DE2620289C2 - Production of a lipase-rich enzyme preparation - Google Patents

Description

35

For the treatment of the insufficiency of the digestive juices in the gastrointestinal tract as well as digestive disorders Ferment preparations with high activity of digestive enzymes, in particular lipase activity, are required. Fresh or frozen tissue of the pancreatic glands, in particular, serves as the starting material of porcine pancreas. To reduce the burden on the organism with ineffective components in the The aim of keeping preparations as low as possible is to increase the fermentation activity required for the manufacture of the pharmaceuticals required products should be as high as possible.

The German patent specification 24 08 379 describes a process for the production of a lipase-rich enzyme preparation described, in which the comminuted pancreatic tissue first with a mixture of Parts by volume of chloroform and 1 part by volume of butanol is partially defatted, whereupon by letting the stand partially defatted tissue material is autolyzed at 0-4 ° C for 24-26 hours and then treated is further degreased with acetone, whereupon the enzyme activity is extracted with 5% aqueous ethanol solution and a precipitate is obtained from the extract by adding acetone, which after drying becomes a Preparation with high lipase activity is based on an older proposal, as it is in the German Patent application P 25 05 887.1 was filed, you can by extraction with the aqueous The active ingredient extract obtained in the ethanol solution can also be lyophilized directly into a dry enzyme preparation convict.

Even if these products are better in terms of their activity than the previously known lipase preparations, so one must always strive to simplify both the manufacturing process and the Enzyme yield from the glandular material and in particular the activity of the products obtained continue to increase increase.

It is the object of the invention to develop a method which can be carried out more easily and leads to an enzyme preparation with higher activity

This object is achieved by the method specified in the main claim. Claims 2 and 3 name embodiments of this process.

Surprisingly, it was found that the necessary main degreasing of the comminuted pancreatic tissue and the measure of autolysis can be combined into one stage, with the duration the autolysis can also be shortened significantly. During the 12 to 40 hours, expediently 14 to Standing for 16 hours, the suspension can be stirred a few times if necessary. at During this autolysis, the gland cells release their aqueous cell content, which is immiscible with water organic solvent mixture then forms an emulsion. When left to stand still, three are formed Layers, whereupon the middle phase, which consists of the emulsion of the protein-containing tissue fluid Pancreatic glands and the solvent mixture consists of the lower and upper phase in usual Way is separated. The upper phase consists essentially of the defatted tissue fibers, while the lower phase consists essentially of the fatty solvent. By pressing the isolated upper fiber layer or by centrifugation can also be used to determine the yield of emulsified Increase tissue fluid up to 10%. The United Emulsions are broken by adding acetone, whereby a lipase-rich precipitate separates out. After precipitation, the enzyme concentrate is allowed to settle, the solution is decanted and washed End product several times by stirring in acetone. The product is with the help of a sieve device or a Separated centrifuge, then dried in vacuo and freed from solvent residues. the Particle size distribution of the precipitation and thus the bulk density of the enzyme preparation can be determined by the Surprisingly, the rate of mixing of the emulsion with the acetone within certain limits influence. To achieve a coarse-grained material, the emulsion is first coated with the amount of acetone necessary for precipitation and then quickly stir both layers together. A very finely divided grain, on the other hand, can be achieved by slowly adding acetone with stirring.

As halogenated hydrocarbon are, inter alia, the trichlorotrifluoroethane, which boils at about 47 ⁰ C, as well as chlorinated hydrocarbons having a boiling point up to 121 ° C or mixtures of halogenated hydrocarbons.

In the organic solvent mixture to be used according to the invention, trichlorethylene is particularly useful Proven to be beneficial because the use of this chlorinated hydrocarbon not only leads to one leads particularly high activity of the preparation obtained, but because beyond that after implementation the liquid mixture obtained from the process of halogenated hydrocarbons, alcohol, acetone and water can easily be separated again by distillation

The recovery of the solvents used is steep in a large-scale process is an important economic component. In particular, due to the formation of aceotrope, some solvent mixtures is known to be difficult to separate during distillation and recovered as a pure substance will.

The use of a chlorinated hydrocarbon with a density above 1.3 is important because thereby separating the suspension into the three desired phases when carrying out the Preparation is facilitated or made possible. 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.

The water-immiscible alcohol (see claim 1) supports the exposure of the Process according to the invention in the middle phase occurring enzyme activity

The autolysis under degreasing conditions and the release of the enzyme activity from the cell tissue can be promoted by a slight warming. Therefore, in a preferred embodiment of the method, the autolysis is carried out in two stages. In this case, the crushed pancreatic tissue is first added to the solvent mixture in an amount of 1-2 l / kg gland material and treated at 15 ° C for 20 ⁰ C12 to 15 hours Then, the settling fatty solvent is separated, the lower layer that is as far as possible and the residue is again mixed with 1-21 solvent mixture per kg of fabric material, whereupon the temperature to 25-35 ⁰ C, suitably 25-30 ⁰ C is increased. After about 1 hour of stirrer, during which vigorous post-autolysis takes place, Vy tet is worked up as described, ie it is left to stand and the middle emulsion layer is worked up.

The method according to the invention not only leads to a significant simplification of the manufacturing process, but also to preparations with increased ■ "' Enzyme activity The combination of degreasing and autolysis with the organic solvent mixture allows the enzyme activity of the cell tissue to accumulate in the form of a highly concentrated emulsion, from which immediately the enzyme can be precipitated. This eliminates the otherwise necessary extraction measures, where special solvents, e.g. B. aqueous alcohol or the like can be used. The kind of Precipitation with the acetone also enables the enzyme powder to be given the desired granules tion degree is conferred, 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 make it possible also their easy separation and recovery, which is essential for the large-scale operation of Meaning is

The invention is illustrated in more detail by the following examples

example 1

150 kg of frozen pancreas are in the cutter mixer Chopped to the size of rice grains or lentils and placed in a double-walled container with a stirrer. Cooling water at 15 ° C flows through the jacket of the container.

The solid is mixed with 150 liters of trichlorethylene, which may contain up to 0.6% by weight of acetone, and 25 liters of butanol (l), and the solid and solvent are mixed with the aid of the stirrer. The solid is thawed after about 1 hour. Cover the container and allow the stirrer to run for 15 hours. The stirrer is then switched off, the solid floats and the yellow, fatty solution underneath is drained off. The cooling water flowing through the container is replaced by warm water at 30-32 ° C, the solid is again treated with 1651 degreasing mixture and the mixture is stirred for 1 hour. The mixture achieved during this time a final temperature of 28-30 ⁰ C. It turns off the stirring motor and allows emulsion and solid float. First of all, some of the solvents are drawn off as a clear, yellowish solution, which is used for solvent recovery. The brown emulsion which then runs off is collected. The thick sludge emerging towards the end of the drain is pressed out on a screw sieve press over a gate box. The press drain is added to the emulsion

The emulsion is returned to the stirred tank, covered with 1 to 1.51 acetone / kg pancreas, the acetone may be contaminated with up to 3% by weight trichlorethylene but less than 1% water, and the two phases are mixed by turning on the agitator. After 15 minutes, the stirrer is switched off. The product sinks as a solid to the bottom of the stirred tank. The supernatant solution is suctioned off, the solid is suspended for washing in 03-0.5 l acetone / kg pancreas, stirred for 5 minutes, allowed to settle again and the solution is suctioned off. The washing process is carried out a total of 4-5 times, whereby the settling process in the last wash can be dispensed with when using a centrifuge to separate the pancreatin. The solid pulp can also be drained into a sieve basket, which naturally increases the acetone losses. The moist material is then dried in a vacuum oven at 45-50 ⁰ C heating temperature and 30 Torr or Wirbelschichtlrockner at 50 ° C inlet and 40 ⁰ C air outlet temperature dried, the material may decompose by sieving into various grain sizes. Fine grain can be agglomerated in a spray granulation. Coarse grain can be crushed by grinding. The results of the tests using glands of the same provenance are shown in the table below.

PANCREATINE yield Lipase activity
(FIP-E./g) Manufacturing process Glandular origin 7.3
10.1 96,200
100,000 State of the art
this registration A.
A.

continuation yield 26 20 289 6th 5 Gland origin 6.3 Manufacturing process B. 7.4 Lipase activity B. 7.8 (FIP-EVg) State of the art C. 10.1 71,000 this registration C. 9.2 86,000 State of the art C. 106,000 this registration 119,000 this registration 120,000

Table 1: Comparison of the yield and lipase activity of pancreatin, which according to the state of of the technology and this application.

yield

Bulk density lipase activity

(g / l)

(FIP-E./g)

Type of acetone addition during precipitation. Grain size distribution
0.315 0.315-0.800

0.800

360
417
440
463
460
514
521
482

84,000
93,000
76,000
93,000
76,500
81,500
83,000
79,200

slowly while stirring

Overlay method 98.1
97.5

46.4

8.0

1.2
2.1

52.6

42.9

49.1

Table 2: Comparison of the bulk density and the grain size distribution of pancreatin from glands of the same provenance when using various methods of adding acetone during precipitation

Example 2

150 kg of deep-frozen pancreas are comminuted in a cutter as in Example 1 and placed in a double-walled 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 151 butanol- (l) and degreased with stirring for 15 h. A further 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. Put a further 1,501 methylene chloride and 151 butanol (l) is added, the mixture heated to 3O ⁰ C (1 h) and separates the clear solution layer and the fibrous layer.

After pressing out the fiber, 140 liters of emulsion are obtained, which are cooled to 15 ° C. and mixed with 140 liters of acetone is overlaid. Mix the emulsion and acetone, decant from the settled pancreatin and obtained in accordance with example 1 a yield of enzyme concentrate of 93%. based on used Pancreas.

Lipase activity of the enzyme concentrate: 86 500 FIP-E7g.

Example 3

150 kg of deep-frozen pancreas are comminuted according to the example and cooled to 15 ° C. in a Given a stirred tank. To this 150 l carbon tetrachloride and 151 n-amyl alcohol are added and that

40

45

50 pancreas degreased at 15 ° C. (15 h) _{. 75 l CCl 4} and 100 l acetone are added, the stirrer is switched off and the fatty lower phase is separated off. The mixture is then warmed to 29 ° C., 151 amyl alcohol and 1501 CCU are again added, the mixture is stirred for 1 hour and, as in Example 1, 1001 emulsions are separated off. The emulsion is covered with 100 l of acetone and the enzyme concentrate is precipitated. After washing and drying, 10.1% enzyme concentrate with 91,000 FIP-EVg lipase activity is obtained.

Example 4

150 kg of deep-frozen pancreas are comminuted as in Example 1 and placed in a cooled to 15 ° C Given a stirred tank. One uses 151 butanol (1) and 1501 1,1,1-trichloroethane added and leads at 15 ° C (15 h) degrease while stirring. Then you give 751 Acetone is added and the fatty solution is separated off. 15011,1,1-trichloroethane is added again, and the mixture is heated 25 ° C and separates after 1 hour 100 liters of emulsion. Man covers the emulsion with 100 liters of acetone and the enzyme concentrate precipitates. After washing and drying 9.8% yield of a product with 88,500 FIP-E7g lipase activity is obtained.

Example 5-13

150 kg of frozen pancreas are accordingly Example 1–4 crushed and degreased The type and amount of degreasing solution used are given in Table 3 at. Likewise, a possible acetone addition Vi after

1. Degreasing before the first separation between pulp and clear fat solution is recorded in Table 3.

In each case 50 l of halogenated hydrocarbons are added to the residue and 15 liters of alcohol are added, the mixture is kept at f ° C for 1 h and V2I emulsion accordingly

Example 1 won. The emulsion is cooled to 15 ° C., covered with V31 acetone and mixed. After washing with acetone - as in Example 1 - and drying, A ⁰ Zo yield of enzyme concentrate with a lipase content of B FIP-EVg is obtained. All figures are given in Table 3.

example
No. Degreasing mixture Acetone-
together <7 am
end of
1. Ent
lubrication 0 temperature
at after
degreasing
and autolysis Emulsion
lot Amount of
Precipitation
acetone Pancreatin
yield Lipase
activity V |
(!) 100 1 |
(O V ₂
(!) V ₃
(!) A.
(%) B.
(F) PF./g) 5 1501CCl ₄
151 butanol (1) 0 31 185 305 10.2 90,000 6th 150 1 trichlorethylene
15 1 n-hexanol 90 30th 165 185 9.7 86,000 7th 1501 trichlorethylene
151 butanol (l) 0 28 145 160 9.9 88,000 8th 1501 trichlorethylene
15 1 η amyl alcohol 80 30th 150 150 10.5 87,500 9 150 1 trichlorethylene
30 liters of n-butanol 60 28 150 150 10.8 90,500 10 1501 chloroform
15 1 n-butanol 100 29 150 180 9.9 86,500 11th 1501 trichlorethylene
7.5 liters of n-butanol 0 28 145 155 10.7 91,000 12th 150 i trichiorethane
15 1 n-hexanol 29 110 120 10.3 87,000 13th 1501 tetrachlorethylene
15 I n-butanol 30th 120 130 10.1 89,000

Table 3: Use of different degreasing mixtures to obtain pancreatin with high lipase activity.

Claims (3)

Patent claims: 1. A process for the production of lipase-rich enzyme preparations from pancreatic tissue by autolysing and degreasing the comminuted tissue using a solvent mixture of halogenated hydrocarbons and alcohol and precipitation of the enzyme from the aqueous phase by adding acetone and drying the precipitate, characterized in that the comminuted pancreatic tissue in one organic solvent mixture of 80-95VoL-% halogenated hydrocarbon whose boiling point at normal pressure is between 47 and about 130 ⁰ C and its density is more than 1.3, and 5-20 vol .-% n-butanol, n-hexanol or n- amyl alcohol are added and allowed to act for 12 to 40 hours at 15-20 ⁰ C, separating the resulting median tissue aqueous emulsion phase of the upper and lower phase and from it the dry enzyme preparation wins 2. The method according to claim 1, characterized in that after about 12-15 hours longer The effect of the solvent mixture at a temperature of 15-20 ° C is the lower fatty one The solvent phase is drawn off and fresh solvent mixture is added again, then another approx. Stir for 1 hour at 25-35 ° C, whereupon sit down leaves and the middle emulsion phase worked up. 3. The method according to claim 1, characterized in that that the emulsion phase is covered with the acetone and then mixed so quickly, that a coarse-grained enzyme concentrate precipitates.