The present invention relates to a method for the preparation of an amino acids-containing solution, as well as to a solution obtained by using such a method. The present invention furthermore relates to the special use of the amino acids-containing solution.

US patent No. 4,292, 334 relates to a process for treating animal feathers so as to provide a food product for animals or a nitrogen source for fertilizer. The method that is known therefrom comprises feeding the feathers into a pressure-resistant vessel, heating the vessel for 2 to 10 minutes under an elevated pressure in the presence of saturated steam at a temperature of 180-240 °C, and subsequently rapidly releasing the overpressure to obtain a product having an structure that readily enables crushing.

US patent No. 4,172, 073 relates to a method for the preparation of water-soluble and edible keratinaceous protein, such as animal hairs, feathers, wool and the like. According to the method that is known therefrom, the starting material keratin is hydrolysed in a medium consisting of saturated steam and from about 5 to about 30 parts of liquid water, which hydrolysing step is carried out under pressure and at an elevated temperature in the absence of added chemicals, such as acids and bases. The preparation time as used in the examples ranges from about 10 minutes to about 30 minutes.

International application WO 98/36782 relates to a bone substitute material based on a porous polymer material,, the surface of which is coated with peptides having an RGD amino acid sequence. The use of such a surface coating is claimed to provide an excellent bio- compatibility.

Japanese patent publication JP 09 009880 relates to protein-rich food products prepared through enzymatic hydrolysis of bones, with the possible addition of tissue and blood from animals.

International application WO 82/00084 relates to a method and apparatus for the hydrolysis of keratinaceous materials, for example feathers, wherein the process is carried out in two separate reactors, viz. a first, non-pressurized reactor and a second, pressurized reactor.

In the first reactor a temperature of less than 100 °C prevails, and steam having a temperature of 130-199 °C is injected into the jacket of the reactor. In the second reactor, in which the hydrolysis is effected, a temperature of 135-166 °C prevails, and the residence time is about 10 to 20 minutes.

International application WO 89/11797 relates to a method and apparatus for continuous hydrolysis of keratinaceous materials, for example feathers, in which said hydrolysis takes place by heating the material by directly injecting steam, wherein the hydrolysis could be completed in about 10 minutes, using a pressure of 10 bar and a temperature of 185 °C.

From British patent No. 828,978 there is known a method for the preparation of esters of amino acids and of peptides, comprising heating a mixture containing a protein, an organic sulphonic acid and an alcohol having at least one hydrogen atom attached to a carbon atom bearing a hydroxyl group, wherein even insoluble proteins, such as feathers, are made soluble in alcohol by such a treatment. By heating the aforesaid mixture, the protein molecules are split up into smaller fragments, with the carboxyl groups being esterified simultaneously therewith. The acid used for the hydrolysis can be removed from the hydrolysis mixture thus esterified, and the amino esters and peptide esters are separated by means of organic solvents. A drawback of such a method is the use of additional chemicals.

Feathers are an important waste product in the poultry industry, in which connection it can be noted that during the period 1997/1998 more than 4 million tonnes of feathers were available worldwide. Said feathers consist of 99% structural proteins, keratins.

After hydrolysis of said proteins, feather meal is obtained, which can be used as an additive in cattle feed.

From Japanese patent publication JP 61040753 there is thus known a method for the preparation of feather meal, wherein feathers having a water content of about 70 wt. % are collected from confined poultry farms and subsequently immersed in hot water. After said immersion, the mixture thus obtained is heat-treated at about 100-110 C. The feathers are softened by such a treatment and simultaneously kneaded and dehydrated to a water content of less than 40 wt. % water by compression. Following that, the dehydrated feathers are heated at 3-4 kg/cm2 pressure at 140-150'C, as a result of which protein is hydrolysed. Subsequently, the end product is dried and ground.

From Japanese patent publication JP 60083542 there is furthermore known a method for the preparation of a high-quality feather meal, wherein the feathers are fed to a closed autoclave, which autoclave comprises a jacket into which hot steam is introduced. The water present in the feathers evaporates as steam and the feathers are hydrolysed by the steam. In this state, an animal or vegetable oil at normal temperature is fed to the autoclave, dispersed in a misty state, after which the high-temperature steam in the autoclave is cooled, condensed, liquefied, and the pressure in the autoclave is reduced. The oil heated by heat exchange with the steam will penetrate the feather tissue and displace the remaining water in the feather tissue, as a result of which the feathers are dried to obtain the desired feather meal. A drawback of such a method is that additional chemicals are required, which is undesirable in practice.

A generally known method for extracting keratins from feathers is to treat the feathers in a slightly alkalic environment by means of 2-mercapto-ethanol for reducing the disulphide bonds and concentrated aqueous ureum solutions for denaturing the keratins. The conditions for extracting up to 75% of the keratins from the feathers comprise a reaction time of 30 minutes at 40 °C, a pH-value of 7.0- 9.0, a ureum content of 5 M or higher and an excess of 2-mercapto-ethanol of ten times the number of disulphide bonds in the keratins. A drawback of such a method is the additional treatment with chemicals.

One aspect of the present invention is to provide a method for the preparation of an amino acids-containing solution, which method does not exhibit the above-mentioned drawbacks of the prior art.

Another aspect of the present invention is to provide a method for the preparation of an amino acids-containing solution, which method can be carried out in relatively mild conditions, with short, soluble peptides being obtained.

Yet another aspect of the present invention is to provide a method for the preparation of an amino acids-containing solution, which method allows the occurrence of side reactions, in particular the dissolution of amino acids, to a limited degree.

Yet another aspect of the present invention is to provide a special use of the amino acids-containing solution, which use has a significant commercial value.

The method as referred to in the introduction is characterized in that hydrogen-bonded biopolymers as the starting material are transferred to a closed vessel, after which the closed vessel is heated to a temperature of at least 180 oC in the presence of water for at least 30 minutes.

One or more of the aforesaid aspects of the present invention are realised by using the present method. In order to obtain an amino acids-containing solution comprising short peptides it is necessary

to maintain the temperature at a level of at least 180 °C for at least 30 minutes. When a lower temperature is used, no hydrolysis will take place, or only to a very small extent. Consequently it is preferable to use a temperature of at least 180 °C, in particular 200 °C, more in particular 220 °C.

Proteins mainly consist of amino acids, and in most embodiments the three-dimensional crystal structure is obtained on account of the hydrogen bond between the molecular chains. The hydrogen bond in proteins, biopolymers in general, is strongly influenced by the pH-value of the solution. The amino acid groups along the molecular chain can function both as hydrogen bond acceptors and donors over a specific range of pH-values and as acceptors or donors (but not both functions) at other pH-values. Water is an active participant in molecular interactions in proteins. Just like the biopolymers, water is a polar molecule, and the water molecules exhibit a high degree of affinity to each other.

Because of its polarity and the hydrogen-bonding capacity, water is a molecule that exhibits a strong interaction with the biopolymers. The consequence is that water weakens the electrostatic and hydrogen-bonding interactions between other molecules. Thus, water is a very effective competitor in said polar interactions. By using the present method the proteins are dissolved, hydrolysed in water.

Preferably, the amount of hydrogen-bonded biopolymers is 5-20 wt. %, based on the total amount of mixture of hydrogen-bonded biopolymers and water. If the amount of hydrogen-bonded biopolymers is less than 5 wt. %, the obtained amino acids-containing solution will contain a low concentration of amino acids, which is undesirable in practice. If on the other hand the amount of hydrogen-bonded biopolymers is more than 20 wt. %, the conversion of proteins in the starting material will be insufficient.

The hydrogen-bonded biopolymers used in the present invention are selected from the group consisting of collagen, silk and

proteins of natural origin, in which connection in particular feathers can be mentioned as proteins of natural origin.

To obtain a solution containing amino acids comprising 5 to 6 monomeric units in length, it is-desirable to effect partial dissolution/hydrolysis by heating the proteins in the presence of water at a temperature ranging from 200 °C to 220 °C.

The dissolution of proteins under relatively mild temperature and pressure conditions in the presence of an environmentally-friendly solvent, such as water, provides a new application in the field of coating and cellular growth. Thus it is possible to increase the biocompatibility of the coated surface with the specific donor of the proteins, such as collagen. The conversion of feathers into amino acids by dissolution thus provides a fully and environmentally-friendly recycling process of waste from the poultry industry.

The present invention furthermore relates to an amino-acids containing solution obtained by using the method as described above, in which connection it is in particular preferable for the amino acids to comprise 5 to 7 monomeric units in length.

The present invention furthermore relates to the use of the amino acids-containing solution as described above, in particular as a starting material for animal feed, as a coating for human implants, as a starting material for the dissociation of amino acids into smaller fragments in the presence of enzymes, which smaller monomeric units are in particular suitable for use in the cosmetic industry.

The present invention will be explained in more detail hereinafter by means of an example and comparative examples, in which connection it should be noted, however, that the present invention is by no means limited to such special examples.

Example 1.

An amount of chicken feathers not having undergone any pretreatment was transferred to a thick-walled, closable vessel, and an excess of water in an amount of about 5-10% dry matter of feathers/water was added. After being closed, the vessel was heated to a temperature of about 210 °C for a period of one and a half hours. After cooling, the vessel was opened and a clear or slightly turbid liquid was obtained.

Upon chemical analysis it appeared that the solution contained short, soluble peptides having a length of 2-5 amino acids.

Comparative Example 1.

The same operations as in the above example were carried out, except for the fact that the heating step was carried out at a temperature of 150 °C. The end product obtained was feather meal, a digestible protein product, which was not soluble in water, however.

Comparative Example 2.

The same operations as described in Comparative Example 1 were carried out, except for the fact that the heating step was carried out at a temperature of 100 °C. No effect on the feathers was observed upon opening the autoclave.

From the above experimental results it can be concluded that, using the correct process conditions, the larger part of the processed feathers has gone into solution. In addition, a so-called PKL- test (average peptide chain length, using the reagent TNBS) has shown that the amino acids are present in the form of small peptides).

Examples 2-12 and Comparative Examples 3-5.

An amount of chicken feathers was cleaned with water and dried, after which the chicken feathers thus pretreated were cut into small parts and weighed. Four metal reactors having an internal volume of 14 ml were filled with the feathers thus pretreated, with the addition of distilled water. The reactors thus filled were placed in a preheated oven for a particular period of time. After having stored in the preheated

oven for the desired period of time, the reactor was removed and cooled under running water. The cooled reactor was opened and the remaining residue, viz. undissolved chicken feathers, was filtered, dried at 80 °C and weighed. The measuring results are shown in the table below.

Table. Example Time (minutes) Starting weight (mg) Weight of residu (mg) reaction temperature 220 °C 2 30 24.03 21.20 3 60 31.6 6.2 4 90 37.6 0.4 5 120 27.79 0.4 reaction temperature 200 °C 6 30 51.76 45.7 7 60 42.47 26.8 8 90 55.77 6.7 9 120 22.64 2.1 reaction temperature 180 °C *) 3 30 19. 75 19.5 10 60 21.44 18.2 11 90 24.25 10.4 12 120 22.06 5.2 reaction temperature 160 °C *) 4 120 25. 05 23.8 *) 5 90 22.8 22.5 *) Comparative Example

From the above table it appears that a residence time of 30 minutes at a reaction temperature of 180 °C had practically no effect on the starting material, because the weight of the residue is about the same as the starting weight of the chicken feathers (see Comparative Example 3). If the reaction time is prolonged from a period of 30 minutes to a period of 60 minutes, using the same temperature conditions, a decrease of the weight of the residue can be observed (see Example 10), which decrease continues when the residence time in the reactor is further prolonged to a period of 90 minutes (Example 11) and a period of 120 minutes (Example 12), respectively. Increasing the temperature to e. g. 200 °C has an advantageous effect on the weight of the residue (see Examples 6-9). When the reaction temperature was further increased to 220 °C, hardly any residue was observed when using reaction times of e. g. 90 minutes (Example 4) and 120 minutes (Example 5).