SK19422000A3 - Process for preparing a polyvinyl alcohol gel and mechanically highly stable gel produced by this process - Google Patents

Abstract

A polyvinyl alcohol gel can be easily and quickly produced by a process having the following steps: (a) use of an aqueous polyvinyl alcohol solution with a degree of hydrolysis >/=98 % by moles; (b) admixture of an additive that is dissolved in the aqueous polyvinyl alcohol solution and forms a separate, finely distributed and aqueous phase after concentration of the solution; (c) drying of the aqueous solution to a residual water content of maximum 50 wt. % to cause the phases to separate and hence the polyvinyl alcohol to gel; (d) rehydrating the polyvinyl alcohol in an aqueous medium.

Description

Process for producing a polyvinyl alcohol biocatalyst and a mechanically high-stability polyvinyl alcohol biocatalyst produced by this process

Technical field

The invention relates to a process for preparing a gel from polyvinyl alcohol. The invention further relates to a mechanically high-stability gel prepared in this manner.

BACKGROUND OF THE INVENTION

It is known that solutions containing polyvinyl alcohol (PVA) exhibit an increase in viscosity on standing. It is also known that PVA solutions can be converted into a gel when the solution is frozen and then thawed again (FR 2 107 711 A). However, the gels thus prepared exhibit relatively low strength.

It is further known from EP 0 107 055 B1 that the freezing strength of the prepared PVA gels is increased by repeating the freezing and thawing process at least once, preferably two to five times. In this case, a PVA solution having a saponification degree of> 95 mol%, preferably> 98 mol%, is used. The upper limit temperature for freezing the solution is -3 ° C, the cooling rate may be between 0.1 ° C / min. and 50 ° C / min, a melting rate between 1 ° C / min. and 50 ° C / min. The PVA used has a degree of polymerization of at least 700. The concentration of PVA in the solution should be above 6% by weight. The PVA gel, prepared several times by repeated freezing and thawing, has good mechanical strength and high water content, which is retained even under mechanical loading. The gel is highly elastic, non-toxic and can be used for many, especially medical purposes.

A variety of substances and materials can be admixed to this gel which, on the one hand, may increase strength, for example glycol, glycerin, sucrose, glucose, agar, gelatin, methylcellulose, etc. By the addition of active substances, such as heparin, medical applications can be carried out in which the active substance is transferred from the gel continuously and uniformly over a long period of time. The gel can be further mixed with microorganisms and enzymes to form a biologically active system.

······· · · · · · · · · · · · · · · · · · · · · · · · · · · · U.S. Pat. No. 4,663,358 discloses that organic solvents are added to an aqueous solution of polyvinyl alcohol to reduce the freezing temperature of the solution. This achieves that water freezing is prevented at gelation temperatures below -10 ° C, preferably at about -20 ° C, thereby achieving a more homogeneous and thus more transparent gel. The low gelatinization temperature is used to form fine-crystalline gels having sufficient mechanical strength.

The preparation of PVA gels by the freezing technique is expensive and time consuming.

DE 43 27 923 C2 discloses a process by which PVA gels can be prepared without the freezing process. By using a PVA solution having a degree of hydrolysis of > 99 mol%. and adding a solubilized additive that exhibits non-aqueous OH- and OH-containing, respectively. NH ₂ -group, PVA gelatinization at temperatures above 0 ° C can be achieved. The gelation process requires a period of several hours, which can optionally be extended by a further storage period of several hours in order to cure to the full stability of the gel bodies. This is naturally disadvantageous for the preparation of larger quantities of gel bodies on an industrial scale.

It is an object of the present invention to be able to prepare PVA gel bodies in a simple and fast manner while still improving the quality of the gel bodies prepared.

SUMMARY OF THE INVENTION

The present invention provides a process for preparing a biocatalyst with biological material in the form of microorganisms, enzymes, spores and / or cells incorporated into a polyvinyl alcohol gel, comprising the following process steps:

(a) use of an aqueous solution of polyvinyl alcohol with a degree of hydrolysis> 98 mol%;

(b) addition of an additive which, when added in an aqueous solution of polyvinyl alcohol, is dissolved and, when concentrated, forms a separate, finely divided and water-containing phase;

c) drying the aqueous solution up to a residual water content of no more than 50% by weight; to induce phase separation and the associated gelation of polyvinyl alcohol,

(d) back-swelling of the polyvinyl alcohol in an aqueous medium.

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Surprisingly, the process according to the invention allows the gelation of polyvinyl alcohol within a few minutes at room temperature or even at higher temperatures. Addition of the water-soluble additive and concentration by evaporation of water induces phase separation into fine particles, which results in the gelation within the PVA phase being achieved in a very short time. The assumption is that the water-soluble additive also forms a water-containing phase, so that by separating the phases, the corresponding proportion of water is withdrawn within a very short time, causing the polyvinyl alcohol to gelatinize. Suitably, the water-soluble additive exhibits an affinity for water which is at least comparable to that of PVA for water.

The PVA phase, which is insufficiently supplied with water during gelation, receives water the next time it is swelled, thereby improving the elasticity and mechanical stability of the PVA gel without abolishing the gelation. It has been shown that, when back-swelling, a certain proportion of electrolytes in the aqueous medium leads to a higher stability of the PVA gel, so that back-swelling preferably takes place in tap water or preferably in brine.

The process according to the invention provides the advantage that it enables the preparation of PVA gel without great expense, in particular without the freezing process and without repeated drying processes, within a short time, so that extremely economical production of PVA-gel bodies is possible. In addition, the gel bodies according to the invention are characterized by high elasticity and stability, in particular tensile strength, and in this respect significantly exceed the PVA gel bodies which can be produced by the prior art.

To further increase the stability and elasticity of the PVA gel bodies, a post-saponified aqueous PVA solution is used to produce them.

A preferred water-soluble additive is polyethylene glycol, which is added at a concentration of 4 to 30% by weight, preferably 4 to 20% by weight, in particular 6 to 16% by weight. starch ethers, polyalkylene glycol ethers, polyalkylene glycols, long-chain alkanols ( _Cn H2n + iOH sn> 8), sugar esters, sugar ethers.

A particularly preferred field of application of PVA-gel bodies is to form them as biologically, physically or chemically active bodies, i.e.

- Biologically, physically or chemically active material into a PVA gel. Thus, the PVA gel is excellent in the production of a chemical or biological catalyst, for example.

The drying of the aqueous solution to separate the phases and the associated gelation is carried out to a residual water content of not more than 50% by weight. It is determined that the prepared PVA gel should still be completely swellable so that below a residual water content of about 10 wt. there is a decrease in the elasticity of the gel bodies and that below the residual water content, possibly incorporated biological materials can be damaged. The preferred range for residual water content is between 10 and 30 wt%.

Drying can conveniently be carried out in a short time by evaporating water in air at ambient temperature if the aqueous solution is divided into small portions, especially those in which the solution has only a small thickness. Thus, it is particularly advantageous to drop the solution onto a hard substrate such that the drop diameter is at least twice as large as the drop height. Something similar can be achieved by pouring the solution into the casting mold and / or as a coating on the carrier material. By forming a thin layer or even a film, evaporation to the desired residual water content is achieved within minutes, for example within 15 minutes. Acceleration of the drying process - and thus the gelation process - can be achieved by performing drying in an oven at an elevated temperature.

The saline solution, preferably used for back-swelling, preferably contains polyvalent anions.

In particular for the immobilization of a biologically active material, it is advantageous in the method according to the invention that it can be carried out in an extremely gentle manner for the biological material, so that the biological material exhibits a significantly higher initial activity compared to other methods of immobilization.

This can be further supported by the fact that the aqueous medium in which the PVA gel is back-swelled is simultaneously a nutrient solution for the biologically active material.

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The density of the PVA gel prepared according to the invention can be modified with suitable additives. Thus, for example, the specific gravity can be increased by the addition of titanium dioxide and reduced by the addition of the smallest possible hollow glass beads.

The gelation according to the invention is, as mentioned above, possible at room temperature, but can be carried out at lower or higher temperatures. The biological material encapsulated in the PVA gel may be enzymes, microorganisms, spores, and cells.

The method of the invention can be practiced in many embodiments. For example, it is possible to dry the droplet to form the phases in the cooling tower during the fall process, so that the gelation after the phase separation occurs before the droplet falls on the substrate. This preparation method is particularly suitable for the production of PVA-gel bodies as a material for chromatography, which may have a diameter for laboratory purposes of 10 to 100 µm and otherwise 100 to 800 µm. Furthermore, it is possible to dry the starting liquid, which is set to a higher viscosity, during the extrusion of the filament, and at the same time to gelatinize the PVA.

The gel body produced by the method according to the invention exhibits improved mechanical stability over the previously known gel bodies, in particular with regard to abrasion resistance and tensile strength.

In particular, these improved mechanical properties make it possible to produce the gel bodies according to the invention in a reaction-kinetically advantageous lens shape, in which the gel bodies known hitherto did not show sufficient mechanical stability, in particular stirring stability. The gel body according to the invention, on the other hand, is stable and abrasion-resistant for many months even in high speed mixing processes. The shape of the lens with a large diameter and low height causes the chemically, physically or biologically active material to always be arranged near the surface, resulting in a reaction-kinetically advantageous situation.

The present invention makes it possible in a simple manner to add a magnetic additive to the polyvinyl alcohol body, so that the gel bodies can optionally be removed from the liquid by the magnets without difficulty.

It has been shown that the porous structure of the polyvinyl alcohol gel bodies of the present invention can be controlled by the molecular weight of the additive phase separation additive. By controlling the molecular weight of the added polyethylene glycol, ·· ····

Whether the molecular weight is preferably between 800 and 1350, the pore sizes of the polyvinyl alcohol goal body can be adjusted between 1 and 15 µm.

For the preparation of an aqueous solution of polyvinyl alcohol and an additive according to the invention, it has been shown that a higher degree of drying is required when using distilled water in order to obtain the same mechanical results. These results are immediately improved when using ordinary tap water of some degree of hardness. As a result, it has to be assumed that a certain salt content in water is advantageous for the process according to the invention.

The invention is explained in more detail below by means of examples.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

To 2 g of PVA and 1.2 g of polyethylene glycol (PEG 1000) were added 16.8 g of water. The solution is heated to 90 ° C until all components have completely dissolved to give a viscous, colorless solution. After cooling to 30 ° C, the polymer solution was dropped onto a polypropylene plate using a syringe using pressure. Dripping is performed by tapping the cannula onto the PP plate at a rate of about 1/2 / s; the droplet size is about 3 mm in diameter and about 1 mm in height. Upon dropping, a white waxy film is formed on the surface of the droplets. After evaporation at room temperature, 89 wt. water, the gel bodies are allowed to swell back in water or in brine. The gel bodies obtained have a diameter of 3 to 4 mm and a height of about 200 to 400 µm.

Example 2

After cooling the polymer slurry (composition: 2 g PVA, 1.2 g PEG 1000 a

15.8 g of water), 1 ml of nitrifying mixed culture (Nitrosomonas europaea and Nitrobacter winogradsky) is added to 20 g of polymer solution and dispersed to obtain a dry biomass (BTM) content of 0.06% by weight. EXAMPLE 1. The gel bodies obtained were re-swelled in standard mineral salt medium (STMN) for nitrifiers. The immobilizates thus prepared show an initial activity of about 70% directly after immobilization for

Nitrosomonas spp. and 100% for Nitrobacter spp. compared to the same amount of free nitrifiers.

When incorporating nitrifiers into PVA cryogels, at -20 ° C the initial activity for Nitrosomonas spp. about 1%, at -10 ° C about 25% with decreasing mechanical stability of the PVA-hydrogels.

Incubation of the immobilisates is performed in the same medium at 30 ° C. When 10 mg of gel bodies are incubated in 30 ml STMN, after 19 days the maximum rate of ammonium degradation is reached between 7 and 8 pmol NH ₄ ⁺ / (gkaťmin).

Example 3

The 12.8 g of H ₂ O are dissolved 1.6 g of polyethylene glycol (PEG 1000), followed by 1.6 g PVA and the further procedure is as in Example 1. After cooling the polymer solution at 30 DEG C., 4 ml of culture strictly anaerobic bacterium Clostridium butyricum NRRL B-1024, which converts glycerin to 1,3-propanediol (PD), which is grown overnight in an oxygen-free atmosphere, dispersed in this solution (number of cells in polymer solution: 6 x 10 ⁷ per ml) . The gel bodies are prepared according to Example 1. After evaporation at room temperature of 70 wt. of water, immobilizates are allowed to swell back in mineral salt medium (20-fold excess). Incubation of the gel bodies containing the cells is performed in the same medium (40-fold excess) at 30 ° C. In order to provide sufficient nutrient supply to the immobilized biomass, the medium is changed several times in the initial growth phase.

When 0.25 g of the immobilized biocatalyst thus obtained is introduced into 40 ml of mineral salt medium with 24.4 g / l glycerin, the concentration of 1,3-PD is increased by 2.8 g / l over 3.25 h. This corresponds to a catalyst activity of 0.14 g 1,3-PD per g cat. and an hour. After counting the activity of the grown cells, a catalyst activity of 0.08 g of 1,3-PD (gcat) is obtained.

Example 4

To 2 g of PVAL and 1.2 g of polyethylene glycol (PEG 1000) were added 15.8 g of water and the procedure was as in Example 1.

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After cooling the polymer suspension to about 30-37 ° C, 1 ml of a defined spore suspension of Aspergillus terreus is added to 20 g of polymer solution and dispersed. The spore suspension is selected such that after 5 days of growth an amount of dry biomass of 0.005% by weight is produced in the growth medium.

After evaporation at room temperature 70 wt. of water, the immobilisates are back-swelled in a mineral salt medium for Aspergillus terreus (20-fold excess).

The immobilisates are incubated in growth medium. For the production of itaconic acid, the growth medium is replaced with the production medium.

Prepared immobilisates show an initial activity of about% compared to the same amount of free fungal cells directly after immobilization. When 0.2 g goal bodies are incubated in 100 ml production medium with 60 g / l glucose, after 7 days productivity of 35 mg itaconic acid / (gka * h) is achieved.

Example 5

Larger amounts of goal bodies are obtained by dripping a polymer solution (composition according to Example 1) with a multi-nozzle system onto a conveyor belt.

According to the belt dryer principle, the PVA droplets are dried in a drying tunnel to a defined residual moisture content and are subsequently collected by a scraper in a collection container, where they are swollen back and washed.

Example 6

In the preparation of Example 1, the polymer solution does not drip, but is poured into preformed semi-open molds having an internal diameter of 1 to 10 mm and any length.

After back-swelling in water, the fibers can be stretched to 3 to 4 times their length without breaking. Lengthwise stretching is irreversible. The fiber produced in this way can be loaded with a weight of 500 g without breaking.

Example 7

The fibers prepared according to Example 6 are mechanically characterized after storage for 14 days in tap water. At this point, the fibers have a width of about

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mm and a height of about 1 mm. The degree of back-swelling takes into account the weight loss after back-swelling and 14-day storage in water, based on the total weight of the polymer solution used before the drying process. The fibers exhibit 40% elasticity until elongation at break.

Mechanical characterization of the fibers produced at different drying stages for a composition of 10 wt. % PVA and 6 wt. PEG 1000:

Residual water content after drying [%] Degree of swelling [%] Elongation at break [%] E-module [N / mm ² ] 27 76 455 0.11 20 74 420 0.11 15 68 410 0.18 13 65 390 0.24 10 63 380 0.27 1 57 360 0.34

Mechanical characterization of the fibers at a drying degree of 80 wt. % for the composition wt. % PVA and 8 wt. PEG for different types of PEG:

Kind of PEG Degree of swelling [%] Elongation at break [%] E-module [N / mm ² ] 400 57 410 0.27 600 66 290 0.22 800 82 360 0.19 1000 84 420 0.11 1350 92 370 0.12

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Mechanical properties of PVA-hydrogel fibers for different concentrations of PVA with addition of 6 wt. PEG 1000 at the degree of drying (amount of water evaporated during the drying process) 80% by weight:

PVAL [%] Elongation at break [%] E-module [N / mm ² ] 8 350 0.09 10 420 0.11 12 420 0.17 14 460 0.19 16 440 0.25

The mechanical properties of the fibers at a drying degree of 80 wt. % for a composition of 10 wt. and 6 wt. PEG 1000 for various back-swelling media:

Back-swelling medium Elongation at break [%] E-module [N / mm ² ] tap water 420 0.11 K ₂ HPO ₄ (100 mmol / L) 410 0.17 K 2 SO 4 (120 mmol / l) 530 0.15 CaCl 2 (120 mmol / l) 360 0.10 KCl (175 mmol / L) 370 0.15

Example 8

The gel bodies were prepared according to Example 1 and allowed to swell back in deionized water (5 pS H ₂ O). The degree of backwashing of the gel bodies is determined directly after the backwashing process for the various drying stages. At

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100% wt. the mass of the gel bodies before and after the back-swelling process is the same as can be seen in the attached drawing in the drawing.

Claims (25)

PATENT CLAIMS A method for producing a biocatalyst with biological material in the form of microorganisms, enzymes, spores and / or cells incorporated into a polyvinyl alcohol gel, comprising the following steps: (a) use of an aqueous solution of polyvinyl alcohol with a degree of hydrolysis> 98 mol%; (b) adding an additive which, when added in an aqueous solution of polyvinyl alcohol, dissolves to form a separate, finely divided and water-containing phase upon concentration; c) addition of biologically active material, d) drying the aqueous solution up to a residual water content of no more than 50% by weight; to induce phase separation and the associated gelation of polyvinyl alcohol, (e) back-swelling of the polyvinyl alcohol in an aqueous medium. Method according to claim 1, characterized in that the polyvinyl alcohol solution has a concentration of 4 to 30% by weight, preferably 6 to 16% by weight. Method according to claim 1 or 2, characterized in that a water-soluble additive is used whose water affinity is at least comparable to that of polyvinyl alcohol to water. Method according to any one of claims 1 to 3, characterized in that the water-soluble additive is selected from the group consisting of cellulose esters, cellulose ethers, starch esters, starch ethers, polyalkylene glycol ethers, polyalkylene glycols, long chain alkanols ( n> 8), sugar esters, sugar ethers. The process according to claim 4, characterized in that polyethylene glycol is used as the water-soluble additive. · · • · ·· ···· · · • • • · • • · • • • · • • · · • • · • • • • • · • · • · · • • • · · · · ·· ·· · · · · Method according to any one of claims 1 to 5, characterized in that the water-soluble additive is used in a concentration of 4 to 20% by weight, preferably 6 to 10% by weight. Process according to any one of claims 1 to 6, characterized in that the drying of the aqueous solution is carried out to a residual water content of at least 10% by weight. Process according to claim 7, characterized in that the drying of the aqueous solution is carried out to a residual water content of 10 to 30% by weight. Method according to any one of claims 1 to 8, characterized in that the drying is carried out after the solution has been dripped onto a hard substrate. Process according to any one of claims 1 to 8, characterized in that the drying is carried out after pouring the solution into the casting mold. Method according to claim 9 or 10, characterized in that the dropping or pouring is carried out so as to form a gel body with a diameter which is at least twice as large as its height. Method according to claim 11, characterized in that the dripping or pouring is carried out so as to form a gel body with a diameter of> 1 mm, preferably between 2 and 4 mm, and a height of between 0.1 and 1 mm, preferably between 0 , 2 and 0.4 mm. Method according to any one of claims 1 to 8, characterized in that the drying is carried out after the solution has been poured into the elongate fiber. A method according to any one of claims 1 to 13, characterized in that the drying of the aqueous solution is carried out after pouring onto the carrier material. ········································ Method according to any one of claims 1 to 14, characterized in that the back-swelling is carried out in tap water. A method according to any one of claims 1 to 14, characterized in that the back-swelling is carried out in saline. Method according to claim 16, characterized in that a nutrient solution for the biologically active material is used as the saline solution. Process according to claim 16 or 17, characterized in that a salt solution containing polyvalent anions is used. A method according to any one of claims 1 to 18, characterized in that the drying is carried out completely when the droplet formed in the cooling tower falls. A mechanically high-stability polyvinyl alcohol biocatalyst produced by the process of any one of claims 1 to 19. 21. A biocatalyst according to claim 20, characterized in that it is made in the form of a lens whose diameter is substantially greater than its height. Biocatalyst according to claim 20 or 21, characterized in that it comprises a magnetic additive. A process for producing a product, characterized in that it is formed by reaction with a biocatalyst according to any one of claims 20 to 22. 24. The process of claim 23 wherein the product is 1,3-propanediol. 25. The process of claim 23 wherein the product is itaconic acid.