DE2620289A1 - Recovering lipase rich fractions from pancreatic tissue - by blending with a mixt. of (halogenated) hydrocarbon and alcohol then pptn. with acetone - Google Patents

Abstract

Preparations rich in lipase are prepd. from pancreatic tissue by first autolysing and defatting the minced tissue with a solvent mixt. comprising 80-95 vol. % of a halogenated hydrocarbon (Ia) or aliphatic or aromatic hydrocarbon (Ib), plus 5-20 vol. % of a water. immiscible 4-6C alcohol. (Ia) has density >1.3 and (Ib) density 0.655-0.88 and both have b.pt. at normal press. 47-130 degrees C. The solvent treatment is at 15-20 degrees for 12-40 hrs. using (Ia) or at 15 degrees C for 4 hrs. using (Ib), then the central emulsion layer contg. the tissue water is separated from the top and bottom layers. The enzymes are pptd. from the emulsion with acetone, then dried. Used for treating digestive disorders. Since autolysis and defatting are now done in a single, stage the method is simpler and quicker. The concn. of enzymes in the prod, is increased and the ppte. is recovered in the required particle size.

Description

description

Process for the production of a lipase-rich enzyme preparation Zur Treatment of the insufficiency of the digestive juices in the gastrointestinal tract as well as in Digestive disorders are fermented preparations with high activity of digestive enzymes, especially lipase activity. Fresh serves as the starting material or frozen tissue of the pancreatic glands, especially of pig pancreas. In order to avoid the burden on the organism with ineffective components in the preparations The aim is to keep the fermentation activity low for the manufacture of the pharmaceuticals required products should be as high as possible. In German patent specification 24 08 379 describes a process for the production of a lipase-rich enzyme preparation, in which the comminuted pancreatic tissue is initially mixed with a mixture of 9 parts by volume Chloroform and 1 part by volume Butanol is partially degreased, whereupon by letting the partially defatted fabric material stand at 0 - 40C It is autolyzed for 24-26 hours, then further degreased by treatment with acetone is, whereupon the enzyme activity is extracted with 5 strength aqueous ethanol solution and a precipitate is obtained from the extract by adding acetone, which after after drying represents a preparation with high lipase activity. After an older one Proposal as it was laid down in the German patent application P 25 05 887.1, you can use the active ingredient extract obtained by extraction with the aqueous ethanol solution can also be converted directly into a dry enzyme preparation by lyophilization.

Even if these products are better than that in terms of their activity already known lipase preparations are, it must always be sought to simplify both the manufacturing process and the enzyme yield the glandular material and in particular the activity of the products obtained to increase.

Surprisingly, it has now been found that the necessary main degreasing of the comminuted pancreatic tissue and the measure of autolysis merged into one stage can be, whereby the duration of the autolysis can also be shortened significantly. According to the invention, an organic solvent mixture is added to the comminuted pancreatic tissue from 80 to 95 Vol.-Oa ° zEqnenwasserstoff, whose boiling point at normal pressure between 47 and approx. 1300C and its density should be above 1.3, as well as 5 - 20 vol. of a water-immiscible alcohol with 4 - 6 carbon atoms with stirring in a added in such an amount until a mobile suspension is formed. The so The tissue suspension obtained is now at 15-200C for 12 to 40 hours, preferably Left to stand for 14-16 hours, with the suspension if necessary. a few times stirred up can be. During this autolysis, the gland cells give up their aqueous cell contents free, those with the organic, which are immiscible with water Solvent mixture then forms an emulsion. Form when left standing quietly three layers, followed by the middle phase, consisting of the emulsion of the proteinaceous Tissue fluid from the pancreatic glands and the solvent mixture consists of the lower and upper phase is separated in the usual way. The upper phase consists here essentially from the defatted tissue fibers, while the lower phase consists essentially of the fatty solvent. By squeezing the isolated upper fiber layer or by centrifugation can also still the Increase the yield of emulsified tissue fluid by up to 10%.

The combined emulsions are broken by adding acetone, a lipase-rich precipitate separates out. After precipitation, the enzyme concentrate is left settle, the solution is decanted and the end product is washed several times by stirring in acetone. The product is cleaned with the help of a sieve device or a centrifuge separated off, then dried in vacuo and removed from solvent residues freed.

The grain size distribution of the precipitation and thus the bulk weight The enzyme preparation can be adjusted by the speed of mixing of the emulsion with the acetone surprisingly influence within certain limits. To the To achieve a coarse-grained material, the emulsion is first coated the amount of acetone required for precipitation and then stir both layers quickly with each other. A very fine grain, on the other hand, can be achieved by that acetone is added slowly with stirring.

Suitable halogenated hydrocarbons include trichlorotrifluorethane, that boils at approx. 470C, as well as the chlorinated hydrocarbons with boiling points up to 121 0C or mixtures of halogenated hydrocarbons.

In the organic solvent mixture to be used according to the invention Trichlorethylene has proven particularly advantageous because the use this chlorinated hydrocarbon not only leads to a particularly high activity of the obtained preparation leads, but because, moreover, after the implementation of the The liquid mixture obtained from the process and composed of aluminum hydrocarbons, alcohol, Acetone and water can easily be separated again by distillation.

The recovery of used solvents is just one large-scale processes represent an important economic component. In particular As is known, some solvent mixtures can cause aceotrope formation during distillation difficult to separate and recover in pure substance.

The use of a chlorinated hydrocarbon with a density above 1.3 is important because it separates the suspension into the three desired phases in the implementation of the preparation facilitated or enabled will. The presence of a small amount of acetone in the organic solvent mixture has proven to be advantageous for the formation of the three layers.

A particularly suitable water-immiscible alcohol is normal butanol, but amyl alcohol and hexyl alcohol are also suitable. These Alcohols support the exposure of the process according to the invention in the middle phase accumulating enzyme activity.

The autolysis under degreasing conditions and the exposure of the Enzyme activity from the cell tissue can be promoted by a slight warming. In a preferred embodiment of the method, the autolysis is therefore in two stages carried out. Here, the comminuted pancreatic tissue is first mixed with the solvent in a crowd of 1 - 2 1 / kg gland material and approx. Treated 150C for 14 to 16 hours. Then the fatty one separates out Solvent, i.e. the lower layer, as much as possible and offsets the Residue again with 1 - 2 1 solvent mixture per kg of fabric material, whereupon the temperature is increased to 25-350C, preferably 25-300C. After about 1 hour Long stirring, during which a vigorous post-autolysis takes place, is as described worked up, i.e. it is left to stand and the middle emulsion layer is worked up.

The method according to the invention not only leads to an essential one Simplification of the manufacturing process, but also to preparations with increased Enzyme activity. The combination of degreasing and autolysis with the organic Solvent mixture leaves the enzyme activity of the cell tissue in the form of a highly concentrated Accumulate emulsion from which the enzyme can be precipitated directly. Through this the otherwise necessary extraction measures, in which special solvents, e.g. aqueous alcohol or the like.

be used. The type of precipitation with acetone makes it possible also that the enzyme powder is already given the desired degree of granulation so that the subsequent granulation to larger particles, if this is desired is no longer necessary. The solvents to be used according to the invention also allow their easy separation and recovery, which is important for the large-scale Operation matters.

The invention is illustrated in more detail by the following examples.

Example 1: 150 kg of frozen pancreas are mixed in a cutter Shredded rice grain to lentil size and placed in a double-walled container with a stirrer given. Cooling water at 15 ° C. flows through the jacket of the container.

The solid is mixed with 150 l of trichlorethylene, which is up to 0.6 % By weight acetone and 25 liters of butanol (1) and mixes solid and solvent with the help of the agitator.

The solid is thawed after about 1 hour. You cover the container off and let the agitator run for 15 h. The stirrer is then turned off, the solid floats up and the yellow, fatty solution underneath is drained off.

The cooling water flowing through the container is replaced by hot water from 30-320C, the solid is again mixed with 165 l of degreasing mixture and stirred the mixture 1 h. During this time, the mixture reaches a final temperature of 28-3o0C. The stirrer motor is switched off and the emulsion and solid are allowed to float. First of all, some of the solvents are removed as a clear, yellowish solution, which is given to solvent recovery. The brown one that then runs off The emulsion is caught. The thick sludge emerging towards the end of its run becomes open a screw sieve press over a sieve box. The press process is added to the emulsion.

The emulsion is returned to the stirred tank and covered with a layer with 1 to 1.5 1 acetone / kg pancreas, the acetone with up to 3 wt.% Trichlorethylene1 but may be contaminated with less than 1% water, and mixes both phases by turning on the agitator. After 15 minutes, the stirrer is switched off. That Product sinks to the bottom of the stirred tank as a solid. One sucks the protruding Solution, suspended the solid for washing in o, 3-o, 5 1 acetone / kg pancreas, stirs for 5 min., lets settle again and sucks the solution off. The washing process will Carried out a total of 4-5 times1 with the use of a centrifuge for separation of Pancreatins can dispense with the sitting down process in the last wash. Man can also drain the solid pulp into a sieve basket, naturally reducing the acetone losses grow. The moist material is then placed in a vacuum drying cabinet at 45-5o0C Heating temperature and 30 Torr or in a fluidized bed dryer at 500C air inlet and an air outlet temperature of 40 ° C. You can sieve the material Break it down into various grain classes. Fine grain can be agglomerated in a spray granulation, Coarse grain can be crushed by grinding. The results of the tests when using of glands of the same provenance is shown in the table below.

PANKREATIN Gland origin Yield lipase activity Manufacturing process (FIP-E./g) A 7.3 96,200 State of the art A 10.1 100,000 this application B 6.3 71,000 state of the art B 7.4 86,000 this application C 7.8 106,000 state of the art Technique C 10.1 119,000 this application C 9.2 120,000 this application Table 1: Comparison of the yield and lipase activity of pancreatin which was produced according to the prior art and this application. Yield bulk density Lipase activity Type of acetone particle size distribution addition to the Precipitation 0.315 0.315-0.800 0.800 (%) (g / l) (FIP.-E / g) (%) (%) (%) 9.4 360 84,000 # - - - 10.1 417 93,000 # slow below 98.1 1.2 0.7 11.5 440 76,000 # agitation 97.5 2.1 0.4 9.8 463 93,000 # 13.7 460 76,500 # method 46.4 52.6 1.0 12.9 514 81,500 # with over - - - 9.1 521 83,000 # layers - - - 14.6 482 79,200 # 8.0 42.9 49.1 Table 2: Comparison of the bulk density and the grain size distribution of pancreatin from glands of the same origin when using different methods of adding acetone during precipitation Example 2: 150 kg of frozen pancreas are crushed in a cutter as in Example 1 and placed in a double-jacketed container with a stirrer. Cooling water at 15 ° C. flows through the jacket of the container. The solid is mixed with 150 l of methylene chloride and 15 l of butanol (1) and degreased with stirring for 15 h. Another 100 liters of methylene chloride are added to the mixture, the mixture is stirred thoroughly and the stirrer is switched off.

The clear sub-phase is drained. Another 150 l of methylene chloride are added and 15 l of butanol (1) are added, the mixture is heated to 3o0C (1 h) and the clear one separates Solution layer and the fiber layer.

After the fiber has been squeezed out, 140 l of emulsion are obtained, which is at 15 ° C cooled and covered with 140 l of acetone. Mix the emulsion and acetone, decanted from the settled pancreatin and obtained according to Example 1 a yield of enzyme concentrate of 9.3%, based on the pancreas used.

Lipase activity of the enzyme concentrate: 86,500 FI-E./g example 3: 150 kg of deep-frozen pancreas are comminuted according to Example 1 and divided into a added to the stirred tank cooled to 15 ° C. This is done using 150 liters of carbon tetrachloride and 15 l of n-amyl alcohol and the pancreas defatted at 150 ° C. (15 h). One sets Add 75 l CCl4 and 100 l acetone, turn off the stirrer and separate the fatty ones Sub-phase.

The mixture is then warmed to 290C and 15 liters of amyl alcohol are again added and 150 1 CCl4 are added, the mixture is stirred for 1 hour and separates as in Example 1 100 1 emulsion. The emulsion is covered with 100 l of acetone and the enzyme concentrate is precipitated.

After washing and drying, 1o, 1% enzyme concentrate with 91, ooo FIP-E./g lipase activity obtained.

Example 4: 150 kg of frozen pancreas are as in example 1 crushed and placed in a stirred tank cooled to 15 ° C. You bet 15 1 butanol (1) and 15o 1 1,1,1 trichloroethane and leads at 150C (15 h) the Degrease while stirring. Then 75 l of acetone are added and the fatty one is separated Solution from 150 1 1,1,1-trichloroethane is added again, heated to 250C and S separates after 1 h 100 1 emulsion. The emulsion is covered with 100 l of acetone and the enzyme concentrate precipitates.

After washing and drying, a 9.8% yield of a product is obtained 88,500 FIPU./g of lipase activity obtained.

Example 5-13: 15G kg of frozen pancreas are correspondingly Example 1 - 5 crushed and degreased. Type and amount of degreasing solution used Table 3 indicates. A possible addition of acetone V1 after the 1st degreasing is also necessary recorded in Table 3 before the first separation between pulp and clear fat solution.

So 1 halogenated hydrocarbon and 15 1 alcohol are added to the residue, the mixture is kept at tOC for 1 hour and V21 emulsion as in Example 1 is obtained. The emulsion is cooled to 150 ° C., covered with V3 1 acetone and mixed. After washing with acetone - as in Example 1 - and drying, an A% yield of enzyme concentrate with a lipase content of B FIP-E./g is obtained. All figures are given in Table 3. Example degreasing mixture Acetone addition temperature Emulsion amount of pancreatin lipase reaction No. at the end of the after-quantity precipitation yield activity 1. Degreasing Degreasing acetone and autolysis V1 t1 V2 V3 AB (1) (° C) (1) (1) (%) FIP-E-./g) 5 150 l CCl4 15 l butanol (1) 0 31 185 305 10.2 90,000 6 150 l trichlorethylene 15 l n-hexanol 100 30 165 185 9.7 86,000 7 150 l trichlorethylene 15 l butanol (1) 0 28 145 160 9.9 88,000 8 150 l trichlorethylene 15 in amyl alcohol 90 30 150 150 10.5 87,500 9 150 l trichlorethylene 30 l n-butanol 0 28 150 150 10.8 90,500 10 150 l of chloroform 15 l n-butanol 80 29 150 180 9.92 86,500 11 150 l trichlorethylene 15 l n-butanol 60 28 145 155 10.7 91,000 12 150 l trichlorethylene 15 l n-butanol 100 29 110 120 10.3 87,000 13 150 l tetrachlorethylene 15 l n-butanol 0 30 120 130 10.1 89,000 Table 3: Use of different degreasing mixtures to obtain pancreatin with high lipase activity.

Claims (3)

Process for the production of lipase-rich enzyme preparations from pancreatic tissue by autolysing and degreasing the minced tissue by means of a solvent mixture of halogenated hydrocarbons and alcohol and precipitation the enzyme from the aqueous phase by adding acetone and drying the precipitate, This is done by removing the shredded pancreatic tissue in an organic solvent mixture of 80 - 95 vol. halogenated hydrocarbons, its boiling point at normal pressure is between 47 and approx. 1300C and its density is more than 1.3 and 5 - 20% by volume of a water-immiscible alcohol mixed with 4 - 6 carbon atoms and left to act at 15 - 200C for 12 to 40 hours, the resulting middle emulsion phase containing tissue water from the upper and lower Phase separates and wins the dry enzyme preparation from it. 2. The method according to claim 1, characterized in that g e k e n n -z e i c h n e t that after about 12-15 hours of exposure to the solvent mixture the lower fatty solvent phase is drawn off and again fresh solvent mixture is added, then stirred for about 1 hour at 25-35 ° C, whereupon it is allowed to settle and the middle emulsion phase worked up. 3. The method according to claim 1 or 2, characterized in that g e k e n n -z e i c It doesn’t mean that you cover the emulsion phase with the acetone and then so quickly mixed that a coarse-grained enzyme concentrate precipitates.