AU2007237175A1 - Synthetic Protein Equivalent Stock Feed - Google Patents

Description

COMPLETE SPECIFICATION FOR A STANDARD PATENT in the name of AGR SCIENCE TECHNOLOGY PTY LTD ABN 200 876 216 27 entitled "SYNTHETIC PROTEIN EQUIVALENT STOCK FEED" Filed by: AGR Science Technology Pty Ltd 160 Musgrave Road Coopers Plains QLD 4108

AUSTRALIA

P.O. Box 210 New Farm QLD 4005

AUSTRALIA

INVENTORS

Z Dr Nicholas James Calos 00 54 Dunstan Street Moorooka QLD 4105

AUSTRALIA

Mr Gregg Lewis Chapple 3/102 Oxiade Drive New farm QLD 4005

AUSTRALIA

TITLE

"SYNTHETIC PROTEIN EQUIVALENT STOCK FEED" 00 5 FIELD OF THE INVENTION This invention relates to a method for producing a non-protein source of nitrogen nutrient as a stock feed ingredient. Furthermore, the invention provides a means of delivering high levels of non-protein nitrogen nutrients with increased safety as r> 10 compared to alternatives such as molasses-urea blends.

BACKGROUTND

Beef cattle production is in perpetual demand for high protein level feeds. Current feed grain requirements for Australia as at 2004 amount to 7.7 million tonnes, estimated to rise to 10 million tonnes by the year 2010, with shortages in protein to be expected. These shortages in protein meals are frequently exacerbated by their seasonal availabilities. Consequently, materials such as urea are inevitably used to boost the protein values beyond those found in grain, cottonseed, copra, etc.

However, the prospect of poisoning places strict limits on the rate at which urea can be fed.

As already indicated, by far the most common non-protein nitrogen source for stock feed is urea, on account of its high nitrogen capacity and low cost. However, urea use must be carefully controlled to minimise the attendant risk of poisoning of livestock.

Furthermore, because of its high rate of nitrogen availability, much of its value may be lost through excretion if the nutritional protocol is not carefully balanced and maintained, through mixing with other protein sources such as grain, cottonseed, or copra. The situation is further complicated by the variable availability and price fluctuation of these other protein sources. Thus, a demand exists for a safe and costeffective alternative non-protein nitrogen source.

-3- By chemically binding urea into larger molecules, its rate of release (and utilisation) 0 can be limited to safe levels. For example, ureaforms such as methylenediurea have been reported as effective feed compounds for ruminants (Kaushal and Swan (1983), 0 SInd. J. Anim. Res., vol. 17, pp. 8 16; Kaushal and Swan (1983), Ind. J. Anim. Res., 00 5 vol. 17, pp. 93 97). However, the range of ureaforms which serve as useful feeds is limited according to these studies.

For instance, the synthesis of methylol derivatives of urea for use as ruminant feeds c are described by McCann (1978) in BG1515280.

O Urea may also be bound to other nutrient molecules as an extension of this concept, so that additional nutritional benefits may be delivered in a controlled fashion for greater conversion efficiency. Sugars are usually the nutrients of choice, so much so that a class of compounds known as ureido-sugars, glycosyl ureas, or glycosylureides exists in the Prior Art as a class of ruminant feeds.

These compounds, of which glucopyranosylamine is one example, have demonstrated the risk-free effectiveness of this class of materials as stock feeds above the use of non-derivatised urea alone (Martin et al, (1982), Can. J. Anim. Sci., vol. 62, pp. 1129 1134; Martin et al, (1983), Can. J. Anim. Sci., vol. 63, pp. 105 116).

Glycosyl ureides are typically synthesised from a reducing sugar ribose, xylose, glucose, cellobiose, lactose, maltose) incubated with urea under acidic conditions, cf Schoorl (1903), Rec. Trav. Chim., vol. 22, p. 31; Goodman (1958), Adv.

Carbohydrate Chem., vol. 13, pp. 215 236; Benn and Jones (1960), J. Chem. Soc., pp. 3837 3841; Helferich and Kosche (1926), Ber. vol. 59B, pp. 69 79.

Disaccharides are equally effective in producing glycoureides when heated in acidic conditions with urea, as in the example given by Belusov et al (1982), Zhurnal Prikladnoi Khimii, vol. 55, pp. 2124 2125.

Hynd ((1926), Biochem. vol. 20, pp. 195 204) reported that the condensation of urea with reducing sugars under ambient conditions is spontaneous, but slow.

SA number of previous workers have utilised variants of this theme to produce a range 0of ureides for stock feeds, namely: Pekkarinen Kurppa (1984), FI830428; aldohexoses and urea with mineral 0 Z acids heated to a temperature of 80 0

C

00 5 Regnault Sachetto (1980), W07800017; glucose, urea and strong acids heated to 68 0 C then mixed with CaCO 3 and possibly further admixed with feedstuffs Vtt to produce a ruminant feed S Takase et al (1983), JP58067148; a glucosylurea is prepared by means similar to FI 83-428 then complexed with additional urea to form an animal feed with extremely 10 high protein score Aizawa et al (1983), JP58067662; glucose is heated with excess urea in acidic conditions to produce a glucosylurea-urea complex Osipow et al (1961), US2967859; glucose and urea heated in strong acid above 0 C, to produce di-D-glucose ureide Smith et al (1978), US4066750; where lactose is heated with acidified urea derivatives to produce ruminant feedstuff Bronze et al (1981), Portugaliae Acta Biologica, Serie A, vol. 16, pp. 267 -268; acidic extract of sugars from carob bean syrup are heated with urea to form ureides.

The same process was used to make slow-release fertilisers, as in Watanabe et al (1983), JP58161987.

In extension of the method, sugars can be sourced from the hydrolysis of higher carbohydrates, as in the following examples: Dudkin et al (1978), Isvestiya Vysshikh Uchebnykh Zavedenii, Pishchevaya Tekhnolgiya, vol. 6, pp. 29 -33; sucrose or molasses is heated with urea and phosphoric acid to hydrolyse the sugar and yield ureide McNeff (1972), US3677767; acidified molasses and urea are heated to produce stock feed Berger et al (1977), US4006253; cellulosic materials are reacted with urea in heated acidic conditions Regnault et al (1980), EP0007136; fodder is made from acidic condensation of urea with hydrolysed cellulosic and lignocellulosic materials Thibault et al. (1985), FR2556566; enzymatic hydrolysis of sucrose followed by thermal acidic condensation with urea Diner and Elofson (1977), US4044156; enzymatic hydrolysis of starch followed Z by thermal acidic condensation with urea.

00 Amines, and particularly amino acids, are able to undergo a condensation reaction It with aldoses or reducing sugars, especially at elevated temperatures and pH values (cf t"- M GP Ellis (1959), Adv. Carbohyd. Chem., vol. 14, pp. 63 164). The products of such t"rC reactions, termed Maillard reactions, with vegetable feed proteins have, in works such r- 10 as Kostyukovsky and Marounek (1995), Animal Feed Science and Technology, vol.

0 55, pp. 201 206, and Cleale et al. (1987), J. Anim. Sci., vol. 65, pp. 1312 1335, been used to control the rumen availability of these feed proteins.

Furthermore, these carbohydrates which undergo condensation reactions with amines, amino acids and amides can proceed along extended reaction pathways, to produce higher condensation products, or other degradation products. Dark coloured highly condensed polymers such as melanoidins may be the end products of Amadori rearrangements or Heyns rearrangements, or heterocyclic compounds such as imidazoles, pyrazines, or diureins may result from Windaus and Knoop-type reactions the review by MR Grimmett (1965), Rev. Pure and Appl. Chem., vol. 15, pp. 101 108). While each of these rearrangement reactions is known to produce colourants and flavourants in the food industry in general, there is no evidence of the application of these reactions to limiting nitrogen availability in ruminant feedstuffs.

These condensation reactions may also be catalysed by metal hydroxides, such as ammoniacal zinc hydroxide (A Windaus and F Knoop (1905), Chem. Ber., vol. 328, p. 1166).

In another example, Wiggins and Wise ((1955), Chem. Ind., vol. 23, pp. 656 657) discussed the production of ammoniated molasses for livestock feed by highly exothermic reaction of inverted molasses with pressurised anhydrous ammonia.

r- Kansas State University Research Foundation (1968) in GB 1127198 and Deyoe et al O (1980) in US4232046 specify the attachment of a non-protein nitrogen feedstuff to a carbohydrate by means of encapsulation or clathration rather than through formal 0 z chemical bonding. Jinderpal Singh and Kaushal (1993), Indian Journal ofAnimal 00 5 Nutrition, vol. 10, pp. 133 138, have also established that similarly clathrated ureas have effectively limited availabilities to degradation. However, it is apparent from the Ilatter study that the process of clathration requires heat and pressure.

Cc Combinations of carbohydrates, urea and formaldehyde (or other aldehydes) are also known, as in the following examples: O* Allied Chemical Corporation (1975) disclosed in GB1380789 the use of ureas, formaldehyde and carbohydrates in the manufacture of a ruminant feed. However, this process still required an acidic-mediated condensation of urea with saccharides; the formaldehyde serving to polymerise and solidify the mixture Protein availability for ruminant nutrition has been limited by treatment with formaldehyde, as taught by Markku (1982) in EP0043202A2 The acidic condensation of urea, and aldehyde, and a carbohydrate to produce stock feed is also known from Dudkin et al (1986), Koksnes Kimija, pp. 105 111, and Snyder (1980), CA1077333.

OBJECT OF THE INVENTION Wherein the Prior Art utilises sugar ureide derivatives derived by pressurised, thermal or acidification processes to limit the availability of protein or non-protein nitrogen sources as a livestock nutrient, the present Inventors have realised that ureides and indeed amines or amides of equal effectiveness may be produced through standard pressure, low-temperature or alkaline processes.

The present Inventors have also realised that a substantial component of the cost of production of ureides, amines, or amides lies in the pressurising or heating requirements of the condensation, which can be eliminated through low- or ambient temperature or pressure processes.

O The present Inventors have also realised that where condensation reactions such as the Maillard reaction have been used to limit the rumen availability of protein feeds, the 0 Z same low-temperature or alkaline processes may be used to limit the rumen 00 5 availability of non-protein nitrogen sources in feeds. Furthermore, extended reaction products of these non-protein nitrogen compounds such as (for example, but not Slimited to) Amadori or Heyns rearrangement products as produced by a low temperature process may be used to limit the rumen availability of said non-protein Cc nitrogen sources in feeds, where previously they were exclusively used to produce r- 10 colourants and flavourants in the food industry in general.

These low temperature or low pressure reactions provide non-protein nitrogen in a form in which it effectively behaves as true crude protein in terms of its pattern of utilisation by ruminants.

The combined protein and energy source of the ureides, amines, or amides, when admixed with a poor quality feed such as straw, allows the more effective utilisation of the latter.

While the ureide, amine, or amide products can be manufactured cost-effectively from virgin ingredients, the present Inventors have also realised that additional cost reductions in production can be achieved through the beneficial reuse of waste or byproduct materials as source ingredients, reagents, or catalysts for such amide derivatives.

Furthermore, the present Inventors have also realised that the condensation and rearrangement reactions mentioned above need not be limited to ureides or their derivatives, but rather may be extended to include any organic hydroxide, ketone, or aldehyde as the counterpart to the amine, imine, amide or carbamate a primary or secondary amino entity).

-8- SUMMARY OF THE INVENTION This invention provides a means of production of a cost-effective and safe non-protein 0 z nitrogen stock feed material, wherein a compound bearing a primary or secondary 00 5 amino entity is bound to an organic polyfunctional hydroxide, aldehyde, or ketone entity, or a polyhydric alcohol, carbohydrate, or deoxy carbohydrate capable of Stautomerising to yield a carboxyl entity or fragment, such that the availability of either nutritional component is limited by said reaction.

Cc- Where a protein is a polymer consisting of peptide-bonded amino acids, the extended nature of this peptide bonding is the limiting factor in the nitrogenous nutrient availability to the rumens. In smaller, non-polymeric fragments or entities, such as amino acids and their oligomers, and other small amine, amide, or imine containing entities, the nitrogen nutrient value of these compounds or fragments is quickly utilised by the rumens microorganisms, at such a rate as to potentially cause the ruminant harm or distress through a condition known as ammonia or urea toxicity.

These non-protein nitrogen compounds and fragments may be bound into higher molecules in order to limit their availability and utilisation to a rate at which the risk of ammonia or urea toxicity is mitigated.

Preferably, the polyfunctional organic hydroxide is a simple carbohydrate such as glucose, fructose, or lactose, and may include deoxy sugars such as fucose or rhamnose (without being limited to these specific examples). Simple sugars may also be derived through hydrolysis (enzymatic or otherwise) of more complex carbohydrates such as starch, cellulose or sucrose. Even more preferably, sugars may be sourced from such waste or by-product sources as (but without being limited to) waste sugars, dextroses, or maltodextrins from out-of-specification fruit juices, whey, soft drinks from dairy and beverage producers, dunder, vinasse, molasses, or cane juice from sugar mills, paper pulp from paper mills, starches, fibres, gums and pentosans, from cereals, tuber, fruit and vegetable, and snack food processors.

Molasses or starch may be used as sources of complex carbohydrates, or they may be hydrolysed to simple sugars by the action of acid or enzymes such as invertase or amylase, respectively.

The non-protein nitrogen components may include the following non-limiting 0examples: urea, biuret, melamine, ureaforms, amino acids, protein fragments and peptide-bonded oligomers, any source of primary or secondary amines, amides, or 0 z carbamates, potentially sourced from (but not limited to) out-of-specification urea, 00 5 biuret, urea-formaldehyde resin wastes, or protein or gluten wastes from cereals, tuber, fruit and vegetable, and snack food processors.

SNon-protein nitrogenous components may also result from the hydrolytic degradation 1r of nitrogenous polymers, including but not limited to proteins, glycosamine

(N

10 derivatives, or melanoidins. Non-protein nitrogenous components may also result 0from the condensation or complexation of nitrogenous molecules or fragments, including but not limited to urea condensates such as biuret, ureaforms, or ureaprotein complexes.

The polyfunctional organic hydroxide, aldehyde, or ketone solution or mixture may additionally contain a solution of a carboxyl-group containing entity or fragment, or a polyhydric alcohol, carbohydrate, or deoxy carbohydrate capable of tautomerising to yield carboxyl entities or fragments.

The contents of polyalcohol, aldehyde, or ketone and amino group entity in aqueous suspension or solution may each range between 0.1% and 75%, while the aldehyde content may range between 0% and 75%. The aldehyde entity may additionally, inclusively, but not restrictively, be contained on the same molecule or fragment as either the alcohol or the amino group entity. Said aqueous suspension or solution may additionally be diluted to achieve the desired concentration.

Preferably, the molar ratio ofpolyalcohol:carboxyl:amino group entity is 1:1:1.

The reaction may be carried out over a broad temperature range, from 0°C to 110°C.

Preferably, the temperature of the reaction is between 6°C and 50°C, and even more preferably, the reaction is carried out within the temperature range of 10°C and The reaction rate is enhanced at elevated pH values and may proceed over the range Sof 6 to 14. Alkaline pH values are preferred, and the pH of the polyalcohol-nitrogen compound solution mixture is more ideally in the range 10 to 13, adjusted or 0 z controlled by the addition of an alkali.

00 Alkali may include (in a non-limiting sense) ammonia, sodium, potassium, t magnesium or calcium hydroxides, sourced from such waste streams as emanating from brickworks, magnesium smelters, cement, margarine, detergent, aluminium or Cc glass production plants and industrial gas scrubbers.

SSaid caustised or alkaline reaction mixture is then aged at the desired temperatures and pH values, to allow the condensation reaction between polyalcohol, optional aldehyde, and amino group entity to be driven to completion.

Ageing time may vary between a matter of hours a minimum of hour) to many months a maximum of 6 months).

Preferably, the ageing period is 3 weeks.

Even more preferably, the mixture is aged for 3 days.

Ageing may be carried out in the presence of a heterogenous or homogenous metal hydroxide catalyst. Preferably, the metal hydroxide may include (but not be limited by) magnesium, calcium, zinc, copper, manganese, or iron. The metal ion may additionally be chelated or ligated by other non-hydroxide groups.

The metal hydroxide may be produced either in-situ or ex-situ, by the addition of a metal salt to an alkaline solution, or to the alkaline reaction mix. The alkaline solution may include (in a non-limiting sense) ammonia, sodium, potassium, magnesium or calcium hydroxide.

Even more preferably, the catalyst will be zinc ammonium chloride or ammoniacal copper acetate (either as a solid or in solution), admixed with caustic soda solution so as to achieve the re-dissolution of the initially produced gelatinous precipitate of -11metal hydroxide. This dissolved ammoniacal metal hydroxide may then be admixed with the alkaline reaction mixture and left to age for the desired length of time.

Such metal salts or chelated metal salts may be sourced either as virgin materials, or as wastes or by-products from other industries. Such sources include (in a nonlimiting sense) galvanising plants, tanneries, metal refiners, extruders, or finishers, or timber treatment plants, from which emanate such waste materials as spent galvanisers acid containing zinc or iron, and waste copper salts.

The catalyst may be added to the reaction mix so as to result in a metal concentration in said reaction mix ranging between 0% and 5% Preferably, the metal concentration will be in the range 0% and 1% (wlw), while more preferably, the concentration will range from 0.02% (wlw) to 0. 1% The catalyst may additionally provide further nutritional value, in that certain trace metals such as magnesium, calcium, zinc or iron (as non-limiting examples) are essential to ruminant metabolic processes.

At the completion of the allocated ageing period, the alkaline reaction mixture is acidified to produce the final non-protein nitrogen stockfeed product.

Acidifiers may include (but not in a limiting sense) such mineral acids as hydrochloric acid, sulphuric acid, or phosphoric acid, such organic acids as acetic acid, propanoic acid, or citric acid, or even Lewis acids such as ferric chloride. Such acids may additionally be sourced from waste or by-product streams, so as to affect an additional cost and environmental benefit. Sources of such waste or by-product acid may include, but not necessarily be limited to, wineries, spent galvanisers' acid, metal surface finishing plants, or wool scourers.

The acidifier may additionally provide further nutritional value, in that acidic sulphates, phosphates, citrates, lactate, or acetates (as non-limiting examples) may be utilised in ruminant metabolic processes.

12- The pH of the product after ageing is preferably adjusted to between 3 and 8 by the admixture of said acid. Alternatively, the pH of the reaction mix may be modified so as to produce a viable, safe, and palatable feed product through its dilution into other Z feed ingredients or water. This dilution is preferably at such a rate as to affect a final 00 5 pH value as per the above specified limits.

The control of the pH of the product through acidification may additionally provide a means of extending the shelf-life of the product, through the bacteriostatic action of high acidity.

r> The control of the pH of the product through acidification may additionally provide a means of controlling the consumption rate of the feedstuff to ruminants, through adjusting the palatability by the souring action of acids.

Throughout this Specification, the ranges stipulated are inclusive rather than exclusive.

The terms "glycosyl" and "carbohydrate" are considered synonymous and are considered to be subsets of the synonymous terms "polyhydric alcohol", "polyfunctional organic hydroxide", and "polyalcohol". These terms as used throughout this Specification refer to any organic molecular entity containing at least one hydroxyl group, and are used inclusively rather than exclusively.

The terms "amino group entity" and "nitrogen compound" are considered synonymous and are used throughout this Specification to refer to any organic molecular entity containing at least one primary or secondary nitrogen group, as defined by the number of hydrogen atoms bound to the nitrogen atom, and are used inclusively rather than exclusively.

The terms "ureide", "condensate" and related terms are considered synonymous and are used throughout this Specification to refer to the product of the reaction between a polyalcohol and an amino group entity, and are used inclusively rather than exclusively.

13 The invention described herein may be understood more clearly by the non-limiting 0 examples listed under the following "Preferred Embodiments".

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Z BRIEF DESCRIPTION OF THE TABLES AND FIGURES 00 Table 1: Initial rate of urease reaction on a range of substrates containing Idifferent urea derivatives.

n Table 2: Feedlot diet (in terms of kg per head per day) for 100 days weight gain, S 10 with introduction of NPN.

Table 3: Daily protein intake (in terms of kg protein per head) from 100 day feedlot diet.

Figure 1: Enzymatic hydrolysis rate curve of starch for production of precursor sugars for glucosyl-ureide derivative manufacture.

Figure 2: Paper chromatogram showing the reaction of glucose, urea, and formaldehyde under different conditions, to yield products different to the starting reagents. Key to annotations: a a-Glucose p p-Glucose f= Fructose F Formic Acid U Ureide.

Figure 3: 3 C NMR spectrum of reaction mixture GU ALK-ACID, showing the presence of unreacted sugars, by-product formic acid and the a-D-glucosylureide derivative.

Figure 4: 'sN NMR spectrum of 5 N isotopically enriched NPN dissolved in

D

2 0. Regions of substituted imidazoles (ca 127 ppm) and amides (ca 83 ppm) are indicated. Note also the complexity and asymmetry of the peak at ca 57 ppm, indicating a substituted ureide.

-14- Figure 5: 15N NMR spectrum of 15N isotopically enriched urea. Note that it Spresents a single, sharp, symmetric peak at 57 ppm 15NH 4 CI), in contrast to the substituted ureide (NPN) spectrum shown in Figure 4.

0 z 00 5 Figure 6: Comparative rates of ammonia production in rumen microbe suspensions fed with free glucose and urea and with glucosyl-ureide derivative.

Figure 7: Comparative rates of glucose availability in rumen microbe Cc suspensions fed with free glucose and urea and with glucosyl-ureide derivative.

PREFERRED EMBODIMENTS Example 1) 4 tonnes of starch was pre-treated with 8 kg of CaCI 2 and approximately 6kg of light soda ash, to adjust its pH to 7.03 and supply the necessary calcium for optimum enzyme activity. The slurry was then cooked to 66 0 C in the presence of 4 kg of an amylase having 240kNU/g activity. The final mixture measuring 12.5 tonnes was a translucent runny liquid, which continued to develop greater translucence as the amylose was being sheared in due course to form lighter sugars.

Glucose levels were monitored over 24 hrs, to assess the completion of enzyme activity prior to adding the remaining ingredients in the next stage of the production.

For the record, the sugar generation curve is presented in Figure 1, showing the plateau in starch hydrolysis rate developing after approximately 4 to 6 hrs.

The final sugar solution (at a glucose reading of 62.8 mmol/l) was transferred to a tonne stirred tank, to which was added 570 kg of urea prills, 713 kg of formaldehyde solution and 404 kg of pearl caustic soda, to achieve a final pH value of 12.11.

This mixture was well stirred, then left to age for 1 month at an ambient temperature of 32 0 C, during which time it developed colour from a pale grey-beige to a bright orange. This colour change signified the onset of the Maillard condensation of the urea with the glycosides.

O After ageing, the pH of this mix was then adjusted with an additional 180 litres of concentrated phosphoric acid sourced as a by-product from an aluminium

O

Z extrusion plant, to achieve a final pH of 7.06. This ultimate glycosyl-ureide product 00 5 was the liquid stockfeed.

SThe product gave a negative Purpald test, indicating the complete reaction of the formaldehyde.

S 10 Example 2) Three syntheses were carried out to demonstrate the effects of order of mixing of reagents on the success of the reaction.

In the first synthesis, 16.2 g of glucose and 6.0 g of urea were dissolved in 50 ml water, followed by 7.5 g of a 40% formaldehyde solution, mixed well. This mixture was labelled GU. The second mixture (labelled GU ALK) was prepared as per GU, but the pH was adjusted to 11.98 with the addition of 10 ml of a 19% caustic soda solution sourced as a by-product from an aluminium extrusion plant. The third mixture (GU ALK-ACID) was as per the second, with addition of 0.25 ml of concentrated phosphoric acid after 2 days.

All samples were left to age for I week.

The first mixture without caustic soda addition produced a fine white precipitate and a clear supernatant solution with dissolved solids. After 3 weeks, the two caustised samples turned an amber colour and went syrupy through water evaporation.

These products were both analysed by paper chromatography against pure glucose and urea, with results shown in Figure 2. The chromatography solvent was 50:50 propanol:ethanol, and chromatograms were developed with 12 vapour, contrasted under UV illumination. Reagents and products were identified by their characteristic mobilities (Rf values) as measured from the chromatograms.

-16r- With reference to Figure 2, both glucose and urea are light molecules, with large Rf O values (mobilities in solvent) measured as 0.757 and 0.806, respectively. Both compounds showed purple stains on 12 exposure.

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00 5 Without caustisation a mixture of free glucose, free urea and insoluble ureaformaldehyde resin is produced.

Again with reference to Figure 2, both GU ALK and GU ALK-ACID samples n behaved similarly to each other, showing white bands at Rf 0.423 on exposure to 12.

S 10 The lower Rf is consistent with a molecule of larger molecular weight and/or greater Spolarity, as expected from being carbamated by urea. Furthermore, the decolourisation of the 12 indicated a strongly reducing compound, and the ability to give a reading on the glucose meter showed that the product remained a derivative.

The paper chromatograph of glucose-bonded urea (Figure shows distinct differences in the product bands (GU ALK GU ALK-ACID) from the starting materials, glucose and urea Some residual glucose and urea is found in the GU band, when illuminated under UV. Likewise, when illuminated under UV, some residual urea is found in the GU ALK sample.

Condensation of urea to glucose through formaldehyde appears to only take place effectively under alkaline conditions. In other circumstances, urea-formaldehyde resins are formed, leaving the glucose unreacted. When the initial condensation reaction is mostly completed in alkali, the condensation can then be "mopped up" by re-acidifying the mixture.

The soluble components of GU ALK-ACID were further unequivocally characterised by 3 C Nuclear Magnetic Resonance (NMR) spectroscopy, as shown in Figure 3.

In this NMR spectrum (Figure two forms of unreacted glucose were identified as a-D-glucopyranose and P-D-glucopyranose, along with a glucose epimerisation product, p-D-fructofuranose. In addition to these, a side product of formaldehyde oxidation, formic acid, was found. However, significantly, an ac-D-glucopyranosyl -17- 1 methyloylurea condensate was clearly identified, commensurate with related 3

C

SNMR spectra reported in Literature (cfBlank et al. (1998), Acta Cryst., vol. C54, pp.

1992 1994; Helm and Karchesy (1993), J Carbohydrate Chemistry, vol. 12, pp. 277 Z- 290).

00 Example 3) A sample of 'N isotopically labelled NPN was prepared as follows: tI 0.2532 g of natural abundance urea, 0.2625 g 98% enriched 5 N urea, 1.5040 g glucose was dissolved in water and caustised to pH 13.04 with NaOH solution.

¢C After aging 2 days, the deep yellow-orange solution was acidified with concentrated 10 hydrochloric acid to pH 4.98 to produce a sample of NPN for N NMR Sspectroscopic study, to ascertain the reaction pathways of the urea group.

The 5 N NMR spectrum of this sample dissolved in heavy water (for a deuterium spin-lock reference point) is shown in Figure 4. The 5 N NMR spectrum is referenced to 5

NH

4 Cl. The spectrum illustrated in Figure 4 also shows regions characteristic of substituted imidazoles (ca 127 ppm) and amides (ca 83 ppm). The complexity and asymmetry of the urea/ureide peak at ca 57 ppm should also be noted, indicating a substituted ureide. For comparison, the 15N NMR spectrum of free, unreacted or unsubstituted urea normally presents as a single, sharp, symmetric peak in the range of 54 ppm to 59 ppm w.r.t. 'SNH 4 CI, as shown in Figure The complexity of the ureide 5 N NMR spectrum is due to H-D exchange on this group, consistent with an asymmetrically substituted urea.

As for the remaining spectral peaks in Figure 4, with reference to the 5 N NMR review by Warren and Roberts ((1974), J. Phys. Chem., vol. 78, pp. 2507 2511), these other minor peaks also indicate the formation of substituted amides, carbamates, pyrazines, and imidazoles, consistent with the possible side-reactions melanoidin formation, Amadori rearrangements, and Maillard-type reactions) that are known to occur in alkaline sugar-amino compound systems (see, for instance, the review by MR Grimmett (1965), Rev. Pure andAppl. Chem., vol. 15, pp. 101 108).

-18- Example 4) A waste fruit juice, as sourced from a snack beverage producer, 0 provided the carbohydrate source for the manufacture of another variant of the nonprotein nitrogen stockfeed.

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00 5 20 litres of a waste fruit juice was characterised as containing 320 grams of total sugars, of which 18% was a-D-glucopyranose, 32 was P-D-glucopyranose, V was P-D-fructopyranose, 12% was p-D-fructofuranose, and 3% was a-D- Sfructofuranose. The fruit juice additionally contained approximately 2% mannitol and l^c glycerol, along with 0.46% acetic acid, 0.32% citric acid and 0.79% lactic acid.

S STo this liquid was mixed 120 grams of urea prills and 150 grams of formaldehyde solution. The pH of the mixture was then brought to 12.09 with the admixture of 2.3 litres of waste caustic soda solution, as sourced from an aluminium extrusion plant.

The alkaline mixture was then aged for 1 month, then neutralised to pH 6.5 with 307 ml of waste concentrated phosphoric acid, as sourced from an aluminium extrusion plant, to make the final stockfeed product.

This product consisted of sodium salts of acetic, citric and lactic acids, a-D-glucopyranose, P-D-glucopyranose, P-D-fructopyranose, P-D-fructofuranose, and an a-D-glucopyranosyl methyloylurea condensate.

This feed product proved to give effective control of nutrient availability to rumen microbes, as in the following Example.

Example 5) The metabolic breakdown of a sample of glucosyl-ureide derivative prepared as per Example 3 was compared against equivalent concentrations of free glucose and urea in rumen microbe suspensions.

Rumen ammonia release rates and glucose degradation rates were monitored for the glucosyl-ureide condensate feed product and the free sugar and urea nutrients, and the relevant reaction rate curves are shown in Figures 6 and 7, respectively.

-19- O In each case, the condensate showed steadier and less extreme production or availability of the ammonia or the sugar than the free components, thereby indicating

O

z less risk of causing urea or ammonia toxicity or acidosis to ruminants. With reference 00oO 5 to Figure 6, the ammonia release rate from the ureide was kept below rumen toxicity levels, compared to that from an equivalent dose of free urea.

Example 6) The activity of urease on a range of substrates was determined to demonstrate the effectiveness of the condensation reaction. The rate of ammonia S 10 release (as measured by increase in pH) from the cleavage of urea compounds by the urease enzyme is a function of the effective molecular size of the substituent on the (Ni urea molecule. Thus, urea as a substrate shows the greatest rate of cleavage by urease, while a massive urea-formaldehyde polymer would show a substantially slowed reaction. Biuret, for instance, being effectively a dimer, would also show a slowed reaction, but not to the extent of a massive polymer.

Non-protein nitrogen (NPN) was produced as in the following procedure, for comparison of its activity against a simple solution of 4% urea in molasses, and of 4% biuret in molasses.

To a 17,000 1 stirred tank of molasses (with 62% total sugars in the form of sucrose, fructose and glucose) was added 238 1 of 33 hydrochloric acid, to achieve a pH of 4.52. This acidified molasses was then hydrolysed using an invertase of 10,000 SU/ml activity.

After hydrolytic inversion of the sugars, the molasses was caustised to pH 10.45 with 2,800 1 of 39 NaOH solution. 4,000 kg of urea was then stirred in and the reaction mixture aged for 4 days.

At the end of the aging period, the resultant NPN solution was neutralised to pH 7.61 using concentrated hydrochloric acid.

As a demonstration of the effectiveness of the condensation reaction described above, the activity of 0.001% urease within this material was measured over time to measure a reaction rate. Similarly, the rates of urease acting on solutions of 4% urea and 4% Sbiuret in molasses were also measured, and all three are reported in Table 1 for comparison.

O

00 5 Table 1: Substrate Initial Rate (ApHemin') Urea molasses solution 3.92 Biuret 1.32 NPN 0.92 It is clear from Table 1 that the condensation of urea in NPN has progressed such that the activity of urease upon the resultant product is substantially reduced compared to the reactions with either simple urea or biuret solutions.

Example 7) To a 17,000 1 stirred tank of molasses (with 62% total sugars in the form of sucrose, fructose and glucose) was added 238 1 of 33 hydrochloric acid, to achieve a pH of 4.52. This acidified molasses was then hydrolysed using an invertase of 10,000 SU/ml activity.

After hydrolytic inversion of the sugars, the molasses was caustised to pH 10.45 with 2,800 1 of 39 NaOH solution. 4,000 kg of urea was then stirred in and the reaction mixture aged for 4 days.

At the end of the aging period, the resultant NPN solution was neutralised to pH 7.61 using concentrated hydrochloric acid.

This process provided a 32 tonne batch of NPN with 35.8 crude protein value, for inclusion into a feedlot diet.

A herd of 300 head of beef cattle was finished for 100 days on a graded diet as outlined in Table 2. The diet was designed to provide between 10.6 MJ/kg and 12.0 MJ/kg metabolic energy, greater than 30 fibre and a gradation in the protein content from 16 initially, down to 14 after 20 days, as protein utilisation efficiency improved.

-21 The gradation in the diet consisted of a replacement of grain and seed protein by NPN over the first 7 days of the diet, followed by a 13 day acclimatisation period, then stepping up of total protein intake over the remaining 80 days (see Table 3 for the effective protein intake over these periods).

Consequently, the daily weight gain was expected to be graduated, starting at 1.6 kglheadlday, and finishing at 2.1 kg/headlday. The overall average was expected to be 2.1 kg/headlday.

Weigh-in at the end of the 100 days feedlotting showed an average weight gain of approximately 2.1 kg/headlday, precisely in accordance with expectations.

These data show that NPN can directly substitute for true protein in beef cattle diets with highly efficient utilisation of its crude protein and energy values, without interrupting the weight gain program.

Table 2: Days Barley CSM* Molasses NFN Lucerne Rhodes Grass Hay 1.0 0.5 2.4 2.4 2.0 1.0 0.0 1.6 1.2 1.2 2.0 0.0 Barley Wt Straw Gain 0.0 1.63 2.0 2.01 1.0 2.15 0-7 8 -20 21 -100 CSM 6.0 1.4 10.3 0.0 12.6 0.0 Cotton Seed Meal Table 3: Days Barley CSM* Molasses NPN Lucerne Rhodes Barley Total Wt Grass Straw Protein Gain Hay 0-7 0.72 0.59 0.04 0.31 0.38 0.17 0.07 2.21 1.63 8-20 1.24 0.00 0.08 0.61 0.00 0.11 0.10 2.14 2.01 21 -100 1.51 0.00 0.05 0.73 0.32 0.00 0.05 2.66 2.15 Average 1.42 0.04 0.05 CSM Cotton Seed Meal 0.69 0.28 0.03 0.05 2.57 2.10 22

Claims (3)

1. mixing together a polyfunctional organic hydroxide, aldehyde, or ketone entity with alkali and non-protein nitrogenous entity, sufficient to raise the pH to 0cause a reaction between the former and the latter entities,

2. ageing said mixture for a period sufficient to drive the reaction between the two reacting entities to equilibrium, and

3. acidifying said aged mixture sufficiently to achieve the desired pH for consumption by ruminants. A method according to Claim 9, whereby the polyfunctional organic hydroxide, aldehyde, or ketone entity is a polyhydric alcohol, carbohydrate, or deoxy carbohydrate capable of tautomerising to yield carboxyl entities or fragments. 11) A method according to Claim 9, whereby the non-protein nitrogenous entity is a primary or secondary amine, a carbamate, an amide, or an imine. 12) A method according to Claim 9, whereby the polyfunctional organic hydroxide, aldehyde, ketone, carbohydrate, or deoxy carbohydrate is sourced from a waste or by-product stream. 13) A method according to Claim 9, whereby the polyfunctional organic hydroxide, aldehyde, or ketone entity is a polyhydric alcohol, carbohydrate, or deoxy carbohydrate entity is the result of hydrolysis of more complex molecules or fragments. -24- 14) A method according to Claim 9, whereby the non-protein nitrogenous entity is Sthe result of hydrolysis, condensation, or complexation of nitrogenous molecules or fragments. 0 00 5 15) A method according to Claim 9, whereby the reaction mixture may alternatively include additional carboxyl-containing entities, or polyhydric alcohol, tIt carbohydrate, or deoxy carbohydrate entities capable of tautomerising to yield carboxyl entities or fragments. (N 10 16) A method according to Claim 9, whereby the reaction mixture may alternatively include additional alkaline metal salt catalysts. 17) A method according to Claim 16, whereby the alkaline metal salt catalyst may contribute additional nutritive properties to the resultant feedstuff. 18) A method according to Claim 9, whereby the acidification agent may contain additional nutritive properties. 19) A method according to Claim 9, whereby the acidification agent may contain additional preservative properties. A method according to Claim 9, whereby the acidification agent may control the consumption rate of the resultant feedstuff. DATED: this twenty-seventh day of November 2007. AGR SCIENCE TECHNOLOGY PTY LTD