The present invention relates to a protein hydrolysate derived from animal tissue having an endothelial or mucosal component (hereafter "mucosa"), a process for its preparation, and the use of such a protein hydrolysate.
Protein hydrolysate by definition consists of a mixture of amino acids and short chain peptides obtained by the hydrolysis of various animal and vegetable proteins. Protein hydrolysates (also known as "peptones") are used as sources of amino acids, seasoning agents and in nutrition, among other things.
For both economic and environmental reasons, productive use is now being made of an increasing percentage of the waste material generated as a result of the slaughter of animals, such as livestock. A major use of livestock waste or other by-products (livestock "offal") is in the production of the blood anti-coagulant heparin.
It has been estimated that over 90% of the heparin currently used as a blood anti-coagulant is obtained from porcine intestinal mucosa. An aqueous solution containing the mucosa from the livestock offal is chemically (acid or alkaline) or enzymatically hydrolyzed, and the heparin is extracted from the hydrolyzed mucosa by well-established techniques, such as selective sorption using an ion exchange resin. The solution containing the digested tissues includes high concentrations of salt. This high concentration of salts in the digest solution prevents constituents other than certain anionic and polyanionic materials (such as heparin) from sticking to the resin during the sorption of these materials. Although the cost of such resin can be high, an advantage of this process is that it requires only a minimal amount of resin, since only enough resin is required to selectively remove the desired anionic or polyanionic materials from the digest solution.
The mucosa and the digest solution also generally contain an additional salt component. This additional salt component is introduced into the solution in the form of an oxygen scavenger, bacteriostat or bacteriocide, typically sodium bisulfite, added to stabilize the raw material and to prevent bacterial growth.
The high residual concentration of salt in the digest solution renders the un-sorbed portion of the digest, which includes most of the proteins, largely useless for most practical purposes. These salt and sulfite levels not only make this protein sidestream inedible, but also potentially toxic for prolonged usage such as in agriculture as a source of nitrogen. The sidestream may also be toxic to those animals or humans allergic to sulfites.
The heparin-depleted digest solution is typically discarded or spread on farm land, since cost-effective ways have not been found to separate the organic components in the solution from the dissolved salt. The discarded solution, however, includes many high quality proteins in the form of protein hydrolysate. Rather than discarding these proteins, it is desirable to utilize them as a source of protein for human or animal nutrition. Additional use can be made of the proteins in microbial nutrition, such as vat fermentation.
Waste disposal costs of solutions of animal waste products continue to increase, and environmental regulations govern the manner in which high BOD materials, such as the heparin-depleted digest, may be disposed. The costs associated with disposal add not only to the cost of processing the livestock, but also to the cost of the heparin produced by this process.
Protein hydrolysates may be produced by either chemical or enzymatic methods. In acid hydrolysis, strong acids at elevated temperatures are used to break the glycosidic bond in the protein molecule. This relatively harsh treatment can result in damage to the heparin as well as some loss of essential amino acids. The treatment can also result in undesirable side reactions. Similarly, alkaline hydrolysis requires fairly extreme conditions for producing this reaction. Furthermore, the large amount of residual acid or alkali in the hydrolysate must be neutralized. This neutralization increases the salt content of the hydrolysate, and thereby further limits its potential use in nutritional formulations seeking minimum salt content.
Enzyme hydrolysis is an effective alternative to chemical treatment. This process is mild in comparison to acid or alkali hydrolysis. Additionally, the inherent specificity of several proteolytic enzymes can control the nature and extent of hydrolysis, and thus the functional properties of the end product. An important use of enzymatically hydrolyzed proteins is in human nutrition. Additionally, the proteins may be used in medical nutrition for undernourished persons, or those persons unable to properly digest and absorb whole protein. For example, it has been postulated that in cases of severe pancreatic insufficiency or malabsorption, that amino acids are better absorbed from hydrolyzed protein than from intact protein.
An initial source of pre-digested protein was milk, which has drawbacks such as poor palatability and high cost. Recently, individual crystalline amino acids have been formulated to mimic the amino acid profile of the protein hydrolysate obtained by hydrolysis of casein. Medical studies, however, have shown that di- and tripeptides such as can be produced by protein hydrolysis, are absorbed through the intestinal mucosa more effectively than the individual crystalline amino acids. Aside from the potential danger of allergic reactions to such crystalline amino acids, often produced by fermentation, such protein hydrolysate formulations are extremely expensive and out of reach for the world population at large.
Nutritional uses of the protein hydrolysate of the present invention include such specialty feeds as milk replacers for calf, piglet and other weaning mammals; protein extender for animal feed; and as an amino acid supplement, flavor or protein enhancer for human food and pet food. Research has shown that the high ash in peptone, or hydrolysate, obtained by traditional processes, significantly depresses appetite and weight gain at moderate inclusion rates. See, e.g. Journal of Dairy science, 75(1): 267; 1992, incorporated herein by reference. Medicine to which this invention may be applied includes total parenteral nutrition, peritoneal dialysis fluid as an alternative to glucose, and as a protein extender in enteral nutrition. Additional use may be found in microbial nutrition for vat fermentation.
British Patent 992,201 describes the conventional procedure for producing heparin described above. The procedure involves the addition of cross-linked copolymers with quaternary functional groups to a heparin-containing digest, using an alkali, alkali earth metal, or ammonium salt as a catalyst. At least 0.1 mole of a dissolved salt must be present. The recommended salt is sodium chloride. This patent focuses on the isolation of heparin and certain other anionic and polyanionic impurities from the remaining constituents in the digest. The isolation of protein hydrolysates, in a contaminated form in the digest, is not addressed in this patent. The examples in the patent teach a 0.5 molar salt concentration to accomplish the separation of the heparin, representing the midpoint in a claimed range of 0.1-1.0 molar.
After the anion exchange resin-heparin copolymer is harvested according to the procedure described in the British patent, the resulting protein-containing sidestream contains not only sodium chloride as a contaminant, but also the original sodium metabisulfite stabilizer, and the various biological materials (including protein hydrolysate) present in the raw materials that are not sorbed by the resin. These impurities left in the protein mother liquor after the harvesting of the resin-heparin copolymer render it largely useless for most practical applications.
Another commercial process presently in use for the production of heparin is based on the purification procedure described in British Patent 889,648. This process consists of treating a heparin-containing digest, which has previously been filtered to clarity, with 2 to 5% of salt and sufficient water soluble quaternary ammonium salt to selectively precipitate substantially all the heparin, but insufficient to precipitate other animal components. The protein sidestream of this process also contains a high percentage of salt. Furthermore, most quaternary ammonium salts are effective bacteriocides thus rendering any resulting protein unsuitable for fermentation. Process waste waters containing quaternary ammonium salts also have an adverse effect on municipal sewage operations.
A common thread of the two processes described above is that only limited use may be made of the heparin-depleted protein sidestream due to its high residual salt content. A need exists for a process for treating animal tissue, and particularly livestock offal, that minimizes the waste of that tissue and enables beneficial use to be made of the protein hydrolysate that may be derived from the tissue.