United States Patent 19 11 3,982,028 Bellingham (45) Sept. 21, 1976

(54) PRESERVATIVES FOR FORAGE, HAY, 3,806,600 4/1974 Lapore...... 424/317 GRAN AND ANIMAL FEEDS FOREIGN PATENTS OR APPLICATIONS 75) Inventor: Francis Bellingham, 1,276,677 6/1972 United Kingdom...... 99/8 Stockton-on-Tees, England OTHER PUBLICATIONS (73) Assignee: Imperial Chemical Industries Condensed Chemical Dictionary - Hawley, 8th Ed., Limited, London, England Van Nostrand, May 1971, p. 481. 22 Filed: Sept. 13, 1973 Technical News Letter, Ministry of Agriculture, 1 1/68, 10/4/68. 21 Appl. No.: 396,666 H. D. Young, Chem. Abs., v.24, 1930, p. 3595. (30) Foreign Application Priority Data Primary Examiner-Norman Yudkoff Sept. 4, 1972 United Kingdom...... 2002 Attorney,Assistant ExaminerAgent, or Firm-Cushman,Hiram H. Bernstein Darby & 52 U.S. Cl...... 426/69; 424/317; Cushman 426/807; 426/335; 426/532; 426/636; 426/635 57 ABSTRACT (51 Int. Cl.'...... A23K 1100 Feedstuffs and silage for ruminants contain mixtures 58) Field of Search ...... 426/15, 227, 310, 221, of isobutyraldehyde (IBD) with mineral or fatty acids, 426/331, 335, 210, 69,807, 635, 636,532; formalin, trioxane, or urea plus , derivatives of 424/317; 252/.407; 260/526 IBD selected from (alone or in admixture with isobutyric acid or salts thereof), isobutyl halides 56 References Cited or esters of fatty acids, isobutyrates, di-isobutyrates, UNITED STATES PATENTS and chlorinated isobutylidene diurea. 3,642,488 2/1972 Watchorn...... 426/69 7 Claims, No Drawings 3,982,028 2 Table 1-continued PRESERVATIVES FOR FORAGE, HAY, GRAIN Additives which showed preservative properties in AND ANIMAL FEEDS compound animal feeds 5 Treatment The present invention relates to substances suitable Additive level 7 ww for feeding to animals (particularly to ruminants) in 4 IBDisobutyric acid mixtures (e.g. 3:1 to i:3) viv 2 admixture with other feedstuffs, for example in com 5 IBD facetic acid (conc.) mixtures pound feeds, feed blocks, liquid feed supplements, (c.g. 3:1 viv) 2 drenches, slow-release pellets, or the like. The sub 6 sobutanol 2 O stances may each fulfil one or more useful functions; *AGS - a mixture of adipic, glutarie and succinic acids, a by-product in the manu for example, they may act as methane inhibitors, they facture of nylon. may control rumen fermentation (which, in turn, leads to enhanced animal performance), they may act as sources of energy, they may act as preservatives for Table 2 animal feedstuffs (e.g. compound feedstuffs containing 5 Compound animal feed composition molasses, and ensiled products such as green fodder, Barley 2000g hay, grain). Wheat 1500g Wheat-feet 480g In our co-pending British patent application No: . Groundnut 308g 31045/72 filed July 3, 1972, (corresponding to U.S. Moasses 337g application Ser. No. 373,783) we have described the 20 Salt 40g Limestone 18g use of isobutyric acid (IBA) or salts or esters thereof, Dicalcium phosphate 70g either alone or in admixture with other substances, e.g. Barley - vitamin premix 250g formalin and/or sulphuric acid, as preservatives for 5000g stored products, such as compound animal feeds con taining molasses, green fodder, hay, grain. 25 Encouraged by these results, we examined isobutyr Additives were incorporated into the above composi aldehyde (IBD), which is a very cheap material, to see tion, using molasses as carrier, and the treated concen whether it, or easily-prepared derivatives thereof, ei trate was incubated at 25°C under conditions where ther alone or in combination with each other or with temperature changes could be accurately monitored. In other compounds, would be useful as a preservative, as 30 this way, microbial activity was detected by observa a methane-inhibitor, as an agent for controlling (reduc tions of rapid temperature rises during the first six ing or stimulating) rumen fermentation, and/or as a weeks of storage. In addition, chemical and microbio source of energy. It was hoped that at least one such logical analyses were carried out to assess the extent of compound or mixture would be useful for at least one deterioration which might have occured during stor such purpose, and this proved to be the case. 35 age. The present invention accordingly provides animal Table 3 feedstuffs, in particular compound feeds, feedblocks, liquid feed supplements, drenches, slow release pellets Temperature response of compound animal feeds treated with and ensiled green fodder, hay and grain, containing additives, and incubatcd at 25°C 40 Control IBD:AGS(1:1) IBD:i-butyric acid : 1) mixtures of isobutyraldehyde with other substances, (no additive) 5%. , , "...... 2% derivatives of isobutyraldehyde and mixtures of said Day Temp.*C Temp...“C Temp.C derivatives with each other or with other substances. 7 27 24 24 In general moulds and yeasts are the main organisms 14 29.5 26 25 responsible for the deterioration of stored products 2 29 25 24 45 28 - 28.5 25 . . . 24.5 such as forage, hay, grain and compound animal feeds. 35 27 25.5 24.5 Thus in a screen for possible chemical additives, pre 42 25.5 25 24 liminary experiments were carried out to determine the antifungal and preservative properties of several simple mixture and compounds derived from IBD. The compound feed of Table 2 was tested, containing These preliminary in vitro experiments indicated that 50 various potential preservatives incorporated using mo certain of these compounds, alone or in mixtures inhib lasses as carrier. This material was moistened to ited the development of microbial growth and permit achieve a very high water content (s.50%) and then ted satisfactory storage of forage, hay, grain, com stored in lagged glass jars fitted with thermometers to pound animal feeds, etc. at high moisture contents (e.g. monitor self-heating, and incubated at 25°C for 6 up to 50%). 55 weeks. At the start and end of the experiment the mate The additives listed in Table hereunder showed rial was assayed for various constituents including dry preservative properties when incorporated into com matter (dim), ash, oil, energy, fibre, reducing sugar and pound animal feeds using molasses as carrier for the nitrogen and checked visually for mould growth. additive (see Table 2 for composition of feed). The results obtained are summarised in Table 4. The 60 material treated was of a very high moisture content Table (about 50% m.c.) and stored at a relatively high tem Additives which showed preservative properties in perature, optimal for the growth of many typical stor compound animal feeds - age moulds (i.e. 25°C). Treatment Additive level wiw Therefore it was considered that any additive which 1 BD/Sulphuric acid (40% wiv) 65 resulted in a 30% to 50% reduction in any of the pa mixtures, e.g. 3 vol acid: v BD 2 rameters of Table 4 would be worthy of further consid 2. BDIAGS* (1.1) wiw 5 eration even if they showed signs of visible moulding, 3 BD/formalin (3: viv) 2 provided this was less than in the case of the control. 3,982,028 3. 4 The treatments which emerged as of high potential on age potential the incorporation of water may be limited this basis are as follows: ...... by the proportion of oil present and is normally in the range 8-10% (w.fw). It would be of considerable advan tage for feed compounders if they could extend the BDiisobutyric acid (3:1) viv IBD/isobutyric acid (l: ) viv range of permissible water contents up to about 18%. BDiisobutyric acid (1:3) wiv At this level spoilage through moulding would be al BDI AGS acid (1: ) viv most inevitable. In addition the use of plastic bags for BDiAcetic acid (3:1) viv the storage of feeds would represent an economic sav BDIFormalin (3:1) w/v. ing over the currently used paper bags. However, even 10 at low moisture contents it has been found that pelleted Table 4 The effect of IBD and derivatives, alone and in admixture, on the storage stahility of approx. 50% m.c. compound feed for six weeks. Application Scif %, losses . rate heating during storage Visible Treatment % (v?w) index DM Energy Moulding Control (c) 56 53 49 -- Control 46 7 72 -- Control f 41 66 57 -- B) 2 64 64 64 - 5 50 59 6 -- BD/IBA (3:1) 2 3 ( : ) . . 2 () () () (1:3) 2 () () () IBD/HSO, (25% v/v) 3: 2 6 37 38 ( :) 2 24 37 36 -- 1:3 2 14 13 7 -- BD/AGS ( 1:) 5 3 5 5 BD/Acetic acid (3:1) 2. () 4 l -- BD/Formalin (3:1) . 2 20 () () lsobutanol 2 28 65 54 -- lsobutyl isobutyrate. 2 18 ' ' 54 36 + lsobutanolfsodium, - isobutyrate 2 25' 79 7() -- icates growth, *self-heatinghis index=the growth , sum, , ,of the rise in temperature above ambient due to self-heating on alternate days throughout storage period. In the manufacture of compound feeds the compound feed industry at the present time has to specify thc use of ingredients having a high dry matter, content, i.e. 10-14%. If this range is exceeded there are problems of 40 storage due to the formation of fungal and bacterial species. Thc toxins associated with such microflora feeds "sweat' during storage and cause condensation then limit intakc and animal performance falls off. to build up on thc inner surface of the bag with result In the process of compounding the feed the manufac ing mould growth on the pellets. turer adds steam to the mix prior to pelleting. This has 45 Some of the additives mentioned hereinabove, even the effect of raising the temperature of the mix and though they showed visible moulding in Table 4, were results in gelatinization of the starch, a process which tested with compound feed pellets having the composi results in the formation of stable pellets. If, however, tion of Table 2, at a typical "farm' m.c. (approx. 12%), the mix already contains appreciable quantities of stored at ambient temperature in the open air in bins, in water the opportunity to add steam is limited and pellct 50 papcrbags, and in plastic bags of varying thickness quality suffers. To Svercome these limitations, com (150 u, 175 u and 200 pu). The pellets were examined pounders are now adding small quantities of propionic in detail chemically and microbiologically during stor acid to the mix, via the molasses, and this allows higher age. The results are shown in Tables 5 to 8. levels of steam to be used. Work has been conducted to Table 5 examine several of the additives mentioned herein- 55 Microbiology of compound pelleted feeds (12% m.c.); above for this purpose. . . . . stored in air (open bins, plastic bags) for 3-4 weeks Microbial spoilage of feedstuffs may occur on materi - - - count/g.f.w. als stored with moisture contents which give rise to Yeasts - - - equilibrium relative humiditics of approximately 70% 4 moulds Moulds Aerobic or greater. The highest m.c. which will permit safe 60 Treatment %. x(). x(). Bacteria storage depends upon the nature of the feedstuff, e.g. Control . . . . Zero 7)4()(). 48 380 isobutyl isobutyrate () , 5 88 cereals 13-14%, oil seeds such as groundnuts, soya and ... 2.() 28()() ()2 24() linseed 6-8%. The oil content of a concentrate thus has isobutanol () 29 , () - 40 a marked effect on the m.c. which can be permitted for 2.() 18 14 5 lsobutanol: BA(2:1 wiv) . . . .() , () 7 35 mould free storage. Typical compound feeds using 65 2.0 32 35 7 concentrates such as cereals, soya, groundnuts and sobutanol: BA( 1:3 wiv) () . . .35() 2 77 added oil as tallow or vegetable oils may have lipid contents from 2-8% (w/w). Thus with respect to stor

3,982,028, 6 Table 6, Table 9-continued Microbiology of compound pelleted feeds (12% m.c. - Self-heating acid mould contamination of wet hay . . . . ; ; in open bins for 5-6 months. in the presence and absence of additives. countlg.f. W. Mixture:- Treatment Total Yeasts ...... Aerobic Composition. 2. . Rate TC rise, ...... i-moulds Moulds. Bacteria (v/v)''. Additive %(v/w) A B Treatment % . . .xl ()' , Xi ()? . x(). --- lume ...... " --- IBD , 0.25 44 Control zero 400. 5 7500 1 AGS ().5 43 Isobutyl isobutyrate 1.0 14 () 70 (w/w) ().75 44 ...... ; ; 2.0 40 3 30 . () () 12() isobutanol () 150 (). 26 -- BD () 173 117 2.() 6 2. 9. Isobutanol: BA(2:1 viv) () 2. : : () 13 The total temperature rise = the sum of (obs. TC-25°C) at 2 day intervals for (A) . - . . .", 2, () 63 : 3 8 42 days (B) 24 days . isobutanol: BA( 1:3v/v) () 9 23 2.() 62 l 6() 15 Ruminants obtain energy from plant material via the fermentative activity of the ruminal microflora. Micro Table 7 Mould contamination on compound feeds after 1 1 week storage (m.c. 12%) Mould count/g at 25°C Mould count/g at 37°C Plastic Plastic Treatment %. Paper 150p. 175p. 200u Paper 150p. 175u 200p. Control zero 40 3() 246() 3355 25 () () 5 Isobutyl isobutyrate 2 () () 5 583 : () 20 O . 0 isobutanol 2 5 () () 15 () () O 80 Isobutano: BA(2:1 w/v) 2 () () () () () () 35 () lschutanol:BA (1:3v/v). 2 () () 5 O () 5 () . ()

Table 8 Mould contamination on compound feeds after 21 weeks storage (m.c. 12%) . . - Mould countig at 25°C - Mould countle at 37°C Treatment % Paper bags 'Plastic bags Paper bags Plastic bags Control zero 810 393 50 15 Isobutyl isobutyrate 2.0 503 220 () () Isobutano... 2.0 123 ... 10 () . . . . ; 5 ; Isobutanol: BA(2:1 v/v) 2.0 55, 268 0 . . . () ..

Isobutanol: BA(l:3v/v) 2.0 O 53 . . 0 ()

. organisms metabolize celluloze, hemicelluloses and Experiments were carried out in which moist hay 40 other carbohydrates of plant tissues to end products (50%. m.c.) was stored for up to 6 weeks treated with such as volatile fatty acids, dioxide, methane various potential hay preservatives. The hay was exam and . Volatile fatty acids are absorbed ined at the termination of storage by chemical and through the rumen walls and are utilized by the animal microbiological techniques. Normally the additives as primary energy sources. Most of the fermentation were tested at four treatment rates, 0.25%, 0.5%, 45 gas is lost from the rumen by eructation. It has been 0.75% and 1.0% (w/w) (equivalent to rates of 4, 1, 1% estimated that 5-10% of the total energy intake of and 2 gallons per ton) at least for the first 2 to 3 weeks ruminants is converted to methane and hence lost in and the results plotted to give the typical results in this way. Thus the elimination of wasteful methane Table 9. production would represent a considerable economic Table 9 50 saving. Self-heating acid mould contamination of wet hay Methane is formed in the rumen by the reduction of : in the presence and absence of additives. the end products of carbohydrate fermentation, partic Mixture Treatment Total ularly CO2, probably by a single bacterial species, Composition Rate TCrisc Methanobacterium ruminantium. Thus selective inhibi (w/v) Additive % (w.fw) A B 55 tion of this organism would prevent methane synthesis. --- Dry hay -- 7 7 The substrates used in methane formation would then --- Wet hay m 19 85 3 Formalin 0.25 181 5() be available for conversion into fatty acids which would l IBD 0.5 179 31 be of greater potential value to the ruminant. 0.75 134 86 The inter-relationships between rumen microorgan () 26 73 60 3 BD ().25 97 83 isms represent an ecological entity and any attempt to Isobutyric acid ().5 142 3 reduce methane production must be selective and not ().75 149 15 1.() 89 75 cause too much disturbance in the rest of the rumen BD 0.25 35 fermentation. However, since inhibition of methane lsobutyric acid 0.5 34 synthesis will release hydrogen which might be used for ().75 2 65 the reduction of carbohydrate metabolism intermedi BD ().25 99 ates, one might expect an increase of fatty acid produc 3 isobutyric acid ().5 154 ().75 () tion. In this way methane inhibitors may play a central 1.() 4 role in providing a means of controlling rumen fermen 3,982,028 7 8 tation and obtaining selective alterations in the pattern under test. During the time that the various compo of volatile fatty acid formation. nents are being added the mixture is continually gassed Compounds and mixtures were tested with the object with oxygen free nitrogen to maintain anaerobic condi of obtaining a material which will inhibit methane pro- tions. Equilibration is carried out for 15 minutes before duction without creating potential residue problems by 5 adding 40 ml of the mixture to a 100 ml glass gas sy entering the normal energy metabolism of microorgan- ringe fitted with a 3-way tap. Air is expelled from the isms in the rumen and thereby possibly also modifying syringe and replaced with 10 ml of nitrogen and the the end products of fermentation to achieve a situation control and test syringes incubated at 37°C in a water more advantageous to the host animal, e.g. stimulating bath for 6 hours. Periodically the total gas evolved in propionic or butyric acid production. 10 the 100 ml syringe is measured and aliquots are re In preliminary studies a range of derivatives of IBD moved via the 3-way tap and a small gas syringe for were tested since this material is cheap and frecly avail- analysis of the evolved gas composition. The evolution able. The following showed encouraging results: of total gas reflects the effect of an additive on overall Table 10 Ref. Physical Level % inhibition No: Formulation State % of methane AF 20 isobutyl bromide liquid ().5 30 21 isobutyl acetate liquid 2 ()() 22 isobutyl chloride liquid ().5 9() 23 Chrinated BDU solid 85 24 BD : Trioxane (2: ) viv liquid 3() 25 IBD : Trioxane (3*)viv liquid 30 26 BD : Trioxane (4:1) viv liquid 9() 28 BD : Trioxane (6: ) v.v. liquid ().5 2() 30 Methylene dioxymethylene liquid ().5 90 diisobutyrate *32 (3D - urea) chlorine derivative solid ().5 6() *Made by heating l'Aign. mcles urea it 55C, mixing with ligm. mole IBD and saturating with chlorine.

These, and other, compounds and mixtures were fermentation and the CH levels indicatc the potential further examined by the following procedure: effectiveness of an additive as a methane inhibitor. The results of these tests are shown in Table l, in Procedure which the following code is used: Rumen fluid is removed from a sheep via a rumen 35 CODE cannula. The fluid is strained through several layers of ra . . . . ." muslin to remove larger food particles; if necessary. Total gas CH% reduction The strained rumen fluid is then mixed l: i (v/v) with O identical artificial saliva and the mixture supplemented with it -3.36, - 39 further nutrients: 0.25% (w/v) of each of the following: ++ - 5-iö0% - 0 to 25%. isurea, prepared sucrose, without starch, additive,cellulose. and A controla test preparation 0 stimulationtimulation red.reduction A (),+) stimulationstimulat containing additive at an appropriate concentration Table Total gas % reduction Ref. % % w control in CH v control No. Compound . concentration 2 hours during 2-4 hrs AF 6 isobutanl 7 lsobutyl isobutyrate l isobutyl succinate 2 isobutyl adipate 13 Isobutyl AGSate 14 Isobutyl glutarate 5 isobutyl phosphate 6 Isobutyl amide 17 N propyl isobutyrate 18 lsobutyl in propionate 19 lsohutyl iodide isobutyl iodide lsobutyl iodide 20 Isobutyl bromide 21 lsohutyl acetate isobutyl acetate isobutyl acetate 2 2 isobutyl chloride Isobutyl chloride lsobutyl chloride 8 23 Chlorinated IBDU 1.() --- Chlorinated BDU - ().5 - Chlorinated BDU (). 24 BD - trioxane mixture (2:1) - - ().5 - (). - 25 BD -- trioxane mixture (3: ) -- ().5 o

3,982,028 9 10 Table 1 1-continued Total gas % reduction Ref. % %. v control in CH v control No. Compound concentration 2 hours during 2-4 hrs 26 BD + Trioxane mixture (4:1) 1 --- ().5 ------0. ------27 BD -- rioxane mixture (5:1) l 0 - ().5. -- -- (). ------28 BD + trioxane mixture (6:) -- --- ().5 -- --- 0.1 --- 29 Methylene di-isobutyratic ------().5 -- - 30 Methylene dioxymehtylenc (). -- - di-isobutyrate - --- - ().5 (). ------32 (IBD + urea)Cl. derivs t ----mm ------

Whereas it is always desirable to inhibit the formation Tables 10, ll. Methane formation in ruminants is of methane, it is not necessarily advantageous to inhibit reduced or inhibited by isobutyl adipate, isobutyl gluta ruminal fermentation (i.e. to reduce the total amount rate, isobutyl iodide, isobutyl chloride, isobutyl bro of gas formed). Stimulation of ruminal activity may mide (i.e. isobutyl halides), isobutyl acetate, chlori decrease the absorption of natural proteins, minerals 25 nated IBDU, mixtures of IBD with trioxane, methylene and trace elements by the host animal, but on the other di-isobutyrate, methylene dioxymethylene di-isobuty hand it enhances utilisation of structural carbohydrate, rate, and chlorinated mixtures of IBD and urea. e.g. cellulose, by increasing the production of C2-C4 I claim: acids which are precursors of milk fat and protein (for 1. Animal feedstuffs selected from the group consist milk production) and body tissue and fat (for beef 30 ing of compound feeds, feedblocks, liquid feed supple production). Thus some substances which are methane ments, drenches, slow release pellets, ensiled green inhibitors may play an additional role in providing a fodder, hay and grain, containing a mixture of isobutyr means of controlling rumen fermentation and obtaining aldehyde with a substance selected from the group selective variations in the pattern of volatile fatty acid consisting of AGS (Adipicglutaric-succinic) acid, ace formation. In the foregoing Table ll, we are primarily 35 tic acid, formalin, sulphuric acid, and trioxane or a interested in compounds showing a reduction in meth chlorinated mixture of isobutyraldehyde with urea. ane than in reduction or stimulation of rumen fermen 2. Animal feedstuffs as claimed in claim 1, compris tation. We would consider as useful any compound ing a mixture of isobutyraldehyde and AGS acid in the showing a methane reduction of about 25% or more ratio l:l w/v. (i.e. depicted with two or three "minus' signs beside 40 3. Animal feedstuffs as claimed in claim 1, compris it). ing a compound feed containing a mixture of isobutyr Reverting to the foregoing Tables 3 to 11, the indi aldehyde and acetic acid in the ratio 3:1 V/v. vidual Tables show: 4. Animal feedstuffs as claimed in claim 1, compris Table 3. IBD:AGS and IBD:IBA exhibit a preserva ing a compound feed containing a mixture of isobutyr tive effect on compound animal feeds. 45 aldehyde and formalin in the ratio 3:l v/v. Table 4. This preservative effect is exhibited by IBD 5. An animal feedstuff as claimed in claim 1, com :IBA, and IBD:AGS (as above) and also by IBD/acetic prising ensiled green fodder, hay or grain treated with a acid and IBD/formalin, and to a lesser extent by IBD/H- mixture containing isobutyraldehyde and AGS acid in SO4, isobutanol, isobutyl isobutyrate and isobutanol/- the ratio 3:1 to 1:3 v/v. sodium isobutyrate. 50 6. An animal feedstuff as claimed in claim 1, contain Tables 5 to 8. Microbial inhibition or reduction, lead ing a mixture of isobutyraldehyde and trioxane in the ing to decrease in microbial spoilage, is exhibited by ratio 2:1 to 6:1 v/v. isobutyl isobutyrate, isobutanol, and isobutanol IBA. 7. An animal feedstuff as claimed in claim 1, contain Table 9. Self-heating acid mould contamination of ing a chlorinated mixture of isobutyraldehyde with wet hay is inhibited by mixtures of IBD:IBA and IBD-55 2. :AGS. ck k - k xk sk

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