| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 5, 2005 |
| PATENT TITLE |
Protein hydrolysates |
| PATENT ABSTRACT |
The present invention describes protein hydrolysates: obtainable by the hydrolysis of a protein containing substrate; comprising free amino acids and peptides; and wherein the molar fraction of at least one free amino acid, present in the protein hydrolysate is at least a factor 2.5, preferably at least a factor 3, more preferably at least a factor 3.5 times higher than in a hydrolysate of the same protein containing substrate which has been completely hydrolysed to free amino acids, wherein the molar fraction of the at least one free amino acid in the protein hydrolysate is at least 25%; and wherein the Amino Acid Quotient (AAQ) in the protein hydrolysate is at least 10%. These protein hydrolysates can be used in the preparation of food compositions, wherein these protein hydrolysates provide for novel and unexpected flavours. Moreover these protein hydrolysates are applicable in personal care applications. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | October 16, 2003 |
| PATENT CT FILE DATE | October 17, 2001 |
| PATENT CT NUMBER | This data is not available for free |
| PATENT CT PUB NUMBER | This data is not available for free |
| PATENT CT PUB DATE | April 25, 2002 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Hurrell, R. F., "Maillard Reaction in Flavour" Chapter 6 In Food Flavours Part A: Introduction Morton, I. and Macleod, A. (eds.) Elsevier Scientific Publishing Company pp. 399-437 (1982). Minagawa, E. et al., "Debittering Mechanism in Bitter Peptides of Enzymatic Hydrolysates from Milk Casein by Aminopeptidase T" Journal of Food Science 54(5):1225-1229 (1989). Voigt, J. et al., "In Vitro Studies on the Proteolytic Formation of the Characteristic Aroma Precursors of Fermented Cocoa Seeds: The Significance of Endoprotease Specificity" Food Chemistry 51:7-14 (1994). |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A protein hydrolysate obtainable by the enzymatic hydrolysis of a protein-containing substrate; which hydrolysate comprises free amino acids and peptides; wherein the molar fraction of at least one free amino acid present in the protein hydrolysate is at least a factor 2.5 higher than in a hydrolysate of the same protein-containing substrate which has been completely hydrolyzed to free amino acids; and wherein the molar fraction of the at least one free amino acid in the protein hydrolysate is at least 25%; and wherein the Amino Acid Quotient (AAQ) in the protein hydrolysate is at least 10%. 2. A protein hydrolysate of claim 1, wherein the AAQ is from 15 to 50%. 3. A protein hydrolysate of claim 1, wherein the enzymatic hydrolysis is by at least one endoprotease and at least one exoprotease. 4. A protein hydrolysate of claim 1, wherein the at least one free amino acid is selected from Glu, Leu, Ile, Val, Phe, Tyr, Pro, Met, Lys, Arg, His, Gly, Ala, Ser and Thr. 5. A protein hydrolysate of claim 1, in the form of a concentrated liquid, a paste, a powder or a granulate. 6. A protein hydrolysate of claim 1, wherein the protein-containing substrate is selected from whey protein, hemoglobin, bovine serum albumin, casein, rice, gluten and soy protein. 7. A protein hydrolysate of claim 1, wherein the protein hydrolysate is capable of improving the flavour of a food composition. 8. A process for preparing a protein hydrolysate, which process comprises: incubating a protein-containing substrate under aqueous conditions at a temperature of 5 to 75.degree. C., and at a pH of 3 to 9, with at least one endoprotease and at least one exoprotease, for a time sufficient to obtain said protein-containing hydrolysate, wherein the molar fraction of at least one free amino acid present in the protein hydrolysate is at least a factor 2.5 higher than in a hydrolysate of the same protein containing substrate which has been completely hydrolyzed to free amino acids, and wherein the molar fraction of the at least one free amino acid in the protein hydrolysate is at least 25%; and wherein the Amino Acid Quotient (AAQ) in the protein hydrolysate is at least 10%. 9. A food composition comprising a protein hydrolysate of claim 1. 10. A food composition of claim 9, wherein the food composition is a fermented food product. 11. A food composition of claim 9, wherein the food composition comprises a reacted flavour. 12. A protein hydrolysate characterized in that it is either (a) a whey protein hydrolysate comprising a molar fraction of free Leu of at least 25%, or a molar fraction of free Lys of at least 25%; (b) a maize protein hydrolysate comprising a molar fraction of free Leu of at least 25%, (c) a soy protein hydrolysate comprising a molar fraction of free Arg of at least 25%, or a molar fraction of free Lys of at least 25%, (d) a BSA protein hydrolysate comprising a molar fraction of free Glu of at least 25%; or (e) a hemoglobin hydrolysate comprising a molar fraction of free Leu of at least 25%. 13. A process for preparing a fermented protein hydrolysate which comprises fermenting the protein hydrolysate of claim 1 with a food grade microorganism. 14. The protein hydrolysate of claim 4, wherein the at least one free amino acid is selected from Glu, Pro, Ala, Gly, Leu, Ile, Val, Met, Pro, Phe, Lys and Arg. 15. The protein hydrolysate of claim 14, wherein the at least one free amino acid is Glu, Leu, Pro, Phe, Lys or Arg. 16. The protein hydrolysate of claim 1, wherein the at least one free amino acid is at least a factor of 3 times higher than in a hydrolysate of the same protein-containing substrate which has been completely hydrolyzed to free amino acids. 17. The protein hydrolysate of claim 16, wherein the at least one free amino acid is at least a factor of 3.5 times higher than in a hydrolysate of the same protein-containing substrate which has been completely hydrolyzed to free amino acids. 18. The protein hydrolysate of claim 2, wherein the AAQ is 15-40%. 19. The fermented food product of claim 10, which is cheese and wherein the at least one free amino acid is Met. 20. The fermented food product of claim 10, which is fermented sausage and the at least one free amino acid is Leu or Phe. 21. The fermented food product of claim 10, which is beer and the at least one free amino acid is Leu, Val or Ile. 22. The fermented food product of claim 10, which is wine and the at least one free amino acid is Arg or Leu. 23. The fermented food product of claim 10, which is whiskey and the at least one free amino acid is Val. 24. A method to prepare a food composition which method comprises adding the protein hydrolysate of claim 1 to additional food components. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention describes protein hydrolysates and their use in food compositions. BACKGROUND OF THE INVENTION The present invention relates to protein hydrolysates and their use in foodstuffs. Hydrolysed proteins from a variety of sources are used widely in the food industry. For instance, they are commonly employed as a component in dehydrated soups, as flavourings and in other processed foodstuffs to obtain e.g. food flavourings after a Maillard reaction. They also find medical use as dietary supplements for patients suffering from a variety of diseases and metabolic disorders. Relatively new developments are their use in products for consumers with non-medical needs as athletes or people on a slimming diet and in personal care applications. Furthermore in situ hydrolysed proteins play an important role in the development of flavours in fermented food products. In the latter products the microbial starter cultures used usually excrete proteolytic enzymes responsible for hydrolysis of the raw material into amino acids. Metabolic transformation of these aminoacids leads to potent flavour compounds and volatiles characteristic for e.g. fermented dairy products such as cheese or yogurts, various meat products, beers and wines. Although conventional protein hydrolysates are prepared by subjecting the protein source to harsh chemical conditions, there has been an increased interest in obtaining such hydrolysates by enzymatic hydrolysis. Both the chemical and the enzymatic route aim to release high levels of amino acids from the protein source with maximum efficiency and lowest cost. For that reason cheaply available protein sources like soy meal and wheat gluten are popular substrates for preparing hydrolysates. To liberate as many amino acids as possible, the enzymatic route employs either complex mixtures of several endo- and exoproteases (e.g. International Patent Application WO94/25580) or it combines endoproteases with a single but broad spectrum exoprotease (International Patent Application WO-A-98/27827). In all cases the aim is to obtain a high degree of hydrolysis and an end product that contains a large variety of free amino acids. Though free amino acids as such, can elicit a number of taste impressions, these taste impressions are very basic (bitter, sweet, sour and "umami") and the amino acid concentration required for perceiving these tastes are high. Threshold values for individual amino acids can range from 0.3-80 millimoles/liter. Despite these high threshold values, free amino acids can create major sensory effects at much lower concentration ranges through a number of mechanisms. One of these mechanisms involves free glutamate and can create strong savoury enhancing effects because of the synergy between glutamate and 5'-ribonucleotides. If combined with proper concentrations of 5'-ribonucleotides such as 5'-IMP and 5'-GMP, the detection threshold of the umami taste generated by glutamate is known to be lowered by almost two orders of magnitude. Another flavour enhancing mechanism involves Maillard reactions. Compared with free amino acids, Maillard products in which free amino acids have been reacted with sugars exhibit much more impressive taste and odour characteristics. In Maillard reactions overwhelmingly complex flavour and odour systems can develop with threshold values that are several orders of magnitude lower than those recorded for the free amino acids. Maillard products are formed at elevated temperatures usually during cooking, baking or roasting when preparing food. During these treatments both colour and a large array of aromas develop. In these reactions amino groups react with reducing compounds as a first step and ultimately leading to a whole family of reaction pathways. In foods the amino compounds involved are predominantly free amino acids and proteins and the reducing compounds primarily represent reducing sugars. Factors that influence the Maillard reaction include the type of sugar and amino acid involved as well as physical factors such as the pH, temperature, water activity (aw), reaction time, and so on. Both mono as well as disaccharides can take part in the Maillard reaction. Generally speaking aldoses are more reactive than ketoses and pentoses more than hexoses or disaccharides, and so whereas the type of sugar strongly influences the amount of flavouring compounds generated, the amino acid involved in the reaction largely determines the nature of the flavour formed. For example, the inclusion of pure methionine in Maillard reaction systems often leads to vegetable or stewed notes, pure cysteine leads to meat-like flavours, pure proline, hydroxy proline and leucine to bakery aromas (R. F. Hurrell, Food Flavours, Part A: Introduction, Elsevier Scientific Publishing Company, Eds.: I. D. Morton and A. J. Macleod). Since these results have been obtained using pure amino acids rather than mixtures of several amino acids, as occur in food ingredients, it is evident that the outcome represents only a gross simplification of the natural situation. Likewise, the sugars that naturally occur in food will have an impact, and further complicate and affect the development of taste and aroma. Apart from Maillard reactions, amino acids can also undergo important chemical transitions at ambient temperatures. The latter type of transitions are enzyme dependent and are quite common in fermented foods such as beer, yogurt, cheese ripening and meat and wine maturation processes. In these fermentation processes, free amino acids are liberated from the raw materials used by proteolytic enzyme activity from either the raw material or the microbial starters used. During the maturation phase microbial metabolic activity then converts the free amino acids into derivatives with increased sensoric properties. For example, L-leucine, L-isoleucine and L-valine lead to the formation of valuable fusel alcohols like amylalcohols and isobutanol in beer fermentation. L-leucine is known as the precursor for cured meat compounds such as 3-methylbutanal and 3-methylbutanol, whereas L-phenylalanine can lead to benzacetaldehyde. Similarly cheese volatiles such as methanethiol and dimethyldisulphide have been traced back to the occurrence of methionine in cheese as well as methylpropanoic acid and methylpropanal to valine. Accordingly the "sur-lie" method used in wine making and known to generate tastier wines, can be ascribed to the increased presence of amino acids such as aspartic acid, arginine, alanine, leucine and lysine. Prior art processes for protein hydrolysis (WO94/25580 and WO98/27827) aim at releasing all available amino acids and the presence of so many different amino acids will blur the desired pronounced taste or aroma note in the final product. WO98/14599 refers to certain polypeptides obtained from Aspergillus oryzae and to hydrolysates prepared with these polypeptides in combination with (specific or unspecific) endopeptidases and (specific or unspecific) exopeptidases. WO98/14599 mentions hydrolysates that have an increased content of Leu, Gly, Ala and/or Pro, such as 1.1 times greater but uses for such hydrolysates are not mentioned. European patent application EP-A-799577, describes a whey protein hydrolysate wherein the Phe (phenylalanine) content is reduced. This whey protein hydrolysate is used as food for patients suffering from PKU (phenylketonuria). Voigt et al (Food Chemistry 51 (1994) pp. 7-14) describes the production of the cocoa-specific aroma precursors by in vitro proteolysis of seed proteins. Cocoa-specific aroma precursors can only be obtained by specific hydrolysis of only one substrate, which is cocoa vicillin-class globulin proteins. DESCRIPTION OF THE INVENTION According to the present invention, the desired flavour effect can be obtained if the molar fraction of a single desired free amino acid in the protein hydrolysate according to the invention is at least 2.5 times higher (enrichment factor) than would have been obtained by acid hydrolysis of the same protein containing substrate. It is demonstrated by the present invention that such protein hydrolysates can be obtained by certain combinations of enzymes, often with a selected protein containing substrate. The present invention provides amongst others for a protein hydrolysate: obtainable by the enzymatic hydrolysis of a protein containing substrate; comprising free amino acids and peptides; wherein the molar fraction of at least one free amino acid, present in the protein hydrolysate is at least a factor 2.5, preferably at least a factor 3, more preferably at least a factor 3.5 times higher than in a hydrolysate of the same protein containing substrate which has been completely hydrolysed to free amino acids, thus the enrichment factor is at least 2.5, preferably at least 3 and more preferably at least 3.5; wherein the molar fraction of the at least one free amino acid in the protein hydrolysate is at least 25%; and wherein the amino acid quotient (AAQ) in the protein hydrolysate is at least 10%. The invention further relates- to a food composition comprising a protein hydrolysate according to the invention. The present invention further relates to a process for preparing a protein hydrolysate, which process comprises: hydrolysing under aqueous conditions at a temperature of 5 to 75.degree. C., and at a pH of 3 to 9, a protein containing substrate with an endoprotease and an exoprotease, whereby the combined action of endoprotease and exoprotease releases at least one free amino acid from the protein containing substrate, and incubating the endoprotease and exoprotease for a period of time suitable to obtain a protein hydrolysate, wherein the molar fraction of at least one free amino acid present in the protein hydrolysate is at least a factor 2.5, preferably at least a factor 3, more preferably at least a factor 3.5 times higher than in a hydrolysate of the same protein containing substrate which has been completely hydrolysed to free amino acids; wherein the molar fraction of the at least one free amino acid in the protein hydrolysate is at least 25%; and wherein the amino acid quotient (AAQ) in the protein hydrolysate is at least 10%. Further the present invention relates to a protein hydrolysate characterized in that it is either: (a) a whey protein hydrolysate or corn protein hydrolysate comprising a molar fraction of free leucine of at least 25; (b) a whey protein hydrolysate comprising a molar fraction of free lysine of at least 25%; (c) a soy protein hydrolysate comprising a molar fraction of free arginine of at least 25%; (d) a soy protein hydrolysate comprising a molar fraction of free lysine of at least 25%; (e) a hemoglobin hydrolysate comprising a molar fraction of free leucine of at least 25%; or (f) a bovine serum albumin (BSA) hydrolysate fraction of free glutamate of at least 25%. In general the AAQ will be less than 40%, preferably the AAQ is between 15 and 30%. In general the molar fraction of the at least one amino acid is less than 80%, preferably this molar fraction is between 25 and 50%, more preferably between 25 and 40%. Preferably the hydrolysis of the protein containing substrate is an enzymatic hydrolysis, more preferably an enzymatic hydrolysis by an endoprotease and an exoprotease. We have found that food compositions comprising the protein hydrolysates according to the invention, obtain an improved flavour e.g. after it has been fermented, processed, cooked and/or reacted with reducing sugars. The molar fraction of a certain free amino acid in a composition is defined as the molar concentration of that free amino acid in the composition, divided by the sum of the molar concentrations of all free amino acids Alanine, Arginine, Asparagine, Aspartic acid, Glutamine, Glutamic acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tyrosine and Valine in that same composition, .times.100%. By total or all amino acids throughout this specification is meant the total of Alanine, Arginine, Asparagine, Aspartic acid, Glutamine, Glutamic acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tyrosine and Valine. The determination of the molar fraction of a certain free amino acid in the enzymatically hydrolysed compostion as well as of a certain free amino acid in a composition obtained by completely hydrolysing the protein containing substrate is described in the Materials and Methods section. Furthermore the total of the amounts of free Gln and Glu, and free Asn and Asp, respectively, which are liberated during enzymatic hydrolysis were taken to enable comparison with the amount of free Glu and Asp obtained after complete hydrolysis with strong acid (acid hydrolysis deamidates Gln and Asn residues, resulting in "additional" free Glu and free Asp). In general, for Glu and Gln, the sum of the molar fractions of Glu and Gln is at least a factor 2.5 (enrichment factor), preferably at least a factor 3, more preferably at least a factor 3.5 times higher than the sum of the molar fractions of Glu and Gln in a hydrolysate of the same protein containing substrate which has been completely hydrolysed to free amino acids. By the enrichment factor of an free amino acid is meant the molar fraction of the free amino acid present in a protein hydrolysate divided by the molar fraction of the free amino acid in protein hydrolysate which is completely hydrolysed to free amino acids. In case the Glu/Gln ratio is known, for example in case of single chain protein (see for instance Example 8) Glu and Gln can be calculated seperately. For Asp and Asn, the sum of the molar fractions of Asp and Asn is at least a factor 2.5, preferably at least a factor 3, more preferably at least a factor 3.5 times higher than the sum of the molar fractions of Asp and Asn in a hydrolysate of the same protein containing substrate which has been completely hydrolyzing a protein containing substrate to free amino acids. In the present invention the term "completely hydrolysed to free amino acids" or "completely hydrolyzing a protein containing substrate to free amino acids" refers to the acid hydrolysis of the protein containing substrate carried out according to the method of Waters (Milford Mass., USA), which method has been described in the Materials & Methods section . Protein hydrolysates according to the invention are characterised by the presence of certain free amino acids in relatively high molar fractions. Therefore the overall degree of hydrolysis of these protein hydrolysates is still limited. Throughout this patent application the Amino Acid Quotient (AAQ) is used to quantify a measure of degree of hydrolysis. AAQ is the total amount of free amino acids present in a hydrolysate obtained by enzymatically hydrolysing a protein containing substrate relative to the total amount of amino acids present in a hydrolysate obtained by completely hydrolyzing the protein containing substrate to free amino acids. AAQ is expressed as a percentage. AAQ can be calculated by dividing the sum of the molar concentrations of all free amino acids present in a hydrolysate by sum of the molar concentrations of all amino acids present in the hydrolysate when the protein containing substrate is completely hydrolysed to free amino acids (.times.100%). The protein hydrolysates of the invention may have AAQ's ranging from 10 to 50%, preferably from 10 to 40%. Preferred amino acids are those amino acids capable of generating desirable, food-compatible flavour (taste and aroma) compounds. For example it is known that upon heating with the appropriate sugar glycine can give rise to a beef broth like flavour profile, leucine to a chocolate or bread crust like flavour profile, phenylalanine to a chocolate or caramel like flavour profile, and lysine to potato or boiled meat like flavour profile. The protein hydrolysates enriched in a specific amino acid or set of amino acids according to the invention in combination with a selected sugar can be used to impart a specific flavour profile on a food or food ingredient. This specific flavour profile can be generated during the cooking, baking or roasting phase of the food preparation as occurs for example, during the baking of bread especially on the crust (wherein a protein hydrolysate of the invention has been added to the dough or on top of the dough). However, the protein hydrolysate and an appropriate sugar can also be pre-reacted (in the absence of the other food ingredients) to obtain a suitable flavouring ingredient. The conversion of free amino acids into flavour compounds by the action of microorganisms is considerably less well documented than conversions involving Maillard reactions. Although many amino acids have been indicated in the aroma development of fermented products like cheese, fermented sausages and beers, hydrophobic amino acids like valine, leucine, isoleucine and phenylalanine as well as sulfur containing amino acids like methionine are known to be of particular importance. During aging of wine the high levels of free arginine, lysine and alanine are particularly striking. So particularly Gly, Leu, Val, Pro, Phe, Met and Lys are compounds known to provide, upon heating with an appropriate sugar, desirable flavour compounds. Furthermore Val, Leu, Ile, Phe, Met, Arg, Lys and Ala, are described as amino acids which upon fermentation by the appropriate microorganism, can be converted in flavour compounds as well. So in a preferred embodiment of the invention, the free amino acid is selected from Glu, Leu, Ile, Val, Phe, Tyr, Pro, Met, Lys, Arg, His, Gly, Ala, Ser or Thr, more preferably the amino acid is selected from Glu, Pro, Ala, Gly, Leu, Ile, Val, Pro, Met, Phe, Lys or Arg, most preferably the amino acid is selected from Glu, Leu, Pro, Phe, Lys or Arg. Protein containing substrates which may be used according to the invention are protein containing materials suitable for human or animal consumption. Preferably the protein content of the protein containing material is substantial, which means that at least 20% w/w is protein. Preferably the protein containing substrate contains at least 40% protein, more preferably at least 50%, most preferably at least 70% proteinbased on weight/dry weight. Examples of protein containing substrates which may be used according to the invention include vegetable proteins such as soy protein, wheat gluten, rape seed protein, pea protein, alfalfa protein, sunflower protein, fabaceous bean protein, cotton or sesame seed protein, maize protein, barley protein, sorghum protein, potato protein, rice protein, coffee proteins, and animal derived protein such as milk protein (e.g. casein, whey protein), egg white, fish protein, meat protein including gelatin, collagen, blood protein (e.g. haemoglobin), hair, feathers and fish meal. Preferred protein containing substrates include whey protein, hemoglobin, gelatin, casein, maize protein, soy protein and gluten. Since the protein hydrolysates according to the invention are selectively enriched in specific amino acids, it is evident that protein containing substrates which also belong to the regular ingredients of the relevant end products (food compositions) are preferred. Additionally protein containing substrates which are exceptionally rich in specific amino acids are preferred sources for the protein hydrolysates. Typical examples of the latter protein containing substrates are whey protein (rich in Leu and Lys), wheat gluten (rich in Gln and Pro), chemically deamidated wheat gluten (rich in Glu and Pro), maize protein (rich in Leu and Pro), hemoglobin (rich in Leu, His and Val), fish concentrate or casein (rich in Met and Lys), peanut (rich in Arg), rice protein (rich in Phe and Arg) or protein fraction as for example the {character pullout}-lactabumine fraction of whey which is rich in tryptophane. The protein hydrolysates obtained may provide flavours to food compositions, and preferably these flavours are not associated with the protein containing substrate. Eg. a cocoa protein hydrolysate is expected to be able to provide a cocoa flavour, whereas it is not expected that a whey or rice protein hydrolysate may provide for a cocoa flavour. So preferably the protein hydrolysates according to the invention provide for novel and unexpected flavours that do not relate to the source of the protein containing substrate. For example, a whey protein hydrolysate provides eg. a meat, cheese or cocoa flavour, instead of a whey protein flavour. Protein hydrolysates according to the invention can be obtained by hydrolysing the protein containing substrate with suitable endo-proteases and exo-proteases. Whereas prior art enzymatic protein hydrolysis processes typically use crude mixtures of several endo and exoproteases with broad specificity (WO9425580; WO9827827), the hydrolysis process according to the present invention requires careful selection of combinations of endo and exo protease(s). The endo and exo proteases suitable for generating a protein hydrolysate by enzymatic hydrolysis of a protein containing substrate, which protein hydrolysate is at least 2.5 times enriched in certain amino acids compared with the acid hydrolysate of that protein containing substrate, preferably have a preference for cleaving adjacent to a certain or selected set of amino acid residues. Furthermore both the endo and exo proteases suitable for generating a protein hydrolysate by enzymatic hydrolysis of a protein substrate, which protein hydrolysate is at least 2.5 times enriched in certain amino acids compared with the acid hydrolysate of that protein containing substrate, preferably are pure, which means that the endo and/or exoprotease consists mainly, at least for 60%, preferably at least for 75%, more preferably at least for 90% and most preferably at least for 95%, of a single proteolytic activity. Depending on the degree of selectivity of the exopeptidase, a pure or less pure endoprotease is required. Depending on the degree of selectivity of the endoprotease, a pure or less pure exopeptidase is required. Selectivity of the endo and exo protease should at least overlap to obtain the desired protein hydrolysates. So for instance for obtaining a protein hydrolysate of which the free amino acid leucine is at least a factor 2.5 times greater than for the acid hydrolysate of the same protein containing substrate, endo and exoproteases that have some selectivity towards leucine are required. Proteases of high purity can for instance be obtained by purification of crude proteolytic enzyme preparations, or by producing the enzyme using overproducing recombinant DNA strains. The proteases suitable in the present invention are preferably recombinant and/or commercially available for food grade applications. Proteases produced using rDNA-techniques, that is cloning the gene encoding the proteolytic activity in a host organism that over-expresses this gene, usually provide for enzyme preparations comprising less contaminating enzymatic activities and thus may not require costly recovery steps. Well known host organisms that over-express cloned genes include yeasts, fungi or bacteria (for example Saccharomyces, Kluyveromyces, Aspergillus, Trichoderma, E. coli, Bacillus etc.) In order to obtain protein hydrolysates according to the invention, the protein containing substrate may be hydrolysed using a combination of an endo and an exoprotease, wherein at least one of the endo or exoprotease, preferably both the endo and exoprotease, are pure and selective towards a specific set of amino acid(s) or preferentially release the amino acid(s), which is/are intended to be enriched in the protein hydrolysate. Suitable endoproteases can originate from animal, plant or microbial material. They include recombinant enzymes, eg. enzymes obtained by genetic engineering techniques. Preferred selective endoproteases, which have a preferance for cleaving adjacent to certain amino acids, include trypsin (EC 3.4.21.4), elastase (EC 3.4.21.36), chymotrypsin (EC 3.4.21.1), thermolysin (EC 3.4.24.27), prolyl oligopeptidase (EC 3.4.21.26), glutamyl endopeptidase I (EC 3.4.21.19), microbial collagenase (EC 3.4.24.3), peptidyl-Asp metallopeptidase (EC 3.4.24.33), glycyl endopeptidase (EC 3.4.22.25), saccharolysin (EC 3.4.24.37), neutral protease (EC 3.4.24.28), streptogrisin B (EC 3.4.21.81), glutamyl endopeptidase 11 (EC 3.4.21.82), engineered proline-specific petidyl-prolyl cis-trans isomerases and enzymes with rennet-like specificity, for example microbial rennet, eg. Mucor pepsin (EC 3.4.23.23). Preferred non-selective endo proteases, which do not have a strong preference for cleaving adjacent to specific amino acids, but which cleave almost adjacent to a selected group of amino acids, include for instance subtilisin (EC 3.4.21.14) and papain (EC 3.4.22.2). Suitable exopeptidases (or exoproteases, the terms are interchangeable) can include carboxypeptidases and/or aminopeptidases. These exoenzymes can originate from animal, plant or microbial material. They include recombinant enzymes, eg. obtained by genetic engineering techniques. Preferred selective carboxypeptidases, which have a preference for cleaving adjacent to certain amino acids, include carboxypeptidase B (EC 3.4.17.2), CPD-1 (pep G) and CPD-II (pep F) from A. niger (Dal Degan, et al, Appl. Environ Microbiol, 58(7):2144-2152, 1992). Preferred non-selective carboxypeptidases, which do not have a strong preference for cleaving adjacent to certain amino acids, but which cleave almost adjacent to any amino acid residue include CPD-S.sub.1, from P. janthinellum and CPD-Y from S. cerevisae (Dal Degan, et al, Appl. Environ Microbial, 58(7):2144-2152, 1992). Preferred selective aminopeptidases, which have a preference for cleaving adjacent to certain amino acids, include prolyliminopeptidase (EC 3.4.11.5), bacterial leucyl aminopeptidase from Aeromonas proteolytica (EC 3.4.11.10) or leucyl aminopeptidase from Aspergillus species, and methionyl aminopeptidase (EC 3.4.11.18) and the phenylalanine specific aminopeptidases as described in EP 773990. Preferred non-selective aminopeptidases, which do not have a strong preference for cleaving adjacent to certain amino acids, but which cleave adjacent to almost any amino acid, include thermophilic aminopeptidase (EC 3.4.11.12). Preferred combinations of endo- and exoproteases include: (a) streptogrisin B or trypsin or papain endoprotease with CPD II (to release Arg or Lys); (b) chymotrypsin or thermolysin or neutral protease with CPD I (to release Tyr, Phe or Trp); (c) thermolysin or neutral protease with bacterial leucyl aminopeptidase or leucyl aminopeptidase from Aspergillus (to release Leu, Ile, Phe or Val); (d) neutral protease or subtilisin with CPD I (to release Phe or Ala); (e) elastase with CPD I (to release Ala); (f) rennet-like proteases with or leucyl aminopeptidase from Aspergillus or methionyl aminopeptidase (to release Met); and (g) engineered proline-specific peptidyl-prolyl cis trans isomerase (cyproase) with prolyl amino peptidase (to release Pro). (h) Proline specific endoprotease with malt enzymes or CPD-Y (to release Pro) (i) Glutamyl endopeptidase with CPD-1 (to release Glu) To obtain protein hydrolysates with amino acids in their most enriched form and with optimal taste characteristics the preferred combination of endo- and exoprotease could be combined with the appropriate or selected protein containing substrate. By incubating preferred selective enzyme combinations with protein containing substrates which are relatively rich in one or more desired amino acids, preferred enzyme-substrate combinations may be established. Preferred combinations of enzymes and substrates, to obtain enriched protein hydrolysates, include: (a) whey protein with trypsin or papain plus a carboxy peptidase CPD II (to enrich in Lys or Arg), or whey protein with thermolysin plus an Aeromonas proteolytica aminopeptidase or other leucyl aminopeptidase (to enrich in Leu); (b) maize protein with thermolysin plus a leucyl aminopeptidase (to enrich in Leu) (c) rice protein with thermolysin plus leucyl aminopeptidase from Aspergillus (to release Phe and Leu); and (d) gluten with engineered proline-specific peptidyl-prolyl cis trans isomerase (cyproase) and prolyl amino peptidase (to release Pro) (e) corn protein with thermolysin plus a leucine aminopeptidase (to enrich in Leu) (f) soy protein isolate with trypsin or papain plus a carboxy peptidase CPD II (to enrich in Arg and Lys). (g) BSA with glutamyl endopeptidase and CPD-I (to release Glu) (h) Hemoglobin with thermolysin plus leucyl aminopeptidase (to enrich in Leu) Combinations of substrate and enzymes (endo and exo proteases) can for example result in the following protein hydrolysates: a whey protein hydrolysate comprising a molar fraction of free leucine of at least 25%, preferably at least 27%, more preferably at least 30%, or a molar fraction of free lysine of, preferably at least 25%, preferably at least 35%, more preferably at least 40%; a corn protein hydrolysate comprising a molar fraction of free leucine of at least 25%, preferably at least 30%, more preferably at least 35%; a soy protein hydrolysate comprising a molar fraction of free arginine of at least 25%, preferably at least 30%, or a molar fraction of free lysine of at least 25%; BSA hydrolysate comprising a molar fraction of free glutamate of at least 25%; a hemoglobin hydrolysate comprising a molar fraction of free leucine of at least 25%, preferably at least 30%, more preferably at least 35%. The protein hydrolysates according to the present invention may be prepared by incubating the protein containing substrates with the appropriate enzymes, endo- and exoprotease, under pH and temperature conditions suitable for protein hydrolysis. Suitable pH and temperatures depend on the proteases optimum conditions which may vary from about pH of 3 to 9 and temperature from about 5 to 75.degree. C. Exceptionally conditions outside these ranges may be optimal for the enzymes. Enzymes and conditions for protein hydrolysis are selected to obtain the desired protein hydrolysate of the invention. E.g. when it is desirable to obtain a whey protein hydrolysate enriched in leucine, then a whey protein substrate is selected and hydrolysed with leucine specific endo and exoproteases under conditions suitable for the endo and exoproteases to specifically release the leucine from whey protein. E.g. whey protein is hydrolysed for five hours with thermolysin and Aeromonas proteolytica leucyl aminopeptidase at a pH of 8 at 40.degree. C., to obtain a whey protein hydrolysate wherein the molar fraction of free leucine present in the protein hydrolysate is at least a factor 2.5 times higher than in a hydrolysate of the same whey protein substrate which has been completely hydrolysed to free amino acids. To obtain for comparison a whey protein hydrolysate which has been completely hydrolysed to free aminoacids, whey protein was acid hydrolysed according to Waters (Milford Mass., USA; see Materials & Method section). To obtain a protein hydrolysate according to the invention, the protein containing substrate is added to water to obtain an aqueous suspension. The protein containing substrate can be added in an amount ranging from 1 to 18%, preferably from 3 to 15%, more preferably from 5 to 13% dry weight protein containing substrate/total weight suspension. The amount may depend on the solubility of the protein containing substrate in water. The endo and exoprotease may already be present in the water wherein the protein containing substrate is added, or the endo and exoprotease can be added after the aqueous suspension of the protein containing substrate has been prepared. Protein containing substrate, endo and exoprotease may be incubated together, or the exoprotease may be incubated after incubation of the protein containing substrate with the endoprotease, wherein optionally the endoprotease has been inactivated before the exoprotease incubation has been started. Conditions of temperature, pH and the like are preferably met before the enzymes are added to the water or suspension. Once the enzymes (endo and exoprotease) and the protein containing substrate are in direct contact, the protein hydrolysis starts. During the protein hydrolysis, the pH may or may not be regulated at a constant pH. If the pH is regulated at a constant pH (or at a pH being kept in a prescribed range), the pH may be adjusted e.g. by addition of any food compatible acid or alkali. For instance sodium or potassium hydroxide may be used as food compatible alkali, and hydrochloric acid may be used as food compatible acid. Once the desired protein hydrolysate has been obtained in the aqueous suspension, preferably the protein hydrolysis is stopped by inactivating the enzymes present. Inactivation may be effected by lowering the pH to below 5 or increasing the pH to above 8 in combination with heating the suspension above at least 70.degree. C., preferably at least 90.degree. C. Exceptionally conditions outside these ranges may be required to inactivate the proteases. However inactivation should not affect the amino acids and peptides present in the aqueous suspension. A person skilled in the art is able to select the best conditions for inactivating the proteases. The aqueous suspension comprising the protein hydrolysate according to the invention preferably will be further treated in order to obtain a protein concentrate in the form of for example a powder or paste. The aqueous suspension comprising the protein hydrolysate may e.g. be centrifuged and/or (ultra) filtrated, then concentrated by e.g. evaporation, and optionally dried in any convenient way, such as spraydrying, freeze-drying, fluidised-bed treatment, or a combination of these methods. The person skilled in the art will understand that the method chosen will depend on the formulation of the product (and it's further use). E.g. if the protein hydrolysate is intended to be combined with a specific sugar to induce a specific reaction product upon heating, then the protein hydrolysate will not be exposed to harsh conditions that would induce unwanted (premature) Maillard reactions. If the protein hydrolysate is intended to be used in a food fermentation process, then the conditions used for recovery of the protein hydrolysate should not affect the free amino acids that upon fermentation will generate the desired flavours. The final protein hydrolysate product is preferably formulated in a concentrated form such as for example a concentrated lquid, a paste, a powder or a granulate. The concentrated product comprises at least 20% dry matter (weight dry matter/total weight), preferably at least 30% dry matter (weight dry matter/total weight), more preferably at least 40% dry matter (weight dry matter/total weight. A granulate or powder comprises at least 80% dry matter (weight dry matter/total weight), preferably at least 90% dry matter (weight dry matter/total weight). Alternatively the aqueous suspension obtained after protein hydrolysis comprising the protein hydrolysate may be directly used in the preparation of reacted flavours or fermented flavours. For instance in the preparation of a reacted flavour, the aqueous suspension comprising the protein hydrolysate e.g. after centrifugation, or concentration may be supplemented with a sugar and thereupon heated. After the reaction, a reacted flavour composition can be obtained, e.g. by concentrating and/or drying of the heated product. In the preparation of a fermented flavour, the aqueous suspension comprising the protein hydrolysate e.g. after centrifugation, or concentration may be incubated with a food grade microorganism, under conditions suitable for the microorganism to ferment the protein hydrolysate. After fermentation, a fermented flavour may be obtained, e.g. by concentrating and/or drying of the fermented product. Protein hydrolysates comprising free amino acids and peptides obtained after enzymatic hydrolysis of a protein containing substrate, using endoprotease and exoprotease, wherein the molar fraction of at least one free amino acid present in the protein hydrolysate is at least a factor 2.5, preferably at least a factor 3, more preferably at least a factor 3.5 times higher than in a hydrolysate of the same protein containing substrate which has been completely hydrolysed to free amino acids, can be used in several applications including foodstuffs. The protein hydrolysates of the invention can for instance be used in cosmetic formulations for treating hair or skin or can be added to sport drinks to enhance physical endurance recovery. Particularly the protein hydrolysates according to the invention are suitable for use in the preparation of foods, fermented foods and fermented food flavours, and for use in the preparation of reacted flavour compositions, wherein the protein hydrolysate is reacted with a sugar to obtain a reacted flavour composition. Remarkable and outstanding flavour characteristics can be obtained when the protein hydrolysates of the invention are used in the above food preparations. So a protein hydrolysate according to the invention is preferably capable of improving the flavour of a food composition. The protein hydrolysate of the invention may be used in the preparation of fermented food ingredients or products as described above. Fermented food ingredients or products are prepared by at least one fermentation step, wherein either enzymes endogenous to the food treated are actively involved or food grade microorganisms are incubated with a foodstuff to obtain the fermented foodstuff. Examples of fermented foodstuffs are for instance meat products such as hams or sausages or yogurt, cheese, beer, whiskey, wine and champagne. Fermented food flavours, are flavours obtained from a fermented foodstuff. These fermented food flavours can be obtained by incubating the protein hydrolysate of the invention with a food grade microorganism, which will ferment the protein hydrolysate to desirable food flavours. A process for producing a fermented food product, wherein a protein hydrolysate according to the invention has been used, will involve the addition of the protein hydrolysate before, or during the fermentation of that fermented food product. The product's endogenous enzymes or the fermenting organisms will convert the specific amino acid in additional flavours characteristic for this type of fermented food. Also flavours not-characteristic for this type of fermented food products may be produced in this way, resulting in fermented food products with surprising new flavours The fermented food product can be for example cheese and the free amino acid of which the molar fraction is at least 2.5 times greater than of the acid hydrolysate is Met, Leu or Phe. In this case the amino acid enriched protein hydrolysate may be added to the milk just before or during the milk clotting process. To minimize the losses of the free amino acids present, the hydrolysate may be added somewhat later in the production process. For example in the production of cheddar cheese the hydrolysate may be added directly together with the salt to the chipped curd. Similarly the protein hydrolysates according to the invention may be incorporated into Enzyme Modified Cheeses to further accelerate the formation of natural cheese aroma's. For the improvement of such dairy products the protein hydrolysate is preferably derived from milk proteins such as casein or whey. The fermented food product may be beer and the free amino acid of which the molar fraction is at least 2.5 times higher than of the acid hydrolysate is Leu, Ile or Val. In this case the protein hydrolysate may be obtained from rice or barley or corn protein (e.g. for lager beers to improve the aroma profiles of the final product) or it may be obtained from wheat gluten (e.g. for wheat beers to improve the aroma profile of the final product). In the production of beers, the amino acid enriched protein hydrolysates may be added just after the whirlpool or centrifuge and upstream of the vessel where the fermentation by the brewer's yeast takes place. The fermented food product may also be a fermented or cured meat product for which the protein hydrolysate may be obtained from a meat or blood protein and the free amino acid of which the molar fraction is at least 2.5 times higher than the acid hydrolysate may be Leu, Ile, Phe, Lys, His, Pro or Gly. With fermented meat products the protein hydrolysate may be incorporated together with the other compounds like salt and dextrose or with the microbial starter culture. With cured meat products the protein hydrolysate may be used to produce a "brew" by dissolving the hydrolysate together with appropriate quantities of glucose, salt and nitrite. Inoculation with a suitable mixture of acid and aroma forming microorganisms such as Lactobacilli and Staphylococcus carnosus generates a aromatic brew after a few days of incubation at temperatures between 10 and 22.degree. C. Upon the addition of diphosphate to neutralize the acid pH value, the brew may be injected into the meat, e.g. a ham. Cooking of the meat, preferably in a bag to keep the aroma's in, finally results in a safe and tasteful product. To stimulate the formation of volatile flavour compounds from the amino acid enriched protein hydrolysate, suitable microbial starter strains are indispensable. The protein hydrolysate according to the invention may also be used in combination with nitrite salt to prepare dry cured ham. The protein hydrolysate of the invention may be used in the preparation of reacted flavours. The protein hydrolysate and a sugar comprising composition are then reacted to obtain the reacted flavour composition. Sugars may e.g. be selected from aldoses and ketoses as well as disaccharides such as xylose, xylulose, glucose, fructose, maltose, sucrose or mixtures thereof. The protein hydrolysate and at least one reducing sugar are heated to start a series of reactions known as the Maillard reactions. Amino groups (particularly of free amino acids) react with reducing compounds as a first step. A whole family of other reaction pathways will follow, and finally results in a (complex) reacted flavour composition. By the use of the protein hydrolysate according to the invention, novel types of flavours can be obtained. These (reacted) flavours can be added to foodstuffs to improve the flavour of foodstuffs. To prepare the reacted flavour the amino acid enriched protein hydrolysate and the desired sugar are dissolved in water in an appropriate ratio and then heated. Upon the dissipation of all water the heating process may be stopped immediately or may be continued to reach temperatures of around 120.degree. C. or even 180.degree. C. The latter incubation conditions lead to vastly different flavour and aroma profiles. Ultimately the dry, reacted product may be recovered as a powder and used as a flavouring ingredient. Alternatively protein hydrolysate and sugar may be reacted in mixtures containing water and e.g. oil or fat, or in the total absence of water e.g. by dissolving sugar and hydrolysate in an essentially water-free system such as for example a polyalcohol. Advantage of this last approach is that even at temperatures above 100.degree. C. the reaction takes place in a liquid and also the final product is a liquid which facilitates the dosing of the flavouring ingredient. Materials and Methods Isolated soy protein was obtained as Soyamin 90 HV from Lucas Meyer (Hamburg, Germany) Corn gluten (Maize gluten) was obtained as Maisin 13875 from Cerestar (Krefeld, Germany). Whey protein was obtained as either WPC 80 or WPC 75 from Havero Hoogwegt (Gorinchem, the Netherlands). Hemoglobin was obtained as HGP po-feed (approx. 90% protein) from Harimex (Loenen, The Netherlands). Bovine serum albumine was obtained as 98% pure Fraction V powder from Sigma-Aldrich. K. lactis (ATCC 8585) was obtained from ATCC: American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA ; Bitec LS-25 Plus starterculture contains a mixture of lactobacilli and Staphylococcus camosus and is commercially available from Gewurzmuller; Doetinchem, The Netherlands. Yeast used in bread baking experiments was obtained as fresh block yeast from DSM Gist, Delft, The Netherlands. Cheese starter culture DS 5 LT1 was also obtained from DSM Gist. Enzymes were either purchased and used as such or obtained as laboratory samples with or without additional purification steps. Aeromonas aminopeptidase and thermolysin were purchased from Sigma. The Aeromonas aminopeptidase as purchased had an activity of 50-150 (Sigma) units per mg protein. A stock solution was prepared containing 15.3 mg solids (as obtained) per ml water, which corresponds with an activity of 630 Sigma units/ml. The thermolysin as purchased had an activity of 50-100 (Sigma) units per mg protein. A stock solution was prepared containing 50 mg solids (as obtained) per ml water which corresponds with an activity of 2200 Sigma units/ml. Trypsin (200 FIP-U/g) was purchased from Merck (Darmstadt, Germany) and was dosed as the dry powder. Thermoase C160 (thermolysin) (lot nr.P9EB761) 1,650,000 PU (protease units)/gram was obtained from Daiwa Kasei K K (Osaka, Japan). Corolase LAP (leucyl aminopeptidase from Aspergillus) (batch 1999-07-20) with an activity of 350,000 LAP units/gram was obtained from Rohm Enzyme (Darmstadt, Germany). Collupuline Liquid (papaine) with an activity of approx. 5000 NF/milligram was obtained from DSM Gist (Seclin, France). Glutamylendopeptidase from B. intermedius was isolated from a B. subtilis culture as described by Shevelev, A. B. et al in Plasmid (2000), 43(3), 190-199. The activity of the purified enzyme towards Z-Glu-pNA under the described conditions was approx. 1 unit per milligram. Carboxypeptidase CPD-II (PepF) was obtained from the culture filtrate of an overproducing A. niger strain containing multiple copies of the pepF gene. Using the pepF sequence information as published (van den Hombergh et al. (1994) Gene 151, 73-79) oligonucleotide primers were designed that directly fuse the A. niger glucoamylase (glaA) promoter plus 5'-noncoding sequences, to the pepF structural gene from the ATG startcodon until the TAA stopcodon, using PCR reactions known to persons skilled in the art. Analogous examples of fusions of structural genes to the glucoamylase promoter have been described (EP-A-0 420 358, EP-A-0 463 706 and WO 99/38956). First, the pepF structural gene was PCR amplified from A. niger GAM4 (CBS513.88) chromosomal DNA and purified. Second, the promoter region of the glaA gene was PCR amplified using, at the 3'-end, a primer that overlaps the 5'end of the pepF structural gene. Third, the two PCR fragments were fused via fusion-PCR with a oligonucleotide primer 5' of the glaA promoter, and a oligonucleotide primer overlapping the stopcodon of pepF in the reverse direction. Fourth, the resulting fusion fragment was cloned in A. niger expression vector pGBTOP8 (WO 99/38956), resulting in a fusion plasmid containing the glaA promoter, PepF structural gene and the glaA terminator. This plasmid was digested with NotI and co-transformed with pGBBMS-1, digested with XhoI, to A. niger GAM4 (CBS513.88), essentially as described in WO 99/38956. Transformants selected for growth on acetamide plates were analysed using colony PCR to check for the presence of the pepF expression cassette, using known techniques. A. niger PepF transformants were cultivated in shake flask using the method as described previously (WO 99/38956). After growth for 6 days at 34.degree. C., the culture was ultrafiltrated to remove the mycelium. The CPD II enzyme was purified using a simplified version of the method described by Dal Degan et al (Appl. Environ. Microbiol. 58 (7) pp 2144-2152 (1992)). After dilution with an adequate quantity of buffer A, the ultrafiltrate was applied on a Q Sepharose FF column in an Akta system. Buffer A was 50 mM phosphate buffer pH 6.0 and buffer B 50 mM phosphate pH 6.0 containing 1 M NaCl. Elution was performed with a gradient from 10% buffer B (in buffer A) to 50% buffer B (in buffer A). Active fractions were pooled on the basis of enzymatic activity towards the synthetic peptide FA-Ala-Lys-OH (Bachem) at pH 4.1. The activity of the final, substantially purified enzyme was 1176 U/ml and assayed on 5 mM FA-Ala-Lys-OH in 50 mM sodium acetate/1 mM EDTA pH 4.1. The rate of hydrolysis was was measured at 331 nm during minutes at 25 degrees C. One unit is defined as 1 micromol of substrate cleaved per minute. Carboxypeptidase CPD-1 (PepG) was isolated from a culture broth of A. niger also according to F. Dal Degan et al.(Appl. Environ. Microbiol., 58 (7), pp. 2144-2152 (1992) with the exception that the CABS-Sepharose step was omitted. The activity of the final, substantially purified enzyme was established to be 150 units/ml on FA-Phe-Ala-OH (Bachem) at pH 4.5 and 25 degrees C. using the activity measurement protocol for this enzyme as provided. Aminopeptidase II from Bacillus stearothermophilus (strain NCIMB 8924) was isolated according to the procedure described by Stoll et al., (BBA 438 (1976) 212-220). The purity of the enzyme was tested by gel electrophoresis under native and denaturing (SDS) conditions. The identity of the enzyme isolated was confirmed by Edman degradation of 9 amino acids of the aminoterminal end of the enzyme. The enzyme was activated by the addition of CoCl2. Using the Lowry reagent the protein concentration of the final enzyme solution was estimated to be 7.2 mg protein per ml. Using leucine-p-nitroanilide as the substrate, the exopeptidase activity of this solution was established as 420 units per ml. The activity test was carried out at pH 7.2 with 3 millimol per liter of substrate for 15 minutes at 25.degree. C., and the absorption was measured at 400 nanometer against a blank. Determination of the Molar Concentrations of the Free Amino Acids in a Hydrolysate. The enzyme incubations with the various protein substrates were carried out with shaking under pH and temperature conditions specified in the Examples. Incubations were terminated after the indicated time intervals by centrifugation at maximum allowable speed for 15 minutes in either an Eppendorf centrifuge or in an Hereaus Megafuge 3.0 R to remove any non dissolved protein substrate. Subsequently the pH of the clear supernatant was adjusted and then heated at 95.degree. C. to destroy any residual proteolytic activity. Any additional precipitate formed was removed by another centrifugation. After removal, the clear supernatant was kept frozen until amino acid analysis could be carried out. Alternatively the clear supernatant was lyophilized to enable further testing in application experiments. Amino acid analysis was carried out on the clear supernatant according to the PicoTag method as specified in the operators manual of the Amino Acid Analysis System of Waters (Milford Mass., USA). Amino acid analysis took place immediately after thawing the sample material. To that end a suitable sample was obtained from the molten liquid added to dilute acid and homogenized. From the latter solution a new sample was taken, dried and derivatised using phenylisothiocyanate. The various derivatised amino acids present were quantitated using HPLC methods. Determination of the Molar Concentrations of the Free Amino Acids in a Hydrolysate which is Completely Hydrolyzed to Free Amino Acids. Acid hydrolysis of the protein hydrolysates to obtain free amino acids, was achieved by vapour phase hydrolysis over 6 N HCl, also according to Waters. In brief this procedure is the following. A sample of the clear protein containing supernatant obtained after enzyme hydrolysis is homogenized in a dilute HCl solution. The resulting solution is then subjected to a vapour phase hydrolysis according to Waters. After this acid hydrolysis the amino acids are derivatised and analysed according to the Picotag method (see above). Since during acid hydrolysis Trp and Cys are destroyed, these amino acids are not included in the data presented. However, Gln and Asn residues are converted into Glu and Asp during acid hydrolysis so that the values for Glu and Gln, and for Asp and Asn were usually summed together to allow comparison with the data obtained before acid hydrolysis. Only in Example 8 a different procedure was followed |
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