DE946061C - Process for the enzymatic hydrolysis of proteins and autolysis of animal tissues - Google Patents

Description

Process for the enzymatic hydrolysis of proteins and autolysis of animal tissues In the processes involved in the enzymatic hydrolysis of Proteins or in the autolysis of animal organs have been used so far, it is not possible to reliably develop microorganisms to prevent. The commercial preparations used as hydrolysis agents, such as pepsin, In fact, papain and pancreatin are seldom completely free from microbes. Self using the pancreatic gland, which allows very rapid hydrolysis, it is necessary to resort to fresh organs and proceed with particular caution, to exclude germs. In the procedures for the autolysis of tissues, it is necessary to remove the tissues to be autolyzed as aseptically as possible, even this procedure does not give absolute certainty and is an obstacle for the practical application of this procedure forms.

The almost inevitable intake at the beginning of the above mentioned procedures caused by germs that the resulting cleavage products of the Proteins very often amino acids as breakdown substances, such as histamine, various poisonous Contain substances and fever-inducing substances that are involved in the transformation of microorganisms form in the course of hydrolysis.

These toxic substances show up, especially in the case of the peptones and the injectable protein hydrolysates, deleterious properties in their industrial and scientific application, in particular at their therapeutic Use in humans or animals. Their extensive elimination after various Procedure is often difficult.

The invention relates to a method for the hydrolysis of proteins, any commercially available enzymatic preparation as the hydrolysis agent or used fresh or frozen organs without developing microorganisms even if you use these remedies without special precautions. End products never obtained in this way are free from toxic substances.

Furthermore, the autolysis of the process according to the invention Tissue is a practically easy process. Furthermore, after the Process according to the invention drive the hydrolysis of protein substances to such an extent that that the ratio of amide nitrogen to the total nitrogen of the conversion products 72 ° / o.

Finally, the pancreatic glands can be used according to the method according to the invention generally use as a hydrolysis agent.

This process can be used to produce end products directly without further purification that are free from histamine and toxic substances.

According to the invention, in addition to the hydrolyzing agent, a mixture of a volatile antiseptic agent such as toluene or chloroform with two antibiotic substances such as penicillin and streptomycin is used in the desired hydrolysis. This mixture prevents the development of microorganisms without hindering the fermentative mechanism. The proportions of these three substances depend on the raw materials used in each case. It can be in a preparation z. B. the following amounts are used: Chloroform. . ¢ °% Penicillin ... 8,000 oxford units Streptomycin o, o8 g at. another the following quantities: Chloroform. . 2 ° / o Penicillin ... 5000 oxford units Streptomycin 0.03 g Depending on the type of substance to be hydrolyzed and the desired degree of hydrolysis, the ratio of hydrolysis agent to protein can be changed. The conditions of the hydrogen ion concentration, the temperature, the duration and the dilution can also be changed here. Optimal working conditions can be selected here without having to fear that these optimal conditions would be particularly obstructive for the development of the microorganisms.

The method of hydrolysis is generally exactly the same as that in the known method, the protein substances in an aqueous medium with various hydrolysis agents under optimal conditions of dilution, temperature and the p11 value are brought into contact. But the procedure is through that Addition of mixtures of antiseptic agents from penicillin, streptomycin and in particular the chloroform-penicillin-streptomycin mixture.

Each hydrolysis is carried out up to a constant pH, its value on the type of substance to be hydrolyzed and the desired degree of hydrolysis depends. The unconverted proteins can optionally be cooked removed. After filtering, the liquids obtained can optionally can be decolorized with charcoal or filtered through special adsorbent substances. That The filtrate can be used as is, concentrated or evaporated to dryness will.

The powdery products obtained show the following characteristics: You are hygroscopic powders with a characteristic smell and taste, their moisture content is about 5 ° / o. They are wholly or partially soluble in water and are, what is important, free from toxic products, especially feverish ones Substances or histamine.

The total nitrogen content is between 12 and 14%. Your amide nitrogen content, which was determined by the Sorensen method, fluctuates between 5 and 90%. The ratio of amide nitrogen to total nitrogen fluctuates between 25 and 72% It is noteworthy that the level of 7z "/, can be achieved while the best previously known processes do not exceed 651) [,.

The invention is applicable, first, to most enzymatic ones Hydrolysis process of proteins, the hydrolysis agent being commercially available Pancreatin, pepsin or erepsin or the fresh or frozen pancreas Pancreas can be of different species; second, with autolysis of various organs and animal tissues using the method described above makes the aseptic removal of the tissue unnecessary.

One can use most animal or vegetable products by this method Treat proteins, especially casein. Paracasein, lactalbumin, blood, muscle, Gelatine, fibrin, fish protein, vegetable protein, various animal organs, z. B. liver, spleen, mucous membrane of the intestine and stomach.

As already described above, the main advantages of the process according to the invention consist in the possibility of using fresh or frozen organs as hydrolysis agents which have been obtained without special precautions may possibly be present in the starting products of the hydrolysis experiments, do not prevent them. The invention also allows working under optimal conditions of temperature, pH, dilution and duration. The end products obtained are free from toxic, fever-inducing breakdown products or histamine. They are very often free of activity on guinea pig intestines, on the blood of the dog and do not cause a shock in humans or animals. Finally will. the amide nitrogen of the proteins is set free in the form of non-racemic amino acids, ie in their optically physiologically active form and, as described above, in amounts that can reach 720 % of the total nitrogen.

In the proteolysates produced by this process, in particular from casein and paracasein, are indispensable for the growth of animal organisms Amino acids are present in the necessary equilibrium quantities. These preparations, which are made only from protein nitrogen, the growth and the Ensure proper nutrition for the organism.

The following examples explain the process according to the invention and show that the present process is as easy to carry out as it was before known procedures.

Example i Peptones of Casein or Parakasein The following mixture was subjected to fermentative degradation Casein or Paracasein .... iooo; og Fresh or frozen Pig or beef pancreas ..... 2oo, og Distilled water. . . . . . . . 8,000 o ccm Chloroform. . . . . . . . . . . . . . . 12.0 cc Penicillin. . . . . . . . . . . . . . . . 2oooo, o Oxford units Streptomycin. . . . . . . . . . . . . - o, io g Fermentative degradation of proteins means the conversion of proteins into smaller molecules under the influence of ferments, so-called hydrolases.

The pii value of the above mixture was measured with a buffer, e.g. B. sodium phosphate or sodium carbonate, adjusted to 6.8-7. The mixture was 8 to 48 hours subjected to fermentative degradation, the temperature being 38 to 40 °. the obtained liquid was heated to boiling for several minutes. Then she became filtered off, concentrated and evaporated to dryness.

Example 2 Peptones of the Muscle A mixture of the following composition was broken down by fermentation: Defatted and chopped Ox meat. . . . . . . . . . . iooo, og Fresh or frozen Pig or beef pancreas ..... 2oo, og or commercial pancreatine .... io, og Distilled water. . . . . . . . 5000.0 cc Chloroform. . . . . . . . . . . . . . . 10.0 cc Penicillin. . . . . . . . . . . . . . . . 2oooo, o Oxford units Streptomycin. . . . . . . . . . . . . 0.20 g The pH of the mixture was adjusted to 7 using the procedure described in Example i. Example 3 Casein or Parakasein hydrolyzate The following mixture was subjected to fermentative degradation: Casein or Paracasein .... iooo, og Fresh or frozen Pig or beef pancreas ..... 2ooo, og Distilled water. . . . . . . . ioooo, o ccm Chloroform. . . . . . . . . . . . . . . 40.0 cc Penicillin. . . . . . . . . . . . . . . . 75,000 o Oxford units Streptomycin. . . . . . . . . . . . . 01759 The preparation is adjusted to a pH of 6.8 at a temperature of 40 ° for 5 to 7 days. The product obtained is then boiled up, filtered and the filtrate used directly or used in a concentrated form or in a form that has been evaporated to dryness. In the case of acid-precipitated casein, the hydrolyzate is decolorized with animal charcoal before it is filtered.

Example 4 Liver hydrolyzate The following mixture was subjected to fermentative degradation Pork, ox or veal liver in fresh or frozen renem condition. . . . . . . . . . -iooo, og Fresh or frozen Pig or beef pancreas ....... 250.09 Distilled water. . . . . . . . . 2ooo, above Chloroform. . . . . . . . . . . . . . . . 4.0 cc Penicillin. .... ... ... .... . . iooo, o oxford units Streptomycin. . . . . . . . . . . . . . 0.0259 The mixture was adjusted to a pH of 6 at a temperature of 40 ° within 12 hours. The product obtained was boiled and filtered through infusorial earth. The liquid obtained can be used directly after the filtration or it can be used in a concentrated form or in a form which has been evaporated to dryness.

Example 5 Autolysate of the liver The following mixture was subjected to fermentative degradation in a sterile container, the ferments contained in the relevant organ itself being used as hydrolases: Ox, pork or veal liver in hacked state iooo, og 8% sodium chloride solution in distilled water .... 5oo, og Chloroform. . . . . . . . . . . . . . . . 2.0 cc Penicillin. . . . . . . . . . . . . . . . . 8ooo, o Oxford units Streptomycin. . . . . . . . . . . . . . o, o6 g This mixture was treated for an average of 24 to 72 hours. The autolysate obtained was filtered through linen. After squeezing, the resulting liquid was boiled, filtered through infusorial earth, concentrated and evaporated to dryness.

All products obtained by this process are free from protein degradation products, caused by microorganisms, especially histamine.

Claims (2)

PATENT CLAIMS: I. Process for the enzymatic hydrolysis of proteins and autolysis of animal tissues, characterized in that the hydrolysis liquid a mixture of a volatile antiseptic, preferably toluene or Chloroform, with penicillin and streptomycin is added. 2. The method according to claim 1, characterized in that commercial preparations or animal, preferably frozen animal organs or tissues are used without special aseptic precautionary measures have been established or obtained. Into consideration Drawn publications:. Boli, Soc. Ital. Biol. Sper., - Vol. 25, 1948, p. Z295, = 296 (referenced Chemical Abstracts, Vol. 43, 1949, column 9145a); Journ. Bact., Vol. 52, 1946, pp. 89 to 98 (referenced Chemical -Abstracts, Vol. 40, 1946, column 612o); magazine for paediatrics, vol. 69, 1951, pp. 26 to 36; The Lancet, Vol. 259, 1950, p. 46 until 50; M. Morin, J. N. Nehlil and R. Pichon, La Streptomycin, Verlag Masson & Cie, Paris, 1949, pp. 45 to 51; Comptes rendus hebd., Vol: 232, 1951, pp. 96, 97.