PL150899B2 - Biotechnical process for obtaining beta fructofuranosidase - Google Patents

Abstract

Yeast cells in suspension are continuously broken up in a spindle mill by micro-glass spheroids, the invertase- containing solution thereby obtained is deslurryed to provide a clear fiitrate by use of a sheet filter, whereafter the clear solution is passed to a combined ultra- and microfiltration plant in such a manner that the solution first runs through an ultrafiltration separation unit, and thereafter is passed over a microfiltration unit. The invertase concentrate solution so obtained is stabilised, e.g. by the addition of glycerol, standardised and packed in known manner. <IMAGE>

Description

^THE REPUBLIC PATENT ^DESCRIPTION 150 899

POLAND OF THE PROVISIONAL PATENT

OFFICE

PATENT

RP

Provisional patent additional to patent No. - Pending: 87 11 16 (P. 268 837)

Int. Cl. ⁵ C12N 9/26 C12M 1/06

Priority: 86 11 20 German

Democratic Republic

Application announced: 88 07 07

Patent description published: 1990 12 31

Inventors: Oleg Kirjuschkin, Ilona Schón, Helmut Kluge,

Alfred Schellenberger, Joachim Herrman, Erhard Kuhn,

Peter Willner

Holder of a provisional patent: VEB Ingenieurtechnik Halle,

Halle (German Democratic Republic)

Biotechnical method of producing invertase

The subject of the invention is a biotechnical method for the production of invertase from active baker's yeast, preferably used in the food industry.

Numerous methods for producing yeast invertase are known.

These methods are characterized by the following working steps: dissolution of yeast cells to release invertase, separation of invertase from cellular components, concentration and purification of the invertase-containing solution, and stabilization of invertase.

For example, it is known to dissolve the cells with organic solvents, e.g. with toluene. After the plasmolysis / autolysis is completed, the undissolved yeast cells are separated, e.g. with a filter press. After adjusting the pH in the autolysate to an appropriate pH value, invertase is obtained by repeated acetone precipitation. After further separation and dissolution steps, the obtained invertase solution is freed of residual solvent and concentrated, for example, by means of a vacuum evaporator. Then it is stabilized with glycerine [compare Waldschmidt - Leitz, E. Und Balls, A. K. in Handbuch der Lebensmittelchemie vol. II / 2 part, ed. Springer Verlag Berlin 1935, pages 734-738].

The disadvantages of this method are, above all, that the poisonous toluene sometimes remains in the invertase solution, the dissolution of the cells takes a long time, which increases the risk of infection, and the yields and activities achieved are low, and the energy input is quite high.

Also post-treatment with papain of the autolysate obtained by adding toluene, and further technical measures, such as adsorption on aluminum hydroxide or calcium phosphate, elution with lactate solution, water or sucrose solution, precipitation with alcohol or picrate solution, dialysis and other purification operations do not lead to significant efficiency of this method [Hartmeier, W. GORDIAN, 78, 320-324 (1978)].

A further known method is described in German Democratic Republic of the DD-PS Patent No. 1401.

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According to conventional yeast culture, accumulation of the active ingredient (hexosidases) in the yeast mass is carried out. Concentration is then carried out by separation, followed by addition of releasing substances, such as wet malt or its diastatic active substances, including cytase.

Cell decomposition occurs by the action of ultrasound on the rotating yeast mass. After the release of specific yeast substances, the invertase solution is separated from cell debris and other solid components by separation. Purification of the cell juice is carried out with a weakly acidic ion exchange resin. The disadvantage of this method consists mainly in the fact that the high technical and technological expenditure and the associated complicated process implementation and high energy expenditure and relatively high expenditure of time for the release of specific yeast substances associated with too low efficiency and achievable activity of the obtained invertase make this method possible. uneconomical.

In addition, a method for the preparation of soluble invertase from yeast is known, as is described in German Patent Specification No. 2,626,626. This method is essentially distinguished by the fact that plasmolysis and autolysis occur due to the interaction of cellulases, proteases and lipases on yeast cells. The starting yeast used is spray-dried yeast from which it is resuspended. After the plasmolysis / autolysis has taken place, the insoluble cell fragments are separated and the filtrate is then separated by ultrafiltration into yeast extract and invertase. Later, the invertase is stabilized or standardized with a glycerin solution or a sorbitol solution. Also in this method it turns out that the energy inputs are quite high. As before, there are also lengthy (8-20 hours) digestion times, which can lead to contamination and thus loss of activity. Moreover, when spray-drying yeast and the associated high temperatures (up to 240 ° C) are used, there is a risk of partial inactivation of the enzyme.

German Democratic Republic Patent Nos. DD-PS Nos. 110 425 and 116135 discuss methods of obtaining yeast extracts, yeast glycan, a yeast protein isolation product with reduced nucleic acid content.

The yeast (brewer's or baker's yeast) cells are then digested in a manner known per se, e.g. by high-pressure homogenization, grinding in a sand or colloid mill, grinding by sound waves, repeated freezing-thawing cycles, lytic enzymes and the like. Heat treatment takes place to separate the nucleic acid from the protein.

Thereafter, a not discussed separation of the insoluble protein product fraction from the soluble fraction then takes place. The preparation of yeast extract, yeast glycan and yeast protein isolation product with reduced nucleic acid content takes place by centrifugation, concentration under vacuum and drying.

These methods are primarily used to obtain protein isolation products from yeast with reduced nucleic acid content, to obtain yeast glycans and yeast extracts. Although these stages of the procedure are essentially similar to those used for the preparation of invertase, there are no indications of the suitability of the proposed solutions for the preparation of invertase in these publications.

In particular, the proposed preferred method of high pressure homogenization for cell cleavage and the proposed methods of heat treatment raise doubts as to the efficient production of invertase.

The patent specification of the Federal Republic of Germany discusses methods for the separation of enzymes, in which these enzymes are solubilized by dissolution, e.g. brewer's yeast cells and / or by treatment with detergents, and dispersed together with the cell debris or with the cells between the various phases of an aqueous multiphase system with a content of at least one macromolecular compound selected from the group consisting of optionally substituted polyalcohols, polyethers, polyesters, polyvinylpyrrolidones and polysaccharides, and at least one inorganic salt, or having at least two macromolecular compounds, and these phases are separated from each other, e.g. by precipitation, ultrafiltration , dialysis, gel filtration, adsorption, ion exchange or electrophoresis.

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In the proposed solution it is said that any enzymes can be obtained, but specifically only pullulanases or maltases are referred to. The results in terms of activity and productivity achieved with the proposed method cannot, however, be satisfactory in view of the required expenditure on resources.

The object of the invention is to provide an energy-saving, biotechnical method for the production of high-purity and high-purity invertase from baked yeast, which method would not use any organic solvent or other chemicals and / or enzymes and achieve short production times. Moreover, the method should be easy to control and uncomplicated by the use of appropriate equipment, and be capable of being operated continuously, while at the same time achieving a high yield of concentrated invertase and being able to further process the obtained waste or by-products.

The method according to the invention consists of the following technical measures.

The yeast cells of a suspension of active baker's yeast, containing about 10% dry yeast substance (abbreviated as HTS) and having a temperature of 8-15 ° C, are continuously ground in a spindle mill, the glass microbeads used have a diameter of 0.5 um, and the speed is the rotational speed of the agitator shaft is 2500-4000 rpm. In the successive separator, the coarse separation of the cell debris and the invertase-containing solution is continuously carried out. To avoid loss of enzyme efficiency, the slurry feed is interrupted prior to demining and the impeller is rinsed with water for approximately 5 minutes, the sludge chamber being emptied only when clean water appears at the product outlet.

The subsequent uninterrupted clarifying filtration of the invertase-containing solution takes place via an in-line filter, the filter layers having a permeability of 425-475 liters / hm ² . The fines of these filter layers are additionally lined with filter paper, the filter layers being rinsed with water for approximately 20 minutes before the start of the clarifying filtration. The pressure difference at the inlet and outlet of the filter during this filtration must not exceed 200-250 kPa.

The in-line filter is switched on so that circulation is possible in addition to the filtration process, thus avoiding overpressure which could lead to damage to the filter plates. The residue of the clarified invertase solution obtained by the circular motion process is separated again.

Purification, concentration and decontamination of the invertase solution takes place in a complex ultra- and microfiltration device. The ultrafiltration separation unit is arranged relative to the microfiltration separation unit such that the solution is first purified and concentrated by means of the ultrafiltration separation unit and then passed through the microfiltration separation unit for decontamination.

The permeability of the UF membranes used in the ultrafiltration separation unit is 80-140 liters / hm ² , the output pressure being about 0.15-0.4 MPa and the output pressure about 0.1-0.3 Mpa. In order to increase the mechanical durability of the flat membranes, a support fabric, for example a non-woven material, is introduced between the membranes and the filter return plates.

The invertase solution is concentrated to about 1/10 by means of an ultrafiltration separation unit. For further purification of this invertase solution concentrate, additionally a diafiltration is carried out via an ultrafiltration separation unit. This concentrate is diluted 1: 1 with water and diafiltered into the circuits until the original volume of the concentrate is reached again.

After this ultra- and diafiltration, the purified invertase concentrate solution is passed through a microfiltration separation unit in order to separate the denatured proteins and microorganisms contained in this solution. The pore diameter of the microfiltration material used is 0.2 μπι, the input pressure should not exceed 0.3 MPa. In order to increase the mechanical durability of the filter material used, as in the case of the ultrafiltration separation unit, a support fabric is used.

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By means of the subsequent, usual stabilization, the solution is adjusted to an activity of about 1500 U / ml, rendering it stable.

The process according to the invention is particularly suitable for the preparation of invertase preparations with a high specific activity [400-800 U / mg], for which there is particular interest, especially for analytical purposes, in which the diafiltration process is repeated in the above-mentioned way 3 to 3. 8 times.

The yield of the glycerin-stabilized invertase solution obtained by the procedure described is 75%, based on the active bakery yeast used.

The following embodiment is explained based on the schematic diagram shown as the Fig. In the drawing.

From 120 kg of active baker's yeast (with 30-33% HTS content) stored at 2-4 ° C, a mixture of 240 liters of water is mixed in a thermostatic mixer 1. After 30 minutes, a homogeneous yeast suspension with a content of 10-15 is formed. % HTS, which is kept at 10-12 ° C.

The cooled suspension is then fed via a metering pump 2 to a continuously operated spindle mill 3. The yeast cells are digested in a water-cooled, approximately 3 liter mill, filled with approximately 2400 g of glass beads with a diameter of 0.8 to 1 mm. The speed of rotation of the agitator shaft is approximately 3000 rpm and the slurry capacity is 16 liters / h. The dissolved yeast suspension flows through the upper outlet of the mill into the thermostatic mixer 4. The dosing pump 5 supplies this suspension to the separator 6. Here, the majority of cell debris is separated from the invertase-containing solution. From the product effluent from the separator 6, the phase flows, almost free of cellular debris, into the intermediate tank 7. The flow rate of the suspension to be separated is about 50 liters / h. The separator 6 is degreased after approximately 0.75 hours in each case. In order to avoid major losses in enzyme yield, the feed of the yeast slurry is interrupted prior to degumming and the rotor is rinsed with water until clear water appears at the product outlet. The control water is then turned on and the sludge is drained.

From the intermediate tank 7, the inverted solution is led by a dosing pump 8 to an in-line filter 9 for clarifying filtration. Clarification takes place thanks to the use of a KP-10 A (40 X 40 cm) flow-through filter with a permeability of about 450 liters / hm ² . In order to increase the quality of the filtrate, the clarifying side of the filter layers is put on a filter paper called Niederschlag 291 (40 x 40 cm). Before starting this filtration, the arranged filter layers are rinsed with water for about 20 minutes.

The filtration speed is adjusted to about 80 to 100 liters / hour. The maximum difference between the pressure at the inlet and the outlet of the filter must not exceed 200-250 kPa. The in-line filter 9 is connected in such a way that there is a fluid circulation next to the filtration process, so that overpressure is prevented, which could lead to premature destruction of the filter material. The residual of the clarified, invertase-bearing solution obtained thanks to the circulating method of motion is fed back to the separator after the clarifying filtration has been completed. The solution to be processed further is introduced into the intermediary tank 10. Further purification, concentration and decontamination of the invertase-containing solution takes place in a special, complex ultra-and microfiltration 11

The plate pack, located on the frame, uses 5 m ² of the filter area for ultrafitration and 4 m ² for microfiltration. For the ultrafiltration separation unit 12, UF membranes with a permeability of 80-140 liters / hm ² of the CAM-UF-120-CP type (from VEB Zellstoff- und Zellwollewerke Wittenberge) are used. This type gives an invertase selectivity of more than 99%. In order to increase the mechanical durability, a support fabric made of non-woven material is additionally placed between the membrane and the filter outlet plate.

The microfiltration separation unit 13 is equipped with a polyester microfiltration microfiltration (from ZfK AdW Rossendorf) with a pore diameter of 0.2 µm. This filter material is also supported with a non-woven material to increase durability.

From the intermediate tank 10, the inverted solution is forced by a metering pump 14 through a tube-in-tube cooler 15 to the ultrafiltration separation unit 12.

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The overflow solution is passed through the filter plates, particles with a mass greater than 150,000 being retained. The effluent rate of the permeate is approximately 80 to 140 liters / h at an initial pressure of 0.1 to 0.3 MPa. The invertase activity in the filtrate is less than 0.5%.

The solution obtained after the clarification filtration is concentrated to 1/10 volume by ultrafiltration. The concentrate obtained after ultrafiltration is then diluted 1: 1 with water and, for further purification, circulated diafiltered through the ultrafiltration separation unit 12 until the original concentrate volume is reached again.

Due to the blocking of the ultrafiltration circuit, the ultra- or diafiltered concentrate in the intermediate tank 10 is transferred by a dosing pump 16 through a cooler 15 to a microfiltration separation unit 13.

The denatured proteins and microorganisms that reduce the quality of the product contained in the concentrate are separated in this unit. Said active filter surface is set at a throughput of about 20 liters / h, the input should not be greater than 0.3 MPa.

The purified, concentrated and decontaminated invertase solution is placed in a thermostatic mixer 17 and mixed with glycerin in a 1: 1 ratio, thereby stabilizing the solution. The yield of the stabilized invertase solution is 90 kg (75% with respect to the introduced active baker's yeast).

Claims (1)

Patent claim Biotechnical method for the production of invertase from active baker's yeast, in which active baker's yeast containing 30-33% dry yeast substance (abbreviated as HTS) is slurried with water with a content of about 10% HTS and its temperature is adjusted to 10-12 ° C method, characterized in that the yeast cells of this suspension are continuously crushed in a known spindle mill with glass microbeads with a diameter of 0.5-1.2 mm and a stirrer shaft rotational speed of 2500-4000 rpm, and then in a known separator it is obtained invertase-containing solution, the slurry feed is interrupted before desliming and the impeller is rinsed with water until clear water appears at the outlet of the product, then the solution is subjected to the usual clarification filtration by means of an in-line filter, the filter layers having a permeability of 425-475 liters / hm ² , the clarifying sides of these filter layers are additionally supported with filter paper and d the start of the clarifying filtration, these filter layers are rinsed with water for about 20 minutes, the pressure difference between the inlet and outlet of the filter during this filtration is not greater than 200-250 kPa, the in-line filter is turned on in such a way that, apart from the filtration process, circulation is possible the mode of movement and the obtained residue of the clarified invertase solution are separated again, after which the solution is fed to a complex ultra- and microfiltration device in such a way that the solution first passes through the ultrafiltration separation unit, is subjected to a diafiltration circuit and then passes through the unit microfiltration separation, the permeability of the membranes used in the ultrafiltration separation unit is 80-140 liters / hm ² , the input pressure is 0.15-0.4 MPa, the output pressure is 0.1-0.3 MPa, and between the membranes and the plates a supporting fabric, for example a non-woven material, is arranged to discharge the filter, this solution is first concentrated to about 1/10 by means of an ultrafiltration separation unit, the concentrate is then diluted 1: 1 with water and recirculated diafiltered until the original concentrate volume is again obtained, then this purified invertase concentrate solution is passed through a microfiltration separation unit, the pore diameter of the microfiltration material used is 0.2 μπι, the input pressure is not more than 0.3 MPa, and the filter material, as in the case of the ultrafiltration separation unit, is provided with a support fabric, and then a concentrate solution invertases are stabilized, standardized and packaged in the usual way. 150 899 ι ~~ ι OJ Department of Publishing of the UP RP. Circulation 100 copies. Price 1500 PLN