DESCRIPTION

Technical field

The present invention relates to a food and feedstuff for hu- mans and animals comprising keto acids.

The object of the present invention is to obtain a food and _, feedstuff, which having a reduced content, of proteins sti ll might fulfi l the demand of the animals as to access to protein forming and energy providing substances. Furthermore, the feed¬ stuff shall fulfi l the demand for access to biomolecules deriv¬ ing from non-essential amino acids. The invention further aims to reduce the excretion of nitrogen from the organism.

Background of the invention

It is well known that water living carnivores, such as salmon, and land based carnivores, such as mink, live under natural conditions, on a diet consisting of 30 to 55 % of proteins (90 % DS) . The choice of food is depending on live phase and spe¬ cie. A great deal of this protein can be oxidized to form en¬ ergy providing compounds, such as ATP ⁽ adenosine t r i phos phate ) , which results in excretion of nitrogen to the envi ronment. At an intense cultivation of e.g. fish and mink this might cause an environmental problem.

During natural conditions omnivores live on a diet consisting of 15 to 30 % of proteins (90 % DS) . During certain conditions this protein might be used for providing energy.

the most energy demanding tissue in the body is the muscles, wh ^' ich obtains its carbohydrate energy from the food directly via the blood or from the liver via the blood in the form of glucose. Glucose is formed in the liver from inter alia decom- ponents of the proteins of the muscles. The compounds which he- reby form g lucose are substantially alanine and its correspond¬ ing keto acid. It has also been shown that the corresponding keto acids of some essential amino acids, such as alpha keto

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isocaproate CKIC), may be precursors of amino acids, e.g. IC of leuci ne .

It is previously known from EP-A1-0 237 061 and EP-A1-0 237 959 to add alpha-keto socaproate CKIC) to egg producing hen to in¬ crease the quantity of eggs and to improve the quality of the eggs produced, to milk producing domestic animals to increase the quantity of milk and to improve the quality of the milk produced-. KIC is thereby added in an amount of 0.01 to 2 % by weight, preferably 0.05 to 1.5 % by weight calculated on the dry weight of the feed-stuff composition. KIC is the only pure ketogenic keto acid of the keto acids formed from the amino ac¬ ids .

From US-A-4,548,937, and US-A-4,645,764 it is known to admini¬ ster to humans and ani als a therapeutically effective amount of pyruvate. At the ad inistration of this compound a reduced gain in weight is obtained, decreased fat depots, and an in¬ creased amount of glycogen in the liver. None of these patents has disclosed an increased efficiency of the protein of the food .

It is previously known from US-A-4,361 ,570 to add a pyridoxine- a Ipha-ketog lutarate (PAK) to a feed-stuff in order to treat hy- per lacti caci demia, whereby PAK is a conjugate between pyridoxi- ne and alpha-ketog lutarate . This molecule can not be compared chemically with free a Ipha-ketog lutarate .

Amino acids which are not used for the synthesis of proteins can not be stored in the organism. They are then reformed to mainly fat or via keto acids, to carbohydrates. Both these mac- romolecules can take part in the energy metabolism. The meta¬ bolic interaction (cf. FIG. 1) between liver and muscle shows the importance of carbon bodies from amino acids. KIC is the only pure ketogenic keto acid of the keto acids formed from amino acids. Alpha-ketog Iutarate (KG) and pyruvate are classi¬ fied as glycogenic keto acids. The latter keto acids have a

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central position in said interaction. The catabolism of KIC is schemati cally evident from FIG. 2. In FIG. 3 the connection be¬ tween the different amino acids and their metabolic products of the citri c acid cycle is shown.

KG is an important part of the metabolism which is exemplified by the o I lowi ng :

(i) NH. is coupled to amino acids via the reaction NH. + KG +

4 ₊ 4 NADPH ==== Glutamate + NADP + H-0

Glutamate provides the alpha amino group in the synthesis of most non-essential amino acids; (ii ) the carbon skeletons present in the synthesis of non-es¬ sential amino acids are all intermediates of the glucoly- sis, the pentose- onophosp ate shunt or the citric acid cycle and may hereby be derived from pyruvate or KG (cf FIG. 5) (iii) at physiological stress, too large energy draft, repro¬ duction conditions and other low energy situations, the oxidation of amino acids increases whereby a deamination with KG or PY as amino receptors belongs to the starting processes (cf. FIG. 6); (iv) at the synthesis of collagen KG is essential to the hyd- roxylation of proline.

Pyruvate i s a centre of the metaboli sm (cf. FIG. 4) , where the following steps can be distinguished:

(i) the formation of lactate to provide the glucolysis with NAD during aerobic conditions; (ii) formation of acetyl-CoA for the synthesis of fatty acids or digestion in the citric acid cycle; (iii) a high concentration of acetyl-CoA increases the forma¬ tion of oxa loacetate , which either provides the citric acid cycle with intermediates (at a low concentration of ATP (adenosine t r i p hosphat e ) or becomes a substrate in the g I uc oneogenes i s (at high ATP concentrations);

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Civ) at the g luconeogenes s pyruvate might possibly contribute to the pentose-monophosphate shunt, where NADPK is formed (essential in several anabolic processes, i .a. the syn¬ thesis of the non-essential amino acids) and ribose-5- -phosphate, which is a substrate at the synthesis of nuc- leoti des; Cv) the formation of alanine as well as other non-essential amino acids where pyruvate contributes with the carbon skeleton .

Description of the present invention

It has now surprisingly been shown possible, with a maintained formation of proteins, to be able to reduce the excretion of nitrogen from animals and man by replacing a part of the prote- in of food and feedstuffs by means of the present invention. This is chara terized in that the ingoing protein is replaced by alpha keto glutaric acid; a combination of alpha keto gluta- ric acid and pyruvic acid; oxaloacetate or oxaloacetate in com¬ bination with alpha keto glutaric acid or pyruvate or another keto acid according to the formula R-C0-C00X, wherein R denotes a part of a non-essential amino acid and X denotes hydrogen, alkali metal, alkaline earth metal or organic base or non-toxic precursors or intermediates of these.

The keto acids above can generally be defined by the formula R- C0-C00X, wherein R denotes a part of a non-essent al amino acid and X denotes hydrogen, alkali metal, alkaline earth metal or - organic base or non-toxic precursors or intermediates of such keto acids. These compounds will in the following for the rea- son of simplicity be calLed keto acids. These keto acids are present in an amount of 1 to 20 % by weight, preferably 1 to 10 % by weight, of the dry substance of the ingoing proteins.

Further characteristi s will be evident from the accompanying cla s.

Administration can be made to carnivores, omnivores and herbi-

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vores to reduce, in applicable cases, the excretion of nitrogen but also to achieve a protein saving effect.

By means of the present invention thus substrate in the form of keto acids of non-essential amino acids is supplied for the formation of proteins or other biomolecules deriving from these amino acids. The keto acids, which can metabolize, during aero¬ bic conditions, in the mitochondria of the cells, -give raise to energy providing and reducing compounds. Both these types of compounds are essential in active life processes. In the mito¬ chondria intermediates are formed during the metabolism of keto acids, i .a. succ inyl-CoA . Succinyl-CoA is present in the syn¬ thesis of the porfyrine molecule in haemoglobin and myoglobin. When keto acids are added to the organism a protein saving ef- feet is obtained as the organism can use keto acids for non-es¬ sential amino acids in stead of using amino acids for other purposes than the formation of protein. Thereby the efficiency of the protein added increases. KG, one of the above described keto acids has a central position in the binding of NH, and later in the synthesis of non-essential amino acids. ^' Different keto acids of non-essential amino acids are furthermore a more effective substrate than the amino acids themselves as no de- amination needs to take place and the energy demanding excre¬ tion of nitrogen from the organism decreases. As evident from the Example 1 below, where fish spawns having obtained a feed¬ stuff being supplemented with 10 % keto acids, reduces the ex¬ cretion of nitrogen with more than 20 %.

The present invention can also be used for parenteral nutrition at catabolic situations, e.g. at large trauma, such as burn da¬ mages etc. but also at kidney damages.

During the - rep roduc t i ve phase when the fish mainly uti lizes its muscle proteins added keto acids in stead be utilized, e.g. py- ruvate, optionally in combination with KG. In that way the oth¬ erwise great loss of muscle protein can be reduced. Further¬ more, the excretion of nitrogen is reduced.

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The present invention will in the following be described more in detail with reference to the Examples given, however, with¬ out being restricted thereto.

Example 1

Growth test.

1-summer spawns of salmon were fed during 5 weeks with partly a control feedstuff intended for very young salmon, partly a feedstuff according to the present invention based upon the same raw materials as the control feedstuff but moreover con¬ taining 5 % of pyruvate Na-salt, and 5 % alpha keto glutaric acid (KG), whereby 10 % of the protein in the form of fish meal present in the control feedstuff had been replaced by keto ac¬ ids. The test was carried out in such a way that 500 spawns in one trough obtained the control feedstuff and 500 spawns in an¬ other trough obtained a feedstuff according to the present in¬ vention. The spawn of salmon had an average weight of about 4 g each at the beginning of the test. Growth, nitrogen excre-tion, total analysis and protein analysis were carried out. Macro¬ scopic investigation of the spawns was carried out as well.

Composition of the feedstuff ControI Test feedstuff Vitamin and mineral premix 2.0 % 2.0 %

Binding agent, IignosuIphate 2.0 % 2.0 %

Wheat, boiled in an autoclave 18.8 % 18.8 %

• Soya, finely ground 4.0 % 4.0 %

BLood meal 2.0 % 2.0 % Fish meal, whole meal 63.2 % 53.2 %

Lodda oil, lecithin 10.0 % 10.0 %

Alpha keto glutaric acid - 5.0 %

Na-pyruvate - 5.0 %

Total 100.0 % 100.0 %

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Feedstuff ana lysis

N-c ont ent

Cys t e i ne

Met h i on i ne Aεparginic acid

T hreoni ne

Ser ine

Glutamic acid

Pro I ine G lyci ne

Alanine

Va I i ne

Isoleucine

Leuc i ne Tyros ine

Phenyl alanine

Histidine

Lys i ne

Argl ni ne Hydroxyp ro I i ne

Sum

Ammon i a Total

The keto acids of the test feedstuff thus were 10 % of the feedstuff and substituted 20 % of the amino acids and thus re¬ duced the nitrogen content with about 16 %. Amino acid analyses of the feedstuff and the fish are carried out in accordance with official EEC-method.

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Sum 534.7 517.4

Ammon ; 8.2 7.3 Total 542.9 524.7

The results are mean values of three analyses based on 50 spawns each. The differences between the groups are non-sig- nifi cant.

Nitrogen excretion tests

In- order to determine the excretion of nitrogen 10 spawns of each group were allowed to live in a closed trough filled with oxygenised water. The spawns lived in this environment during 90 minutes, whereupon they were killed, weighed and dissected. All spawns had their stomachs as well as intestines well filled with feedstuff. The excretion of NH, and NH, were analysed in water samples from the two troughs in such a way that the time

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differences between feeding, ki lling and final sampling was uniform. Ammonia was analysed in accordance with Parsons et al, A Manual of Chemicaland Biologi cal Methods for Seawater Analy¬ sis, Pergamon Press, pages 15-16 ⁽ ISBN-0- 08- 030288-2) . The test whi ch was carried out as a final of an earlier growth test was carried out when the fishes were about 20 weeks of age. No dif¬ ference in mortality between control and test group could be noted during the added test periods.

Results

G roup of fishes Weight Excretion of nitrogen (g+SEM ⁾ C .ug NH*/g BW~ ¹ " 1 'hr ^"1 ')

Control feedstuff 3.75+_0.15 8.00+_0.69 Test feedstuff 3.77+0.21 5.71 +0.37

The difference in excretion of nitrogen between the test groups is 28% and statistical significance in accordance with students t-test i s n=20, p 0.01.

Macroscopic analysis of the spawns.

Dissection of the spawns showed no difference between the groups with regard to liver status, stomach -i nte st i na I fi lling and accumulation of visceral fat.

The spawns of the control group had at the beginning of the test an average weight of 3.40 g and at the end of the test an average weight of 3.95 g, whi le the group fed a feedstuff ac¬ cording to the present invention had an average weight of 3.56 g at ^' the beginning of the test, and 4.11 g at the end of the te ^' st. The growth was thus equal .

Example 2

In a test using recently veined rats according to Eggum (Eggum, B.O.; A Study of Certain Factors Influencing Protein Uti liza¬ tion in Rats and Pigs, National Institute of Animal Science, (1973), p.406) where a control feedstuff consisting of ce.reals,

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other carbohydrates, fish protein, fat, mineral, and vitamins was compared with a test feedstuff where 0.6 % by weight of py¬ ruvate and 0.8% by weight of a Ifa-ketog luta rate was allowed to replace 1.4% by weight of fish protein and where 0.15% by weight of soya oil was added to the test feedstuff in order to compensate for the difference in fat content appearing by the reduced content of fish protein, the following results were ob- tai ned :

Table 1

Cont roI group Test group

Fecal digestibility (TD), % 91.2 (1.6) 92.4 C1.0)

Biological value (BV), % 92.8 (1.1) 94.6 ^' (0.5)

Net pr ote in ut i l i za t i on ( NP U ) , % 84.7* (1.9) 87.4* (0.7)

G r ow t h , g r a m s 13.8 (2.3) 13.6 (1.7)

Protein of feedstuff, % DS 10.39 9.16

Standard deviation within parenthesis

* Significant difference according to Student's t-test p<0.02

The test, which was carried out in such a way that the total amount of feedstuff (g DS) was equal to the two groups of ani¬ mals, showed no significant growth differences in spite of the fact that 1.4 % by weight of the protein in the control feed¬ stuff had been replaced by keto acids.

The net protein utilization which shows the animals' possibi¬ lity of uti lizing the protein added for building up a protein of its own shows as evident from Table 1, significant differen¬ ces, mainly depending on better biological value (BV) of the test group. The protein improving effect is explained by the addition of, in this case, a combination of alfa-keto glutarate and pyruvate.

Macroscopical investigations of liver, kidneys, and other in¬ ternal organs showed, moreover, no differences between the

groups of animals.

At a composition of a feedstuff according to the present inven¬ tion where large parts of the protein have been replaced by keto acids in accordance with above, the amount of salt should be restricted in order to avoid e I ect ro lyt i c a I disturbances. Thus other salts than sodium salts of the keto acids might be present, such as potassium, magnesium, and calcium salts. Free acids may be used as well.

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