BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is broadly concerned with processes for utilizing the enzymatic activity remaining in peptone solutions for carrying out various hydrolysis processes, as well as preserved mucosa tissue products and methods for preserving these products. More particularly, the mucosa tissue is hydrolyzed by conventional processes with an excess quantity of enzymes to yield a product comprising heparin (which is preferably extracted) and a peptone solution. A protein-containing material (e.g., soybeans, animal liver) is then hydrolyzed using the enzymatic activity remaining in the peptone solution. In another embodiment, the mucosa tissue is preserved by mixing the tissue with a preserving agent selected from the group consisting of hydrogen peroxide and phosphoric acid to yield a preserved product. The mucosa product preserved by hydrogen peroxide has a low ash count and reduced odor.
2. Description of the Prior Art
Peptone is a hydrolysate mixture derived from the mucosa tissue of swine, cattle, and other animals. Peptone is produced by hydrolyzing mucosa tissue with proteolytic enzymes to produce a digest solution containing, among other things, protein hydrolysates (i.e., peptone) and heparin. The heparin, which has great commercial value, is then extracted from the digest solution, typically by anion exchange resins. These processes and the resulting products have been described in U.S. Pat. No. 5,607,840 and GB 992,201, incorporated by reference herein.
When hydrolyzing mucosa tissue, large quantities of proteolytic enzymes are used in order to increase the heparin yield. For example, during mucosa hydrolysis, proteolytic enzymes are typically added at a rate of about 1-1.5 g of enzyme per kg of mucosa tissue. This is equivalent to about 13-20 g of enzyme per kg of protein present in the tissue as compared with about 0.2-3 g of enzyme per kg of protein utilized in other hydrolysis processes. Such large quantities of enzymes increase the costs of carrying out these processes and results in a substantial amount of enzymatic activity remaining after these processes have concluded. This activity is then deactivated by lowering the pH of the product (which is reversible) or by irreversibly denaturing the enzyme with heat. There is a need for a process which would provide commercially viable uses for this excess enzymatic activity.
There are numerous protein-containing materials which are hydrolyzed for various purposes which could benefit from this excess enzymatic activity. For example, soybeans are an inexpensive source of essential amino acids. However, anti-nutritional factors such as protease inhibitors and antigenicity are also present in soybeans. These factors can be substantially reduced by hydrolyzing the soybean proteins. Similarly, whey proteins are an important source of proteins for young animals, but whey proteins have a drawback in that they may cause allergic reactions. This risk of reaction can be reduced by hydrolyzing the whey proteins. Finally, enzymatic hydrolysis processes have other useful purposes, including reducing the viscosity of blood products, increasing the bioavailability of feed-grade meat isolates, and increasing the quantity of soluble materials which can be extracted from by-products such as fish offal.
On a dry basis, typical peptone derived from heparin production processes includes about 50-55% by weight crude protein and about 20-30% by weight ash. Due to this protein content, peptone is commonly used as a nutritional supplement for animals. However, currently available peptone suffers from two disadvantages—a high ash level and an unappealing flavor. High ash contents in peptone have been shown to adversely affect animal weight gain (see e.g., Journal of Dairy Science, Vol. 75(1):267 (1992)). These high levels are predominantly caused by the current mucosa preservation methods wherein sodium metabisulfite or calcium propionate is added to the mucosa product or wherein the pH of the product is lowered. There is a need for a mucosa preservation method which is effective yet does not generate high levels of ash in the resulting peptone solution, thus reducing the unappealing flavor in peptone.
SUMMARY OF THE INVENTION
The instant invention overcomes the problems of the prior art by providing improved mucosa tissue preservation methods as well as methods of utilizing the enzymatic activity remaining in the peptone solution after mucosa hydrolysis and heparin extraction.
Broadly, the enzymatic activity in peptone solution can be utilized to hydrolyze other protein-containing materials, either as the sole source of enzymatic activity or in conjunction with another enzyme for either supplementing the available activity or hydrolyzing a constituent other than protein (e.g., fat). These processes comprise first hydrolyzing a quantity of mucosa tissue (which inherently contains proteins) with at least one proteolytic enzyme so as to yield a hydrolyzed product which includes heparin and peptone. Preferably the heparin is then extracted by conventional techniques (e.g., by anion exchange resins) leaving the peptone solution which includes hydrolysates, salts, and possibly phosphorous. While in prior art processes the enzymatic activity of the peptone solution is deactivated by the application of heat, this is not necessary with the inventive processes. Rather it is preferred that the remaining enzymatic activity in the peptone solution be used for further hydrolysis processes.
In order to be commercially worthwhile, the peptone solution should retain at least about 30%, preferably at least about 40%, and more preferably at least about 50% of the enzymatic activity of the starting proteolytic enzymes used to hydrolyze the mucosa as determined by the enzymatic assay utilized by the manufacturer of the enzyme. This solution can then be used to hydrolyze other proteins by simply contacting the peptone solution with the proteins under the appropriate hydrolyzing conditions so as to yield a final hydrolyzed product. The peptone solution and proteins or protein-containing material should be mixed in quantities such that the level of peptone solution utilized is less than about 50% by weight on a solids basis, and preferably from about 15 to 30% by weight on a solids basis, based upon the total solids weight of both the protein or protein-containing material and the peptone solution taken as 100% by weight. Examples of protein-containing materials which can be hydrolyzed with the enzymatic activity remaining in a peptone solution include animal liver, animal viscera, wheat, soybeans, products comprising blood, whey products, animal offal, meat isolates and mixtures of the foregoing. The quantity of peptone solution utilized depends upon the enzymatic activity, product combination, nutritional factors, and other factors.
While the above processes utilize the peptone solution remaining immediately after heparin formation and extraction, it will be appreciated that these processes will also work on peptone solution purchased from peptone manufacturers provided the enzymatic activity in the solution was deactivated by pH adjustment rather than by the irreversible denaturation of the enzymes by heat. In these instances, it would first be necessary to reactivate the enzymatic activity by adjusting the pH of the acidic peptone solution to at least about 6.5, and preferably at least about 7.5. The pH-adjusted peptone solution would then simply be contacted with the proteins or protein-containing material as described above.
Furthermore, while the foregoing processes were described with respect to peptone solutions hydrolyzed from mucosa, other hydrolyzation products can also be used so long as they are obtained by processes using an excess quantity of proteolytic enzymes (e.g., at least about 10 g of enzyme per kg of protein). The remainder of the process would be carried out as described above.
In another embodiment, mucosa tissue can be preserved by contacting the tissue with a preserving agent selected from the group consisting of hydrogen peroxide and phosphoric acid. In applications where phosphoric acid is utilized as the preserving agent, the preservation process is carried out by simply mixing the phosphoric acid with the mucosa tissue in sufficient quantities to maintain the tissue pH at a level of about 2-4, and preferably about 2.5-3. The mixing is carried out by mechanical agitation and under ambient conditions. The use of phosphoric acid has the benefit that it can eliminate the need for supplemental dietary phosphorous by increasing the phosphorous content in the mucosa tissue product, and thus in the resulting peptone solution, to a level of about 4% by weight on a dry basis.
In applications where hydrogen peroxide is utilized as the preserving agent, it is preferably to first heat (such as by direct steam injection) the mucosa tissue to a temperature of from about 50-105° C., and preferably from about 65-75° C. prior to mixing the hydrogen peroxide with the mucosa tissue. Only very small quantities of hydrogen peroxide are required to preserve the mucosa tissue. Therefore, the hydrogen peroxide should be mixed at a level of less than about 1% by weight hydrogen peroxide, and preferably less than about 0.5% by weight hydrogen peroxide, based upon the total weight of the mucosa tissue taken as 100% by weight.
The quantity of hydrogen peroxide remaining in the final preserved product, without any treatment to remove excess hydrogen peroxide, is less than about 0.04% by weight, and preferably less than about 0.01% by weight, based upon the total weight of the preserved product taken as 100% by weight, and would even more preferably be determined to be undetectable by standard methods conventionally used in the art. If there is hydrogen peroxide remaining after the process, it can be removed by peroxidase hydrolysis processes or other known methods.
The preserved mucosa tissue has an ash content of less than about 10% by weight, and preferably less than about 7% by weight, based upon the total weight of the preserved tissue taken as 100% by weight. Those skilled in the art will appreciate that this low ash content substantially reduces the poor flavor generally associated with peptone and peptone-supplemented products derived from mucosa hydrolysis processes. Finally, the preserved mucosa tissue products have very low bacterial counts. For example, the products will have a standard plate count of less than about 20,000 cfu/g, and preferably less than about 5,000 cfu/g about 7 days after the tissue is treated with the preserving agent. The Coliform count will be less than about 10 cfu/g, and preferably less than about 3 cfu/g about 7 days after the tissue is treated with the preserving agent. Also, the E. Coli count of the product will be less than about 10 cfu/g and preferably less than about 3 cfu/g about 7 days after the tissue is treated with the preserving agent. As a result of these low counts, the unappealing odor of mucosa tissue is substantially reduced. This process is particularly useful for preserving mucosa tissue which will not be hydrolyzed or otherwise processed for a number of days.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The following examples set forth preferred methods in accordance with the invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.
Mucosa Preservation with H2O2 Followed by Hydrolysis
Porcine mucosa was heated with steam injection under agitation to 73° C. The steam was stopped, and the solution was mixed for 13 minutes. Hydrogen peroxide (0.5% of pure hydrogen peroxide, which is equivalent to 1.5% of 34% hydrogen peroxide) was added to the solution followed by mixing for 10 minutes. The color of the solution changed so that it was similar to the color of a whey protein solution. The solution was analyzed to determine whether there was any hydrogen peroxide remaining. This was carried out by adding 0.01 M KmnO4 to the diluted solution (1:150) and observing whether a color change took place. This test indicated that there was no hydrogen peroxide remaining in the product. The analytical data of the solution was as follows: protein (76% on a dry basis), ash (7.5% on a dry basis) and moisture (86% by weight). The micro counts after 7 days at regular storage conditions were: on SPC—300 cfu/g; Salmonella—negative/25 g; E. Coli—3 cfu/g; and Coliforms—3 cfu/g. After 20 days at regular storage conditions, the counts were: SPC—600 cfu/g; Salmonella—negative/25 g; E. Coli—3 cfu/g; and Coliforms—3 cfu/g. The mucosa solution had no off-odor after 7 days.
Six days later, the above porcine mucosa was transferred to a stainless steel container. A commercially available bacterial alkaline protease enzyme having a minimum activity of 580,000 DU/g was added to the solution at a rate of 1 g of enzyme per kg of mucosa. The solution was heated in a water bath at a temperature of 55° C. The pH of the solution was adjusted to 9.2 with sodium hydroxide. The viscosity of the solution was significantly reduced after about 30 minutes of hydrolysis, indicating that hydrolysis was taking place. The complete hydrolysis process lasted 5 hours after which the solution was divided into three portions. The pH of the first portion of the solution was adjusted to 5.0 with 6 N HCl as is conventional in the prior art preservation processes. The second portion of the solution was treated by the addition of hydrogen peroxide (0.2% of pure hydrogen peroxide, which is equivalent to 0.6% of 34% hydrogen peroxide) according to the instant invention. The third portion of the solution was used for heparin extraction with an anion ion exchange resin. The analytical data of the peptone after heparin extraction were as follows: protein (66.2% on a dry basis); ash (13.6% on a dry basis); and moisture (86% by weight).
Enzyme activity was determined by under the different processing conditions. The results were as follows:
After 5 hours hydrolysis w/mucosa
After treatment with HCl to a pH of 5
After treatment with 0.2% H2O2