DK146630B - PROCEDURE FOR GELATINE PREPARATION BY PROCESSING COLLAGENIC MATERIAL WITH PROTEOLYTIC ENZYMES - Google Patents

Description

146630

The invention relates to a novel process for the preparation of gelatin which is of the kind specified in the preamble of the claim and is characterized by the characterizing part of the claim.

Chemical Abstracts 68-96816 y discloses a two-step process for preparing gelatin comprising treating collagen-containing material, calfskin, with a proteolytic enzyme, pronase, in the presence of CaCl 2, and heating for denaturation to β-chain molecules.

Chemical Abstracts 73-78571 p discloses a three step process for the preparation of gelatin, comprising a partial hydrolysis of calfskin in acidic medium, enzymatic hydrolysis at pH 2.9 with an enzyme produced by Aspergillus niger, and neutralization of the medium and denaturing agent. Heating the collagen by heating for 2.5 hours.

In Chemical Abstracts 78-160919 v, the various methods for preparing gelatin from pigskin collagen are compared. In this comparison, pronase yields higher yields of gelatin than any other enzymatic method known to date, but this disclosure refers to the preparation of gelatin from collagen-containing material not from cartilage or meat waste.

From Chemical Abstracts 79 “122997 g, a treatment of calfskin with proteases from Actinomyces fradiae 25 119 is known.

Chemical Abstracts 82-107985 v discloses the action of proteases from Actinomyces fradiae 119 which have been fractionated on carboxymethyl cellulose, where the first three fractions were found to possess both elastolytic, proteolytic and collagenolytic properties, whereas the fourth fraction had only proteolytic and collagenolytic properties. A further fractionation of the first fraction yielded very strong proteases which hydrolyze collagen and elastin to four free amino acids and small peptides. The other enzymes contained in this enzyme mixture have a peptidic activity (the enzymes in the 2).

146630 2 and 3 fraction hydrolyzes peptides having a molecular weight less than 25,000). However, cartilage has a molecular weight of about 130,000, and therefore these enzymes are unlikely to have any effect on cartilage for the preparation of gelatin.

German Patent Specification No. 1.1 ^ 5,904 discloses a method of improving the yield of collagen, which is compared to the method known from French Patent No. 1.1 ^ 9,555 wherein 10 cowhides are treated with a proteolytic enzyme such as takadiastase or pancreatin, followed by heating in acidic medium. The higher yield obtained according to German presentation specification 1.1U5-90U is obtained only when the proteolytic treatment is supplemented with an acid hydrolysis.

15 German Patent Specification No. 1,298,211 discloses a process for producing soluble collagen precipitated in the form of fibers, and in this process, bones are treated with an enzymatic solution produced by cultivation of the strain Aspergillus niger and the resultant viscous solution is neutralized and the soluble collagen precipitates. Thus, this process does not deal with the preparation of gelatin, but rather soluble collagen.

None of these known methods for the preparation of gelatin use cartilage, cuts of meat and / or meat waste as a starting material, and none of these known methods use an enzymatic mixture prepared from nutrient media of Streptomyces fradiae no.

2019 or Streptomyces fradiae No. 1998 from the Museum d'Histoire Uaturelle de Paris, and none of these known methods perform the procedure in one step, but additional treatment is always required in addition to the enzymatic.

The enzymes from Streptomyces fradiae 1998 or from Streptomyces fradiae No. 2019 are known, but have so far only been used for therapeutic purposes, and it is therefore not obvious to use these enzyme products for the preparation of gelatin.

Thus, the process of the invention comprises a novel one-step process for the preparation of gelatin, using a process for the preparation of novel gelatin and an unrelated enzyme mixture.

The process of the invention furthermore has the advantage of obtaining the gelatin in a very large yield of about 70%, in contrast to the known methods, where the gelatin is obtained in a yield of about 15%. or less, depending on the starting material used.

Without being bound by some theoretical considerations 15, it must be assumed that the enzymatic mixtures containing Streptomployes no. 1998 or Streptomyces fradiaenr.

In 2019, the glycoproteins and the mucopolysaccharides bound to the collagen attack attack, thereby obtaining the gelatin in the amount far greater than the amount obtained by the known methods.

Furthermore, the gelatin prepared according to the invention is of a very fine quality with a very low mineral content of less than 1%. The process of the invention is described in more detail below, wherein: the enzymatic treatment is carried out at a temperature between + 10 ° C and 50 ° C, preferably between 15 and 25 ° C, the enzymatic treatment is carried out for a period of time of 30 minutes. to 25 hours depending on the raw material used, preferably from 17 to 20 hours,

the enzymatic treatment is carried out at a pH of from 3 to 9, preferably from 5 to 5 S

the isolation of the gelatin is carried out by filtration or centrifugation of the reaction mixture and extraction of the gelatin from the undissolved hot water residue. The temperature of the hot water can vary from JO to 9 ° C. The extraction of the gelatin from the undissolved residue is effected by contact between the undissolved residue and hot water for a period of time of from 30 minutes. to 90 minutes, the isolation of the gelatin is carried out by evaporation of the aqueous extract to dryness either under reduced pressure or by lyophilization or by atomization, the enzymatic mixture being preferably that obtained by concentration of a nutrient medium containing Streptomyces fradiae, strain 2019.

The gelatin thus prepared can be used in the food industry as a gelling agent, e.g. in canned food, desserts, confectionery, jam or jam. It can also be used in the pharmaceutical industry for the preparation of pharmaceutical compositions containing gelatin, e.g. injectable solutions of gelatin, and it can also be used as binding material in the preparation of tablets or microbedlets.

Furthermore, the soluble fraction of the reaction mixture usually contains from 10 to 20% soluble material which can be recovered by evaporation, spray drying or lyophilization of the aqueous solutions to dryness. The dry residue thus prepared can be used as animal feed as it has a high content of proteins.

It can e.g. mixed in compound feed.

The following example illustrates the process of the invention in more detail.

Example.

Step A

The starting material may be cartilage, meat slices or 30 meat waste. This raw material is pre-washed, but it is not mandatory. The raw material is mashed out and then suspended with stirring in the aqueous solution or suspension of the 2019 enzymatic mixture of Streptomyces fradiae.

The nature of the water used is immaterial, which can be used distilled water, deionized water or tap water, without affecting the performance of the enzymatic reaction.

The reaction mixture is kept under stirring and the pH is adjusted to a value between k and 5 which appears to be optimal. From time to time, pH 5 is checked to keep it within these limits.

The reaction mixture is kept at a temperature of 20 ° C - 5 ° C for 17 hours with continued stirring. Afterwards, the undissolved material is filtered off by suction and washed three times with cold water. The filtrate is evaporated to dryness and used as food or as animal feed.

The undissolved residue is suspended with gentle stirring in hot water, preheated to 80 ~ 90 ° C, to decompose the enzymatic mixture and dissolve as much gelatin as possible each time. After one hour of contact, the suspension is filtered by suction, the undissolved material is dried and the aqueous phase is combined with the solutions of the previous gelatin recoveries.

These aqueous solutions are clarified and decolorized by the addition of small amounts of charcoal, filtered and evaporated in vacuo. The dry residue gives a very pure gelatin.

The undissolved residue from the filtration is then suspended in hot water under the same conditions. After an hour, the solution is separated, the undissolved residue is washed again with warm water and the wash water is collected with the filtrate. The aqueous solution is distilled under reduced pressure to give a different yield of gelatin. Yield in total JO%.

Step B.

To the residue left in step A, a new amount of cartilage or meat waste is added. The entire mixture 30 is mixed with water at 20 ° C - 5 ° C and an additional amount of enzymatic mixture is added. The mixture is kept under stirring and the pH is adjusted to between 5 and 5. The reaction is carried out for 17 hours as indicated in step A and the undissolved residue is extracted as described in step A.

35 In this way, all cartilage will be transformed into gela tine.

6 146630

The number of treatments can be repeated as many times as necessary until no gelatin is recovered.

The enzymatic mixture of Streptomyces fradiae has the following analytical specifications: 5 Total proteolytic activity 7000 Anson units / mg proteolytic activity not inhibited with aprotinin 5160 Anson units / mg proteolytic activity not inhibited by trypsin-1o inhibitor from soybeans 5570 Anson units / mg protein content (Lowry's method) ^ 5.7% based on the dry material.

Electrophoresis: major component component 2, as defined by Morihara and Coll. (Bio. Chem. Biophys. Acta 139 (1967) 382).