AU3531900A - Method for determining amino acid content in foodstuffs - Google Patents

Description

P/00/01i1 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE

SPECIFICATION

STANDARD PATENT Invention Title: Method for determining amino acid content in foodstuffs The following statement is a full description of this invention, including the best method of performing it known to us: Freehilis Carter Smith Beadle MELC600368770.7 1A- METHOD FOR DETERMINING AMINO ACID CONTENT IN FOODSTUFFS TECHNICAL FIELD This invention relates to methods for determining the content of an amino acid in foodstuffs and more particularly both the reactive lysine digestibility co-efficient and the digestible reactive lysine content in foodstuffs.

BACKGROUND

Recently there has been renewed interest in the effects of food processing on the availability of amino acids, in particular lysine (Knipfel 1981, Barterham 1992) in foodstuffs.

Lysine is of particular interest because it is an essential amino acid and is often the first 15 limiting amino acid in diets, particularly pig and poulry diets. In foodstuffs that have.

:i undergone processing or prolonged storage, the e-amino group of lysine can react with other compounds present in feedsruffs the Maillard reaction) to become structurally altered and nutritionally unavailable (Hun-ell and Carpenter 1981). Consequently, numerous assays have been developed to allow determination of the chemically reactive lysine content of foods (Hendriks et al., 1994).

One such assay is the l-fluoro-1,4-dinirnobenzene (FDNB) assay (Carpenter 1960). In this assay reactive lysine in a foodstuff to be tested is detennined by reacting the foodstuff with FDNB resulting in the production of Dinin-ophenyl lysine (DNP-lysine) which is 25 then detected by absorbance at 435 nm and the corresponding reactive lysine content in the foodstuff calculated.

An ileal amino acid digestibility assay to predict the availability of amino acids, including lysine, in a foodstuff is also known and in current use. This ntaditional ileal amino acid digestibility assay is described comprehensively in Moughan et al., 1990 and Burts el al., 1991 incorporated herein by reference. Briefly, the traditional assay comprises firstly calculating the amino acid content of a foodstuff. Secondly, calculating the amino acid digestibility co-efficient from both the amino acid content of a diet formulated from the foodstuff, and the amino acid content of the digesta of a subject fed that diet. Finally, the content of digestible amino acids in the foodstuff is calculated by multiplying the amino acid content in the foodstuff by the digestibility co-efficient.

-2- However, it has recently been noted that not all of the chemically reactive lysine in heattreated proteis is absorbed from the small intestine (Schmitz 1988, Desrosiers et a., 1989, Mougan 1991). Consequently, methods for determining chemically reactive lysne such as the FDNB method are inappropriate for assessment of available lysine as they incorrectly assume that all of the reactive lysine present in a feedstuffis dige.ted and absorbed. Furthermore, it has been shown that the traditional true ileal amino acid digestibility assay does not always accurately predict the availability of lysine in heat processed feedstuffs (Barterham !992). The explanation for this is that during the acid hydrolysis step of amino acid analysis, an integral part of the digestibility assay, a portion of the structurally-altered nutritionally unavailable lysine derivatives in processed feedstuffs can break down, reverting back to lysine and leading to an overestimate of unaltered lysine in diets and digesta samples and therefore inaccuracy in digestibility co- efficients.

Accordingly, there is a recognised need for an accurate method for determining both the foodstuff, especially heat processed foodstuffs.

It is therefore an object of this invention to provide a more accurate method for determining the content of an amino acid in a foodstuff, as well as methods for determining both the reactive lysine d i estibility co-efficient and the digestible reactive lysine content in a foodstuff, or at least to provide the public with a useful choice.

SUMMARY OF THE INVENTION 25 Accordingly, in a first aspect the present invention can broadly be said to consist in a method for determining the reactive lysine digestibility co-efficient of a foodstuff which S method comprises the steps of: introducing a marker into the foodstuff to be analysed; feeding the foodstuff to a subject for a predetermined period of time obtaining a sample of the foodstuff digesta from the subject; determining the digestible reactive lysine content of the foodstuff by: introducing a lysine derivatising agent into the foodstuff; and (ii) determining the digestible reactive lysine content of the foodstuff by measunng the equivalent derivatised lysine content in the foodstuff; determining the digestible reactive lysine content in the foodstuff digesta by: introducing a lysine derivatising agent into the foodstuff digesta; and (ii) determining the digestible reactive lysine content of the foodstuff digesta by measunng the equivalent derivatised lysine content in the foodstuff digesta; -3measuring the marker concentration in both the foodstuff and foodstuff digesta; expressing the reactive lysine content of both the foodstuff and foodstuff digesta per gram of the marker; and calculating the reactive lysine digestibility co-efficient.

In a further aspect, the present invention provides a method for determining the digestible reactive lysine content of a foodstuff which method comprises the steps of: calculating the reactive lysine digestibility co-efficient using the above defined method; and determining the digestible reactive lysine content of the foodstuff by multiplying the value for the reactive lysine content of the foodstuff by the reactive lysine digestibility co-efficient.

In a preferred embodiment of both methods, the foodstuff is formulated into a diet before 15 adding the marker and feeding to a subject.

Conveniently, the foodstuff digesta sample is taken from the terminal ileum. It is also preferred that the foodstuff digesta is dried, before treatment with a derivatising agent.

In a further aspect, the present invention provides an assay for determining the content of an ammo acid in a foodstuff which method comprises determining the amino acid S digestibility co-efficient and the digestible amino acid content in a foodstuff according to the methods detailed above, wherein the amino acid of interest is substituted for lysme.

25 The term foodstuff as used herein refers to a foodstuff comprised at least partially of protein.

The term foodstuff digesta as used herein means digesta derived from an animal, or test animal fed that foodstuff.

Although the present invention is broadly as defined above, it will be appreciated by those persons skilled in the art that the invention is not limited thereto and that it also includes embodiments of which the following description gives examples. In particular preferred aspects of the invention will now be described in relation to the accompanying drawings in which: -4- Figure 1 is a graph illustrating the homoarginine (reactive lysine) content of heated lactose/casein cubated with 0.6 M O-methylisourea pH 10.6 in a shaking waterbath at 21 0 C for 1 to 14 days, with the reagent to lysine ratio greater than 1000.

Figures 2/1 and 2/2 provide graphic illustrations of the amount ofhomoarinine (reactive lysine) present in the digesta of rats fed unheated casein and heaed lactose/casein determined using the guanidination reaction.

For Figure 2/1 the reaction time was varied. The guanidination conditions were incubation in 0.6M O-methylisourea pH 10.6 at 2:2°C for 1 to 21 days with the reagent to lysine ratio being greater than 1000.

For Fig. 2/1, n=13 for unheated casein, 1 day incubation: n=8 for heated lactose/casei 1 day incubation: n=7 for unheated casein 3 and 7 day incubations and heated lactose/casei, 7 day incubation: n=5 for heated lactose/casein, 7 day incubation: n=3 for both proteins at 14 and 21 day incubations.

F

S•For Figure 2/2 the pH of the reaction mixture was varied. Guanidination conditions for the digesta of rats fed unheated casein were incubation for 1 day at 21:2°C in 0.6M 0methylisourea at pH 9.8 to 11.4, with the reagent to lysme ratio being greater than 1000.

The guanidiation conditions for the digesta of rats fed heated lactose/cascin were incubation for 7 days at 21±2 0 C in 0.6M O-methylisourea at pH 9.8 to 11.4, with the reagent to lysine ratio being greater than 1000.

25 For Fig. 2/2, n=3 for all analyses.

Values were means

SE.

igure 3 illustrates by means of bar graphs the recovery of amino acids from various protein sources after guanidination with O-methylsourea. Recoveres were calculated as follows: Recovery Moles of amino acid detelmned in uanidiated protein X 100 Moles of amino acid determined in unreacted protein 1 Figure 4 illustrates by means of graphs the mean food intakes for the first five meals for rats on the last day of trial.

Figure 5 is a flow chart illustrating a method of detemiining endogenous amino acid flow.

Figure 6 is a flow chart illustrating the present methods for determining both the digestible reactive lysine co-efficient and digestible reactive lysine content of a foodstuff.

Figure 7 is a flow chart illustrating the traditional ileal amino acid digestibility assay.

Figure 8 is a flow chart illustrating a current process for determining the reactive lysine content of a foodstuff.

Figure 9 is a bar graph comparison of the true ileal digestability of amino acids (other than lysine) as determined using conventional amino acid analysis or following the guanidination reaction DETAILED DESCRIPTION OF THE INVENTION 15 As the reader will appreciate, while the present invention is discussed for convenience with respect to lysine, the methods of the invention are equally applicable to other amino acids of interest, -and therefore -represents an alternative to the traditional aminc acid assays.

In a first aspect, the present invention provides methods for determining both the reactive lysine digestibility co-efficient and digestible reactive lysine content of a foodstuff.

Essentially, the method involves coupling a derivatising reaction (which converts chemically reactive lysine to an equivalent stable derivative) to the traditional true ileal I amino acid digestibility assay. An example of a derivatising reaction is to use 25 .guanidination to convert lysine to the equivalent acid stable homoarginine. The guanidination reaction is described in Rutherfurd and Moughan 1990 incorporated herein by reference.

a In the method of the present invention a denvatising reaction is used in a method for determining the reactive lysine content of both the foodstuff being tested and the foodstuff digesta of subjects fed that foodstuff. When coupled with the ileal digestibility assay the true ileal reactive lysine digestibility co-efficient of the foodstuff can be determined. The co-efficient is calculated according to the following equation: True ileal reactive lysine digestibility Reactive Ivsine in diet (Reactive Ivsine in digesta Endoenous Ivsine) Reactive lysine in diet -6- Once the reactive lysine digestibility co-efficient has been calculated the true digest reactive lysine content of the foodstuff can be calculated by multiplying he reci sine content of the foodstuff by the reactive lysine digestibility co-efficient The foodstuffs analysed according to the methods of the present invention will be comprised at least partially of protein, and may be wholly comprised of protein. The foodstuffs may be naturally occurrin or processed foodstuffs. Naturally occurring foodstuffs include crude protein such as mcats and rilk. Processed foodstuffs range from relatively unprocessed proteins such as sozyme, soy protein isolates, im mil powder, lactic casein, whey protein concentrate, soy protein concentrate wheatmeal, and soyabean meal amongst others, through to c r ht ad b meal amongst others, through to severely processed foodstuffs such as meat and bone meal, cottonseed meal, bloodmeal, commercial animal feeds, and foods produced for human consumption as examples only.

5 In a preferred embodiment the foodstuff is formulated into a diet before feedig to a subject. Formuation of a diet may include mixing a selected protein source with additives such as water, carbohydrates, vitamins and minerals but not limited thereto. Formulation of a diet may also comprise physical treatment such as freeze drying, heating, and pulverising but again is not limited thereto.

A marker or markers are introduced into the diet as a me measuring te degee of ie underans for measuring the deeonve t of' cocentration the diet undergoes as it is convered ito digesta. Selected markers will be indigestible and of low toxicity. Suitable markers include spectrophotomeric markers 25 such as chromic oxide or radiopaque markers amongst others. Chromic oxide is the 25 presently preferred marker.

uThe diet formulated together with the chromic oxide marker is then fed to a selected subject for a predetermined period of tinme The term subjects as used herein generally refers to non-human animals. Suitable "subjects" include by way of example only rats, mice, pigs, chickens and dogs. "In vitro subjects" are possible. These subjects constitute in viro environments which simulate an animal ut, and produce iro dist Subjects may be selected according to the diet to be tested. However rats are a convenient test subject. t a The period over which the diet is fed to the subject may be varied according to the diet to be tested, and the subject selected. For example, a diet will be fed to a rat for between to 20 days, and most preferably 14 days. The rat may be fed the diet i one or more meals a day for a set time period. It is preferable on the 14th day to adopt a feeding -7regime whereby the rats are fed hourly from 8.30 h and 16.30 h for a total of nine meals is appropriate. Generally the feed will be available for 5 to 15 minutes and preferably minutes only.

To obtain a sample of the foodstuff digesta from the subject it is generally necessary to sacrifice the subject, although live sampling techniques are possible. The digesta sample may be selected from any part of the ileum. However, in a preferred embodiment the digesta sample is taken from the ileum immediately anterior to the ileal-caecal junction.

For a rat the ileum portion dissected out would be in the order of 10 to 30 cm preferably 20 cm.

The digesta sample is obtained by dissecting out a portion of the ileum and then flushing out the digesta. Conveniently, the digesta may be flushed out of the ileum using a syringe filled with distilled, deionised water. The digesta obtained can then he analysed directly 15 but is preferably freeze-dried.

Samples of in vitro digesta are readily obtainable from the in vitro environment.

SThe reactive lysine content of the diet and digesta is then required to be determined. As a first step it is necessary to convert reactive lysine into a stable derivative. This is conveniently accomplished by reaction with a derivatising agent. Any derivatising agent used to determine the reactive lysine in diets and digesta must be specific for the e-amino group of lysine, to allow the determination of both free and bound reactive lysine that may be present in digesta. Secondly, the derivatised lysine compound must be acid stable.

Thirdly, derivatisation must be quantitative.

FDNB, the most commonly used reagent for determining reactive lysine, fails as a suitable reagent as it can also react with the a-amino group of lysine and will therefore not detect free reactive lysine. Furthermore, DNP-lysine is not particularly acid stable necessitating the use of correction factors.

The derivatising agent, O-methylisourea, is specific for the E-amino group of lysine. In a preliminary study, 95% of lysine was recovered as homoarginine or residual unguanidinated lysine from a mixture of lysine and 0.6M OMIU at pH 10.6 incubated for 24 hr at 20 0 ±2 0 C in a shaking waterbath. Furthermore, O-methylisourea appears to be acid stable. In a preliminary study, 96% of homoarginine was recovered when homoarginine was incubated in 6M HCL in a sealed -evacuated glass tube for 24 hr at 110°C±2°C. Therefore, O-methylisourea shows promise as a suitable reagent for the ileal -8reactive lysine digestibility assay, and is currently the dervatising agent of choice. 0methylisourea acts as a guanidinating agent converting reactive lysine to acid stable homoarginine. The homoarginme content as measured is therefore equivalent to the reactive lysine content. Previous guanidination techniques are described in Rutherfurd and Moughan 1990.

It is also preferred that the guanidination reaction be carried out under optimised conditions to ensure maximum guanidination.

Conversion of lysine to homoarginine in an unprocessed protein source may be calculated as shown below: Conversion of lysine to homoarginine Moles of homoarginine 1 Moles of unreacted lysine Moles of homoarginine x 1 The homoarginine level is measured by any suitable means known in the art. A preferred method is amino acid analysis As a parallel step in the methods of the invention it is also necessary to determine the marker content of both the diet and digesta. In the case of a chromic oxide marker the chromium content of the diet and digesta are determined spectrophotometrically following the method of Costigan and Ellis (Costigan and Ellis, 1987) incorporated herein by 25 reference. The reactive lysine content of both the diet and digesta must then be expressed in terms of reactive lysine per gram of the marker according to the following equations: Reactive lysine content in diet Reactive Ivsine in diet Chromium in diet Reactive lysine content in digesta Reactive lysine in digesta X Chromium in diet Chromium in digesta It will also be appreciated by the skilled worker that fractions analysed may include endogenous peptides and proteins. It is therefore necessary to calculate the endogenous amino acid flow, and to correct the apparent figure for ileal reactive lysine digestibility flow by subtracting from it the endogenous amino acid flow. One scheme suitable for determining the endogenous amino acid flow is illustrated in Figure 5. Generally, any peptides or proteins with molecular weight greater than 10 000 Daltons will be regarded as of endogenous origin. The following equations are used to calculate the endogenous amino acid flows and true digestibility co-efficient.

Endogenous amino acid flows at the terminal ileum were calculated using the following equation (units aie ug dry matter intake (DMI)): Ileal amino acid flow Amino acid concentration in ileal digesta X Chromium in diet Chromium in digesta True ileal amino acid digestibility was calculated using the following equations (units are gg DMI): True digestibility Dietary amino acid intake (Ileal amino acid flow endogenous amino acid flow) x 100 Dietary amino acid intake 1 True ileal reactive lysine digestibility was calculated using the following equation (units are pg DMI): o* True reactive lysine digestibility Dietary reactive lvsine intake (Ileal reactive lvsine flow endogenous Ivsine flow) x 100 Dietary reactive lysine intake The second method of the invention relates to the determination of the digestible reactive 25 lysine content of a foodstuff In this method the reactive lysine digestibility co-efficient is determined in accordance with the first method of the invention. As a second step the reactive lysine content of the foodstuff is determined using the guanidination reaction set S out above. In the case of a foodstuff which has not been formulated into a diet in any specific way this calculation will be the same for the reactive lysine content of the foodstuff calculated in the first method.

From these two methods the digestible reactive lysine content of the foodstuff can be calculated by multiplying the digestible reactive lysine content of the foodstuff by the true reactive lysine digestibility co-efficient.

As the reader will appreciate, the methods of the invention apart from having general applicability to any amino acid of interest, may also be performed in vitro. For in vitro applications of the methods of the invention the subject comprises an in vitro environment which simulates the gut of an animal of interest. The foodstuff to be analysed is fed into the in vitro environment, digested and analysed in the same manner as for an animal subject.

The present invention will now be further described with reference to the following specific non-limiting examples.

EXPERIMENTAL

Materials l-fluoro-l,4-dinitrobenzene (FDNB), Dinitrophenyl-lysine (DNP-lysine) and Omethylisourea were obtained from Sigma chemicals, St Louis, MO. Barium hydroxide octahydrate and lactose were obtained from BDH Laboratory Supplies, Poole, England.

Lactic casein, skim milk powder and whey protein concentrate were obtained from the New Zealand Dairy Board, Wellington, New Zealand. Soy protein isolate and concentrate were obtained from Columbit (New Zealand) Ltd, Auckland, New Zealand. Wheat meal, blood meal, meat and bone meal and soybean meal were obtained from the Feed Processing Unit Massey University, New Zealand and cottonseed meal from Cargill Oilseed Ltd, Brisbane, Australia. The enzymatically hydrolysed casein was obtained from New Zealand Pharmaceuticals LTD, Palmerston North, New Zealand and contained 20 peptides no larger than 2000 Daltons. Centriprep 10 disposable ultrafiltration devices were obtained from Amicon, Inc, Beverly, MA. Laboratory rats were sourced from the Small Animal Production Unit, Massey University, Palmerston North, New Zealand.

EXAMPLE 1 25 FDNB Method .I -fluoro-1,4-dinitrobenzene (FDNB)-reactive lysine was determined according to the method of Carpenter (1960) using the modifications described by Booth (1971). Samples :containing approximately 10 mg of reactive lysine (estimated previously using amino acid analysis), were reacted with FDNB in ethanol/NaHCO, at room temperature for 2 hr. The resulting Dinitrophenyl (DNP)-lysine was liberated from the protein by hydrolysis in 5.8M HCI for 16 hr under reflux conditions. The unreacted FDNB was removed by diethylether extraction and the remaining DNP-lysine detected by absorbance at 435nm.

EXAMPLE 2 Preparation of 0.6M O methvlisourea solution A 0.6M O-methylisourea solution was prepared by a modified procedure based on the methods of Chervenka and Wilcox (1956), Shields et al, (1959), Mauron and Bujard (1964) and Kassell and Chow (1966). Four grams of barium hydroxide octahydrate were -11added to approximately 16 ml of preboiled boiling distilled deionised water which had been preboiled for 10 min to remove carbon dioxide. The solution was heated to near boiling then added to 2 g of O-methylisourea (sulphate salt) in a 40 ml centrifuge tube.

The solution was left to cool for 30 min before centrifuging at 64 0 0g for 10 min. The supernatant was retained and the precipitate was washed with approximately 2 ml of distilled deionised water before recentrifuging. The washings were added to the supernatant and the pH checked. If the pH of the solution was lower than 12 then it was assumed that conversion of the sulphate salt to the free base was incomplete and the solution was remade. However, if the pH was above 12 then the pH was adjusted to the appropriate pH for guanidination (pH 10.6-11.2), and made up to 20 ml with distilled deionised water.

EXAMPLE 3 A. Preparation of a heated lactose/casein mixture A heat treated lactose/casein mixture, which contained 250 g lactose and 750 g lactic casein, was prepared by mixing the two components mi 4 litres of distilled deionised water then freeze drying the suspension and autoclaving the dried mixture for 3.5min at 12 1 0

C.

The autoclaved sample was ground through a Imm mesh. The resultant mixture simulated a protein having undergone early to late Maillard damage (Gall 1989).

B. Preparation of protein sources To ensure that at least one of the protein sources was sufficiently heat damaged to allow sizeable differences between the assays, approximately I kg of skim milk powder was autoclaved for 3 nun at 121 °C before use. The autoclaved skim milk powder along with 25 a selection of readily available feedstuffs including wheat meal, blood meal, soybean meal, meat and bone meal, dried maize, cottonseed meal and a pelleted lucerne based mix containing 55% lucerne, 10% meat and bone meal and 5% each of blood, wheat, barley, maize, sorghum, soybean, broil meals, were each ground through a 0.5 mm mesh. The blood meal, soybean meal and wheat meal represented processed feedstuffs which were expected to be of high quality whereas the other materials, being subjected to a higher degree of processing during manufacture, were expected to have a lower overall protein quality.

EXAMPLE 4 Optimisaion of reaction time for uanidination of unheated partially purified proteins 5-10 mg samples were incubated for 1, 2 and 3 days in 0.6M O-methylisourea, pH 10.6, at 21°C 2°C in a shaking waterbath with the reagent to lysine ratio being greater than 12- 1000. The samples were then reduced to dryness and the homoargiine and ysine contents determined.

In preliminary studies investigating the optimal time for guanidination of two unheated protein sources (lysozyme and unheated casein), carried out over 1, 2 and day incubation periods, near complete conversion of lysine to homoarginine (greater than 98%) was achieved in all cases.

EXAMPLE Optimisation of the reaction time for euanidination of heated lactose/casein 5-10 mg samples of heated lactose/casein were incubated for 1, 3, 7 and 14 days in 0.6M O-methylisourea, pH 10.6, at 210 2 0 C in a shaking waterbath, with the reagent to lysine ratio greater than 1000. The samples were subsequently reduced to dryness and the homoarginine content was determined.

The conversion of lysme to homoarginine in heated lactose/casein was investigated using incubation times ranging from 1 to 14 days. The yield of homoarginine over the 14 day period is shown in Fig. Maximal guanidination was achieved after 3 to 7 days Sincubation in the O-methylisourea solution, although there was no significant difference between homoarginine yields observed after 1, 3, 7 or 14 days incubation.

EXAMPLE 6 ptimisation of the reaction time and pH for guanidination of dipesta The optimal incubation time was determined after incubating 5-10 mg samples of rat ileal digesta in 0.6M O-methylisourea, pH 10.6, at 21±2 0 C in a shaking waterbath, for 1, 3, 7, ~14 and 21 days, with the reagent to lysine ratio greater than 1000. The samples were reduced to dryness and the homoarginine content was determined. The ileal digesta had been obtained from rats given either an unheated casein based diet or a heated lactose/casein based diet.

The optimal reaction mixture pH was determined after incubating 5-10 mg samples of ileal digesta from rats fed unheated casein and from rats fed heated lactose/casein in 0.6M O-methylisourea at pH 9.8, 10.2, 10.6, 11.0, I 1.4 at 211 °C in a shaking waterbath, with the reagent to lysine ratio being greater than 1000 (or for 1 to 7 days in 0.6M O-methylisourea at pH 10.6 in a shaking waterbath). The samples were then reduced to dryness and the homoarginine content was determined.

13 The optimum incubation times for maximal guanidination of digesta from rats fed unheated casein and heated lactose/casein were determined (Fig.2a). Maximal conversion of lysine to homoarginine in the digesta of rats fed unheated casein was achieved with a 1 day incubation, after which the levels appeared to decline slightly, although this trend was not statistically significant. In contrast, a 7 day incubation time was required to achieve maximal guanidination of lysine in the digesta of rats fed the heated lactose/casein, although there was no significant difference between homoarginine yields for the reaction mixtures after 3, 7 or 14 days incubation. There was also no significant difference in the amount of the homoarginine determined in the heated lactose/casein digesta after incubation in 0.6M O-methylisourea for I and 3 days.

The optimum reaction mixture pH for maximal guanidination of digesta of rats fed unheated casein and heated lactose/casein was also determined (Fig.2b). The pH optimum for the guanidination of lysine in digesta of rats fed the unheated casein was approximately 10.6, although the amounts of homoarginine obtained from guanidination mixtures at pH's ranging from 10.2 to 11.4 were not significantly different. From the pH range examined in this experiment, the pH required for optimal guanidination of digesta from rats fed the heated lactose/casein was between I 1.0 and 11.4.

S 20 EXAMPLE 7 Determination of endogenous amino acid loss Enzymatically hydrolysed casein (EHC), containing peptides no larger than 5000 Daltons, are fed to a group of animal in the same manner as described above for animals fed test foodstuffs. The EHC fed animals are slaughtered and digesta sampled from the tenninal 25 ileum. The digesta is centrifuged at 6500 x g for 10 min. The supernatant is then ultrafiltered using a Centriprep 10 ultrafiltration device. The resulting filtrate (containing peptides smaller than 10000 Daltons) is discarded, while the retentate is pooled with the precipitate from the previous centrifugation step. This fraction containing large endogenous peptides and protein, is then dried down and analysed for amino acids and chromium. This measure of endogenous loss is then used to correct apparent amino acid digestibility in a test foodstuff to true amino acid digestibility as shown in the equations below and Moughan 1991, and Butts ei al., 1991.

Endogenous ileal amino acid flow Amino acid concentration in ileal digesta X Chromium in diet Chromium in digesta 14- EXAMPLE 8 Digestibility study Sprague-Dawley male rats, of approximately 15 0g bodyweight, were housed individually in stainless steel wire-bottomed cages in a room maintained at 22 2 0 C, with a 12 h light/dark cycle.

A. In a first study, two semi-synthetic test diets were formulated to each contain 1OOg/kg crude protein. Two enzymatically hydrolysed cascin (EHC) based diets were also formulated to allow determination of endogenous ileal amino acid flows (Moughan et a/, 1990, Butts et al, 1991). Chromic oxide was included in each diet as an indigestible marker. The ingredient compositions of the diets are given in Table 1 below.

B. In a second study, eight semi-synthetic test diets were formulated to each contain 100g/kg crude protein. An EHC based diet was also formulated to allow determination of endogenous ileal lysine flow as above. Chromic oxide was included in each diet as an indigestible marker. The ingredient compositions of the diets are given in Table IA below.

*ee 15 TABLE 1. Ingredient compositions (g kg' air dry weight) to the laboratory rat.

of the experimental diets given EHC' Unheated casein EHC2- Heated lactose/casein 3 Wheat starch Soybean oil Purified cellulose Sucrose Vitamin/mineral mix' Lactose Casein Heated lactose/casein

EHC

Chromic oxide 625.7 50 50 100 39.3 130 5 EHC Unheated casein 639.7 50 50 100 39.3 116 5 583 50 50 100 39.3 42.7 130 5 584.9 100 39.3 170.8 All diets were formulated to contain equal crude protein contents.

'Enzymatically hydrolysed casein containing diet for determining endogenous amino acid loss for the unheated casein diet.

'Enzymatically hydrolysed casein containing diet for determining endogenous amino acid loss for the heated lactose/casein diet.

-Heated lactose/casein was prepared as described in Experimental example 3.

'Vitamin/mineral mix was formulated to meet the requirements for vitamins and minerals as described by the National Research Council (National Academy of Sciences, 1972).

r :4 0%

I

TABLE IA. Ingredient comnpositions I (g kg- I air dry weight) of the experimental diets.

Blo)od WIheat Meat and Soyea Hete skimr Dried Lucerne Cottned meal meal bone meal mreal milk Powder maize based mrix meal Wheat starc 625. 646. 1 572.7 542!.7 4 95T7 -355.7 504.7 Soybean oil 50 50 50 50 50 50 5 Purified cellulose 50 50 50 50 50 -50 Sucrose 1oo 100 20.7 100 100 100 -100 100 Vitaininh/nineral mnix' 39.3 39.3 39.3 39.3 39.3 39.3 39.3 39.3 39.3 EHC 130 Blood meal 109 Wheat meal Meat/bone meal 88 18- Soybean meal 213 Heated skim milk Powder 260 Dried maize 3 955.7 Lucernie based mix' 400 Cottonseed m eal Chromic oxide 5 5 5 5 5 55 5 51 'All diets were formulated to contain equal crude protein contents.

'Enzymatically hydi-olysed casein diet used for determi-ning endogenous ami~no acid losses at die terminal ileum, the EHC contained free ainio acids aid sinall peptides (<2000 Da).

'The lucerne based inix consisted of 55% lucerne, 10% meat and bone meal and each of blood, wheat, barley, maize, sorghum, soybean and broil meals and was initially in a pelleted form..

'Vitamin/mnneraj niix was formulated to meet the requirements for vitamins and minerals in the final diets as described by the National Research Council (National Academy of Sciences, 1972).

17- For both studies A and B the diets were randomly allocated to the rats such that in study A there were six rats on each diet, and in study B there were a minimum of five rats on each diet. The rats were given the diets for a 14 day period. On each day each rat received its respective diet as nine meals given hourly (0830h to 1630h). At each meal time the diet was freely available for a ten minute period. The feed containers were weighed after each meal. Water was available at all times. On the fourteenth day of the study, from 5.5 to 7 hours after the start of feeding, the rats were asphyxiated in carbon dioxide gas and then decapitated. The 20cm of ileum immediately antenor to the ileocaecal junction was dissected out. The dissected ileum was washed with distilled deionrsed water to remove any blood and hair and carefully dried on an absorbent paper towel. The digesta were then gently flushed from the ileum section with distilled deionised water from a syringe. The digesta from the rats fed the test diets were then freeze-dried ready for chemical analysis. The pH of the digesta of rats fed the EHC diet was adjusted to approximately pH 3 with 6M HCI, to minimise protease activity. The EHC digesta were then centrifuged at 6 4 00g for 30 min at 3+1°C and the precipitate was washed and recentrifuged. The washings were pooled with the supernatant. the supernatant underwent ultrafiltration in a Centriprep 10 disposable ultrafiltration device after which the filtrate was discarded and the retentate washed and underwent ultrafiltration for a second time. The resulting retentate was added to the precipitate from 20 the centrifugation step and freeze-dried ready for chemical analysis.

CHEMICAL ANALYSIS Study A Amino acids contents were determined in triplicate 5 mg digesta samples and S 25 quadruplicate 5 mg diet samples using a Waters ion-exchange HPLC system, utilising post-column o-phthalaldehyde derivatisation and fluorescence detection, following hydrolysis in 6M glass-distilled HCI containing 0.1% phenol for 24 hr at II 10±2C in evacuated sealed tubes. Cysteine, methionine, proline and tryptophan were not determined. Where appropriate, the weight of each amino acid was calculated using free amino acid molecular weights.

For the determination of reactive lysine, the samples were incubated for 7 days in 0.6M O-methylisourea pH 10.6(pH 11.0 for the digesta samples), at 21"C in a shaking waterbath, with the reagent to lysine ratio being greater than 1000, before being dried down and analysed for amino acid content as described above.

18 Study B Amino acid contents were determined in duplicate 5 mg diet and digesta samples and quadruplicate 5 mg semi-synthetic diet samples using a Waters ion-exchange

HPLC

system, utilising post-column ninhydrin derivatisation and detection using absorbance at 570nm and 4 4 0nm, following hydrolysis in 6M glass-distilled HCI containing 0.1% phenol for 24 hr at 110±2 0 C in evacuated sealed tubes. Cysteine, methionine and tryptophan were not determined as they are destoyed during acid hydrolysis. The weight of each amino acid was calculated using free amino acid molecular weights.

Reactive lysie contents were detemined in duplicate 5 ing feedstuff and digesta samples and quadruplicate 5 mg diet samples by incubation for 1, 7 and 7 days respectively in 0.6M O-methylisourea, pH 10.6 (pH 11.0 for the digesta samples), at 21 C in a shaking waterbath, with the reagent to lysine ratio being greater than 1000. After incubation, the samples were dried down using a Speedvac concentrator (Savant Instruments, Inc Farmingdale, NY, USA) and analysed for amino acid content as described above.

The chromium contents of diet and ileal digesta samples for both study A and study B were determined in duplicate on an Instrumentation Laboratory atomic absorption spectrophotometer following the method of Costigan and Ellis (1987).

REACTIVE LYSINE CONTENT STUDY A SThe reactive lysine contents of the unheated casein and the heated lactose/casein were compared using the guanidination method (where homoarginine levels were equated to reactive lysine levels), the FDNB-reactive lysine method and conventional amino acid 25 analysis. The guanidination conditions used were incubation for 24 hr in 0.6M Omethylisourea, pH 10.6 at 21 0 C±2 0 C in a shaking wvaterbath, with the reagent to lysine ratio being greater than 1000. The reactive lysine level of the heated lactose/casein was then extrapolated using Fig. I to deteimine the reactive lysine content using the optimal 7 day incubation period. The results are shown in Table 2 below V V

V

19- TABLE 2. Reactive lysine content in unheated casein and heated lactose/casein determined using the FDNB and guanidination methods, and "total lysine" levels determined by conventional amino acid analysis.

Lysine content (mmol g' casein) FDNB' Guanidination 2 Total 3 Unheated casein 0.51 0.55 0.50 Heated lactose/casein 0.31 0.33 0.38 'FDNB analyses used a correction factor of 1.05 for both samples.

2 Guanidination analyses consisted of 24hr incubation in 0.6M O-methylisourea, pH 10.6 in a shaking waterbath at 21±2 0 C with the reagent to lysine ratio being greater than 1000, followed by conventional acid analysis. Reactive lysine values were corrected to an optimal 7 day incubation time using Fig. 1.

'Total analyses consisted of conventional acid hydrolysis and amino acid quantitation.

Values are means of triplicate analyses.

REACTIVE LYSINE CONTENT STUDY B The reactive lysine content for five of the protein sources was determined using the guanidination method and te FDNB-reactive lysine method and compared to total lysine content determined using conventional amino acid analysis. The guanidination conditions are set out above. The results are shown in Table 2A below.

TABLE 2A. Reactive lysine contents (mg g-I sample) of several protein sources determined using the FDNB or Guanidination methods in comparison with total lysine contents (mg g-1 sample) determined using conventional amino acid analysis.

35 Reactive lysine Total lysine FDNB Guanidination Blood meal 84.4 88.0 89.1 Wheat meal 3.1 3.1 Meat and bone meal 30.4 34.6 36.5 Soybean meal 27.1 32.3 32.3 Cottonseed meal 14.7 14.4 20.6 The correction factors used for the FDNB method were 1.06 for blood meal, 1.03 for wheat meal, 1.08 for meat and bone, 1.04 for soybean meal and 1.05 for cottonseed meal and were determined as described in the materials and methods.

Results of Study A The amount of reactive lysine in the unheated casein ranged from 0.51nmmol g- casei determined using conventional amino acid analysis to 0.55mmol casein detenmined using the guanidination method. Generally the amounts of lysine in the unheated casein where it can be assumed that all the lysine is available, compared quite well between methods, with a less than 10% difference between the three methods. The reactive lysine content of the heated lactose/casein determined using the FDNB and guanidination methods also agreed well (0.3 rmnol g casein and 0.33 mmol g' casein respectively) and differed by less than In contrast, the total lysine level detenmined usin conventional amino acid analysis was considerably higher (almost than for the other two methods.

Results of Study B Reactive lysine determined using guanidination was generally similar or higher than FDNB-reactive lysine content for all five protein sources.

Generally good agreement was found between the reactive lysine contents of the Sfeedstuffs determined using the FDNB and guanidination methods, especially for blood meal, wheat meal and cottonseed meal. For soybean meal and meat and bone meal the 20 reactive lysine contents determined using the FDNB method were lower than those determined using guanidination. Since theoretically the guanidination method camnot overestimate reactive lysine, it would appear that these differences are most likely an artefact of the FDNB method in which correction factors must be used.

In an unprocessed protein source the reactive lysine content should be equivalent to the total" lysine content, where total lysine is the lysine determined by conventional amino acid analysis. In contrast, in a protein source which has sustained early heat damage the total lysine content may be higher than the reactive lysine content due to reversion of lysine during acid hydrolysis. In some processed protein sources where more severe 30 processing damage has occurred, structurally altered lysine derivatives may be acid-stable.

In this case reactive and total lysine values would be expected to be similar. For the blood meal, meat and bone meal and soybean meal in Study B the reactive lysine content determined using the guanidination method was similar to the total lysine content, suggesting that these protein sources either did not contain structurally-altered lysine derivatives, or if they were present, they were in a form that is stable to acid conditions.

For wheat meal the reactive lysine content was lower than the total lysine content suggesting that some reversible modification of lysine may have occurred. For dried maize, the lucerne based mix, cottonseed meal and heated skim milk powder, the reactive -21 lysine content was considerably lower than total lysine, reflecting protein sources m which lysine had undergone early Maillard type reactions during processing. Cottonseed meal undergoes considerable heat processing, in order to reduce the toxicity of the anti-nutritional factors known to be present (Berardi and Goldblatt, 1980), while the skim milk powder in the present study was subjected to controlled heating in our laboratory.

Determination of true ileal amino acid digestibility in unheated casein and heated lactose/casein of Study A.

The rats appeared healthy throughout the 14 day study. There was no sign of faecal particles in the stomach contents of the rats at post-mortem, indicating that coprophagy had not occurred, at least on the last day of the study. Meal intakes were relatively constant over the first five meals on the last day of study and therefore a relatively constant flow of digesta through the gut should have been achieved (Fig.4).

The endogenous amino acid flows at the terminal ileum, determined using the "lactosefree" EHCI diet, were used to correct apparent amino acid digestibilities to true ones for the unheated casein diet. The lactose-containing EHC.2 diet was used to correct apparent "digestibilities to true digestibilities for the heated lactose/casein diet. Although the 20 absolute endogenous amino acid flows appeared to be higher with the lactose-containing EHC diet compared to the lactose-free EHC diet, there was no statistical difference (p<0.05) between the two diets for all amino acids determined, with the exception of histidine (Table The variation in endogenous flows observed in the rats fed the lactose-containing EHC diet was considerably greater than that observed for the rats fed the lactose-free EHC diet.

eeo.

22 TABLE 3. Endogenous amiino acid flows of rats fed an enzymatically hydrolysed casein diet with and without lactose.

EHC with lactose EHC without lactose Aspartic acid 501 7 2 3 Threonine 391 448 Glutami'c acid 58551- Glycie 307 .305 Alanine 225 33 6 Valine 296 418 Isoleucine 226 337 os euine 307 405 Prse172 2 3 Penylalanine 2 10 209 Histidine 156 3)52 Lysine, 2 29 285 Arginine 175 207 The true ileal amino acid digestibility (conventional assay) of the unheated casein was very high with a mean digestibility (excluding lysine) of about 95% (Table 4 below). The digestibility of the heated lactoselcasein was significantly lower for all amino acids except glycine, alanine, phenylalanie and arginine. The mean decrease in digestibility between the unheated casein and the heated lactose/casein (excluding lysine) was 3% units but was as high as 9% units for aspartic acid (Table 4).

00.* 23 TABLE 4. Mean true ileal amino acid digestibility of unheated casein and heated lactose/casein in the growing rat determined using an ileal amino acid digestibility assay based on conventional amino acid analysis.

Unheated casein Heated Overall SE Significance lactose/casein' Aspartic acid 96.0 87.3 1.37 Threonine 93.6 88.0 0.96 Serne 89.7 85.0 1.03 Glutamic acid 93.1 89.4 0.74 Glycine 86.0 81.2 2.70

NS

Alanine 97.2 93.0 0.67

NS

Valine 96.7 92.4 0.73 Isoleucine 94.8 90.1 0.82 Leucine 99.1 97.1 0.27 Tyrosine 100.4 97. 1 0.29 Phenylalanine 100.4 98.0 0.30

NS

Histidine 95.8 86.0 0.88 Arginine 98.1 96.1 1.68

NS

'Heated lactose/casein was prepared as described in Experimental example 3.

NS P>0.05, P<0.05, P<0.01, P<0.001.

25 The true ileal digestibility of lysine determined using the traditional ileal digestibility assay which utilises conventional amino acid analysis was very high in the unheated casein (Table 5 below). The same coefficient in the heated lactose/casein was considerably lower than 71%. In contrast, the true ileal digestibility of reactive lysine determined using the traditional ileal amino acid digestibility assay coupled with the 30 guanidination reaction (new method) yielded a digestibility of 86%, significantly higher than the digestibility found using conventional methods.

The amino acid digestibility data were subjected to a one-way analysis of variance for each amino acid singly (GLM Procedure, SAS Institute Inc. USA).

-24- TABLE 5. Mean true ileal lysine digestibility for an unheated casein detenrined using the rat ileal digestibility assay and based on conventional amino acid analysis and for a heated lactose/casein using the rat ileal digestibility assay coupled with either conventional amino acid analysis or the guanidination method (reactive lysine digestibility coefficient).

Unheated Heated lactose/casein' Overall Significance casein Conventional Guanidination amino acid method analysis Digestibility of lysine 98.8 70.5 85.92 2.01 Endogenous amino acid flows at the terminal ileumwere corrected for, using the EHC/ultrafiltration method (Butts et al, 1991) and the appropriate EHC diet (See Table 1).

'Heated lactose/casein was prepared as described in Experimental example 3.

2 The reactive lysine digestibility was calculated as follows: Reactive lysine in the diet (Reactive lvsine in the di,,esra Endoenous vsine) X 100 SReactive lysine in the diet 25 Where units are pg g' DMI.

Comparison of true ileal lysine digestibility (conventional assay) with true ileal reactive lysine digestibility for Study B.

30 The rats appeared healthy throughout the 14 day digestibility study. Meal intakes were relatively constant over the first six meals on the last day of study and therefore a relatively constant flow of digesta through the gut should have been achieved. Mean meal intakes ±SE for the first six meals on the last day were 1.

7 ±0.08g for the wheat meal diet, 1.8+0.05g for the cottonseed diet, 1.

9 ±0.06g for the meat and bone diet, 2 .0+0.06g for the soybean diet, 0.

8 ±0.04g for the blood meal diet, 1.7±0.120 for the heated skim milk powder diet, 0.9±0.15g for the dried maize diet, 1.9-0.28g for the lucere based mix diet and 1.

7 ±0.0 7 g for the EHC based diet.

True ileal digestibility values based on "total" lysine as determined using conventional amino acid analysis were compared with true ileal digestibility values for reactive lysine, determined following the guanidination reaction, for eight different protein sources (Table 6 below).

TABLE 6. Comparison of the mean true ileal lysine digestibility determined using conventional amino acid analysis (total) and true ileal lysine digestibility based on determined reactive lysine (reactive).

Lysine digestibility Total' Reactive' Overall SE Blood meal 96.3 96.7 0.41

NS

Wheat meal 92.6 92.1 0.45

NS

Meat and bone meal 88.9 91.5 0.76

NS

Soybean meal 94.5 96.5 0.41

N

Dned Maize 80.5 84.3 1.54 Heated skim milk powder 69.1 94.0 1.11 Cottonseed meal 62.1 71.9 1.75 Lucerne based mix 74.2 86.3 0.63 'For blood meal, wheat meal, soybean meal, meat and bone meal, heated skim milk powder and cottonseed meal n=8, for the dried maize, and lucerne based mix 2 Lysine digestibility was determined using a true ileal amino acid digestibility assay trat) and conventional amino acid analysis was used to quantitate total lysine in the diets and S: 25 digesta.

:-Lysine digestibility was determined using a true ileal amino acid digestibility assay (rat) and the guanidination reaction was used to quantitate reactive lysine in the diets and digesta.

For blood meal, wheat meal and meat and bone meal the digestibilities of total lysine and reactive lysine were high (generally greater than 90%) and there was no significant difference between total lysine digestibility and reactive lysine digestibility. For soybean meal, the total lysine digestibility, which was also high, was statistically significantly lower than the reactive lysine digestibility, although the actual difference was less than 35 three percentage units.

These results again reflect protein sources containing minimal amounts of acid-labile "damaged" lysine derivatives, and as such indicates that the new true ileal reactive lysine digestibility assay, may be a suitable alternative method for determuning lysine digestibility.

For the dried maize, lucerne based mixed diet, cottonseed meal and heated skim milk powder, there were significant differences between the total lysine digestibilities -26and the reactive lysine digestibilities. Differences between the digestibility values for the two approaches were four, twelve, ten and 25 percentage units respectively.

Further, total lysine digestibility underestimated the actual digestibility of reactive lysine This result indicates that the conventional true ileal amino acid digestibility assay appes to be unsuitable for assessing lysine availability in heat processed feedstuffs as it underestimates the digestibility of structurally unaltered lysine. This result also shows that the new true ileal reactive lysine digestibility assay may provide a more accurate assessment of digestibility of structurally unaltered (available) lysine in processed protein sources.

Digestible lysine (based on total lysine determined by conventional analysis) and digestible reactive lysine contents are shown in Table 7 below.

TABLE 7. Mean' digestible total lysine and mean digestible reactive lysine contents (g kg-1 sample) in several protein sources Digestible lysine 20 Total' Reactive' Overall SE S Blood meal 89.9 85. 0.34 NS *heat meal 3.2 2.9 0.02 Meat and bone meal 32.5 31.6 0.24

NS

25 Soybean meal 30.6 31.2 0.12 Dned maize 2.6 1.9 0.04 Heated skim milk powder 19.8 16.6 0.30 Cottonseed meal 12.9 10.3 0.29 Lucerne based mix 14.4 10.8 0.10 S: 'For blood meal, wheat meal, soybean meal, meat and bone meal, heated skim milk powder and cottonseed meal n=8, for the dried maize, and lucerne based mix 'Digestible total lysine was calculated from true ileal lysine digestibility (rat), with lysine determined by conventional amino acid analysis and from the total lysine 35 content in the protein source, also determined using conventional amino acid analysis.

-Digestible reactive lysine was calculated from true ileal reactive lysine digestibility (rat, guanidination analysis), and the reactive lysine content of the protein source, also determined using guanidination.

For blood meal and meat and bone meal, there were no significant differences between the two values. In contrast, there were statistically significant differences between the two values for the six remaining protein sources. However, for soybean meal there was less -27 than a 2% difference between digestible total lysine and digestible reactive lysine. For the lucerne based mix, the dried maize, cottonseed meal and heated skim milk powder, all of which had undergone more severe heat processing, the differences 37%, and 19%, respectively) between digestible total lysine and digestible reactive lysine were quantitatively significant. This result indicates that digestible reactive lysine more accurately reflects available lysine in these protein sources than the conventional true ileal lysine digestibility assay which is known to overestimate lysine availability in processed feeds (Batterham 1990).

EXAMPLE 9 Recovery of acid stable amino acids during guanidination The amounts of other acid stable amino acids were compared when using the guanidination method and conventional amino acid analysis for a range of protein sources, with the aim of determining if the guanidination method interfered with the quantitation of these other amino acids. The recovery of acid stable amino acids is shown in Fig.3.

Again conversion of lysine to homoarginine in the relatively unprocessed proteins (lysozyme, soy protein isolate, skim milk powder, lactic casein, whey protein concentrate, soy protein concentrate, rotary dried bloodmeal and soyabean meal), was high, ranging 20 from 97 to 100%. In contrast, in wheatmeal and the more severely processed protein sources such as meat and bone meal and cottonseed meal the conversion of lysine to homoarginine was considerably lower. The recoveries of almost all of the amino acids in all protein sources examined were close to 100%. The main exception was histidine, where, in skim milk powder, wheat and soybean meals, recoveries well above 100% were 25 observed. This may have been associated with the chromatographic procedure and may have been due to column ageing.

-28- EXAMPLE Comparison of the true ileal digestibility of acid-stable amino acids in protein sources determined using conventional amino acid analysis or following guanidination of the diet and digesta prior to amino acid analysis.

The true ileal digestibilities of amino acids, other than lysine, were determined using the true ileal digestibility assay applied to unguanidinated diet and digesta samples or to samples which had undergone guanidination and the results are given in Figure 9. For most of the protein sources tested, including wheat meal, soybean meal, blood meal, dried maize, meat and bone meal, skim milk powder and a lucerne based mix, there was either no statistically significant or practical difference (less than 3 percentage units) for most of the amino acids between digestibility determined using conventional amino acid analysis with or without prior guanidination. In contrast for cottonseed meal there were significant and practical differences for nine of the amino acids examined. There was no one amino acid for which digestibility differed between the two approaches for all protein sources. Depending on the level of accuracy required it may be possible to obtain digestibility coefficients for amino acids other than lysine following guanidination of the diet and digesta samples.

20 EXAMPLE 11 Comparison of the true ileal amino acid (including reactive lysine) digestibility of a heated S.lactose/casein determined in the rat and pig.

Four kilograms of lactic casein and 1.25kg of lactose were mixed in suspension then 25 freeze dried. The dried mixture was then autoclaved at 121 °C for 15mmin and ground through a 0.5mm mesh.

Semi-synthetic test diets were formulated to each contain 100 g kg-1 crude protein and to meet the nutrient requirements (except for the amino acids) of the growing rat (National 30 Research Council, 1972) and growing pig (Agricultural Research Council, 1981)(Table 8).

-29- TABLE 8. Ingredient compositions (g kg' air dry weight) of the experimental diets for the interspecies comparison (rat and pig) of the true ileal reactive lysine digestibility of a heated lactose/casein mixture.

Rat Pig EHC Heated lactose/casein EHC Heated lactose/casein Cornstarch 561.9 598.7 555.7 592.5 Soyabean oil 50.0 50.0 50.0 50.0 Cellulose 50.0 50.0 50.0 50.0 Sucrose 100.0 100.0 100.0 100.0 Vit/Min mix 39.3 39.3 45.5 45.5 Lactose 70.0 -70.0 EHC 123.8 -123.8 Unheated casein Heated lactose/casein 157.0 157.0 Chromic oxide 5.0 5.0 5.0 'Vitamin/mineral mix was formulated to meet the rats requirements for vitamins and minerals in the final diets as described by the National Research Council (1972) and for the pigs requirements as described by the Agricultutral Research Council (1981).

The heated lactose/casein was used as the sole source of protein. An EHC based diet was also prepared for each species of animal to allow determination of endogenous ileal amino acid flows.

Forty 150g bodyweight Sprague Dawley male rats were housed as described previously.

Forty entire-male Landrace X (Landrace X Large White) pigs of 30 kg bodyweight were housed individually in steel metabolism crates at an ambient room temperature of approximately 23°C. The rats and pigs were randomly allocated to their respective diets such that there were eight animals on each of the diets. The animals underwent a 14 day experimental period. On each day each rat or pig was given its respective diet at nine hourly meals (0830 to 1630h). For the rats, the diet was available for ten minutes at each meal time. Water was freely available. The pigs were given a set and equal amount of food at each meal time. The daily intake of the pig was proportional to their ad lib energy 35 intake, based on the ratio of energy intake for the rats on the 13th day of study to the rat's ad lib energy intake. Prior to each meal the pig diets were mixed with water w/v).

Water was freely available between meals.

On the fourteenth day of study the rats and pigs were killed 6 hours after the first meal and digesta were immediately collected from the terminal 20cm of ileum. The rats were killed and digesta collected and processed as described previously. The pigs were killed by an intra-cardial injection of sodium pentobarbitone administered with the animal under halothane anaesthesia. The ileal digesta samples for the pigs given the test diets were freeze dried and stored frozen (-20C) while awaiting chemical analysis. The EHC digesta samples were processed as described previously. Amino acids were determined on duplicate digesta samples and quadruplicate diet samples while chromium content was determined in duplicate.

The apparent and true ileal amino acid digestibilities for the heated lactose/casei given to the rat and pig are presented in Tables 9 and 10. There was no significant difference in apparent digestibility values determined between the two species. Further, for only alanine was there a significant difference in true digestibility estimates determined in either the rat or the pig.

TABLE 9. Mean apparent ileal amino acid digestibility in a heated lactose/casein mixture, for the rat and pig Pig Rat Overall SE Significance Aspartic acid 90.4 88.3 0.89

NS

Threonine 87.4 86.8 1.10

NS

20 Serine 82.9 87.5 1.99

NS

6:*i Glutamic acid 92.3 93.0 0.85

NS

Proline 94.6 93.5 055

NS

6 7 Glycine 67.3 61.2 5.32

NS

Valine 87.8 90.8 1.07

NS

Alanine 92.2 92.4 0.80

NS

Isoleucine 90.1 90.9 1.08

NS

Leucine 95.5 96.2 0.38

NS

S Tyrosine 96.6 95.7 0.36

NS

SPenylalanine 96.3 96.0 0.32

NS

30 Histidine 89.7 88.5 0.70

NS

Arginine 90.0 90.6 2.10

NS

Reactive lysine 94.6 94.1 0.47

NS

too* NS, non significant, P> 0.05.

-31- TABLE 10. Mean true ileal amino acid digestibility in a heated lactose/casein mixture, for the rat and pig 5 Pig Rat Overall SE Significance Aspartic acid 96.6 95.0 0.89

NS

Threonine 95.6 94.0 1.10

NS

Serine 89.9 92.5 1.99

NS

Glutamic acid 95.6 95.7 0.85

NS

Glycine 78.5 74.4 5.32

NS

Valine 96.4 96.9 0.80

NS

Alanine 95.1 99.2 1.07 Isoleucine 94.7 95.5 1.08

NS

Leucine 98.8 99.6 0.38

NS

Tyrosine 99.3 99.3 0.36

NS

Phenylalanine 98.9 99.4 0.32

NS

Histidine 93.8 94.6 0.70

NS

Arginine 95.0 94.3 2.10

NS

Reactive lysine 98.2 98.0 0.47

NS

NS, non significant, P> 0.05; P<0. These results demonstrate the applicability of the guanidation assay for feedstuffs for a range of animals. From these results it is also apparent that the laboratory rat is a suitable 25 model animal for the growing pig.

V EXAMPLE 12 Evaluation of the accuracy of the true ileal digestible reactive Ivsine assay using a pig growth study In this experiment two comparisons were made: comparison of the lysine and protein depositions and liveweight gain in pigs fed heated skim milk powder with that in pigs fed an EHC/free amino acid diet (EHC diet A) formulated to contain a lysine level equal to the digestible lysine level based on the traditional true ileal digestibility assay (reactive lysine content x total lysine digestibility); and comparison of the lysine deposition in pigs fed the heated skim milk powder with that in pigs fed an EHC/free amino acid diet (EHC Diet B) formulated to contain a lysine level equal to the digestible lysine level based on the new true ileal reactive lysine digestibility assay (reactive lysine content x reactive lysine digestibility).

Approximately 130kg of skim milk powder was autoclaved in 5kg batches at 121oC for approximately 10min then ground through a 1mm mesh. Samples were taken and ground -32 to 0.5mm before analysis for total and reactive lysine contents. Three semi-synthetic diets containing either unheated skim milk powder (basal diet), heated skim milk powder or EHC as the sole source of nitrogen were formulated in order to determine the digestibility of the heated skim milk powder (Table 11). The EHC diet was used to determine the endogenous lysine flow. Chromic oxide was added as an indigestible marker.

Sixteen pigs were housed in metabolism crates as described previously and allocated to either a basal skim milk powder diet or an EHC based diet. After 7 days the pigs on the basal skim milk powder diet were changed to the heated skim milk powder diet and fed for a further 7 days. After a 14 day trial period the pigs were slaughtered and ileal digesta collected, processed and true ileal total lysine and reactive lysine digestibilities determined as described previously.

TABLE 11. Ingredient compositions (g kg- air dry weight) of the experimental diets for the determination of true ileal reactive and total lysine digestibility of heated skim milk powder for the evaluation of the new assay using a pig growth study.

Basal skim milk powder Heated skim milk powder

EHC

Cornflour 488.2 492.4 551.2 20 Maize Oil 50.0 50.0 50.0 SCellulose 50.0 50.0 50.0 Sucrose 100.0 100.0 100.0 SVit/Min mix 1 50.0 50.0 50.0 Lactose Skim milk powder 256.8 700 Heated skim milk powder 252.6 EHC 123.8 Chromic Oxide 5.0 5.0 Vitamirnmineral mix was formulated to meet the pigs requirements for vitanins and minerals in the final diets as described by the Agricultural Research Council (1981).

The true ileal reactive lysine digestibility for the heated skim milk powder was significantly higher than the true ileal total lysine digestibility Consequently the 35 heated skim milk powder used as a test feedstuff to evaluate the accuracy of the true ileal reactive lysine digestibility assay.

Based on the true ileal total lysine and reactive lysine digestibility coefficients for heated skim milk powder determined above, three test diets were formulated (Table 12) to contain similar net energy levels and fibre levels and similar amino acids balances. The heated skim milk powder was the sole source of nitrogen.

.33 TABLE 12. Ingredient compositions (g kg-' air dry weight) of the experimental diets for the pig growth study.

Basal skim milk Heated skim milk EHC Diet A EHCDietB powder powder Cornflour 365.0 345.0 401.4 372.1 Maize Oil 50.0 50.0 50.0 50.0 Cellulose 50.0 50.0 50.0 50.0 Sucrose 100.0 100.0 100.0 100.0 Vit/Min mix' 50.0 50.0 50.0 50.0 Citric acid 6.4 6.4 Sodium citrate -0.8 0.8 DiCalcium phosphate 2.0 Magnesium oxide 0.007 0.007 Potassium chloride 1.2 1.2 Lactose -191.7 191.7 Skim milk powder 385.0 Heated skim milk powder 385.0 EHC -90.5 108.6 Asparagie 1.7 25 Aspartic acid -1.7 Threonine 2.0 2.4 Serine -1.8 2.1 Glutamic acid 8. 1 9.7 Glycine 11.0 9.7 11.6 30 Alanine 9.0 8.3 10.0 Valine 1.9 2.3 Cysteine 0.6 0.7 Methionine 0.6 0.7 Isoleucine -1.3 35 Leucine 4.5 5.4 Tyrosine 3.2 3.9 Phenylalanine -3.2 3.9 Tryptophan 2.2 2.6 Hisidine 1.4 1.7 40 Arginine 1.0 1.2 Proline 2.9 Vitaminin neral mix was formulated lo mneei tih pigs requirements for vitamins and minerals in the final diets as described by the Agricultural Research Council (1981).

Heated skim milk powder: Heated skim milk powder based diet.

EHC Diet A: EHC free amino acids formulated to contain a lysine level equal to the digestible lysine content of the heated skim milk powder determined using the conventional ileal digestibility assay (reactive lysine in heated skim milk powder x true 34 digestibility of total lysine (determined using conventional methods) for the heated skin milk powder).

EHC Diet B: EHC free amino acids formulated to contain a lysine level equal to the digestible lysine content of the heated skim milk powder determined using the new ileal reactive lysine digestibility assay (reactive lysine in heated skim milk powder x true digestibility of reactive lysine (determined using the new method) for the heated skim milk powder).

The heated skim milk powder based diet was also tested independently to ensure that lysine was limiting. Six 25kg liveweight entire male pigs were housed in metabolism crates designed for complete urine collection. After a six day acclimatisation period, during which the pigs were fed a basal skim milk powder based diet, the diet for three of the pigs was changed to the heated skim milk powder diet which was fed for a further six days. The remaining three pigs were fed the heated skim milk powder diet supplemented with lysine. The total daily urine volume for each pig was determined by collecting the daily urine output for each of the first three days of the six day test period. Total urine was collected for each day of the last three days of this six day period into a bottle containing 25ml of 1.8M H2S04 per litre of urine. The walls of the metabolism crates 20 were also washed down with distilled water. After this 6 day period the pig's diets were swapped over and again total urine was collected for each day of the last 3 days of the 6 day period. Each individual pig's daily urine sample was analysed for creatinine content, after which, the samples were pooled and analysed for total nitrogen and urea content.

The daily urinary total nitrogen excretion of the pigs fed the heated skim milk powder diet 25 were then compared to the total nitrogen excretion for the pigs receiving the heated skim milk powder diet supplemented with lysine.

Growth trial Entire littermate Landrace x (Landrace x Large white) male pigs were housed at the Pig 30 Research Unit in a temperature controlled room maintained at 22±2oC. The pigs were fed at 10.2% of their metabolic bodyweight and were given their respective daily allowance as three equal meals. Any feed refusals were collected and weighed. At the end of the acclimatisation period the pigs were weighed and the feed level recalculated, again to equal 10.2% of their metabolic weight. At this time 8 pigs were slaughtered and their body lysine content determined to provide a baseline lysine level. When the pigs were slaughtered, the entire gut, gall bladder and bladder contents were removed and care was taken to recover any blood that was lost from the body. The pig bodies were stored at until they could be ground. The remainder of the pigs were fed there respective test diets in a similar manner as during the acclimatisation period and every 7 days the pigs were reweighed and the feed level was adjusted accordingly. At the end of the 19 day test period the pigs were slaughtered and the bodies were processed as described above. After grinding the pigs, whole body samples were taken and freeze dried. Fat was then extracted using the Soxhlet fat extraction technique before amino acid analyses were carried out. The deposition of lysine was calculated over the trial period after subtraction of the lysine contents calculated to be present in the animals at the start of the trial period from the lysine content measured at the end of the trial period. The lysine content of the animals at the start of the trial was estimated using a regression of the lysine content of the baseline" pigs on liveweight.

The urinary nitrogen excretion in the pigs when fed the heated skim milk powder diet was significantly higher (P=0.021) than when fed the lysine supplemented diet Ig/day and 6.6g/day respectively), demonstrating that lysine was limiting in the heated skim milk powder diet. Furthermore, since the EHC diets were formulated with the same ratio of amino acids to lysine as present in the heated skim milk powder then these diets too were deemed to be limiting in lysine.

The pigs appeared to be healthy during the acclimatisation period and during the first few 20 days of the 19 day trial. However, the majority of the pigs fed the EHC control diets then began to suffer from dian-hoea. The diarrhoea lasted on average for about two to three days after which the pigs appeared normal. Apart from during the acclimatisation period, the pigs generally consumed their diets readily and there were negligible food refusals during the trial period. The mean initial liveweight of the pigs fed the heated skim milk powder diet was 29.2kg which was significantly higher (P<0.025) than that for the pigs S. fed the EHC diet A (25.7kg), but was not significantly different from that for the pigs fed the EHC diet B (28.0kg). Further, there was no significant difference in initial liveweights between the pigs fed either of the EHC diets. Consequently, lysine and protein depositions as well as weight gain were compared statistically using initial 30 liveweiht as a covanate. The lysine and protein contents of the pigs at the beginning of the trial period were determined by regression of the lysine and protein depositions against liveweight for the baseline pigs slaughtered at the onset of the trial.

Over the 19 day trial period the pigs (for all diets) on average increased their body lysine content by 38.5%. The lysine depositions of the pigs fed the heated skim milk powder were significantly higher than for the pigs fed the EHC control diet A containing a lysine level equivalent to the digestible lysine in the heated skim milk powder determined using the traditional digestibility assay (reactive lysine content x total lysine digestibility)(Table -36- 13). In contrast, the lysine depositions of the pigs fed the heated skim milk powder were similar and not significantly different to those for the pigs fed the EHC control diet B containig a lysme level equivalent to the digestible lysine in the heated skim milk powder determined using the new assay (reactive lysine content x reactive lysine digestibility) The protein depositions and liveweight gain of the pigs fed the heated skim milk powder diet were significantly higher than that found in both groups of pigs fed the EHC diets.

Though the pigs fed EHC diet B deposited more protein and grew faster than those pigs given EHC diet A.

TABLE 13. Mean lysine deposition (g day-1), protein deposition (g day-1) and bodyweight gain (g day-1) determined over 19 days in pigs fed a heated skim milk powder diet, an EHC diet A and EHC diet B.

Heated skim EHC Diet A' EHC Overall SE Significance milk powder Diet B 2 Lysine Deposition 9.6' 5.1b 8.1 1.46 Protein Deposition 140.8" 80. 1 107.7 13.41 20Weight gain 764.5' 539.0 b 627.5c 46.48 S'E C Diet A: EHC free amino acids formulated to contain a lysine level equal to the digestible lysine content of the heated skim milk powder determined using the conventional ileal digestibility assay (reactive lysine in heated skim milk powider x true digestibility of total lysine (determined using conventional methods) for the heated skim milk powder).

"2 'EHC Diet B: EHC free amino acids formulated to contain a lsine level equal to the digestible lysine content of the heated skim, milk powder determined using the new ilcal reactive lysine digestibility assay (reactive lysine in heated skim milk powder x trIue digestibility of reactive lysine (determined using the newi method) for the heated skim milk powder).

Since most of the pigs fed the EHC based diets suffered from diarrhoea in the early part of the trial, the lysine and protein depositions and weight gains of the pigs over the last 12 days of the trial were also examined. The mean initial liveweight at the commencement of this 12 day period for the pigs fed the heated skim milk powder diet was 33kg which was significantly higher (P<0.023) than that for the pigs fed the EHC diet A (28.8kg), but was not significantly different from that for the pigs fed the EHC diet B (31.3kg). Further, there was no significant difference in initial liveweights between the pigs fed either of the EHC diets. Consequently again, lysine and protein depositions as well as weight gain were compared statistically using initial liveweight fitted as a -37covarate. The lysine and protein contents of the pigs at the beginning of the 12 day period were determined by regression of the lysine and protein depositions against liveweight for the baseline pigs slaughtered at the onset of the trial.

Over the 19 day trial period the pigs (for all diets) on average increased their body lysine content by 26.9%. Again, lysine depositions of the pigs fed the heated skim milk powder were significantly higher than for the pigs fed the EHC control diet A (Table 14), the lysine depositions of the pigs fed the heated skim milk powder were similar and not significantly different to those for the pigs fed the EHC control diet B. The protein deposition and liveweight gain of the pigs fed the heated skim milk powder diet was significantly higher than that found in both groups of pigs fed the EHC diets.

TABLE 14. Mean lysine deposition (g protein deposition (g day and bodyweight gain (g day') determined over 12 days in pigs fed a heated skim milk powder diet, an EHC diet A and EHC diet B.

S

S.

Heated skim EHC Diet A' EHC Overall SE Significance milk powder Diet B: level Lysine Deposition 10.2' 5.0b 8.7 2.23 Protein Deposition 156.4' 86.8h 109.2" 22.45 Weight gain 790.0' 575.8" 647.9 98.08 'EHC Diet A: EHC free amino acids fornulateCd to contain a lysine level equal to the digestible lysine content of the heated skim milk powder determined using the conventional ieal digestibility assay (reactive /vsine in heated skim milk powder x true digestibility of total lysine (determined using conventional methods) for the heated skim milk powder).

'EHC Diet B: EHC free amino acids forulated to contain a lysine level equal to the digestible lysine content of the heated skim milk powder determined using the new ileal reactive lysine digestibility assay (reactive lysiie in heated skim milk powder x true digestibility of reactive lysine (determined using the newi method) for the heated skim milk powder).

Lysine and protein depositions and liveweight gains were also determined over the final 12 days on an initial liveweight basis instead of using initial liveweight as a covariate (Table 15). The results calculated in this manner showed similar trends as those that use initial liveweight as a covariate.

-38 TABLE 15. Mean lysine deposition (g day-I kg-I initial bodyweight), protein deposition (g day-I kg- initial bodyweight) and bodyweight gain (g day-i kg-i initial bodyweight) determined over 12 days in pigs fed a heated skim milk powder diet, an EHC die A and EHC diet B.

Heated skim EHC Diet A' EHC Overall SE Significance milk powder Diet B- level Lysine Deposition 0.31 0.18b 0.28' 0076 Protein Deposition 4.9" 2.9 b 3 5 b 0.75 Weight gain 25.2' 19.0b 20. 8 3.26 'EHC Die, A: EHC free amino acids formulated to contain a lysine level equal to the digestible lysine content of the heated skin milk powder determined using the conventional i/eal digestibility assay (reactive lysine in heated skim milk powder x true digestibility of total lysine (determined using conventional methods) for the heated skim milk powder).

'EHC Diet B: EHC +free amino acids formulated to contain a lysine level equal to the digestible lysine content of the heated skim m1ilk powder determined using the new ileal reactive lysine digestibility assay (reactive lysine in heated skim milk poiwder x inie 'i digestibility of reactive lysine (determined using the new method) for the heated skim milk powder).

The mean lysine deposition for the pigs fed the heated skim milk powder measured over the total 19 day experimental period was significantly different from that for the pigs fed EHC Diet A. Further, the lysine deposition of the pigs fed the heated skim milk powder was similar and not significantly different from the pigs fed EHC Diet B. Over the final 12 days of the trial (excluding an initial period of two to three days where many of the EHC fed pigs suffered from diarrhoea) the results similar to that observed over the total 30 trial period. The protein deposition and liveweight gains of pigs fed the heated skim milk S. powder was significantly higher than pigs fed either of the EHC control diets although the protein deposition and liveweight gains of pigs fed EHC Diet B were closer to that of the pigs fed the skim milk powder based diet than were those of the pigs fed EHC Diet A.

This result demonstrates firstly, the inadequacy of the traditional assay in predicting available lysine in the heated skim milk powder and secondly, the accuracy of the new true ileal reactive lysine digestibility assay in determining lysine availability in a heated skim milk powder and gives confidence in the suitability of the new assay for accurately predicting lysine availability in processed feedstuffs to be used in least cost feed formulation.

-39-

CONCLUSION

In accordance with the present invention there are provided methods for determining the digestibility co-efficient of an amino acid in a foodstuff. More especially, the present invention provides methods for determining both the reactive lysine digestibility coefficient and the digestible reactive lysine content in a foodstuff. The methods of the present invention represent an alternative amino acid assay for many amino acids and for lysine, a significant advance on conventional assays which do not adequately take account of the effects of processing on lysine bioavailability. The approach taken in the present invention was to regard altered lysine residues as "lost" to a subject for protein synthesis and to attempt to directly determine the absorption of reactive lysine residues remaining in a foodstuff.

The present methods detected that as much as 40% of the original lysine in a heated foodstuff may be destroyed or modified by heat treatment.

By determining the reactive lysine content of the diet and digesta rather than the total lysine content (as in the traditional digestibility assays) the problem of overestimated :lysine values due to interference from lysine destroyed or modified by heat treatment, and o* o heat induced lysine derivatives reverting back to lysine during acid hydrolysis is avoided.

.Lysine was found to be highly digestible in the unheated or unprocessed foodstuffs in the order of 90 to 100% digestible. The determined lysine digestibility co-efficient in the heated or processed foodstuffs, when lysine content was qualified using conventional amino acid analysis, was some 25 to 35 percentage units lower than for the unheated foodstuffs. This decrease in digestibility was considerably greater than the 2-10 percentage unit decrease observed for the other amino acids. In contrast, the true reactive lysine digestibility co-efficient in heat damaged or processed foodstuffs obtained using guanidination was significantly higher at 80 to 90%, approximately 13 percentage units lower than in the unheated or unprocessed foodstuffs. This shows better agreement with 30 the decrease in digestibility observed for the other amino acids.

It is clear that the use of conventional amino acid analysis significantly underestimates lysine digestibility. The reactive lysine digestibility estimate obtained using the guanidination method therefore clearly provides more reliable estimates than those obtained using conventional amino acid analysis.

There are many methods for determining reactive lysine in feeds, all with their inherent advantages and disadvantages, but until now there has been no ideal method that allows for the routine determination of digestible reactive lysine in processed feeds, especially heat processed feeds. The new true ileal reactive lysine digestibility methods described here are means by which this can be achieved. Furthermore, the use of the rat as an experimental animal may allow for a relatively routine assessment of available lysine in processed feedstuffs.

This is an economically important assay where lysine is often the first limiting amino acid particularly in pig and poultry diets. Accurate analysis of available lysine in feedstuffs will allow for the formulation of feedstuffs designed to met the nutritional requirements of the subjects to which they are to be fed. The methods of the invention can also be used to determine optimal lengths of time over which feeds can be stored. Both factors will allow for reduction in feed costs, where the feeds utilised will have a higher nutritional value through optimised available lysine content.

It will be further appreciated by those persons skilled in the art that the present description is provided by way of example only and that the scope of the invention is not limited thereto.

e*

S

*o -41-

REFERENCES

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Comparison of the availability and ileal digestibility of lysine in cottonseed and soyabean meals for grower/finisher pigs. Brit J. Nut 1990, 64, 663-677.

Batterham, E.S. Availability and utilisation of amino acids for growing pigs. Nuitr. Res.

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Liener, J.E. ed. Academic Press, New York, USA. 1980, 183-273.

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Butts, Moughan, Smith, W.C. Endogenous amino acid flow at the terminal ileum of the rat determined under conditions of peptide alimentation. J. Sci. FoodAgric.

1991, 44, 175-187.

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Chervenka, and Wilcox, P.F. Chemical derivatives of chymotrypsinogen. 11 Reaction with O-methylisourea. J. Biol. Chen. 1956. 222, 635-647.

25 Costigan, and Ellis, K.J. Analysis of fecal chromiium derived from controlled release marker devices. NZ J Tech. 1987, 3, 89-92.

Desrosiers, Savoire, Bergeron, and Parent, G. Estimation of Lysine Damage in Heated Whey Proteins by Furosine Detenninations in Conjunction with the Digestion 30 Cell Technique. J Agric. Food Chem. 1989, 37. 1385-1391.

Gall, M.P.J. Nutritional and physiological effects of short-termn feeding of an early Maillard browned casein to growing pigs. Ph.D. Dissertation, Massey University, Palmerston North, New Zealand, 1989.

Hendriks, Moughan Boer, and van der Poel, A.F.B. Effects of extrusion on the dye-binding, fluorodinitrobenzene-reactive and total lysine content of soybean meal and peas. Animn. FeedSci. Tech 1994, 48, 99-109.

42 Hurrell, REF., and Carpenter, K.J. The estimation of available lysine in f6odstuffs after Maillard reactions. Prog. Fd Nuir. Sci. 1981, 5, 159-176.

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Ed.; Pergarnon Press, Oxford, 1981;- Vol. 5 N umbers 1-6, Chapter 2. 1.

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Moughan, P.J. Towards an improved utilisation of dietary amino acids by the growi .ng pig. In Recent1 Adi'ances in Animal Nutrition: Haresign, WV., and Cole, Eds.; Butterworth-H einema, 1 in Ltd, Oxford, 1991;- pp 45-64.

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Claims (8)

1. A method for determining the reactive lysine digestibility co-efficient of a foodstuff which method comprises the steps of: introducing a marker into the foodstuff to be analysed; feeding the foodstuff to a subject for a predetermined period of time; obtaining a sample of the foodstuff digesta from the subject; determining the digestible reactive lysine content of the foodstuff by: introducing a lysine derivatising agent into the foodstuff; and (ii) determining the digestible reactive lysine content of the foodstuff by measuring the equivalent derivatised lysine content in the foodstuff; determining the digestible reactive lysine content in the foodstuff digesta by: introducing a lysine derivatising agent into the foodstuff digesta; and (ii) determining the digestible reactive lysine content of the foodstuff digesta by measuring the equivalent derivatised lysine content in the foodstuff digesta; measuring the marker concentration in both the foodstuff and foodstuff digesta; 20 expressing the reactive lysine content of both the foodstuff and foodstuff digesta per gram of the marker; and calculating the reactive lysine digestibility co-efficient.

2. A method for determining the digestible reactive lysine content of a foodstuff which 25 assay comprises the steps of: calculating the reactive lysine digestibility co-efficient according to the method of claim 1; and determining the digestible reactive lysine content of the foodstuff by 30 multiplying the value for reactive lysine content of the foodstuff by the reactive lysine digestibility co-efficient.

3. A method according to claim 1 or claim 2 wherein the foodstuff is formulated into a diet before adding the marker and feeding the foodstuff to the subject.

4. A method according to any one of claim 1 to 3 whereing the foodstuff is a heat processed foodstuff. -44- A method according to any one of claims 1 to 4 wherein the marker is a spectrophotometric marker. o A method according to claim 5 whereing the spectrophotometric marker is chromic oxide.

7. A method according to any one of claims 1 to 6 wherein foodstuff digesta is extracted from the terminal ileum.

8. A method according to any one of claims 1 to 7 wherein the foodstuff digesta is dried before treatment with the derivatising agent.

9. A method according to any one of claims 1 to 8 wherein the dervatising agent is O- methylisourea. A method for determining the digestibility co-efficient of an amino acid in a foodstuff which method comprises the steps of: introducing a marker into the foodstuff to be analysed; feeding the foodstuff to a subject for a predetermined period of time; obtaining a sample of the foodstuff digesta from the subject; determining the digestible amino acid content of the foodstuff by: introducing an amino acid denvitising agent into the foodstuff; and (ii) determining the digestible amino acid content of the foodstuff by 25 measuring the equivalent derivatised amino acid content in the foodstuff; determining the digestible amino acid content in the foodstuff digesta by: introducing an amino acid derivatising agent into the foodstuff digesta; and 30 (ii) determining the digestible amino acid content of the foodstuff digesta by measuring the equivalent derivatised amino acid content in the foodstuff digesta; measuring the marker concentration in both the foodstuff and foodstuff digesta; expressing the amino acid content of both the foodstuff and foodstuff digesta per gram of the marker; and calculating the amino acid digestibility co-efficient.

11. A method for determining the digestible content of an amino acid in a foodstuff which method comprises the steps of: calculating the digestibility co-efficient of the amino acid according to the method of claim 10; and determining the digestible content of the amino acid in the foodstuff by multiplying the value for the amino acid content of the foodstuff by the digestibility co-efficient. S S