of the sum of the HMBA, butyric acid, lactic acid, and phosphoric acid content; the content of the butyric acid is from about 10% to about 30% of said sum; the content of the lactic acid is from about 10% to about 30% of said sum; and the content of the phosphoric acid is from about 20% to about 40% of said sum. Even more preferably, the content of HMBA is about 30% of said sum; the content of the butyric acid is about 20% of said sum; the content of the lactic acid is about 20% of said sum; and the content of the phosphoric acid is about 30% of said sum. Alternatively, the content of HMBA is from about 20% to about 40% of said sum of the 2-hydroxy-4- (methylthio) butanoic acid, butyric acid, lactic acid, and phosphoric acid content, the content of the butyric acid is from about 5% to about 25% of said sum, the content of the lactic acid is from about 10% to about 30% of said sum, and the content of the phosphoric acid is from about 25% to about 45% of said sum; more preferably, the content of HMBA is about 30% of said sum, the content of the butyric acid is about 15% of said sum, the content of the lactic acid is about 20% of said sum, and the content of the phosphoric acid is about 35% of said sum.

[0594] In yet another more preferred embodiment, the compositions and combinations described herein comprise HMBA, butyric acid, formic acid, lactic acid, and phosphoric acid. Preferably, the content of HMBA is from about 10% to about 30% of the sum of the HMBA, butyric acid, formic acid, lactic acid, and phosphoric acid content; the content of the butyric acid is from about 2% to about 22% of said sum; the content of the formic acid is from about 20% to about 40% of said sum; the content of the lactic acid is from about 8% to about 28% of said sum; and the content of the phosphoric acid is from about 10% to about 30% of said sum. Even more preferably, the content of HMBA is about 20% of said sum; the content of the butyric acid is about 12% of said sum; the content of the formic acid is about 30% of said sum; the content of the lactic acid is about 18% of said sum; and the content of the phosphoric acid is about 20% of said sum.

[0595] In yet another more preferred embodiment, the compositions and combinations described herein comprise HMBA, butyric acid, lactic acid, propionic acid, and phosphoric acid. Preferably, the content of HMBA is from about 10% to about 30% of the sum of the HMBA, butyric acid, lactic acid, propionic acid, and phosphoric acid content; the content of the butyric acid is from about 2% to about 22% of said sum; the content of the lactic acid is from about 8% to about 28% of said sum; the content of the propionic acid is from about 20% to about 40% of said sum; and the content of the phosphoric acid is from about 10% to about 30% of said sum. Even more preferably, the content of HMBA is about 20% of said sum; the content of the butyric acid is about 12% of said sum; the content of the lactic acid is about 18% of said sum; the content of the propionic acid is about 30% of said sum; and the content of the phosphoric acid is about 20% of said sum.

[0596] In yet another more preferred embodiment, the compositions and combinations described herein comprise HMBA, butyric acid, formic acid, propionic acid, and

phosphoric acid. Preferably, the content of HMBA is from about 1% to about 20% of the sum of the HMBA, butyric acid, formic acid, propionic acid, and phosphoric acid content; the content of the butyric acid is from about 1% to about 15% of said sum; the content of the formic acid is from about 65% to about 85% of said sum; the content of the propionic acid is from about 1% to about 15% of said sum; and the content of the phosphoric acid is from about 1% to about 15% of said sum. Even more preferably, the content of HMBA is about 10% of said sum; the content of the butyric acid is about 5% of said sum; the content of the formic acid is about 75% of said sum; the content of the propionic acid is about 5% of said sum; and the content of the phosphoric acid is about 5% of said sum.

[0597] In yet another more preferred embodiment, the compositions and combinations described herein comprise HMBA, formic acid, and propionic acid. Preferably, the content of HMBA is from about 20% to about 40% of the sum of the HMBA, formic acid, and propionic acid content; the content of the formic acid is from about 40% to about 60% of said sum; and the content of the propionic acid is from about 10% to about 30% of said sum. Even more preferably, the content of HMBA is about 30% of said sum; the content of the formic acid is about 50% of said sum; and the content of the propionic acid is about 20% of said sum.

[0598] In yet another more preferred embodiment, compositions and combinations described herein comprise HMBA and phosphoric acid. Preferably, the content of the HMBA is from about 5% to about 50% of the sum of the HMBA and phosphoric acid content. In various more preferred embodiments, the content of the HMBA is about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% of said sum.

[0599] In another embodiment of the present invention is provided an animal feed composition comprising a

compound of Formula I as described herein, and an acidulant as described herein.

[0600] In another embodiment of the present invention is provided a method of inhibiting or killing microbes in a subject, comprising treating said subject with a composition or combination as described herein. In a preferred embodiment, the subject to be treated is water or food, preferably selected from the group consisting of human food, livestock food, pet food, or aquaculture food.

[0601] Animals for which the food, food ingredients and/or feed compositions described herein may be provided include humans, ruminants such as dairy cows, lactating dairy cows, dairy calves, beef cattle, sheep, and goats; aquaculture such as fish and crustaceans (including, but not limited to, salmon, shrimp, carp, tilapia and shell fish; livestock such as swine and horses; poultry such as chickens, turkeys, and hatchlings thereof; and companion animals such as dogs and cats.

[0602] The exact formulation of the above-mentioned animal feed composition is not critical to the present invention. Feed ingredients are selected according to the nutrient requirements of the particular animal for which the feed is intended; these requirements depend, inter alia, upon the age and stage of development of the animal, the sex of the animal, and other factors. Feed ingredients may be grouped into eight classes on the basis of their composition and their use in formulating diets: dry forages and roughages; pasture, range plants and forages fed fresh; silages; energy feeds; protein supplements; mineral supplements; vitamin supplements; and additives. See National Research Council (U. S. ) Subcommittee on Feed Composition, United States-Canadian Tables of Feed Composition, 3d rev., National Academy Press, pp. 2,145 (1982). These classes are, to a certain extent, arbitrary, as some feed ingredients could be classified in more than one class. Typically, a feed formulation will also depend upon the costs associated with each ingredient, with the

least-expensive composition of ingredients which gives the needed nutrients being the preferred formulation.

[0603] Silage is a forage product that is produced from the harvest, storage and fermentation of green forage crops such as corn and grain sorghum plants. These crops are chopped, stems and all, before the grain is ready for harvest. The plant material is stored in silos, storage bags, bunkers or covered piles causing the material to ferment, thereby lowering the pH and preserving the plant material until it can be fed. The ensiled forage is subject to changes in pH, temperature, and oxygen levels.

[0604] As noted above, feed formulations depend in part upon the age and stage of development of the animal to be fed. Leeson and Summers (Nutrition of the Chicken, 4th ed. , pp. 502-510, University Books (2001) ) describe several representative poultry diets for pullets, layers, broilers and broiler breeders. For example, most chicken diets contain energy concentrates such as corn, oats, wheat, barley, or sorghum; protein sources such as soybean meal, other oilseed meals (e. g. , peanut, sesame, safflower, sunflower, etc. ), cottonseed meal, animal protein sources (meat and bone meal, dried whey, fish meal, etc. ), grain legumes (e. g. , dry beans, field peas, etc. ), and alfalfa; and vitamin and mineral supplements, if necessary (for instance, meat and bone meal is high in calcium and phosphorous, and thus these minerals do not need to be supplemented in a feed ration containing meat and bone meal). The relative amounts of the different ingredients in poultry feed depends in part upon the production stage of the bird. Starter rations are higher in protein, while grower and finisher feeds can be lower in protein since older birds require less protein. Model diets for swine and other animals are also available, and may be modified according to the particular needs of the animal (s) to be fed.

[0605] The term"inhibit"when used herein in phrases such as"inhibiting bacteria,, means any one or more of (a)

killing bacteria or mold; (b) any decrease in growth of the bacteria or mold, which may be measured in terms of colony counts; (c) any decrease in the concentration of bacteria or mold; or (d) the inability of bacteria or mold to grow on a particular selection medium. Each of these may be determined, for instance, by comparing the bacterial or fungal colony counts or concentration of bacteria or mold present in the absence of the application of the methods of the present invention with the bacterial or fungal colony counts or concentration of bacteria or mold after application of the methods of the present invention.

Application of suitable bactericides or fungicides will show a ten-fold difference in colony counts.

[0606] Certain methods of the present invention call for computing, for instance, the concentration, of a compound of Formula I in an animal feed, necessary to inhibit bacteria or mold present in an animal feed, or the concentration of a compound of Formula I and another organic acid or acids necessary to inhibit bacteria or mold present in an animal feed. Provided herein are Examples 1- 24, which illustrate amounts of compound of Formula I and/or other organic acids that are sufficient to inhibit bacteria or mold. Also provided hereinabove and hereinbelow are acceptable ranges of amounts of compound of Formula I and/or other organic acids, and ratios between the two, which are suitable for use the methods of the present invention. Other suitable concentrations, ranges and ratios can be determined as needed.

[0607] Treatment of the animal feed compositions with the compounds of Formula I and optionally with the other organic acids disclosed herein, or with the compounds of Formula I and optionally with other organic acid (s), may be done by mixing the compound of Formula I (and other organic acid, if present) with the other ingredients in the feed, such as the corn, soybean meal, other feed supplements, etc. , as the feed is being formulated. Alternatively, the compound of Formula I and optional other organic acid (s)

may be applied to a pre-mixed or pre-pelleted feed. In either case, the compound of Formula I and optional organic acid (s) are preferably added as liquids, and uniformly disperse throughout the bulk of the feed composition when applied. When the compound of Formula I and another organic acid are both used in the methods of the present invention, preferably said compound of Formula I and said other organic acid or acids are mixed together before application to the animal feeds. This pre-mixed compound of Formula I/organic acid (s) blend can be applied to the animal feed ingredients during formulation of the feed compositions, or can be applied to pre-mixed or pre-pelleted feed.

[0608] The term"cfu"stands for colony forming units.

[0609] The following examples illustrate the invention.

EXAMPLE 1 [0610] The effects of increasing quantities of formic acid and/or AlimetX on the colony counts of four bacteria (E. coli, S. enteritidis, L. plantarum and C. jejuni) were studied. Varying amounts of formic acid or Alimet were added individually to cultures of these bacteria at pH 4.5 or 6.75 and the cultures were incubated for a length of time, whereupon colony counts were performed.

[0611] The S. enteritidis culture for the in vitro study contained a mixture of S. enteritidis ID-Lelystad (nalidixic acid resistant strain) and S. enteritidis (97.07773 RIVM, isolated from poultry). The E. coli culture contained a mixture of E. coli 0149K91K88 (VA2000-08915, pig pathogen) and E. coli ATCC 25922. The L. plantarum culture studied was L. plantarum Bd 99.00553. The C. jejuni culture studied was C. jejuni C356, ex. ID-Lelystad.

[0612] S. enteritidis and E. coli from fresh overnight cultures in Brain Heart Infusion broth were incubated aerobically in phosphate buffered (0.11 M) salt solution (8.5 g/L NaCl) with peptone (1 g/L), except for S.

enteritidis at pH 4.5. For this culture, medium 5 was used as the broth, and the culture was incubated aerobically for 4 hours at 37°C. Colony counts were performed according to standard operating procedures.

[0613] The fresh overnight culture of L. plantarum in brain heart infusion broth were used to inoculate medium 5.

The test tubes were incubated under reduced oxygen atmosphere for 6 hours at 37°C. Colony counts were performed.

[0614] C. jejuni grown on Campylobacter blood-free selective agar was used for inoculation. Preston broth was incubated under reduced oxygen atmosphere for 6 hours at 37 °C. Colony counts were performed.

[0615] Formic acid and Alimets were added to the bacterial cultures in concentrations of 0.108 g/L, 0.30 g/L and 0.83 g/L. These dosages were chosen based on commercial use of Alimets and an approximate 10-fold dilution in the proximal digestive tract.

[0616] A summary of the results obtained with formic acid and Alimets on S. enteritidis and E. coli is given in Tables 1 and 2, and on L. plantarum and C. jejuni in Tables 3 and 4; the results are illustrated in Figures 1-4.

Table 1 Effect of formic acid and AlimetX on population of S. enteritidis after 4 hours at pH 4.5 and 6.75 initial colony count: 5.23 log cfu/mL Acid (g/L) pH = 4.5 pH = 6.75 Alimets Formic log cfu/mL A log log cfu/mL A log -- 5.03-0. 20 6.62 1.42 0. 108-4. 92-0.31 6.71 1.51 0. 30-4. 96-0.27 6.63 1.43 0. 83-4. 93-0.30 6.53 1.33 - 0.108 5.04-0. 19 6.79 1.59 - 0. 30 4.96-0. 27 6.77 1.57 - 0. 83 4. 86-0.38 6.72 1.52

Table 2 Effect of formic acid and Alimets on population of E. coli after 4 hours at pH 4.5 and 6.75 initial colony count: 5.24 log cfu/mL Acid (g/L) pH = 4.5 pH = 6.75 Alimet# Formic log cfu/mL A log log cfu/mL A log -- 5.36 0.12 7.47 2.23 0. 108-5. 45 0.21 7.33 2.09 0. 30-5. 25 0.01 7.36 2.12 0. 83-3. 96-1.28 7.39 2.15 - 0.108 5.19-0. 05 7.48 2.24 - 0. 30 4.96-0. 28 7.50 2.26 - 0. 83 5.08-0. 16 7.49 2. 25 Table 3 Effect of formic acid and Alimets on population of L. plantarum after 6 hours at pH 4.5 and 6.75 initial colony count: 5.04 log cfu/mL A log = logsample - loginitial Acid (g/L) pH = 4.5 pH = 6. 75 Alimet Formic log cfu/mL A log log cfu/mL A log -- 5.67 0.63 6.10 1.06 0. 108-5. 67 0.63 6.09 1.05 0. 30-5. 57 0.53 6.20 1.16 0. 83-5. 74 0.70 5.70 0.66 - 0.108 5.75 0.71 5.88 0.84 - 0. 30 5.74 0.70 6.23 1.19 - 0. 83 5.56 0.52 6.19 1.15

Table 4 Effect of formic acid and Alimet on population of C. jejuni after 6 hours at pH 4.5 and 6.75 initial colony count: 5.23 log cfu/mL Acid (g/L) pH = 4.5 pH = 6. 75 Alimets Formic log cfu/mL A log log cfu/mL A log -- 3.70-1. 53 6.54 1. 31 0. 108-0. 108 4.07-1. 16 6.44 0. 30-0. 30 3.95-1. 28 6.40 0. 83-0. 83 2.80-2. 43 6.34 - 0.108 3.86-1. 37 6.27 1.04 - 0. 30 2. 63-2. 60 6.38 1.15 - 0. 83 < 1.30 <-3.93 6.25 1.02 [0617] S. enteritidis : Prior to inoculation, the S. enteritidis cultures had a colony count of 5.03 log cfu/mL at pH 4.5, and of 6.62 at pH 6.75. The results obtained at both pH values were similar for Alimet and formic acid: at pH 4.5, neither had a significant effect on inhibiting the growth of S. enteritidis ; at pH 6.75, no inhibition of S. enteritidis was observed.

[0618] E. coli : Prior to inoculation, at pH 4.5, the E. coli cultures had a colony count of 5.36 log cfu/mL; at pH 6.75, it was 7.47. 0.83 g/L Alimets gave approximately a 1.3 log reduction of E. coli growth at pH 4.5, compared to the approximately 0.1 log reduction by the same concentration of formic acid at pH 4.5. Lower concentrations of both Alimets and formic acid showed little or no inhibition. Neither Alimet# nor formic acid inhibited E. coli at pH 6.75.

[0619] L. plantarum : Prior to inoculation, at pH 4.5, the L. plantarum cultures had a colony count of 5.67 log cfu/mL; at pH 6.75, it was 6.10. Neither Alimet# nor formic acid inhibited L. plantarum at either pH studied.

[0620] C. jejuni : Prior to inoculation, at pH 4.5, the C. jejuni cultures had a colony count of 3.70 log cfu/mL; at pH 6.75, it was 6.54. All doses of Alimets inhibited C. jejuni at pH 4.5. 0.83 g/L of Alimets gave approximately a 2.4 log reduction of C. jejuni growth at this pH. Lower dosages of Alimet (0.108 g/L and 0.30 g/L) gave approximately 1.1 and 1.2 log reductions, respectively. Formic acid demonstrated comparable inhibition. No antibacterial activity was shown against C. jejuni at pH 6.75 for any Alimet# or formic acid concentration studied.

[0621] These results are demonstrated graphically in Figures 1A, 1B, 1C, and 1D. Figures 2A, 2B, 2C, and 2D demonstrate the pH dependent effects of formic acid and Alimet@. None of the four bacteria studied were inhibited by either formic acid or Alimet# at pH of 6.75 ; in fact, at this pH, the colony forming unit count of each bacteria increased, with the E. coli count increasing the most, and L. plantarum increasing the least.

EXAMPLE 2 [0622] The effect of higher dosages of Alimet and formic acid on the colony count of S. enteritidis cultures was studied, following the procedure described in Example 1. The results obtained are given in Table 5 and illustrated in Figure 3.

Table 5 Effect of formic acid and Alimet# on population of S. enteritidis after 4 hours at pH 4.5 and 6.75 initial colony count: 5.23 log cfu/mL Acid (g/L) pH = 4.5 pH = 6.75 Alimet° Formic log cfu/mL A log log cfu/mL A log -- 5. 15-0.09 6.92 1.69 1-5. 02-0. 21 6. 61 1.38

3-4. 76-0.48 5.97 0.74 5-2. 37-2.86 5.43 0.20 - 1 5.01-0. 22 6.92 1.69 - 3 4.55-0. 68 6.58 1.35 - 5 3.83-1. 41 6.10 0.87 [0623] Prior to inoculation, the S. enteritidis cultures had a log cfu/mL of 5.15 at pH 4.5, and of 6.92 at pH 6.75. At pH 6.75, the addition of 5 g/L formic acid or 3 g/L Alimet# gave approximately a 1 log cfu/mL growth inhibition. An addition of 5 g/L Alimet stops the growth of S. enteritidis. At pH 4.5, 5 g/L Alimet reduces the growth of S. enteritidis by approximately 2.8 log cfu/mL.

Lower concentrations of Alimet gives a smaller effect.

Formic acid at 5 g/L reduces the growth of S. enteritidis by approximately 1.3 log cfu/mL. Thus, at the dose ranges studied, the antibacterial effect of Alimet against S. enteritidis is greater than that of formic acid. These results are demonstrated graphically in Figure 3.

EXAMPLE 3 [0624] Combinations of Alimet# and formic acid were studied, following the procedure described in Example 1.

The results obtained are given in Table 6 and illustrated in Figures 4A and 4B.

Table 6 Effect of formic acid and Alimet# on population of S. enteritidis after 4 hours at pH 4.5 and 6.75 initial colony count: 5.15 log cfu/mL) Acid (g/L) pH = 4.5 pH = 6. 75 Alimets Formic log cfu/mL A log log cfu/mL A log -- 4. 99-0.17 6.87 1.80 3-4. 76-0.39 5.89 0.82 5-2. 07-3. 08 5.45 0. 38

- 3 4. 57-0.58 6.51 1.44 - 5 3.94-1. 21 6.19 1.12 0.75 2.25 4.78-0. 37 6.27 1.20 1.25 3.75 4.01-1. 14 5. 94 0.88 1.50 1.50 4.73-0. 42 6.11 1.04 2.25 0.75 4.78-0. 37 5.97 0.90 2.50 2.50 2.48-2. 67 5.74 0.67 3 5 1.15-4. 00 5.31 0.24 3.75 1.25 2.11-3. 04 5.54 0.47 [0625] Combinations of Alimet and formic acid having a combined concentration of 5 g/L inhibit growth of S. enteritidis to a greater extent than do combinations having a combined concentration of 3 g/L. Three 5 g/L combinations were prepared, having Alimet@-to-formic acid ratios of 1: 3, 1: 1, and 3: 1.

[0626] At pH 4.5, treatment with 3 g/L of Alimets alone gave an approximately 0.4 log cfu/mL reduction in S. enteritidis growth. Treatment at that pH with 5 g/L of formic acid gave an approximately 1.2 log cfu/mL reduction.

Remarkably, treatment with a combination of 3 g/L Alimet# and 5 g/L formic acid gave a reduction of 4 log cfu/mL, which was higher than expected given the individual results with Alimets and formic acid at those levels. The results obtained suggest that at pH 4.5, combinations of 2.5 g/L Alimet and 2.5 g/L formic acid, and with 3 g/L Alimet5 and 5 g/L formic acid may have a synergistic effect. The latter combination gives the best results of all tested combinations: at pH 4.5, this combination gives 4 log (almost complete) reduction of S. enteritidis.

EXAMPLE 4 [0627] The effects of blends of organic acids (butyric, citric, formic, lactic, and propionic) and Alimet on the colony counts of E. coli (ATCC 25922) grown in trypticase soy broth at 35°C according to the

manufacturer's instructions were studied. Blends of organic acid: Alimets of 2: 1 and 5: 1 were studied, at a total concentration (organic acid + Alimet@) of 6 g/L.

[0628] The pH of the solutions were originally adjusted to pH 5 by addition of HCl and/or NaOH as needed.

Activated E. coli culture solutions were transferred to fresh soy broth twice at 24-hour intervals prior to before addition of the organic acid: Alimets blend. E. coli culture solutions were centrifuged; pellet produced was re- suspended with Butterfield buffer, and the resulting solutions were diluted to approximately 107 CFU E. coli/mL.

[0629] Bottles were inoculated with 100 laL of prepared bacterial suspension and an organic acid: Alimets blend. Samples were taken after five and 24 hours of incubation, serially diluted and spread-plated on trypticase soy agar, and incubated at 35°C for 24 hours.

Populations of E. coli are reported in Table 7 below.

Table 7 Effect of Alimet@/acid blends on E. coli populations in trypticase soy broth initial colony count: 4.97 log cfu/mL A id Acid : Alimet original log cfu/mL Acid ratio pH c ratio pH t=4h t=24h Control--7. 18 9. 22 HC1--7. 85 8. 34 5 : 1 ca. 4. 2 4. 68 3. 98 lactic 2 : 1 4. 1 4. 87 4. 45 5 : 1 3. 1 4. 95 <1 formic 2 : 1 3. 56 4. 95 1. 00 5 : 1 4. 75 6. 38 8. 59 citric 2 : 1 4. 59 5. 90 8. 66 5 : 1 4. 62 4. 77 3. 70 butyric 2 : 1 4. 6 4. 85 3. 80 5 : 1 4. 54 4. 79 4. 57 propionic 2 : 1 4. 53 4. 83 4. 53 [0630] Blends with formic acid were the most effective among the tested blends to control E. coli at both 5: 1 and 2: 1 blends of formic acid: Alimet@. Upon prolonged exposure (after 5 hours), both ratio give nearly complete reduction of E. coli. Blends of lactic, butyric and propionic acids with Alimet suppressed the growth of E. coli, but did not reduce the bacterial population in 24 hours.

EXAMPLE 5 [0631] The effects of hydrochloric acid, formic acid, lactic acid, or Alimets on the colony counts of E. coli were studied. Amounts of formic acid, lactic acid, or Alimet were added to cultures of E. coli, grown in a soy

broth, at pH 4 or 7.3. The cultures were incubated, and colony counts performed at increasing times.

[0632] The results are illustrated in Figure 5.

Formic and lactic acid, and Alimet@, decreased the colony counts of E. coli better than hydrochloric acid. As in Example 1, Alimet showed a better reduction of E. coli colony counts as compared to formic acids, and showed comparable reduction compared to lactic acid.

EXAMPLE 6 [0633] The effect of Alimet on Salmonella in a meat meal premix was studied according to the protocol set forth by Smyser and Snoeyenbos (Poultry Sci. 58 (1979) 50-54).

Meat meal premix (Papillon Ag Products, Inc. , Easton, MD) containing approximately 77% crude protein was used in the assays. Ten grams of premix test sample for each concentration of Alimet studied were measured into a sterile tube (three replicates per sample). Sterile water (1 mL) was added to each tube to assure adequate moisture for salmonella multiplication. A final moisture level of 20% was achieved after inoculation. Each test sample was inoculated with 1.0 mL of a diluted TSB broth culture of nalidixic acid (NA) resistant Salmonella (approximately 103-5 cells/g as determined from spread plate counts of the culture). The inoculated samples were mixed with a sterile tongue blade or equivalent tool and incubated at 37°C for the duration of the trial.

[0634] Salmonella counts were determined using brilliant green agar containing sodium nalidixate at days 1,2 and 3. A 1 g test sample was taken from each tube and transferred to 9 mL sterile water. This test sample was incubated at 4°C for approximately 4 hours, then agitated for 60-90 seconds. Each test sample was serially diluted in 1: 10,1 : 100 and 1: 1000 proportions, and 100 pL of undiluted test sample, 1: 10 diluted sample, 1: 100 diluted sample, and 1: 1000 diluted samples were plated on brilliant green agar plates containing sodium nalidixate. The percent recovery

of Salmonella for different levels of Alimets is reported in Table 8 and Figure 7.

Table 8 Recovery of Salmonella in meat meal premix with 20% moisture; 1: 10 dilution Alimets Alimet % Salmonella recovered conc. conc. (reported) (found) day 0 day 1 day 2 day 3 control (0%) 100% 1990% 971% 267% 0. 275% 0. 056% 100% 102% 67% < 3% * 0. 18% 0. 140% 100% 190% 65% 11% 0. 25% 0. 188% 100% 476% 139% 25% 0. 36% 0. 192% 100% 114% 62% 4% 0. 40% 0. 220% 100% 343% 267% 53% 0. 69% 0. 631% 100% 5% < 3% * < 3% * * below detection limit [0635] The control sample (no Alimets added) showed an initial steep increase in Salmonella population one day after inoculation, indicating multiplication of the bacteria. This multiplication was followed by a gradual decline in the bacterial counts on days 2 and 3. Results from the highest level of AlimetX tested suggest that Alimets is bactericidal for Salmonella in meat and bone meal. As Figure 8 illustrates, AlimetX at this level gives results comparable to treatment with formic acid at 1. 65% (15 kg/ton). See Liu, "Using Organic Acids to Control Salmonella in Poultry Production,"Kemin Industries (Asia) Pte Limited, Singapore, available at http://www. kemin. com.

[0636] Alimet in the range of 0.14-0. 22% likewise showed anti-Salmonella effects, and does not appear to be dose-dependent in this range. However, each sample was from different batches of MBM, which may be responsible for the lack of dose-response. The initial multiplication seen in the control was significantly reduced upon addition of

Alimets at these levels. The subsequent decline occurred faster for these lower levels of Alimet compared to the control.

EXAMPLE 7 [0637] Fungal growth in basal starter mash was studied in compositions supplemented with DLM or Alimets as the methionine source. Total microbial growth was monitored by measuring carbon dioxide (CO2) formation in sealed vessels at 28°C over time. Measurement of CO2 formation does not distinguish between bacterial and mold growth; however, the ability of mold to grow at much lower water activities, compared to bacteria, is well known, and both mold and bacteria play a part in feed degradation.

[0638] The technique of using a closed system and measuring CO2 formation has been verified as an approximation of the conditions found in grain storage bins. See, e. g. , Muir et al. , Trans. ASAE, 28 (5) 1673-1675 (1985), the contents of which are hereby incorporated by reference in their entirety.

[0639] A mash starter mash feed (formulation shown in Table 9, below) was subdivided into three groups: basal (control), 0.2% DLM, or 0.2% Alimet@. The feed studied had no commercial mold inhibitors added, and is representative of a typical broiler feed.

[0640] Initial moisture of the feed was 10.8%. After the addition of Alimets or DLM, the moisture of the samples was adjusted by the addition of 2%, 4%, or 6% sterile distilled water to promote mold growth, achieving three moisture groups: 83.2% dry matter/12. 8% moisture; 85. 2% dry matter/14. 8% moisture; and 87.2% dry matter/16.8% moisture.

[0641] For each study, four replicate samples of each moisture group were mixed, and 600 g of the mixture was placed into 1L containers, sealed, and placed at 28°C in a temperature-controlled room. Draeger Detector tubes (CO2- measuring, obtained from Fisher Scientific) were used to measure the developed CO2 at different days following vessel

sealing (two measurements were made per week). Draeger Short-Term Detector Tubes are glass tubes filled with inert carrier and an indicating reagent. The reagent produces a colorimetric indication in the presence of a particular gas (CO2). The concentration of gas is read directly from the discoloration on the tube's printed scale.

[0642] The specific mold species present were not identified. Statistical analysis was accomplished by using Duncan's multiple range test (SAS). Different letters on individual time points in Figures 9-11 indicate statistical differences of P < 0.05.

Table 9 Basal starter mash formulation Ingredient % by weight of total mix Corn 60.551 Soybean meal 32.254 Fat, animal 3.665 Dical940224PhosfromD (dicalcium phosphate) 1. 861 Limestone 0.811 Novus Vitamin/Mineral premix manufactured 0. 350 by Trouw Nutrition (Highland, Illinois) Salt 0.340 L-lysine HC1 78% 0.097 Threonine 0.051 Santoquin-mix6 Antioxidant preservative sold by Solutia 0.019 Inc. (St. Louis, Missouri) Copper Sulfate 0. 003 [0643] As shown in Figure 9, Alimet effectively inhibited mold growth for up to seven days at the highest moisture level tested (83.2% dry matter/16.8% moisture), while DLM was the least effective, and, in fact, showed mold growth within two days. In fact, DLM-treated starter

mash showed mold growth faster than basal mash (i. e. , feed with no added methionine or methionine analog), and faster than the Alimet@-treated mash, for all moisture levels tested.

[0644] As demonstrated in Figure 10, Alimet@-treated feed showed a slower rate of mold growth in 85.2% dry matter/14. 8% moisture feed, than the DLM-treated feed.

[0645] For feed having 87.2% dry matter/12. 8% moisture, Alimet@-treated feed shows low mold levels for up to sixty days, while DLM-treated feed shows a sharp increase in mold growth after only twenty days (see Figure 11).

[0646] Figures 9,10, and 11 each illustrate that DLM treated mash is more likely to develop mold than either methionine-deficient feed or feed treated with Alimet@, and that Alimets was more effective in inhibiting mold growth than compared to untreated feed, or feed supplemented with DLM.

EXAMPLE 8 [0647] The experiment described above in Example 7 was repeated with blends of 2.0 lb/ton, 1.5 lb/ton, 1.0 lb/ton or 0.5 lb/ton of 65% propionic acid and either 2% AlimetX or 2% DLM. The blends were prepared according to the matrix outlined in Table 8, below. The 65% propionic acid was buffered with ammonium hydroxide to a pH of 5.5.

Table 10 Antifungal blends of Alimet# or DLM with propionic acid - Alimet Propionic DLM (%) (lb/ton) (%) 1 0. 2-- 2-2 0.2 3-1. 5 0.2 4-1. 0 0.2 5-0. 5 0.2 6--0.2 7 0.2 2- 8 0.2 1. 5- 9 0.2 1. 0- 10 0. 2 0. 5- [0648] Statistical analysis was accomplished by using Duncan's multiple range test (SAS). Different letters on individual time points in graphs indicate statistical differences of P<0.05.

[0649] As shown in Figure 12, basal diet having 85. 2% dry matter and containing 0. 2% Alimet# delayed the onset of mold growth by 5 days; the effect of 2.0 lb/ton, 1.5 lb/ton and 1.0 lb/ton 65% propionic acid is about 11 days, i. e, no significant difference was seen when propionic acid was added beyond 1 lb/ton.

[0650] Combinations of Alimet and propionic acid were compared to feed treated with propionic acid alone, and the results are shown in Figures 13-15. The results indicate that, for all moisture levels studied, basal feed containing Alimet plus propionic acid showed improved mold inhibition compared to feed containing only propionic acid.

EXAMPLE 9 [0651] The effect of formic, butyric and lactic acids on Salmonella populations in basal corn soy based broiler starter feed was studied to determine the levels of these acids required for complete bacteriacide.

[0652] Tests were carried out using 1 g feed (crumble ground) with 6% meat and bone meal ("MBM"). Aqueous 25% solutions of formic, butyric, and lactic acids were prepared. The acid solutions were added to the feed as indicated below; water (1 mL) and 150 mM HC1 (1.8 mL) was added to bring the pH to 4.0. Naldixic Acid resistant Salmonella (provided by Dr. Stan Bailey, USDA/ARS, Athens, Georgia) (initial colony count = 40,000 cfu/g) was added, and the feed solutions were incubated at 37°C for 90 min.

Each sample was diluted with 6 mL H20, plated, and counted the following day. Colony counts are reported in Table 11 below.

Table 11 Effect of formic, butyric or lactic acids on Salmonella populations in feed [E log = logsample - logcontrol] Acid g/L vol. (pL) final pH log cfu/g A log control 4. 47 5. 0- 2. 5 10 4. 28 4. 3 0. 7 5. 0 20 4. 18 3. 6 1. 4 formic 7. 5 30 4. 1 1. 0 4. 0 10 40 4 1. 0 4. 0 10 40 4. 31 4. 3 0. 7 butyric 30 120 4. 17 1. 0 4. 0 50 200 4. 04 1. 0 4. 0 10 40 4. 2 4. 1 0. 9 lactic 30 120 3. 92 1. 0 4. 0 50 200 3. 68 1. 0 4. 0 control 4. 44

[0653] Complete bacteriacide was seen at the two highest doses tested for all three acids (7.5 and 10 g/L for formic acid; 30 and 50 g/L for butyric and lactic acids).

EXAMPLE 10 [0654] Following the procedure set forth in Example 9, the effect of blends of formic, butyric and/or lactic acids, with and without Alimet3, on Salmonella counts in basal corn soy based broiler starter feed (described in Table 9, above, crumble feed with 6% MBM) was studied. The blends studied are described in Table 12, and the results obtained in the in vitro study are reported in Tables 13- 15.

Table 12 Acid blend formulations (concentrations reported in g/L) Formic Lactic Butyric o Acid Acid Acid Al 5--- A2 4 4-- A3 4-4- A4 3-8- A5 3 4 4- A6 3 8-- A7 2-12- A8 2 4 8- A9 2 8 4- A10 2 12-- All I-16- A12 1 4 12- A13 1 8 8- A14 1 12 4- A15 1 16--

Table 12, cont'd Formic Lactic Butyric Alimet# Acid Acid Acid A16--20- A17-4 16- A18-8 12- A19-12 8- A20-16 4- A21-20-- A22 5 - - 1 A23 4 4-1 A24 4-4 1 A25 3-8 1 A26 3 4 4 1 A27 3 8-1 A28 2-12 1 A29 2 4 8 1 A30 2 8 4 1 A31 2 12-1 A32 1-16 1 A33 1 4 12 1 A34 1 8 8 1 A35 1 12 4 1 A36 1 16-1 A37 - - 20 1 A38-4 16 1 A39-8 12 1 A40-12 8 1 A41-16 4 1 A42-20-1 A43 5 - - 2. 27 A44 4 4-2. 27

Table 12, cont'd Formic Lactic Butyric o Blend Acid Acid Acid A45 4-4 2.27 A46 3-8 2.27 A47 3 4 4 2.27 A48 3 8-2. 27 A49 2-12 2.27 A50 2 4 8 2. 27 A51 2 8 4 2. 27 A52 2 12-2. 27 A53 1-16 2.27 A54 1 4 12 2.27 A55 1 8 8 2.27 A56 1 12 4 2.27 A57 1 16-2. 27 A58--20 2.27 A59-4 16 2.27 A60-8 12 2.27 A61-12 8 2.27 A62-16 4 2. 27 A63-20-2. 27

Table 13 Effect of formic/butyric/lactic blends without AlimetX on Salmonella populations in feed Blend Final pH log cfu/g A log reduction Al 4.12 3.1 1.6 A2 4.13 2.9 1.8 A3 4.18 3.1 1.6 A4 4.18 3.0 1.7 A5 4.18 3.2 1.5 A6 4.15 2.6 2.1 A7 4.15 2.8 1.9 A8 4.18 2.8 1.9 A9 4.16 2.5 2.2 A10 4.12 1.8 2.9 All 4.16 2.7 2.0 A12 4.17 2.7 2.0 A13 4.16 2.8 1.9 A14 4.14 2.9 1.8 A15 4.1 2.6 2.1 A16 4.16 2.7 2.0 A17 4.21 2.7 2.0 A18 4.18 2.5 2.2 A19 4.16 1.7 3.0 A20 4.13 2.7 2.0 A21 4. 05 1.8 2. 9 control 4. 33 4. 7-

Table 14 Effect of formic/butyric/lactic blends with 1 g/L added Alimets on Salmonella populations in feed Blend Final pH log cfu/g # log reduction A22 4.09 3.5 1.0 A23 4.09 2.1 2.4 A24 4.09 2.9 1.6 A25 4.12 2.2 2.3 A26 4.11 2.3 2.2 A27 4.1 1.9 2.6 A28 4.11 2.3 2.2 A29 4.14 2.3 2.2 A30 4.1 1.9 2.6 A31 4.06 1.6 2.9 A32 4.15 2.4 2.1 A33 4.15 2.3 2.2 A34 4.15 1.4 3.1 A35 4.11 1.7 2.8 A36 4.06 1.8 2.7 A37 4.13 2.0 2.5 A38 4.16 2.0 2.5 A39 4.13 1.8 2.7 A40 4.12 1.8 2.7 A41 4.08 1.7 2.8 A42 4.06 1.7 2.8 control 4.33 4. 7 0.2 control 4.36 4. 5-

Table 15 Effect of formic/butyric/lactic blends with 2.27 g/L added Alimets on Salmonella populations in feed Blend Final pH log cfu/g reduction reduction A43 4.21 2.8 0.7 A44 4.17 2.8 0.7 A45 4.18 3.0 0.5 A46 4.18 2.8 0.7 A47 4.15 3.7-0. 2 A48 4.11 2.0 1.5 A49 4.19 2.7 0.8 A50 4.19 2.8 0.7 A51 4.16 2.8 0.7 A52 4.13 2.4 1.1 A53 4.2 2.8 0.7 A54 4.2 2.7 0.8 A55 4. 14 2. 3 1.2 A56 4.13 1.7 1.8 A57 4.08 1.0 2.5 A58 4.21 2.3 1.2 A59 4.23 2.7 0.8 A60 4.17 2.0 1.5 A61 4.14 2.0 1.5 A62 4.12 0.7 2.8 A63 4.07 0.7 2.8 control 4.37 3.5 0 control 4.45 3. 5- [0655] Addition of 1 g/L Alimets to the blend gave improved results in sixteen of the blends tested.

[0656] The formulations of the blends used in Examples 11-13 are set forth in Table 16.

Table 16 Organic acid/Alimets blend formulations Blend Alimet# Phos. 1 Butyric Formic Lactic Prop.2 A64 30 30 20-20- A65 30--50-20 A66 20 20 12 30 18- A67 10 10-75-5 A68 30 35 15-20- A69 30 5-55-10 A70 20 20 12-18 30 A71 10 5 5 75-5 A72---50-50 A73---75-25 1 Phosphoric acid 2 Propionic acid EXAMPLE 11 [0657] The effects of blends of organic acids on the colony counts of Salmonella in a corn soy based diet as set forth in Table 9, above, (DLM at 0.2%) were studied, following the procedure outlined in Example 9. The results are reported in Table 17.

Table 17 effect of acid blends on Salmonella populations in corn soy based diet after 60 min. Blend g/kg A i reduction control 40, 000 4. 6- 5 29, 700 4. 5 0. 1 A64 10 2, 300 3. 4 1. 2 5 3, 100 3. 5 1. 1 A65 10 40 1. 6 3. 0 5 10, 000 4. 0 0. 6 A66 10 40 1. 6 3. 0 5 3, 000 3. 5 1. 1 A67 10 40 1. 6 3. 0 control 13, 900 4. 1- after 90 min. Blend g/kg A 10g final pH reduction control 46, 000 4. 7-4. 68 5 19, 300 4. 3 0. 4 4. 45 A64 10 2, 400 3. 4 1. 3 4. 32 5 200 2. 3 2. 4 4. 44 A65 10 40 1. 6 3. 1 4. 25 5 13, 500 4. 1 0. 6 4. 37 A66 10 40 1. 6 3. 1 4. 22 5 200 2. 3 2. 4 4. 33 A67 10 40 1. 6 3. 1 4. 02 control 38, 800 4. 6-4. 68 40 cfu/g (minimum detection level) reported when no Salmonella detected.

I Single reading for each treatment.

[0658] Three of the four blends tested showed complete bacteriacide at 10 g/kg (1%) application. Both the A65 and A67 blends showed significant bacteriacide at the lower application rate (0.5%) EXAMPLE 12 [0659] The effect of blends of organic acids on Salmonella counts were studied using model poultry and swine diets. Blends A70 and A71 were tested using the corn soy based diet set forth in Table 9, above, with DLM added at 0.2%. The model poultry diet was a corn soy based layer diet, no meat product; the effects of blends A68 and A69 were tested with this diet.

[0660] Blends were added to 1 g of feed sample.

Salmonella (200 pL, 40,000 cfu) were added to each feed sample, and mixed. The samples were incubated at room temperature, then diluted 1: 10 with water and plated on BG plate. Results are reported in Table 18.

Table 18 effect of acid blends on Salmonella populations in corn soy based diet Final after 1 h after 24 h blend g/kg cfu/g log cfu/g cfu/g log cfu/g control 5. 83 12, 400 4. 1 10, 133 4. 0 (Diet #1) 2 5. 53 15, 000 4. 2 3, 300 3. 5 5 5. 32 3, 900 3. 6 1, 200 3. 1 A70 7. 5 5. 08 3, 400 3. 5 700 2. 8 10 4. 94 1, 500 3. 2 100 2. 0 2 5. 42 9, 600 4. 0 2, 300 3. 4 5 5. 16 1, 300 3. 1 100 2. 0 A71 7. 5 4. 84 200 2. 3 100 2. 0 10 4. 66 100 2. 0 100 2. 0 Table 18 effect of acid blends on Salmonella populations in corn soy based diet Final after 1 h after 24 h blend kg cfu/g log cfu/g cfu/g log cfu/g control control 5. 92 15, 300 4. 2 6, 300 3. 8 2 5. 70 10, 400 4. 0 5, 600 3. 7 5 5. 67 6, 300 3. 8 4, 200 3. 6 A68 7. 5 5. 54 9, 900 4. 0 2, 600 3. 4 10 5. 36 8, 300 3. 9 1, 300 3. 1 2 5. 53 7, 800 3. 9 3, 300 3. 5 5 5. 29 3, 600 3. 6 1, 200 3. 1 A69 7. 5 5. 22 1, 200 3. 1 300 2. 5 10 5. 08 1, 000 3. 0 100 2. 0

EXAMPLE 13 [0661] The antibacterial effect of two organic acid/Alimet blends were compared with blends containing formic and propionic acids, and with no Alimet@, following the procedure set forth in Example 12. Results after 90 minutes are reported in Table 19.

Table 19 Blend g/kg cfu/g log cfu/g final pH control 29,400 4.5 4.54 2 140 2. 1 4.56 A65 5 80 1.9 4.45 10 1 0 4.32 2 900 3.0 4.57 A67 5 1,100 3.0 4.49 10 1 0 4.25 2 2,100 3.3 4.5 A72 5 90 2 4.39 10 1 0 4.15 2 2,700 3.4 4.51 A73 5 600 2.8 4.4 10 1 0 4. 1 control 30, 650 4. 5 4. 68 EXAMPLE 14 [0662] The antibacterial effect of two different batches of Alimet on a corn soy diet (see Table 9, above) were compared. The first batch was of an unknown age and the second batch was freshly prepared (less than two weeks old). The protocol set forth in Example 9 was used, and results are reported in Table 20.

Table 20 effect of two different batches of Alimets on Salmonella populations in corn soy based diet after 90 min. incubation Acid g/kg cfu/g log cfu/g pH padded control 6, 850 3. 8 4. 80 2. 3 800 2. 9 4. 72 AlimetO 5. 7 50 1. 7 4. 66 batch 1 8. 5 120 2. 1 4. 58 11. 4 0-4. 57 2. 3 3, 900 3. 6 4. 68 Alimet 5. 7 300 2. 5 4. 54 batch 2 8. 5 20 1. 3 4. 61 11. 4 20 1. 3 4. 57 control 14, 850 4. 2 4. 80 [0663] The first batch of Alimets showed slightly improved bactericidal effects at lower concentrations. Both batches were bactericidal at higher doses.

EXAMPLE 15 [0664] The antibacterial effect of dry acids (fumaric, tartaric, and sorbic) alone and in combination with Alimets blends were studied according to the protocol of Example 9. The formulations of the blends studied are reported in Table 21. The results after 90 minutes are reported in Table 22.

Table 21 acid concentration (g/kg) Blend Alimete Pumaric Tartaric Sorbic A74 10 0 0 0 A75 0 10 0 0 A76 0 0 10 0 A77 0 0 0 10 A78 5 5 0 0 A79 5 0 5 0 A80 5 0 0 5 Table 22 Blend cfu/g log cfu/g A log reduction control 27900 4.4 A74 40 1.6 2.8 A75 30 1.5 3.0 A76 14450 4.2 0.3 A77 1150 3.1 1.4 A78 0-4. 4 A79 4600 3.7 0.8 A80 170 2.2 2.2 control 13900 4.1 EXAMPLE 16 [0665] The effect of formic acid on Lactobacillus plantarum was studied. As demonstrated in Example 1, addition of Alimets at pH 3.5 to a bacteria-containing broth showed a clear lethal effect on L. plantarum at doses of 3 and 5 g/1. Comparable concentrations of formic acid (technical quality 85%, ex Franklin Products) were also studied and compared against Alimet@.

[0666] Fresh overnight culture L. plantarum in Brain Heart Infusion broth is used to inoculate medium 5 at a log

4.1 cfu/mL at pH 3.5. The tubes are incubated under oxygen reduced atmosphere for 6 hours at 37°C. Colony counts are performed according standard procedures. All analyses were performed in duplicate, and the results are reported in Table 23.

Table 23 Effect of Alimet and formic acid on L. plantarum colony counts in broth (after 6 hours) initial log cfu/mL = 5.61<BR> A log = 10g6 hours-loginitial Acid g/kg log cfu/mL A log control 5.67 0.06 3 3. 02-2.59 Alimet 5 0.83-4. 78 3 3.19-2. 42 formic acid 5-0. 30-5. 91 EXAMPLE 17 [0667] Blends of various acids with Alimet# were studied at pH 4.5. The effect of these blends on S. enteritidis colony counts in broth were studied, using the protocol set out in Example 1. Results are reported in Tables 24-26.

Table 24 Effects of blends of acids and Alimet# on S. enteritidis colony counts in broth Blend Acid Acid g/L Alimet g/L log cfu/mL Control 5. 04 Control 5. 04 Control (Alimet)-0. 30 A81 5 0 3. 93 A82 4. 5 0. 5 0. 74 A83 4 1 0. 24 formic A84 3. 75 1. 25 0. 15 A85 3. 5 1. 5-0. 15 A86 2. 5 2. 5-0. 30 A87 5 0 4. 44 A88 4. 5 0. 5 3. 56 A89 4 1 1. 70 butyric A90 3. 75 1. 25 1. 44 A91 3. 5 1. 5-0. 15 A92 2. 5 2. 5-0. 30 A93 5 0 4. 74 A94 4. 5 0. 5 4. 40 A95 4 1 4. 34 citric A96 3. 75 1. 25 1. 59 A97 3. 5 1. 5 1. 19 A98 2. 5 2. 5 0. 24

Table 25 Effects of blends of acids or formaldehyde and Alimets on S. enteritidis colony counts in broth Blend Acid Acid g/L | Alimet g/L log cfu/mL Control 5. 02 Control (Alimet@) 0. 00 A99 5 0 4. 68 A100 4. 5 0. 5 4. 65 A101 4 1 4. 43 fumaric A102 3. 75 1. 25 4. 38 A103 3. 5 1. 5 4. 36 A104 2. 5 2. 5 2. 32 A105 5 0 4. 44 A106 4. 5 0. 5 4. 48 A107 4 1 4. 43 lactic A108 3. 75 1. 25 4. 51 A109 3. 5 1. 5 4. 75 A110 2. 5 2. 5 4. 55 All1 5 0 5. 01 A112 4. 5 0. 5 4. 71 A113 4 1 4. 77 malic A114 3. 75 1. 25 4. 91 A115 3. 5 1. 5 4. 96 A116 2. 5 2. 5 4. 89

Table 26 Effects of blends of acids or formaldehyde and Alimet on S. enteritidis colony counts in broth Blend Acid Acid g/L Alimet g/L log cfu/mL Control 4. 95 Control (Alimet@) 0. 63 A117 5 0 4. 34 A118 4. 5 0. 5 4. 34 A119 4 1 4. 10 propionic A120 3. 75 1. 25 3. 76 A121 3. 5 1. 5 3. 30 A122 2. 5 2. 5 0. 87 A123 5 0 4. 68 A124 4. 5 0. 5 4. 67 A125 4 1 4. 56 phosphoric A126 3. 75 1. 25 4. 62 A127 3. 5 1. 5 4. 48 A128 2. 5 2. 5 3. 30 A129 5 0-0. 30 A130 4. 5 0. 5-0. 30 A131 4 1-0. 30 formaldehyde A132 3. 75 1. 25-0. 30 A133 3. 5 1. 5-0. 30 A134 2. 5 2. 5-0. 30 [0668] Phosphoric, fumaric, lactic, malic and propionic acids do not show a significant inhibitory effect at 5 g/l. Blends of these acids with Alimet# gave similar results, except for the 50: 50 blend of fumaric and Alimet@, which gave greater than 2 log reduction in colony counts compared to 5 g/L of fumaric alone, and the 50: 50 blend of phosphoric and Alimet#, which gave an approximately 1.3 log reduction compared to 5 g/L of phosphoric alone.

[0669] Blends of formic acid and Alimets performed more favorably than formic acid alone for all the blends studied. Similarly, blends of butyric acid and Alimet@, and citric acid with Alimet@, gave improved bactericidal effects with increasing proportion of Alimet added.

EXAMPLE 18 [0670] The antibacterial effect of acid blends was studied according to the protocol of Example 9. Phosphoric acid (75%) was obtained from Astaris (St. Louis, MO), lot # TK60. L-lactic acid (80%) was obtained from Purac America (Lincolnshire, IL), batch # 015703-A. Butyric acid (99+%) was obtained from Aldrich Chemical Co. (Milwaukee, WI), batch # 0.5110A. The formulations of the blends studied are reported in Table 27. The results are reported in Table 28.

Table 27 acid formulations (% of total) Blend AlimetO Lactic Phosphoric Butyric A135 0.33 0. 67-- A136 0.317 0.633 0. 05- A137 0.267 0.533 0. 20- A138 0.25 0.50 0. 25- A139 0.33 0. 33-0. 33 A140 0.317 0.317 0.05 0.317 A141 0.267 0.267 0.20 0.267 A142 0.25 0.25 0.25 0.25 A143 0. 33--0. 67 A144 0. 317-0. 05 0.633 A145 0. 267-0. 20 0.533 A146 0. 25-0. 25 0.50

Table 28 Blend cfu/g log cfu/g final pH control 7,900 3.9 4.86 A135 20 1.3 4.49 A136 20 1.3 4.39 A137 340 2.5 4.52 A138 160 2.2 4.44 A139 80 1.9 4.50 A140 20 1.3 4.49 A141 160 2.2 4.42 A142 160 2.2 4.43 A143 160 2.2 4.48 A144 40 1.6 4.48 A145 240 2.4 4.53 A146 20 1.3 4.42 control 22, 100 4. 3 4. 73 EXAMPLE 19 [0671] The effects of blend A71 at neutral pH were tested. The blend was added to 1 g of feed sample.

Salmonella (40,000 cfu (65ul)) was added to each 1 g sample, mixed, and incubated at room temperature. Following incubation, samples were diluted 1: 10 with water and plated on a BG plate.

[0672] Two diets were studied, as shown in Table 29.

Results are reported in Table 30.

Table 29 Diet 1 (swine diet) ingredients % of total corn 51.60 SBM, 48 30 DairyLac 80 8.50 Menhaden fish meal (Select) 3.98 Choice white grease 3.00 Dicalcium Phosphate 1.24 Limestone 0.34 Lysine 0.26 DL-Methionine 0.13 Threonine 0.16 Vitamins, TMs, Salt and Mecadox 0.93 Corn Starch to 100 Diet 2 (broiler diet) ingredients % of total corn 60.551 SBM 32.254 Fat, animal 3.665 Dicalcium Phosphate 1.861 Limestone 0.811 Vitamin/Mineral premix 0.350 Salt 0.340 L-lysine HC1 78% 0.097 Threonine 0.051 Satoquin-mix6 0.019 Copper Sulfate 0.003 DL-methionine 0.2

Table 30 Blend tested: A71 g/kg 1 hour 24 hours 48 hours final cfu/g log cru/g log cfu/g log pH Diet 1 control 27,700 4.4 6,432 3. 8 2,530 3.4 5.83 2 4,320 3.6 80 1.9 40 1.6 5.52 5 3, 840 3.6 40 1.6 0-5. 21 7.5 3,080 3.5 80 1.9 0-5. 01 10 1,560 3.2 0-0-4. 87 Diet 2 control 25,000 4.4 6,160 3.8 2,570 3.4 6.06 2 8,080 3.9 40 1.6 40 1.6 5.67 5 7,000 3.8 40 1.6 40 1.6 5.31 7.5 4,400 3.6 40 1.6 0-5. 1 10 900 3.0 0-0-4. 82 EXAMPLE 20 [0673] Following the protocol of Example 19, the effects of blend A69 at neutral pH were tested. Formic acid (85%) was obtained from BASF Corporation (Mt. Olive, NJ), product # 019723, lot # 87656216KO. Two diets were studied, as shown in Table 31. Results are reported in Table 32.

Table 31 Diet 3 ingredients % of total soybean meal 33.40 corn 32.85 wheat hard red 20.00 organic peas meal 5.00 fat, animal 4.80 dicalcium phophate 1.81 limestone 0.98 salt 0.43 Vitamin/Mineral premix 0. 35 Threonine 0.10 Avizymel502 0.10 Santoquin-mix6 0.02 Coban 60 0.05 copper sulfate 0.00 Diet 4 ingredients % of total corn 60.50 soybean meal 28. 46 limestone 7. 76 Dicalcium Phosphate 1.63 animal fat 1.00 Vitamin/Mineral premix 0.35 salt 0.26 Santoquin-mix6 0.02 copper sulfate 0.00 choline Cl-60% 0.00

Table 32 blend tested: A69 g/kg 1 hour 24 hours 48 hours final cfu/g log cfu/g log cru/g log pH Diet 3 control 68, 000 4.8 2,470 3.4 880 2.9 6.05 2 5,600 3.7 120 2.1 40 1.6 5.67 5 8,900 3.9 40 1. 6 40 1.6 5.46 7.5 4,600 3.7 40 1. 6 80 1. 9 5.28 10 10,600 4.0 40 1. 6 0-5. 13 Diet 4 control 68,000 4.8 2,630 3.4 1,380 3.1 5.99 2 8,600 3.9 0-0-5. 8 5 900 3.0 40 1.6 0-5. 55 7.5 2,600 3.4 200 2.3 0-5. 37 10 2,700 3.4 0-40 1.6 5.12 EXAMPLE 21 [0674] Following the protocol of Example 9, the effects of blends of Alimet@, lactic acid, formic acid, and/or butyric acid were studied. Blend formulations are set forth in Table 33. Up to five replicates were performed, and results are reported in Tables 34 and 35.

Table 33 acid formulations (g/kg) Blend A1 L2 F3 B4 Blend Al 2 F3 B4 A147 0 0 0 10 A162 0 0 6.7 3. 3 A148 2 0 0 8 A163 2 0 5.3 2. 67 A149 7.5 0 0 2.5 A164 7.5 0 1.67 0.8 A150 3.3 0 0 6.7 A165 0 10 0 0 A151 2 2.67 0 5.33 A166 2 8 0 0 A152 7.5 0.8 0 1.67 A167 7.5 2.5 0 0 A153 0 0 3.3 6.7 A168 0 6.7 3.3 0 A154 2 0 2.67 5.3 A169 2 5.3 2.67 0 A155 7.5 0 0.8 1.67 A170 7.5 1.67 0.8 0 A156 0 6.7 0 3.3 A171 0 3.3 6.7 0 A157 2 5.3 0 2.67 A172 2 2.67 5.3 0 A158 7.5 1.67 0 0.8 A173 7.5 0.8 1.67 0 A159 0 3.3 3.3 3.3 A174 0 0 10 0 A160 2 2. 67 2. 67 2.67 A175 2 0 8 0 A161 7.5 0.8 0. 8 0.8 A176 7. 5 0 2.5 0 1 Alimet# 2 Lactic acid 3 Formic acid 4 Butyric acid

Table 34 Trial: Average 1 2 3 4 Blend cfu/g log A log cfu/g cfu/g cfu/g cfu/g control 49,200 4. 7-58, 200 43,400 46,000 N/A A147 18,800 4.3 0.4 22,200 15,400 N/A N/A A148 15,700 4.2 0.5 19,800 11,600 N/A N/A A149 70 1.8 2.8 80 200 0 0 A150 12,300 4.1 0.6 1,200 23,400 N/A N/A A151 6,000 3.8 0.9 8,200 3,800 N/A N/A A152 60 1.8 2.9 40 40 120 40 A153 1,600 3.2 1.5 3,200 0 N/A N/A A154 20 1.3 3.4 40 40 0 0 A155 10 1. 0 3.7 40 0 0 0 A156 11,300 4.1 0.6 6,000 16,600 N/A N/A A157 2,900 3.5 1.2 1, 000 4,800 N/A N/A A158 400 2.6 2.1 800 0 N/A N/A A159 0-4. 7 0 0 0 0 A160 0-4. 7 0 0 0 0 A161 20 1.3 3.4 40 40 0 0 control 63, 133 4. 8-56, 800 64, 000 68, 600 N/A N/A = replicate not performed

Table 35 Trial: Average 1 2 3 4 Blend cfu/g log A log cfu/g cfu/g cfu/g cfu/g control 25,666 4. 4-23, 200 25,800 28,000 N/A A162 0-4. 4 0 0 0 0 A163 0-4. 4 0 0 0 0 A164 0-4. 4 0 0 0 0 A165 5,100 3.7 0.7 4,000 6,200 N/A N/A A166 150 2.2 2.2 120 120 120 240 A167 30 1.5 2.9 40 80 0 0 A168 0-4. 4 0 0 0 0 A169 0-4. 4 0 0 0 0 A170 0-4. 4 0 0 0 0 A171 0-4. 4 0 0 0 0 A172 0-4. 4 0 0 0 0 A173 0-4. 4 0 0 0 0 A174 0-4. 4 0 0 0 0 A175 0-4. 4 0 0 0 0 A176 0-4. 4 0 0 0 0 control 20000 4. 3-20200 19800 N/A N/A N/A = replicate not performed EXAMPLE 22 [0675] Following the protocol of Example 9, the effects of blends of Alimet@, lactic acid, propionic acid (99%, obtained from Sigma Chemical Co. , St. Louis, MO, lot P-1386), and/or butyric acid were studied. Blend formulations are set forth in Table 36. Up to five replicates were performed, and results are reported in Tables 37 and 38.

Table 36 acid formulations (g/kg) Blend A1 L2 P3 B4 Blend A1 L2 P3 B4 A177 0 0 0 10 A193 2 0 5.3 2.67 A178 2 0 0 8 A194 7.5 0 1.67 0.8 A179 7.5 0 0 2.5 A195 0 10 0 0 A180 0 3.3 0 6.7 A196 2 8 0 0 A181 2 2.67 0 5.3 A197 7.5 2.5 0 0 A182 7.5 0.8 0 1.67 A198 0 6.7 3.3 0 A183 0 0 3.3 6.7 A199 2 5.3 2. 67 0 A184 2 0 2.67 5.3 A200 7.5 1.67 0.8 0 A185 7.5 0 0.8 1.67 A201 0 3.3 6.7 0 A186 0 6. 7 0 3.3 A202 2 2.67 5.3 0 A187 2 5.3 0 2.67 A203 7. 5 0.8 1.67 0 A188 7.5 1.67 0 0.8 A204 0 0 10 0 A189 0 3.3 3.3 3.3 A205 2 0 8 0 A190 2 2.67 2.67 2.67 A206 7.5 0 2.5 0 A191 7.5 0.8 0.8 0.8 A207 10 0 0 0 A192 0 0 6. 7 3. 3 l AlimetX 2 Lactic acid 3 Propionic acid 4 Butyric acid

Table 37 Trial: Average 1 2 3 Blend cfu/g log A log cfu/g cfu/g cfu/g control 20,067 4.3 17,200 18,800 24,200 A177 6,333 3.8 0.5 5,400 7,000 6,600 A178 500 2.7 1.6 400 600 N/A A179 400 2.6 1.7 200 600 N/A A180 1,400 3.1 1.2 1,400 1, 400 N/A A181 1,500 3.2 1.1 2,000 1,000 N/A A182 100 2.0 2.3 200 0 N/A A183 6,000 3.8 0.5 9,600 2,400 N/A A184 6,900 3.8 0.5 8,800 5,000 N/A A185 1,300 3.1 1.2 1,400 1,200 N/A A186 4,100 3.6 0.7 5,400 2,800 N/A A187 2,400 3.4 0.9 600 4,200 N/A A188 400 2.6 1.7 200 600 N/A A189 4,700 3.7 0.6 1,000 8,400 N/A A190 7,300 3.9 0.4 7,800 6,800 N/A A191 300 2.5 1.8 600 0 N/A control 18, 733 4. 3 20, 000 22, 800 13400 N/A = replicate not performed

Table 38 Trial: Average 1 2 3 4 Blend cfu/g log A log cfu/g cfu/g cfu/g cfu/g control 7,800 3. 9-9, 800 7,200 6,400 N/A A192 6,100 3.8 0.4 6,600 5,600 N/A N/A A193 2,600 3.4 0.8 2,200 3,000 N/A N/A A194 25 1.4 2.8 50 50 0 0 A195 400 2.6 1.6 800 0 N/A N/A A196 300 2.5 1.7 200 400 N/A N/A A197 0-4. 2 0 0 0 0 A198 2,400 3.4 0.8 1,000 3,800 N/A N/A A199 600 2.8 1.4 0 1,200 N/A N/A A200 0-4. 2 0 0 0 0 A201 3,800 3.6 0.6 3,200 4,400 N/A N/A A202 900 3.0 1.2 1,000 800 N/A N/A A203 0-4. 2 0 0 0 0 A204 3,500 3.5 0.6 5,400 1,600 N/A N/A A205 100 2.0 2.2 200 0 N/A N/A A206 0-4. 2 0 0 0 0 A207 0-4. 2 0 0 0 0 control 30,600 4. 5-41, 600 19,400 30,800 N/A N/A = replicate not performed EXAMPLE 23 [0676] The ability of Alimet and DLM to function as palatants for dog and cat food was studied. Alimet and DLM were added into premium-type dog and cat food in the mixer to test the acceptance of the food compared to food lacking either supplement. The food used comprised good quality protein, and was high in CP and fat. A premium palantant was also added to the food. Alimets or DLM was added into the mixer prior to extrusion. The formulation of the feline diet is described in Table 39, and of the canine diet in

Table 40. The Alimet@/DLM supplementation levels, and intake ratios, are described in Table 41. The intake ratio describes the relative incidence of selecting one food over another.

[0677] For the canine study, twenty-one dogs were used (seven small, seven medium, and seven large dogs). For the feline study, twenty mature cats were used. The animals were given two choices of food, placed in separate bowls.

Over a two-day period, the dogs were given access to the food for 30 minutes; cats had access for 22 hours. The food chosen and consumed first was observed.

Table 39 Feline Diet Ingredient % of total diet Corn 14.7 Poultry byproduct (low ash) 20 Soybean Meal 12.5 Corn gluten meal 12.2 Meat and Bone meal 4 Animal fat 11.4 Rice brewer's 19.4 Flavor 2 Fish meal 2 Dried eggs 0.5 Salt 0.5 KC1 0.5 Vitamins 0.2 Choline 0.1 Taurine 0.1 Tocopherol 0.05 Trace Minerals 0.05

Table 40 Canine Diet Ingredient % of total diet Corn 33.0 Barley 15.0 Poultry byproduct (low ash) 13.0 Poultry byproduct 12.0 Soybean Meal 9.0 Animal fat 8.0 Rice brewer's 5.0 Flavor 2.0 Dried eggs 1.0 Salt 0.5 Limestone 0.36 Vitamins 0.2 Choline 0.1 Tocopherol 0.05 Trace Minerals 0.05

Table 41 Foods compared Intake Ratio Canine 0.05% Alimet vs. control 2.07 : 1 0.10% Alimet vs. control 5.58 : 1 0. 15% Alimet control 5.13 : 1 0. 10% DLM vs. control 5.32 : 1 0.15% DLM vs. control 4.95:1 0.10% Alimet vs. 0.05% Alimet 2.54:1 0.15% Alimet vs. 0. 05% Alimet 1.99 : 1 0.15% Alimet vs. 0.10% Alimet 2.54 : 1 0.15% DLM vs. 0.10% DLM 1.57 : 1 0.10% DLM vs. 0. 10% Alimet 1.05 : 1 0.15% DLM vs. 0.15% Alimet 2.5 : 1 Feline 0. 20% Alimet# vs. control 1.67 : 1 D 1. 9 1 : 1 0.30% Alimet vs. control 1.85 : 1 0. 25% DLM vs. control 1.87 : 1 0.30% DLM vs. control 1.63 : 1 0.25% Alimet vs. 0. 20% Alimet# 1.30 : 1 0.30% Alimet vs. 0.20% Alimet# 1.26 : 1 0.30% Alimet# vs. 0.25% Alimet# 1.16 : 1 0.30% DLM vs. 0. 25% DLM 1.04 : 1 0.25% DLM vs. 0.25% Alimet# 1.24 : 1 0.30% DLM vs. 0.30% Alimet 1. 47: 1 EXAMPLE 24 [0678] Acceptance of food containing Alimet or DLM was studied to evaluate dietary consumption under no-choice conditions. Food was offered to the animals (18 dogs: 6 small, 6 medium, 6 large; 15 cats) for one week. The urine pH of six of the cats was also monitored.

[0679] The diets described in Tables 39 and 40 above were used. Diets were supplemented with Alimet or DLM (0.1% for the canine study, 0.25% for the feline study).

Additional urine pH tests were carried out with 0.3% Alimet@.

[0680] Results of the urine pH experiments are given in Tables 42 and 43. Results of the acceptance text are given in Table 44.

Table 42 Urine pH Cat No. Control Alimet 0. 25% DL Met 0. 25% 452 6.35 6.45 6.39 453 6.44 6.2 6.38 457 6.29 6.56 6.39 460 6.25 6.03 6.36 465 6.22 6 6.1 475 6. 56 6. 44 6. 36 Table 43 Urine pH Cat No. Control Alimet 0.3% 450 6.20 6.03 453 6.44 6.10 465 6.04 5.53 468 6.40 5.73 469 6.68 6.31

Table 44 Supplementation Total grams consumed level control supplemented food Canine 0. 05% 5, 046 10, 420 Alimets 0. 1% 2, 101 11, 714 0. 15% 2, 419 12, 978 0. 1% 2, 114 11, 244 DLM 0. 15% 2, 734 13, 542 Feline 0. 2% 1, 111 1, 853 Alimet 0. 25% 955 1, 827 0. 3% 1, 003 1, 858 0. 25% 985 1, 842 DLM 0. 3% 1, 078 1, 754 [0681] In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

[0682] As various changes could be made in the above feed rations and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawing shall be interpreted as illustrative and not in a limiting sense.

[0683] When introducing elements of the present invention or the preferred embodiment (s) thereof, the articles"a, ""an, ""the,"and"said"are intended to mean that there are one or more of the elements. The terms "comprising,""including,"and"having"are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0684] Unless otherwise specified, amounts expressed as percentages are in percent by weight.