NO312081B1 - Formulated proteinaceous feed and use of such feed - Google Patents

Description

This invention relates to a formulated proteinaceous lining, containing an additive consisting of a precisely measured, controllable amount of protease inhibitor.

This feed, which is generally intended for both animals, birds and fish, can in particular take the form of a particle/pellet/flake-shaped main feed for farmed fish.

The invention also relates to the use of such a feed for a special purpose when feeding farmed fish in cages or land-based farming vessels.

When feeding with protein-containing formulated feed, the protein is poorly utilized because the feed is digested faster than the animal, bird or fish is able to make use of the protein. A significant part of the forage's protein content therefore goes to energy instead of muscle building.

The state of the art is represented, among other things, by raw materials that are distinguished by the fact that the protein has different digestion rates.

Such known precursors have a very limited effect when it comes to implementing an increased utilization of protein so that a larger part of the protein in the feed contributes to muscle building and thus growth. In the case of known techniques, the choice of raw material and the production process are complicated.

Modern fish feed consists of finely ground, heat-treated raw materials that are "glued" together using starch, steam and pressure. The protein in such a lining is finely particulate and denatured, which means that the molecule's tertiary structure, its original unique form, has been opened up and changed.

In the fish's natural food, which is live, whole prey, on the other hand, the protein is present intact in its native, unique form bound up in large organs, for example muscles. The digestive processes of these two forms of protein differ relatively greatly from each other, as the speed through the gastrointestinal tract increases proportionally with the degree of processing without affecting the protein's total digestibility.

A protein's native unique form is such that protein-digesting enzymes (proteolytic enzymes) can hardly attack it. All deviations from the one native biological form lead to the emergence of new attack points for the proteases, which explains the much faster breakdown of denatured protein compared to native protein, and the subsequent rapid uptake of amino acids.

In the case of too high a supply of amino acids over a short period of time to the animal's protein synthesis apparatus, the biochemical processes that take place in fish will cause a greater proportion of the digested amino acids to be burned as energy instead of being deposited as fish muscle.

There are reports from the literature of almost twice as high a protein yield in the form of higher deposition as muscle protein when feeding with relatively large pieces of fish compared to feeding with finely ground raw materials.

In modern aquaculture, however, the use of formulated for sole-prevailing practical considerations.

The state of the art is likewise represented by EP-A2-144993. This published European patent application relates to a method for promoting growth in animals or plants, and consists in assigning to the animal, optionally via the feed, or to the plant, optionally via its place of growth, an effective amount of a protease inhibitor such as leupeptin.

Leupeptin, which is a low molecular weight synthetic serine/cysteine protease inhibitor, is preferably an endopeptidase inhibitor with particular affinity towards trypsin and certain cysteine proteases such as papain and cathepsin B. These cysteine proteases are also not central digestive enzymes. Leupeptin also has no effect against chymotrypsin, which is the most central digestive protease.

As examples of animal species and plant species that have been tested using this known method to accelerate their

growth rate, are baby hamsters and small pea plants. These are of course only examples of possible animals and plants that can be treated and positively influenced, but mammals, as in this case, are far away from farmed fish that are preferred

ket animal species in the present invention.

From this known publication, EP-A2-144993, it is known per se to add a protease inhibitor to animal feed, which has been known within the industry. However, in EP-A2-144993 allocation of an effective amount of protease inhibitor is mentioned, and a preferred allocation amount of this inhibitor per kilogram of animal weight is further specified, more precisely equal to

about. 0.002-0.05 grams per day. The use of more than one protease inhibitor (ie in a mixture with one or more other protease inhibitors) is not mentioned in this publication.

In accordance with the present invention, it is recognized that there is a need to increase the utilization of the pre-protein in formulated for, and according to the present invention it is aimed to take the necessary measures and to provide an additive for formulated for the realization of this general purpose.

The protein molecule consists of hundreds to many thousands of amino acids linked together in a chain. Such chains of amino acids are further folded or packed together into completely unique three-dimensional structures, depending on their amino acid sequence. During digestion, this three-dimensional structure is first disturbed by the acidic liquid in the stomach, after which the chain is broken up by proteolytic enzymes and the individual amino acids are released. Some amino acids will exist as di- and tripeptides, but most will appear in free form. This breakdown of the protein takes place with the help of a number of protein-cleaving enzymes. These enzymes can be divided into two main groups: endopeptidases and exopeptidases. The exopeptidases attack inside the peptide chain and split the protein molecule into smaller chains, while the endopeptidases cleave one amino acid at a time from the end of the amino acid chain. For effective digestion, both of these enzyme groups must be present.

Examples of exopeptidase are trypsin and chymotrypsin, while examples of endopeptidase are carboxypeptidase A and B.

More specifically, the purpose of the present invention has therefore been to arrive at a lining which favors the utilization of the protein in the lining, that is to say promotes increased growth per amount of protein measured by normal feeding, feed quantity and feeding frequency.

It is also intended to designate an application for such a formulated for the purpose of realizing a special purpose.

According to the invention, said purpose is realized by formulating the lining in accordance with patent claim 1, with patent claim 9 indicating a special application for such a lining.

In order to increase the utilization of the pre-protein, it is desirable to change the absorption time for amino acids from the intestine. According to the invention, this can be done by blocking part of the protein-digesting enzymes (proteases) found in the intestine.

In accordance with the invention, a formulation distinguishes itself by containing an additive consisting of a mixture of different protease inhibitors, at least one of which shows affinity towards central digestive enzymes, with the intention of giving the feed growth-accelerating properties.

It is known in and of itself that protease inhibitors can appear in certain precursors, but then unintentionally, appearing in completely uncontrolled quantities. An example is the so-called "soy-trypsin" inhibitor found in soybeans. These are actually two inhibitors, namely Bowman-Brink and Kunitz trypsin inhibitor, which are two inhibitors that inhibit the same enzyme, namely trypsin. This soy trypsin inhibitor has, according to several reports, been considered undesirable due to its negative effect as it inhibits only one enzyme.

Wood to formulated to add to a mixture protease inhibitors/inhibitors (enzyme regulators) which can be extracted from vegetable substances, such as plants, for example from potatoes or soy or which can be chemically produced, for example camostat, or in various combinations of these, by adding to the feed a mixture of several different protease inhibitors in very small, precisely controlled quantities - a significant improvement in protein utilization and growth has, however, been shown to occur.

The active substance (protease inhibitor mixture) can, for example, amount to 0.001 - 0.2% calculated on the finished product.

A lining according to the invention is thus distinguished primarily by the fact that it contains an additive consisting of a carefully measured mixture of various protease inhibitors in an amount that is very small in relation to the rest of the weight of the formulated lining.

The protease inhibitors can advantageously be added to the feed in concentrated form. A significant effect that is an inherent property of protease inhibitors that include a concentrated inhibitor isolated or concentrated from potato juice is that

the mixture inhibits both endogenous and exogenous proteases.

The aforementioned percentage share/addition amount of protease inhibitors can be based on a protein source number

> 2. In the case of 3 protein sources, the contribution from binders is included.

In five test series carried out in accordance with the present invention, a mixture containing several different protease-inhibiting components was used as an additive to an ordinarily formulated fish feed. The protease inhibitor mixture was added to the feed in concentrated form, among other things within the aforementioned quantity range of 0.001 - 0.2%.

In these five series of experiments, an attempt was made to map:

Whether the additive inhibited the enzymatic activity in a fish intestine (on salmon);

To ascertain the optimal addition level of the protease inhibitor mixture when feeding salmon;

The digestibility of main nutrients using protease inhibitor mixture in feed for salmon;

The effect of using a protease inhibitor in combination with different protein sources in salmon feed;

The course of uptake of amino acids by means of protease inhibitor mixture.

The results were as follows:

Investigations in vitro showed that a reversible partial inhibition of the enzyme activity could be achieved in an enzyme extract isolated from salmon intestine. This indicates that a temporary immobilization of endo- and exopeptidases occurs when the formulated feed is added to a protease inhibitor mixture according to the invention;

Dose response experiments showed that at an addition level, a growth-promoting effect was achieved by adding the protease inhibitor mixture according to the invention, without any negative impact on the pre-utilization (pre-factor) or total digestion of the protein being found;

Dose response experiments also showed that above a certain mixing level for the protease inhibitor mixture there was a clear reduction in protein digestibility measured as nitrogen digestibility. No effect on the digestibility of fat was recorded;

When using an optimal protease inhibitor concentration, a clear increase in growth was achieved based on formulated, fishmeal-based fish feed compared to the use of identical feed without the addition according to the invention. By replacing some of the fish meal component with hydrolysed protein (fish silage), growth was further enhanced; (Amount of hydrolysed protein added in a for can be measured as TNBS with a binding of free alpha amino groups to trinitrobenzo-sulphonic acid, L-leucine is used as standard.) The TNBS value should lie between 0.4 and 2.0.

In studies of the uptake into the blood of <14>C-labeled intact protein from the intestine, a changed uptake was found when the concentrated protease inhibitor mixture according to the invention was added to the feed.

Subsequent tables illustrate relationships between the addition of protease inhibitor mixtures to those formulated for:

Table 1 illustrates the increase in % daily growth when using a fishmeal-based for added protease inhibitor mixture according to the invention. The increase is 13%.

Table 2 illustrates the increase in % daily growth when some of the fishmeal in the feed is replaced with hydrolysed protein (fish silage). The increase is 32%.

Table 3 graphically illustrates the nitrogen digestion (ordinate) in % as a function of added amount of protease inhibitor mixture (in grams per kg for), and illustrates how nitrogen or protein digestion is affected by increasing the addition of protease inhibitor mixture according to the invention.

It is concluded that results obtained by adding a mixture of different protease inhibitors in the form of endo- and exopeptidase inhibitors to a formulated for will, as intended, cause a reversible inhibition of the protein digestion enzymes. In the case of too high additions of inhibitor mixture, this will exceed the total protein digestibility. With an optimal mixing of the inhibitor mixture, a growth gain is achieved which is enhanced by the use of several protein sources with different absorption processes from the fish intestine.

Uptake studies of isotopically labeled amino acids indicate that there is a combination of increased enzyme production as compensation for the inhibition of some digestive enzymes in the intestine, together with a lifting of the enzyme blockage as a result of the reversible bond between enzyme and inhibitor. This gives a different uptake pattern for amino acids when using a protease inhibitor mixture added to the feed.

Claims (9)

1. Formulated proteinaceous feed, containing an additive consisting of a precisely measured, controllable amount of protease inhibitor, characterized in that the feed contains an additive consisting of a mixture of different protease inhibitors, at least one of which exhibits affinity towards central digestive enzymes, with the intention of providing the feed with growth-accelerating properties. 2. Formulated protein-containing feed as stated in claim 1, characterized in that the feed contains an additive consisting of a mixture of endo- and exo-protease inhibitors. 3. Formulated protein-containing lining as stated in claim 1 or 2, characterized in that each of the protease inhibitors included in the mixture added to the lining was in concentrated form at the time of the mixture. 4. Formulated proteinaceous lining as stated in one or more of the preceding claims, characterized in that the mixture of protease inhibitors added to the lining in concentrated form constitutes between 0.001 and 0.2% of the lining's weight. 5. Formulated protein-containing lining as stated in one or more of the preceding claims, characterized in that one of said various protease inhibitors in the mixture added to the lining is produced on a vegetable basis, for example potato pulp. 6. Formulated protein-containing lining as stated in one or more of the preceding claims, characterized in that one of said various protease inhibitors in the mixture added to the lining is chemically produced, for example camostat. 7. Formulated proteinaceous lining as stated in one or more of the preceding claims, characterized by one or more of the mixture of different protease inhibitors added to the lining being selected with a view to their ability to inhibit both endo- and exoproteases. 8. Formulated protein-containing food as stated in one or more of the preceding claims, characterized in that the protein content essentially consists of fishmeal and hydrolysed protein, where the TNBS value is between 0.4 and 2.0. 9. Use of formulated protein-containing feed as specified in one or more of the preceding claims as main feed when feeding monogastric animals, for example salmon and trout, with the aim, through the addition of the mixture of various protease inhibitors in concentrated form, to achieve an increased utilization of the proteins in the feed, so that a larger proportion of the digested amino acids are deposited as muscle protein while a smaller proportion is burned as energy.