WO1997002489A1 - Method for determining amino acid content in foodstuffs - Google Patents

Abstract

This invention is directed to a method for determining the available amino acid content, particularly lysine, in a foodstuff. A true ileal amino acid digestibility is used in conjunction with an amino acid derivatising reaction to determine the true ileal digestibility of the amino acid. The amino acid concentration in a diet and digesta is then determined. Co-efficients derived using the assay and the amino acid content of the foodstuff can then be used to calculate the available amino acid content of the foodstuff.

Description

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 paiticulariy 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 ( nipfel 1981, Batterham 1992) in foodstuffs.

Lysine is of particular interest because it is an essential amino acid and is often the first limiting amino acid in diets, particularly pig and poultry diets. In foodstuffs that have undergone processing or prolonged storage, the e-amino group of lysine can react with other compounds present in feedstuffs (e.g. the Mailla d reaction) to become structurally altered and nutritionally unavailable (Hurrell 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 1 -fluoro- 1,4-dinitrobenzene (FDNB) assay (Carpenter 1960). In this assay reactive lysine in a foodstuff to be tested is determined by reacting the foodstuff with FDNB resulting in the production of Dinitiophenyl lysine (DNP-lysine) which is then detected by absorbance at 435 nm and the conesponding reactive lysine content in the foodstuff calculated.

An ileal amino acid digestibility assay to predict die availability of amino acids, including lysine, in a foodstuff is also known and in cunent use. This traditional ileal amino acid digestibility assay is described comprehensively in Moughan et ai, 1990 and Butts el al, 1991 incoφorated 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 ofthe 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. However, it has recently been noted that not all of the chemically reactive lysine in heat- treated proteins is absorbed from the small intestine (Schmitz 1988, Desrosiers et al, 1989, Moughan 1991). Consequently, methods for determining chemically reactive lysine 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 feedstuff is 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 (Batterham 1992). 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 reactive lysine digestibility co-efficient and the digestible reactive lysine content in a 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 digestibility 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 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 method comprises the steps of:

(a) introducing a marker into the foodstuff to be analysed;

(b) feeding the foodstuff to a subject for a predetermined period of time; (c) obtaining a sample of the foodstuff digesta from the subject;

(d) determining the digestible reactive lysine content of the foodstuff by: (i) 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; (e) determining the digestible reactive lysine content in the foodstuff digesta by: (i) introducing a lysine derivatising agent into the foodstuff digesta; and (ii) deteiTnining the digestible reactive lysine content of the foodstuff digesta by measuring the equivalent derivatised lysine content in the foodstuff digesta; (f) measuring the marker concentration in both the foodstuff and foodstuff digesta;

(g) expressing the reactive lysine content of both the foodstuff and foodstuff digesta per gram of the marker; and

(h) 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:

(a) calculating the reactive lysine digestibility co-efficient using the above defmed method; and (b) determining the digestible reactive lysine content ofthe 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 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 deteπriining the content of an amino acid in a foodstuff which method comprises determining the amino acid 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 lysine.

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 defmed 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 ofthe invention will now be described in relation to the accompanying drawings in which: Figure 1 is a graph illustrating the homoarginine (reactive lysine) content of heate lactose/casein incubated with 0.6 M O-methylisourea pH 10.6 in a shaking waterbath a 21±2°C for 1 to 14 days, with the reagent to lysine ratio greater than 1000.

Figures 2/1 and 2/2 provide graphic illustrations ofthe amount of homoarginine (reactiv lysine) present in the digesta of rats fed unheated casein and heaied lactose/casei determined using the guanidination reaction.

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

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

For Figure 2/2 the pH ofthe reaction mixture was varied. Guanidination conditions fo the digesta of rats fed unheated casein were incubation for 1 day at 21±2°C in 0.6M O methylisourea at pH 9.8 to 11.4, with the reagent to lysine ratio being greater than 1000 The guanidination conditions for the digesta of rats fed heated lactose/casein wer incubation for 7 days at 21±2°C in 0.6M O-methylisourea at pH 9.8 to 11.4, with th reagent to lysine ratio being greater than 1000.

For Fig. 2/2, n=3 for all analyses. Values were means ± SE.

Figure 3 illustrates by means of bar graphs tiie recovery of amino acids from variou protein sources after guanidination with O-methylisourea. Recoveries were calculated a follows:

Recovery (%) = Moles of arnino acid determined in guanidinated protein X 100 Moles of arnino acid determined in unreacted protein 1

Figure 4 illustrates by means of graphs the mean (±SE) food intakes for the first fiv meals for rats on the last day of trial.

Figure 5 is a flow chart illustrating a method of determining endogenous arnino 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 ofthe true ileal digestability of amino acids (other than lysine) as determined using conventional amino acid analysis (D) or following the guanidination reaction (■).

DETAILED DESCRIPTION OF THE INVENTION

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 amino 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 amino acid digestibility assay. An example of a derivatising reaction is to use guanidination to convert lysine to the equivalent acid stable homoarginine. The guanidination reaction is described in Rutherfurd and Moughan 1990 incorporated herein by reference.

In the method of the present invention a derivatising 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 lysine in diet - (Reactive lysine in digesta - Endogenous lysine)

Reactive lysine in diet Once the reactive lysine digestibility co-efficient has been calculated the true digestible reactive lysine content ofthe foodstuff can be calculated by multiplying the reactive lysine 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 occurring or processed foodstuffs. Naturally occurring foodstuffs include crude protein such as meats and milk. Processed foodstuffs range from relatively unprocessed proteins such as lysozyme, soy protein isolates, skim milk powder, lactic casein, whey protein concentrate, soy protein concentrate, wheatmeal, and soyabean 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.

In a preferred embodiment the foodstuff is formulated into a diet before feeding to a subject. Formulation ofa 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 means for measuring the degree of concentration the diet undergoes as it is converted into digesta. Selected markers will be indigestible and of low toxicity. Suitable markers include spectrophotomeric markers such as chromic oxide or radiopaque markers amongst others. Chromic oxide is the presently preferred marker.

The diet formulated together with the chromic oxide marker is then fed to a selected subject for a predetermined period of time. 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 vitro environments which simulate an animal gut, and produce " « vitro digesta". Subjects may be selected according to the diet to be tested. However, rats are a convenient test subject.

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 5 to 20 days, and most preferably 14 days. The rat may be fed the diet in one or more meals a day for a set time period. It is preferable on the Mth day to adopt a feeding regime 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 10 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 ofthe ileum using a syringe filled with distilled, deionised water. The digesta obtained can then be analysed directly but is preferably freeze-dried.

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

The 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 deteirnine 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 α-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°±2°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 reactive lysine digestibility assay, and is currently the derivatising agent of choice. O- methylisourea acts as a guanidinating agent converting reactive lysine to acid stable homoarginine. The homoarginine 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 x lOO

Moles of unreacted lysine + Moles of homoarginine 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 ofthe diet and digesta are determined spectrophotometrically following the method of Costigan and Ellis (Costigan and Ellis, 1987) incorporated herein by 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 lysine 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 are μg g^"1 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 μg g-' 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 μg g^"1 DMI):

True reactive lysine digestibility =

Dietary reactive lysine intake - (Ileal reactive lysine flow - endogenous lysine flow) x 100

Dietary reactive lysine intake 1

The second method ofthe invention relates to the determination of the digestible reactive 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 ofthe foodstuff is determined using the guanidination reaction set 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 ofthe 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 ofthe methods ofthe 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 1 -fluoro- 1,4-dinitiObenzene (FDNB), Dinitrophenyl-lysine (DNP-lysine) and O- methylisourea 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 enzymaticaUy hydrolysed casein was obtained from New Zealand Pharmaceuticals LTD, Palmerston North, New Zealand and contained 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 FDNB Method

1 -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 HCl 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 methylisourea 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 Bujar (1964) and Kassell and Chow (1966). Four grams of barium hydroxide octahydrate wer added 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 6400g 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 10.6-1 1.2), 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 in 4 lines of distilled deionised water then freeze drying the suspension and autoclaving the dried mixture for 3.5min at 121°C. The autoclaved sample was ground through a 1mm 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 1 kg of skim milk powder was autoclaved for 3 min at 121 °C before use. The autoclaved skim milk powder along with 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 Optimisation of reaction time for guanidination 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 1000. The samples were then reduced to dryness and the homoarginine and lysin contents determined.

In preliminary studies investigating the optimal time for guanidination of two unheate protein sources (lysozyme and unheated casein), carried out over 1, 2 and 3 da incubation periods, near complete conversion of lysine to homoarginine (greater tha 98%) was achieved in all cases.

EXAMPLE 5 Optimisation of the reaction time for guanidination of heated lactose/casein

5-10 mg samples of heated lactose/casein were incubated for 1, 3, 7 and 14 days in 0.6 O-methylisourea, pH 10.6, at 21°±2°C in a shaking waterbath, with the reagent to lysin ratio greater than 1000. The samples were subsequently reduced to dryness and th homoarginine content was determined.

The conversion of lysine to homoarginine in heated lactose/casein was investigated usin incubation times ranging from 1 to 14 days. The yield of homoarginine over the 14 da period is shown in Fig. l . Maximal guanidination was achieved after 3 to 7 days incubation 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

Optimisation of the reaction time and pH for guanidination of digesta

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°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 o 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, 1 1.0, 1 1.4 at 21±2°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. The optimum incubation times for maximal guanidination of digesta from rats fe unheated casein and heated lactose/casein were determined (Fig.2a). Maximal conversio of lysine to homoarginine in the digesta of rats fed unheated casein was achieved with 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 1 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 1 1.0 and 1 1.4.

EXAMPLE 7

Determination of endogenous amino acid loss

EnzymaticaUy 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 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 et al, 1991.

Endogenous ileal amino acid flow = Amino acid concentration in ileal digesta X Chromium in diet

Chromium in digesta EXAMPLE 8

Digestibility study

Sprague-Dawley male rats, of approximately 150g bodyweight, were housed individually in stainless steel wire-bottomed cages in a room maintained at 22±2°C, with a 12 h light/dark cycle.

A. In a first study, two semi-synthetic test diets were formulated to each contain lOOg/kg crude protein. Two enzymaticaUy hydrolysed casein (EHC) based diets were also formulated to allow determination of endogenous ileal amino acid flows (Moughan et al, 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 lOOg/kg crude protein. An EHC based diet was also formulated to allow determination of endogenous ileal lysine flow as above. Chiomic oxide was included (0.5%) in each diet as an indigestible marker. The ingredient compositions ofthe diets are given in Table 1 A below.

TABLE 1. Ingredient compositions (g kg^"1 air dry weight) ofthe experimental diets given to the laboratory rat.

EHC Unheated casein EHC2 Heated lactose/casein³

Wheat starch 625.7 639.7 583 584.9

Soybean oil 50 50 50 50

Purified cellulose 50 50 50 50

Sucrose 100 100 100 100

Vitamin/mineral mix⁴ 39.3 39.3 39.3 39.3

Lactose - - 42.7 -

Casein - 1 16 - -

Heated lactose/casein - - - 170.8

EHC 130 - 130 -

Chromic oxide 5 5 5 5

All diets were formulated to contain equal crude protein contents.

'EnzymaticaUy hydiOlysed casein containing diet for determining endogenous amino acid loss for the unheated casein diet.

²Enzymatically hydiOlysed casein containing diet for determining endogenous amino acid loss for the heated lactose/casein diet.

^:Ηeated lactose/casein was prepared as described in Experimental example 3.

⁴Nitarnin/mineral mix was formulated to meet the requirements for vitamins and minerals as described by the National Research Council (National Academy of Sciences, 1972).

TABLE IA. Ingredient compositions 1 (g kg-1 air dry weight) of the experimental diets.

EHC² Blood Wheat Meat and Soybean Heated skim Dried Lucerne Cottonseed meal meal bone meal meal milk powder maize based mix meal

Wheat starch 625.7 646.7 - 572.7 542.7 495.7 355.7 504.7

Soybean oil 50 50 50 50 50 50 50 50

10 Purified cellulose 50 50 - 50 50 50 50 50

Sucrose 100 100 20.7 100 100 100 100 100

Vitamin/mineral mix⁴ 39.3 39.3 39.3 39.3 39.3 39.3 39.3 39.3 39.3

EHC 130 - - - - -

Blood meal - 109 - - - -

15 Wheat meal - - 885 - - - > Meat/bone meal - - - 183 - -

Soybean meal - - - - 213 -

Heated skim milk powder - - - - - 260

20 Dried maize - - - - - - 955.7 .

Lucerne based mix³ - - . . - - 400

Cottonseed meal - - - . - - - 251

Chromic oxide 5 5 5 5 5 5 5 5

25 'All diets were formulated to contain equal crude protein contents. I

²Enzymatically hydrolysed casein diet used for determining endogenous amino acid losses at the terminal ileum, the EHC contained free amino acids and small peptides (<2000 Da).

³The lucerne based mix consisted of 55% lucerne, 10% meat and bone meal and

5% each of blood, wheat, barley, maize, sorghum, soybean and broil meals and was initially in a pelleted form..

30 ⁴Vitamin/rnineral mix 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).

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 ofthe 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 anterior to the ileo- caecal junction was dissected out. The dissected ileum was washed with distilled deiomsed 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 HCl, to minimise protease activity. The EHC digesta were then centiifuged at 6400g 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 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 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 HCl containing 0. 1% phenol for 24 hr at 1 10±2°C 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 1 1.0 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. Studv 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 440nm, following hydrolysis in 6M glass-distilled HCl containing 0.1% phenol for 24 hr at 110±2°C in evacuated sealed tubes. Cysteine, methionine and tryptophan were not determined as they are destroyed during acid hydrolysis. The weight of each amino acid was calculated using free amino acid molecular weights.

Reactive lysine contents were determined in duplicate 5 mg 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 1 1.0 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

The 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 analysis. The guanidination conditions used were incubation for 24 hr 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 1000. The reactive lysine level of the heated lactose/casein was then extrapolated using Fig.1 to detennine the reactive lysine content using the optimal 7 day incubation period. The results are shown in Table 2 below. 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^'1 casein)

FDNB¹ Guanidination² Total³

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.

²Guanidination analyses consisted of 24hr incubation in 0.6M O-methylisourea, pH 10.6 in a shaking waterbath at 21 ±2° 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.

³Total analyses consisted of conventional acid hydrolysis and amino acid quantitation.

Values aie 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 the 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-1 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.

Reactive lysine Total lysine

FDNB Guanidination B Blloooodd mmeeaall 8 844..44 88.0 89.1

Wheat meal 3.1 3.1 3.5

Meat and bone meal 30.4 34.6 36.5

Soybean meal 27.1 J *^•*_"-**.J -> 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 0.5 lmmol g"¹ casein determined using conventional amino acid analysis to 0.55mmol g^"1 casein determined 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 lmmol g^'1 casein and 0.33 mmol g^"1 casein respectively), and differed by less than 7%. In contrast, the total lysine level determined using conventional amino acid analysis was considerably higher (almost 20%), 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 feedstuffs 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 reactive lysine contents determined using the FDNB method were lower than those determined using guanidination. Since theoretically the guanidination method cannot overestimate reactive lysine, it would appear that these differences are most likely an artefact of the FDNB method in which coπection 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 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 lysine content was considerably lower than total lysine, reflecting protein sources in 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 "lactose- free" EHC1 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 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 3). 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.

TABLE 3. Endogenous amino acid flows of rats fed an enzymaticaUy hydrolysed casein diet with and without lactose.

EHC with lactose EHC without lactose

(n=6) (n=5)

Aspartic acid 501 723

Threonine 391 448

Serine 393 555

Glutamic acid 587 1133

Glycine 307 305

Alanine 225 336

Valine 296 418

Isoleucine 226 337

Leucine 307 405

Tyrosine 172 235 Phenylalanine 210 209

Histidine 156 352

Lysine 229 285

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 digestibiUty ofthe heated lactose/casein was significantly lower for all amino acids except glycine, alanine, phenylalanine 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).

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/casein¹

Aspartic acid 96.0 87.3 1.37 **

Threonine 93.6 88.0 0.96 **

Serine 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 90J 0.82 **

Leucine 99J 97J 0.27 **

Tyrosine 100.4 97J 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, * PO.05, ** P<0.01, *** PO.001.

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 (99%) (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 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). TABLE 5. Mean true ileal lysine digestibility (n=6) for an unheated casein determined using the rat ileal digestibility assay and based on conventional amino acid analysis and for a heated lactose/casein (n=6) 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 SE

Conventional Guanidination amino acid method analysis

Digestibility of lysine 98.8 70.5 85.9² 2.01 ***

Endogenous amino acid flows at the terminal ileum were 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. ²The reactive lysine digestibility was calculated as follows:

Reactive lysine in the diet - (Reactive lysine in the digesta - Endogenous lysine) X 100

Reactive lysine in the diet 1

Where units are μg g^"1 DMI.

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

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 (g) ±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±0.12g for the heated skim milk powder diet, 0.9±0.15g for the dried maize diet, 1.9±0.28g for the lucerne based mix diet and 1.7±0.07g 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 meanl 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' Reactive³ Overall SE

Blood meal 96.3 96.7 0.41 NS

Wheat meal 92.6 92J 0.45 NS

Meat and bone meal 88.9 91.5 0.76 NS

Soybean meal 94.5 96.5 0.41 *

Dried Maize 80.5 84.3 1.54 *

Heated skim milk powder 69.1 94.0 1.11 ***

Cottonseed meal 62J 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 n=5. ²Lysine digestibility was determined using a true ileal amino acid digestibility assay (rat) and conventional amino acid analysis was used to quantitate total lysine in the diets and digesta.

³Lysine 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 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 determining lysine digestibility.

For the dried maize, lucerne based mixed diet, cottonseed meal and heated skim milk powder, there were significant differences (p<0.5) between the total lysine digestibilities and the reactive lysine digestibilities. Differences between the digestibility values for th 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 appear to be unsuitable for assessing lysine availability in heat processed feedstuffs as i underestimates the digestibility of structurally unaltered lysine. This result also show that the new true ileal reactive lysine digestibility assay may provide a more accurat assessment of digestibility of structurally unaltered (available) lysine in processed protei sources.

Digestible lysine (based on total lysine determined by conventional analysis) an 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

Total² Reactive³ Overall SE

Blood meal 89.9 85.1 0.34 NS

Wheat meal 3.2 2.9 0.02 ***

Meat and bone meal 32.5 31.6 0.24 NS

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 * •)- *

'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 n=5. ²Digestible total lysine was calculated from true ileal lysine digestibility (rat), with lysine determined by conventional amino acid analysis and from the total lysine content in the protein source, also determined using conventional amino acid analysis. ³Digestible 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 les 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 (34%, 37%, 25% 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 odier 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 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 observed. This may have been associated with the chromatographic procedure and may have been due to column ageing.

EXAMPLE 10

Comparison of the true ileal digestibility of acid-stable amino acids in protein sources determined using conventional amino acid analysis or following guanidination ofthe 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 ofthe 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 (89%) ofthe 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 ofthe 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.

EXAMPLE 11

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

Four kilograms of lactic casein and 1.25kg of lactose were mixed in suspension then freeze dried. The dried mixture was then autoclaved at 121 °C for 15min 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 me growing rat (National Research Council, 1972) and growing pig (Agricultural Research Council, 1981)(Table 8)* TABLE 8. Ingredient compositions (g kg^'1 air dry weight) of the experimental diets f the interspecies comparison (rat and pig) of the true ileal reactive lysine digestibility a heated lactose/casein mixture.

Rat Pig

EHC Heated lactose/casein EHC Heated lactose/casei

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 5.0

Vitamin/mineral mix was formulated lo meet ihe rats requirements for vitamins an minerals in the final diets as described by lhe National Research Council (1972) and f the pigs requirements as described by the Agricultural Research Council (1981).

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

Forty 150g bodyweight Sprague Dawley male rats were housed as described previousl Forty entire-male Landrace X (Landrace X Large White) pigs of 30 kg bodyweight we housed individually in steel metabolism crates at an ambient room temperature approximately 23 °C. The rats and pigs were randomly allocated to their respective die such that there were eight animals on each of the diets. The animals underwent a 14 da experimental period. On each day each rat or pig was given its respective diet at ni hourly meals (0830 to 1630h). For the rats, the diet was available for ten minutes at ea meal time. Water was freely available. The pigs were given a set and equal amount food at each meal time. The daily intake of the pig was proportional to their ad lib ener intake, based on the ratio of energy intake for the rats on the 13 th day of study to the ra ad lib energy intake. Prior to each meal the pig diets were mixed with water ( 1 : 1, 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 me and digesta were immediately collected from the terminal 20cm of ileum. The rats we killed and digesta collected and processed as described previously. The pigs were kill by an intra-cardial injection of sodium pentobarbitone administered with the animal und halothane anaesthesia. The ileal digesta samples for the pigs given the test diets wer freeze dried and stored frozen (-20 °C) while awaiting chemical analysis. The EH digesta samples were processed as described previously. Amino acids were determine on duplicate digesta samples and quadruplicate diet samples while chromium content wa determined in duplicate.

The apparent and true ileal amino acid digestibilities for the heated lactose/casein give 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 (n=8) and pig (n=8).

Pig Rat Overall SE Significance

Aspartic acid 90.4 88.3 0.89 NS

Threonine 87.4 86.8 1 J0 NS

Serine 82.9 87.5 1.99 NS

Glutamic acid 92.3 93.0 0.85 NS

Proline 94.6 93.5 0.55 NS

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

Tyrosine 96.6 95.7 0.36 NS Phenylalanine 96.3 96.0 0.32 NS

Histidine 89.7 88.5 0.70 NS

Arginine 90.0 90.6 2J0 NS

Reactive lysine 94.6 94J 0.47 NS

NS, non significant, P> 0.05. TABLE 10. Mean true ileal amino acid digestibility (%) in a heated lactose/casei mixture, for the rat (n=8) and pig (n=8).

Pig Rat Overall SE Significance

Aspartic acid 96.6 95.0 0.89 NS

Threonine 95.6 94.0 1J0 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<0.05.

These results demonstrate the applicability of the guanidation assay for feedstuffs for range of animals. From these results it is also apparent that the laboratory rat is a suitabl model animal for the growing pig.

EXAMPLE 12

Evaluation of the accuracy of the true ileal digestible reactive lysine assay using a pi growth study

In this experiment two comparisons were made:

(a) comparison ofthe lysine and protein depositions and liveweight gain in pigs fe heated skim milk powder with that in pigs fed an EHC/free amino acid di (EHC diet A) formulated to contain a lysine level equal to the digestible lysin level based on the traditional true ileal digestibility assay (reactive lysin content x total lysine digestibility); and

(b) comparison of the lysine deposition in pigs fed the heated skim milk powd with that in pigs fed an EHC/free amino acid diet (EHC Diet B) formulated t contain a lysine level equal to the digestible lysine level based on the new tr ileal reactive lysine digestibility assay (reactive lysine content x reactive lysi digestibility).

Approximately 130kg of skim milk powder was autoclaved in 5kg batches at 121oC f approximately lOmin then ground through a 1mm mesh. Samples were taken and grou to 0.5mm before analysis for total and reactive lysine contents. Three semi-synthetic diet containing either unheated skim milk powder (basal diet), heated skim milk powder o EHC as the sole source of nitrogen were formulated in order to determine the digestibilit of the heated skim milk powder (Table 11). The EHC diet was used to determine th endogenous lysine flow. Chromic oxide (0.5%) was added as an indigestible marker Sixteen pigs were housed in metabolism crates as described previously and allocated t either a basal skim milk powder diet or an EHC based diet. After 7 days the pigs on th basal skim milk powder diet were changed to the heated skim milk powder diet and fe for a further 7 days. After a 14 day trial period the pigs were slaughtered and ileal digest collected, processed and true ileal total lysine and reactive lysine digestibilitie determined as described previously.

TABLE 11. Ingredient compositions (g kg^"1 air dry weight) of the experimental diets fo 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

Maize Oil 50.0 50 0 50.0

Cellulose 50.0 50 0 50 0

Sucrose 100 0 100 0 100 0

Vit/Min mixl 50.0 50 0 50 0

Lactose - - 70 0

Skim milk powder 256 8

Heated skim milk powder - 252 6

EHC - 123 8

Chromic Oxide 5 0 5 0 5 0

' VitaminAmineral mix was formulated to meet the pigs requirements for vitamins 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 (88.5%) was significantly higher than the true ileal total lysme digestibility (67.1%). Consequently the 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. TABLE 12. Ingredient compositions (g kg^'1 air dry weight) of the experimental diets fo the pig growth study.

Basal skim milk Heated skim milk EHC Diet A EHC Diet powder powder

Cornflour 365.0 345.0 401.4 372J

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 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

Asparagine - - 1.7 2.0

Aspartic acid - - 1.7 2.0

Threonine - - 2.0 2.4

Serine - - 1.8 2J

Glutamic acid - - 8.1 9.7

Glycine - 1 1.0 9.7 11.6

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 1.5

Leucine - - 4.5 5.4

Tyrosine - - 3.2 3.9 Phenylalanine - - 3.2 3.9

Tryptophan - - 2.2 2.6

Histidine - - 1.4 1.7

Arginine - - 1.0 1.2

Proline - ~ 2.9 3.5

¹ Vitamin/mineral mix was formulated to meet the pigs requirements for vitamins an 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 th digestible lysine content of the heated skim milk powder determined using th conventional ileal digestibility assay (reactive lysine in heated skim milk powder x tru digestibiUty of total lysine (determined using conventional methods) for the heated ski milk powder).

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

The heated skim milk powder based diet was also tested independently to ensure tha 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 o 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 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 die pigs fed the heated skim milk powder diet 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 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 a -20 °C 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 th pigs were reweighed and the feed level was adjusted accordingly. At the end ofthe 1 day test period the pigs were slaughtered and the bodies were processed as describe above. After grinding the pigs, whole body samples were taken and freeze dried. Fat wa then extracted using the Soxhlet fat extraction technique before amino acid analyses wer carried out. The deposition of lysine was calculated over the trial period after subtractio ofthe lysine contents calculated to be present in the animals at the start of the trial perio from the lysine content measured at the end of the trial period. The lysine content of th animals at d e start of the trial was estimated using a regression of the lysine content o the baseline" pigs on liveweight.

The urinary nitrogen excretion in the pigs when fed the heated skim milk powder diet wa significantly higher (P=0.021) than when fed the lysine supplemented diet (9. lg/day an 6.6g/day respectively), demonstrating that lysine was limiting in the heated skim mil powder diet. Furthermore, since the EHC diets were formulated with the same ratio o arnino acids to lysine as present in the heated skim milk powder then these diets too wer deemed to be limiting in lysine.

The pigs appeared to be healthy during the acclimatisation period and during the first fe days ofthe 19 day trial. However, the majority ofthe pigs fed the EHC control diets the began to suffer from diaπhoea. The diaπhoea lasted on average for about two to thre 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 refusal during the trial period. The mean initial liveweight of the pigs fed the heated skim mil powder diet was 29.2kg which was significantly higher (PO.025) than that for the pig fed the EHC diet A (25.7kg), but was not significantly different from that for the pigs fe the EHC diet B (28.0kg). Further, there was no significant difference in initia liveweights between the pigs fed either of the EHC diets. Consequently, lysine an protein depositions as well as weight gain were compared statistically using initia liveweight as a covariate. The lysine and protein contents of the pigs at the beginning o the trial period were determined by regression of the lysine and protein deposition 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 lysin content by 38.5%. The lysine depositions of the pigs fed the heated skim milk powde were significantly higher than for the pigs fed the EHC control diet A containing a lysin level equivalent to the digestible lysine in the heated skim milk powder determined usin me traditional digestibility assay (reactive lysine content x total lysine digestibility)(Tabl 13). In contrast, the lysine depositions ofthe pigs fed the heated skim milk powder we similar and not significantly different to those for the pigs fed the EHC control diet containing a lysine level equivalent to the digestible lysine in the heated skim milk powd 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 powd diet were significantly higher than that found in both groups of pigs fed the EHC diet Though the pigs fed EHC diet B deposited more protein and grew faster than those pi given EHC diet A.

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

Heated skim EHC Diet A¹ EHC Overall SE Significan milk powder Diet B²

Lysine Deposition 9.6^a 5J⁰ 8J^* Ϊ746 ***

Protein Deposition 140.8^a 80J^b 107.7° 13.41 ***

Weight gain 764.5^a 539.0^b 627.5° 46.48 ***

'EHC Diet A: EHC + free ammo acids formulated to contain a lysine level equal to th digestible lysine content of the heated skim milk powder determined using th conventional ileal digestibility assay (reactive lysine in heated skim milk powder x tru digestibility of total lysine (determined using conventional methods) for the heated ski milk powder).

²EHC Diet B: EHC + free ammo acids formulated to contain a lysine level equal to th digestible lysine content ofthe heated skim milk powder determined using the new de reactive lysine digestibility assay (reactive lysine m heated skim milk powder x tru digestibility of reactive lysine (determined using the new method) for the heated skim milk powder).

Since most of the pigs fed the EHC based diets suffered from diarrhoea in the early pa of the trial, the lysine and protein depositions and weight gains of the pigs over the las 12 days of the trial were also examined. The mean initial liveweight at th commencement of this 12 day period for the pigs fed the heated skim milk powder di was 33kg which was significantly higher (PO.023) than that for the pigs fed the EHC di A (28.8kg), but was not significantly different from that for the pigs fed the EHC diet (31.3kg). Further, there was no significant difference in initial liveweights between th pigs fed either of the EHC diets. Consequently again, lysine and protein depositions a well as weight gain were compared statistically using initial liveweight fitted as covariate. The lysine and protein contents of the pigs at the beginning of the 12 d period were determined by regression of the lysine and protein depositions agai 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 lysi content by 26.9%>. Again, lysine depositions of the pigs fed the heated skim milk powd were significantly higher than for the pigs fed the EHC control diet A (Table 14), t lysine depositions of the pigs fed the heated skim milk powder were similar and n significantly different to those for the pigs fed the EHC control diet B. The prote deposition and liveweight gain of the pigs fed the heated skim milk powder diet w significantly higher than that found in both groups of pigs fed the EHC diets.

TABLE 14. Mean lysine deposition (g day^'1), protein deposition (g day ) a bodyweight gain (g day^"1) determined over 12 days in pigs fed a heated skim milk powd diet, an EHC diet A and EHC diet B .

Heated skim EHC Diet A¹ EHC Overall SE Significan milk powder Diet B² level

Lysine Deposition 10.2^a 8.7^a 2.23 ** Protein Deposition 156.4^a 86.8^b 109.2^b 22.45 Weight gain 790.0^a 575.8^b 647.9^b 98.08

'EHC Diet A: EHC + free amino acids formulated to contain a lysine level equal to t digestible lysine content of the healed skim milk powder determined using t conventional eal digestibility assay (reactive lysine in heated skim milk powder x tr digestibility of total lysine (determined using conventional methods) for the heated ski milk powder).

² EHC Diet B: EHC + free amino acids formulated to contain a lysine level equal to t digestible lysine content ofthe heated skim milk powder determined using the new ile reactive lysine digestibility assay (reactive lysine m heated skim milk powder x tr digestibility of reactive lysine

(determined using the new method) for the heated skim milk powder).

Lysine and protein depositions and liveweight gains were also determined over the fin 12 days on an initial liveweight basis instead of using initial liveweight as a covaria (Table 15). The results calculated in this manner showed similar trends as those that u initial liveweight as a covariate. TABLE 15. Mean lysine deposition (g day-1 kg-1 initial bodyweight), protein depositio (g day-1 kg-1 initial bodyweight) and bodyweight gain (g day-1 kg-1 initial bodyweight) determined over 12 days in pigs fed a heated skim milk powder diet, an EHC diet A an EHC diet B.

Heated skim EHC Diet A¹ EHC Overall SE Significanc milk powder Diet B² level

Lysine Deposition 0.3 l^{a "}018°^" 0.28^a 0.076 ** Protein Deposition 4.9^a 2.9^b. 3.5^b 0.75 *** Weight gain 25.2^a 19.0^b 20.8^b 3.26 **

'EHC Diet A: EHC + free amino acids formulated lo 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 digestibility of total lysine (determined using conventional methods) for the heated skim milk powder).

²EHC Diet B: EHC + free amino acids formulated to contain a lysine level equal to the digestible lysine content ofthe 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 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 trial period. The protein deposition and liveweight gains of pigs fed the heated skim milk powder was significantly higher than pigs fed eidier of the EHC control diets although th protein deposition and liveweight gains of pigs fed EHC Diet B were closer to that of th 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 predictin available lysine in the heated skim milk powder and secondly, the accuracy of the ne true ileal reactive lysine digestibility assay in determining lysine availability in a heate skim milk powder and gives confidence in the suitability of the new assay for accuratel predicting lysine availability in processed feedstuffs to be used in least cost fee formulation. CONCLUSION

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

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

By determining the reactive lysine content of the diet and digesta rather than the tota lysine content (as in the traditional digestibility assays) the problem of overestimate lysine values due to interference from lysine destroyed or modified by heat treatment, an heat induced lysine derivatives revelling back to lysine during acid hydrolysis is avoide

Lysine was found to be highly digestible in the unheated or unprocessed foodstuffs in th order of 90 to 100% digestible. The determined lysine digestibility co-efficient in th heated or processed foodstuffs, when lysine content was qualified using conventiona amino acid analysis, was some 25 to 35 percentage units lower than for the unheate foodstuffs. This decrease in digestibility was considerably greater than the 2-1 percentage unit decrease observed for the other amino acids. In contrast, the true reactiv lysine digestibility co-efficient in heat damaged or processed foodstuffs obtained usin guanidination was significantly higher at 80 to 90%, approximately 13 percentage unit lower than in the unheated or unprocessed foodstuffs. This shows better agreement wit the decrease in digestibility observed for the other amino acids.

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

There are many methods for determining reactive lysine in feeds, all with their inhere advantages and disadvantages, but until now there has been no ideal method that allo for the routine determination of digestible reactive lysine in processed feeds, especiall heat processed feeds. The new true ileal reactive lysine digestibility methods describe 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 wiU allow for the formulation of feedstuffs designed to met the nutritional requirements ofthe subjects to which they are to be fed. The methods of me 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.

REFERENCES

Batterham E.S; Anderson, L.M.; Baigent, R.D; Darnell, R.E; Teverner, M.R.A Comparison ofthe availability and ileal digestibility of lysine m 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 Nutr Res Rev. 1992, 5, 1-18.

Berardi, L C, Goldblatt, L.A. 1980 Gassypol In Toxic Constituents of Plant Feedstuffs Liener, J.E. ed Academic Press, New York, USA 1980, 183-273

Booth, V H Problems in the determination of FDNB-available lysine J Sci Food Agric 1971, 22, 658-666

Butts, C A , Moughan, PJ , Smith, W C Endogenous amino acid flow at the terminal ileum ofthe rat determined under conditions of peptide alimentation J Sci FoodAgric 1991, 44, 175- 187

Carpenter, K J The estimation of the available lysine in animal-protein foods Biochem, J 1960, 77, 604-610

Chervenka, C H , and Wilcox, P F Chemical derivatives of chymotrypsinogen II Reaction with O-methylisourea J Biol Chem 1956, 222, 635-647

Costigan, P , and Ellis, K J Analysis of fecal chiomium derived from controlled release marker devices NZJ Tech 1987, 3, 89-92

Desrosiers, T , Savon e, L , Bergeron, G , and Parent, G Estimation of Lysine Damage m Heated Whey Pioteins by Furosme Deteπnmations in Conjunction with the Digestion Cell Technique J Agric Food Chem 1989, 37. 1385- 1391

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

Hendnks, W.H , Moughan P J , Boer, H , and van der Poel, A F.B Effects of extrusion on the dye-bmdmg, fluorodinitrobenzene-reactive and total lysme content of soybean meal and peas Amm Feed Sa Tech 1994, 48, 99-109 Hurrell, R.F., and Carpenter, KJ. The estimation of available lysine in foodstuffs after Maillard reactions. Prog. Fd. Nutr. Sci. 1981, 5, 159-176.

Kassell, B., and Chow, R.B. Modification of die basic trypsin inhibitor of bovine pancreas. The e-amino groups of lysine and the amino terminal sequence. Biochemistry 1966, 5. 3449-3453.

Knipfel, J.E. Nitrogen and energy availabilities in foods and feeds subjected to heating. In Progress in Food and Nutritional Science. Maillard Reactions in Food; Eriksson, C, Ed.; Pergamon Press, Oxford, 1981; Vol. 5 Numbers 1-6, Chapter 2.1.

Mauron, J., and Bujard, E. Guanidination, an alternative approach to the determination of available lysine in foods. Proc. 6th Int. Nutr. Congr. 1964, 489-490.

Moughan, PJ. Towards an improved utilisation of dietary amino acids by the growing pig. In Recent Advances in Animal Nutrition; Haresign, W., and Cole, DJ.A., Eds.; Butterworth-Heinemann Ltd, Oxford, 1991 ; pp 45-64.

Moughan, PJ., Darragh, AJ., Smitii, W.C, and Butts, CA. Perchloric and trichloroacetic acids as precipitants of protein in endogenous ileal digesta from the rat. J. Sci. Food Agric. 1990, 52, 13-21.

National Academy of Sciences. Nutrient requirements of the laboratory rat. In Nutrient requirements of laboratory animals, National Academy of Sciences: Washington, DC 1972; pp 56-93.

Schmitz, M. Moglichkeiten und Grenzen der Homoarginin-Markierungsmethode zur Messung der Proteinverdaulichkeit beim Schwein. Ph.D. Dissertation, Christain- Albrechts-Universitat, Kiel, 1988.

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Claims

CLAIMS:

1. A method for determining the reactive lysine digestibility co-efficient of a foodstuff which method comprises the steps of:

(a) introducing a marker into the foodstuff to be analysed;

(b) feeding the foodstuff to a subject for a predetermined period of time;

(c) obtaining a sample of the foodstuff digesta from the subject;

(d) determining the digestible reactive lysine content of the foodstuff by: (i) 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;

(e) deteimining the digestible reactive lysine content in the foodstuff digesta by: (i) 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;

(f) measuring the marker concentration in both the foodstuff and foodstuff digesta; (g) expressing the reactive lysine content of both the foodstuff and foodstuff digesta per gram of the marker; and (h) calculating the reactive lysine digestibility co-efficient.

2. A method for deteπnining the digestible reactive lysine content of a foodstuff which assay comprises the steps of:

(a) calculating the reactive lysine digestibility co-efficient according to the method of claim 1; and

(b) determining the digestible reactive lysine content of the foodstuff by 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.

5. A method according to any one of claims 1 to 4 wherein the marker is a spectrophotometric marker.

6. 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 derivatising agent is O- methylisourea.

10. A method for determining the digestibility co-efficient of an amino acid in a foodstuff which method comprises the steps of:

(a) introducing a marker into the foodstuff to be analysed; (b) feeding the foodstuff to a subject for a predetermined period of time;

(c) obtaining a sample of the foodstuff digesta from the subject;

(d) deteimining the digestible amino acid content of the foodstuff by:

(i) introducing an amino acid derivitising agent into the foodstuff; and (ii) determining the digestible amino acid content of the foodstuff by measuring the equivalent derivatised amino acid content in the foodstuff;

(e) determining the digestible amino acid content in the foodstuff digesta by: (i) introducing an amino acid derivatising agent into die foodstuff digesta; and (ii) deteimining the digestible amino acid content of the foodstuff digesta by measuring the equivalent derivatised amino acid content in the foodstuff digesta;

(f) measuring the marker concentration in both the foodstuff and foodstuff digesta; (g) expressing the arnino acid content of both the foodstuff and foodstuff digesta per gram of the marker; and (h) calculating the amino acid digestibility co-efficient.

11. A method for determimng the digestible content of an amino acid in a foodstuff which method comprises the steps of:

(a) calculating the digestibility co-efficient of the amino acid according to the method of claim 10; and (b) 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.