Method for stabilizing the enzyme lactoperoxidase in products

Abstract: The present invention relates to a method for obtaining an increased stability at storage of the enzyme lactoperoxidase in particularly dry products, as well as such products, whe­ reby the hydrogen ion concentration is adjusted to 10 -3.25 -10⁻⁶.

Description: The present invention relates to a method for stabilizing the enzyme lactoperoxidase in various products, and such products comprising lactoperoxidase. The object of the present invention is to obtain a possibility of storing the enzyme lactoperoxidase without losses, or more correct, without substantial losses of enzyme activity. Background of the invention. It is for some years known to use the enzyme lactoperoxidase together with a thiocyanate and a hydrogen peroxide donor to prolong the freshness of milk, and to produce antibacterial compositions being active in the gastro-intestinal tract, against diarroea and other intestinal disturbances. The enzyme lactoperoxidase used in these compositions is recovered and isolated normally from bovine milk, or, more common, dried milk products are used as a lactoperoxidase enzyme source. The enzyme is thus heat stable to such an extent that it is not deactivated at the drying process to any substantial extent. However, it has turned out that the enzyme is not stable at storage to a desired extent in the dried products/compositions. Attempts made to stabilize the storability by adding anti-oxidantia have turned out to be of no effect. Hereby, one thought that lack of stability was dependent on an oxidation of the hem-group of the enzyme. Requests for a stable, storable form of lactoperoxidase have however, been raised as various products, feedstuffs and food-stuffs, particularly in dry form, shall possess a fair storage time. Normally, at least 3-6 months will run before the product has gone all the way from producer/production, and, via retailer been completely used by the customer/consumer. The requests for stability of pharmaceutical preparations are still much greater. Description of the present invention. It has now surprisingly turned out possibly to obtain a storage-stable product comprising lactoperoxidase by means of the present invention, which is characterized in that a product comprising enzyme lactoperoxidase is adjusted with regard to pH, so that pH is 3.25-6, preferably 3.25-5, more preferably 3.5-4.5 at the dissolution in water. The term product comprising lactoperoxidase means above, primarily milk products, such a whole milk, nonfat milk, various types of whey, as well as other foodstuffs and feedstuffs, and pharmaceuticals to which the enzyme lactoperoxidase is added. The invention will be described more in detail in the following with reference to some examples of which the stability at storage of enzyme lactoperoxidase is clearly evident. Example 1. Whey was produced by precipitating the casein by adding rennet to fresh milk. The fraction of whey was divided into three parts of which pH was adjusted using 1 M NaOH, and 1 M HCl, respectively, to pH 8, 6, and 4. The samples were freeze-dried and stored at 37°C. The LP-activity of the samples were determined after storage after varying long term storage using a standardized method . The results are given in Table 1. Example 2. A further test was carried out in the same way as in Example 1 above, whereby pH was adjusted to 5, 6, and 8. The results obtained are given in Table 2 below. In a further test series the same freeze-dried whey was used, whereby pH was varied from 2.5 to 4.0. The results obtained are evident from Table 3 below. In a further test series for a further longer period of time the tests above were confirmed. The results are given in Table 4 below. In a third, further test series a spray dried whey powder without LP-activity was tested, which powder was dissolved in water, whereupon pure LP enzyme was added and pH was adjusted, the samples were freeze-dried, and then stored at 37°C. The results obtained from these tests are evident from Table 5 below. As evident from the above the analyses in Example 1 have varied to some extent, but the results still consistantly show that the inactivation of lactoperoxidase takes place considerably slower at a lower pH. It is thus evident from the above given tests that the inactivation of the enzyme lactoperoxidase takes place considerably slower in lower pH, 3.25-6 than at a higher pH. The inactivation at pH 3 is immediate. In all products, even if they should be dried, there is some moisture present, and thus a pH is present in the micro environment. By adding an acid, or a buffering system to the already dry product in such an amount that at the dissolution of the product in water one obtains a pH of 3.25-6, it has however, turned out that the stability is radically improved. A stabilization is thus already achieved by adjusting the micro environment, why a dissolution with accompanying pH adjustment and final drying is not necessary to obtain an effect. In order to adjust pH any inorganic or organic acid can be used, such as hydrochloric acid, sulphuric acid, nitric acid, citric acid, acetic acid, formic acid, and others, as well as acidifying buffering systems, as citric acid-citrate buffering systems, phosphoric acid-phosphate buffering systems, and others. It is evident that in those cases the product should have a pH of below 3.25 prior to addition of lactoperoxidase, an addition of an alkalizing agent of the type NaOH, or NaHCO₃, Na₂CO₃ or buffering system shall take place.

Claims: A method for achieving an increased stability at storage of the enzyme lactoperoxidase in products, characterized in that a product comprising the enzyme lactoperoxidase is provided with a pH adjusting agent and/or buffering system in such an amount that pH becomes 3.25-6 at dissolution in water A method according to claim 1, characterized in that pH is adjusted to 3.5-5, preferably 3.5-4.5. A method according to claims 1-2, characterized in that pH is adjusted by adding, to an already dry product, a dry organic acid, and/or buffering system based upon organic acid/salt. A product comprising an addition of the enzyme lactoperoxidase, characterized in that pH in the product at the dissolution in water is 3.25-6. A product according to claim 4, characterized in that pH is 3.5-5, preferably 3.5-4.5.

Inventors: BJOERCK KARL-ERIK LENNART, BJOERCK, KARL-ERIK LENNART, Björck, Karl-Erik Lennart
Applicants: EWOS AB, EWOS AKTIEBOLAG
Application year: 1987
Main classification: C12N9
IPC codes: C12N9