GB2198731A - Preparation of invertase from yeast - Google Patents

Description

r 2198731 Biotechnical process for the preparation of invertase The

present invention is concerned with a bio-ion of the enzyme technical process for the preparaL. invertase from bakers' yeast, which enzyme is prefer 5 ably used in the foodstuff industry.

A number of processes are known for the prepar ation of invertase from yeasts. The processes are essentially characterised by tha following worki.q steps:

digestion of the yeast cells for the liberation Of Lhe invertase; separation of the invertase from the cell components; concentration and purification OIL the invertasecontaining solution; stabilisation of the invertase.

Thus, for example, it is known to carry out the destruction of the cells by means of organic solvents, for example with toluene. After completion of the plasmolysis/autolysis, there follows a separation o-F the non-dissolved yeast cells, for example by ineans of filter presses. After an appropriate pH adjustment of the autolysate, there follows the recovery of the invertase by a repeated acetone precipitation. After further separation and dissolving steps, the invertase solution obtained is freed from solvent residues, for example by means of vacuum evaporators, and concentrated. Subsequently, it is stabilised with glycerol (see E. Waldschmidt-Leitz and A.K. Balls, in Handbuch der Lebensmittelchemie, Vol. II/2nd part, pub. Springer Verlag, Berlin, 1935, pp. 734 - 738).

The disadvantages of these processes are, in particular, that, amongst other things, residues of the toxic toluene remain in the invertase solution, the cell digestion is laborious, which increases the danger of microbial contamination, and the yields and activities achieved are low, the energy expenditure thereby C) being relatively high. Furthermore, the additional treatment of the autolysate obtained by the addition of toluene by means of papaine, as well as further measures, such as adsorption on aluminium hydroxide or calcium priosphate, elution with lactate solution, water or saccharose solution, precipitation with alcohol or picrate solution, dialysis and other purification operations, do not result in the process becoming sub- W stantially more effective (see W. Hartmeier, Cordian, 78, 320 - 324/1978).

A further known process has been described in German Democratic Republic Patent Specification No.

1401. After the usual yeast culturing, an active 1 1.

i.

material enrichment (hexosidases) is carried out in the yeast mass. There follows a concentration by separation with subsequent addition of liberating materials, such as green malt or its diastatic active materials, including cytase. The cell destruction takes place by the action of ultrasonics on the rotating yeast mass. After the liberation of the component materials of the yeast, there follows the separation of the invertase solution from the cell debris and other solid components by separation. The purification of the cell juice is carried out with the-help of weakly acidic exchanger resins.

The disadvantage of this process is, in particular, that the high technical technological expense and the complicated carrying out of the process involved therewith, as well as the high expenditure of energy and the relatively high expenditure of time for the liberation of the yeast component materials, together with the too low yields or achievable activity of the invertase obtained, make it uneconomic.

Furthermore, a process is known from German Patent Specification No. 26, 626 for obtaining soluble invertase from yeast. This process is essentially characterised in that the plasmolysis and autolysis is completed by the action of cellulases, proteases and lipases on the yeast cells. As starting yeast, there is used spray-dried yeast which is subsequently 1 resuspended. After plasmolysis/autolysis have taken place, there follows the separation of the insoluble cell fragments and subsequently the filtrate is separated into yeast extract and invertase by means 5 of ultrafiltration. Thereafter, there follows a stabilisation or standardisation of the invertase by means of glycerol or sorbitol solutions. In the case of this process, too, the expenditure of energy is found to be relatively high. As previously, the digestion times are also high (8 to 20 hours), which can result in microbial contanination and thus in losses of activity. Furthermore, in the case of the use of the spray drying of the yeas'. and the high temperatures thereby involved, there is a danger of a 7 the enzyme.

partial inactivation of German Democratic Republic Patent Specifications

Jos. 110,1L25 and 116,135 describe processes for obtain ing yeast extracts, yeast glycan and yeast protein 0 1 isolates with a reduced nucleic acid content.

The yeast cells (brewers' or bakers' yeast) are thereafter digested in known manner, for example by 1 high pressure homogenisation, comminution in a sand or colloid mill, disintegration by means of sound waves, repeated freezing-thawing cycles, lytic enzymes and the like. A heat treatment follows for the purpose of separating the nucleic acids from the protein.

Thereafter, there follows a separation of the A insoluble protein product fraction from the soluble fraction in a manner which is not described in detail. The obtaining of the yeast extract, yeast glycan and yeast protein isolate with a reduced nucleic acid content takes place by centrifuging, vacuum concent-ration and drying.

These processes serve, in the first place, for obtaining yeast protein isolates with a reduced nucleic -ent, yeast glycans and yeast extracts.

acid cont Ad-r-iiittedly, the-process steps resemble those which are used for obtaining invertase but, in these inventions, there is no teaching regarding the transfer of the proposed teachings to the obtaining of invertase.

In particular, the preferably proposed high pressure homogenisation process for the destruction of the cells and the suggested heat treatment process give rise to doubt for an effective preparation of invertase. In Federal Republic of Germany Patent Specific20 ation No. 26 39 129, there is described a process for the separation of enzymes in the case of which the enzymes are solubilised by digestion of, for example, beer yeast cells and/or treatment with detergents and partition of the cell debris or cells between different phases of an aqueous multi-phase system with a content of at least one high molecular weight compound selected from the group consisting of optionally substituted polyalcohols, polyethers, polyesters, polyvinylpyrrolidones and polysaccharides and at least one inorganic salt or with a content of at least two high molecular weight compounds, whereafter the phases are 5 separated from one another, for example by precipitation, ultrafiltration, dialysis, gel permeation, adsorption, ion exchange or electrophoresis.

In the suggested teaching, mention is admittedly made that any desired enzymes can be obt--ained but con- io crete reference is only made to pullulanases and maltases. However, the results achieved with the proposed process with regard to activities and yields in comparison with the n2cessary expenditure of agents cannot be satisfactory.

It is an object of the present invention to provide an energy-economic biotechnical process for the preparation of highly active and very pure invertase from bakers' yeast in which no organic solvents or other chemicals and/or enzymes are used and with which short production times can be achieved. Furthermore, by means of the use of appropriate equipment, the process is to be easily controllable and noncomplicated, as well as being capable of continuous operation and, at the same time, a high yield of concentrated invertase is to be achieved and the waste or by-products obtained are to be capable of further working up.

1 1 5.

-7 -Thus, according to the present invention, there is provided a biotechnical process for the preparation or invertase from active bakers' yeast, in which active bakers' yeast with a yeast dry substance (YDS) of 30 to 33% is suspended with the help of water to give a YDS of bout 10% and adjusted to a temperature of 8 to 15'C., wherein the yeast cells of this suspension are contin uously broken up in a spindle mill with the help of micro-glass spheroids with a diameter of 0.5 to 1.2 mm.

and a stirrer shaft speed of rotation of 2500 to 4000 r.p.m., thereafter an invertase-containing solution is obtained in a separator and, before deslurrying, the inflow of the suspension is stopped and the rotor rinsed with water until clear water emerges at the product outlet, thereafter the solution is subjected to a clear filtration with the help of a sheet filter, the filter layers having a permeability of 425 to 475 l./h.m2, the clear sides of the filter layers being additionally underlayed with filter paper and, before commencement of the clear filtration, these filter layers are wetted with water for about 20 minutes, the pressure difference between the filter inlet and outlet during the filtration being not greater than 200 to 250 kPa, the sheet filter being so connected that, besides the filtration process, a circulatory manner of procedure is possible and the remaining residues of the invertase solution to be CD fied are again subjected to the separation, clari whereafter the solution is passed to a combined ultra- and microfiltration plant in such a manner that the solution first runs through an ultra- filtration separation unit, is subjected in circulation to a defiltration and thereafter is passed over a microfiltration unit, the permeability of the ultrafiltration membranes used in theultraffiltration separation unit being 80 to 140 Mh m2, the inlet pressure ')einz 0.15 to O.A MPa and the outlet pressure being 0.1 to 0.3 MPa, between the membranes and the filter leadoff plates there is arranged a supporting fabric, the solution first being concentrated by the ultrafiltration separation unit to about one tenth, whereafter the concentrate is diluted with water in the ratio of 1:1 and diafiltered in circulation until the starting volu-,-.ie of concentrate is again achieved, whereafter the purified invertase concentrate solution obtained is passed over the microfiltration separation unit, the pore diar.,ieter of the microfiltration material used being about 0.2 pm. and the inlet pressure being not greater than 0.3 MPa and the filter material, as in the case of the ultrafiltration separation unit, being provided with a supporting fabric, whereafter the invertase concentrate solution obtained is stabilised, standardised and packed in known manner.

The yeast cells of an active bakers' yeast 1 -9- sus pension with a yeast dry substance (YDS) content of about 10% and a temperature of 8 to 15C. and preferably of 10 to 12'C. are continuously broken up in a spindle mill, the. microglass spheroids used having a diameter of 0.5 to 1.2 mm. and the speed of rotation of the stirring shaft being 2500 to 4000 r.p.m. In a following separator, there takes place continuously a rough separation of1cell debris and invertasecontaining solution. In order to avoid enzyme yield losses, before deslurrying, the inflow of the suspension is stopped and the rotor rinse.d with water for about 5 minutes, the slurrying chamber only then being emptied.when clear water emerges from the product outlet of the separator.

The subsequent continuous clear filtration of the invertase-containing solution takes place via a sheet filter, the filter layers having a permeability of 425 to 475 l./ I h.m 2. The clear sides of the filter layers are thereby additionally underlaid with filter paper, the filter layers thereby being wetted with water for about 20 minutes before commencement of the filtration. The pressure difference between the filter inlet and outlet must, during the filtration, not be greater than 200 to 250 kPa.

The sheet filter is so constructed that, besides the filter process, a circulation is possible so that the formation of an overpressure, which can result in -10 damage to the filter plates, is avoided. The residue of the invertase solution to be clarified due to the cyclic procedure is again subjected to the separation.

The purification, concentration and sterilis- ation of the invertase solution takes place viia combined ultra- and micro-filtration plant. The ultrafiltration separation unit is to be so arranged with regard to the microfiltration separation unit tnat the solution is first purified via F-he u'Lera-i-i!Lration separation unit and concentrated and tlierea-.,Lter, for sterilisation, passed over the microfiltration separation unit.

The permeability of the ultrafiltration membranes used in the ultrafiltration separation unit is 80 to 140 1./h.r.i 2, the inlet pressure being about 0.15 - 0.4 MPa and the outlet pressure being about 0.1 to 0.3 MPa. For increasing the mechanical stability of the flat membranes, between the membranes and the filter lead-off plates there are provided supporting fabrics, for example fleece materials.

The invertase solution is concentrated to about one tenth by means of the ultrafiltration separation unit. For further purification of the invertase solution concentrate, there follows an additional diafiltration over the ultrafiltration separation unit. The concentrate is thereby diluted with water in the ratio of 1:1 and diafiltered in circulation until the 2:

1.

c initial volume of concentrate is again achieved.

After this ultra- and diafiltration, the purified invertase concentrate solution is passed over the microfiltration separation unit for the separation of the denatured proteins contained in the solution, as well as of the micro-organisris. The pore diameter of the micro-filter material employed is 0.2 pm., an inlet pressure of 0.3 MPa not being exceeded. For increasing the mechanical stability of the filter material used, a support4-ig fabric is employed in the inanner described in the case of the ultrafiltration separation unit.

With a subsequent, known stabilisation, the solution is adjusted to an activity of about 1500 U.riil and thus made storage-stable.

The process according to the present invention is especially suitable for producing invertase preparations of high specific activity (400 to 800 U/mg.), for which, for analytical purposes, there is an espec- ial interest, in that the diafiltration is repeated 3 to 8 times in the above-described manner.

The yield of glycerol-stabilised invertase solution achieved according to the above-described process amounts to 75%, referred to the active bakers' yeast employed.

ollowing Example is given for the purpose The f of illustrating the present invention, reference being 1 9_ made to the accompanying drawing, which schematically illustrates a flow diagram. Example.

kg. of active bakers' yeast stored at 2 to 4'C. (YDS 30 to 33%) is placed in a mixing and tempering container 1, together with 240 litres of water. After about 30 minutes, a homogeneous yeast suspension is obtained with a YDS of 10 to 15% which is maintained at a temperature ot ifrom 10 to 12'C.

iu The cooled suspension is tlierea,'er passed via a dosing pump 2 to a continuously operating spindle mill 3. The digestion of the yeast cells thereby takes 0 place in a water-cooled approximately 3 litre capacit y grinding pot which is filled with about 2400 g. of microglass spheroids of 0.8 to 1 mm. diameter. The speed of rotation of the stirrer shaft is about 3000 r.p.m. and the suspension throughput is 16 litres/hour. The digested yeast suspension runs over an upper overflow-of the grinding pot into a mixing and tempering container 4. The suspension is passed via a dosing pump 5 into a separator 6. The preponderant part of the cell debris is here separated from the invertasecontaining solution. From the product flow-off of the separator 6, the phase substantially freed from the cell debris runs into an intermediate container 7. The rate of inflow of the suspension to be separated is about 50 litres/hour. The separator 6 is thereby 1 r, to be freed of slurry after about 0.75 hours. Inorder to avoid comparatively large losses of enzyme yield, before the deslurrying, the inflow of the yeast suspension is stopped and the ro-tor rinsed with water until clear water emerges from the product outlet. The control water is then switched on and the slurry removed.

The invertase-containing solution is passed from the intermediate container 7, with the help of a dosing pump 8, to a sheet filter 9 for the purpose of clear filtration. The clarification takes place by the use of filter layers of the type KP-10 A (40 x 40 cm.) 2 with a permeability of about 450 l./h.m In order to increase the quality of the filtrate, the clear side of the filter layers is underlayed witia filter paper deposit 291 (40 x 40 cm.). Before commencement of the filtration, the introduced filter layers are wetted with water for about 20 minutes.

The speed of filtration is adjusted to about 80 to 100 l./h. The maximum pressure difference between the filter inlet and outlet must thereby not be greater than 200 and 250 kPA, respectively.

The layer filter 9 is thereby so connected that, besides the filtration process, a medium circulation is present so that the formation of an overpressure, which can result in a premature destruction of the filter material, is avoided.

The residue of the invertase-containing solution to be clarified remaining due to the circulatory method of proceeding is, after conclusion of the clear filtration, again passed to the separator 6.

The solution to be further worked up is transferred to an intermediate container 10. The further purification, concentration and sterilisation of the invertase-containing solution takes place in a special combined ultraand microfiltration plant 11.

The plate packet Present on a fr2ne is used..-ith 5 ill 2 filter surface for the ultrafiltration and with 4 m 2 for the microfiltration. For the ultrafiltration separation unit 12, there are used ultrafiltration membranes with a permeability of 80 to 140 1./h.m 2 of the type CAM-UF120-CP (VEB Zellstoff- und Zellwollewerke Wittenberge). This type ensures a selectivity, referred to the invertase, of more than 99%. For increasing the r.,.iechanical stability, between the membrane and filter lead-off plate, there is additionally arranged a supporting fabric of fleece material.

The microfiltration unit 13 is covered with a nucleus trace microfilter of polyester (ZfK AdW Rossendorf) with a pore diameter of 0.2 pin. For increasing the stability, this filter material is also supported with fleece material.

From the intermediate container 10, the invertasecontaining solution is pumped with the help of a dosing pump 14 through a double-tube cooler 15 into the ultrafiltration unit 12. The solution is passed, according to the overflow principle, over the filter plates, molecules with a molecular weight of greater than 150,000 thereby being retained.

The permeate run-off rate is thereby about 80 to 140 1./h. at a starting pressure of 0.1 to 0.3 PMa. The invertase activity in the permeate is thereby below 0.5%.

Lhe solution obtained after the clear filtration -he is concentrated to one tenth of its volume by t ultrafiltration. The concentrate obtained after the ultrafiltration is subsequently diluted with water in the ratio of 1:1 and, for thepurpose of a further purification, diafiltered in circulation over the ultrafiltration separation unit 12 until the starting volume of the concentrate has again been reached.

Due to the blocking of the ultrafiltration circulation, the ultra- and diafiltered concentrate present in the intermediate container 10 is conveyed by means of a dosing puinp 16 via the cooler 15 to the microfiltration unit 13.

The denatured proteins contained in the concent rate, as well as microorganisms, which impair the product quality, are there separated off. The abovementioned filter surface is adjusted to a throughput of about 20 litres/hour, the inlet pressure thereby 16 being not greater than 0.3 14Pa.

The pu-úLfied, concentrated and sterilised invertase solution is introduced into a mixing and tempering container 17 and there mixed with glycerol in a ratio of 1:1 and thereby sterilised. The yield of stabilised invertase solution is 90 kg. (75% referred to the active bakers' yeast used).

Claims (5)

Patent Claims

1. Biotechnical process for the preparation of invertase from active bakers' yeast, in which active bakers' yeast with a yeast dry substance (YDS) of 30 to 33% is suspended with the help of water to give a YDS of about 10% and adjusted to a temperature of 8 to 15'C., wherein the yeast cells of this suspension are continuously broken up in a spindle mill with the help of micro-glass spheroids with a diameter of 0.5 to 1.2 mr.q. and a stirrer shaft speed of rotation of 9-,no to 4000 r.p.m., thereafter an invertase-containing solution is obtained in a separator and, before deslurrying, the inflow of the suspension is stopped and the rotor rinsed with water until clear water emerges at the product outlet, thereafter the solution is subjected to a clear filtration with the help of a sheet filter, the filter layers having a permability of 425 to 475 I./h.m 2, the clear sides of the filter layers being additionally underlayed with filter paper and, before commencement of the clear filtration, these filter layers are wetted with water for about 20 minutes, the pressure difference between the filter inlet and outlet during the filtration being not greater than 200 to 250 kPa, the sheet filter being so connected that, besides the filtration process, a circulatory manner of procedure is possible and the remaining residues of the invertase solution to be -18clarified are again subjected to the separation, whereafter the solution is passed to a combined ultra- and microfiltration plant in such a manner that the solution first runs through an ultrafiltration separation unit, is subjected in circulation to a diafiltration and thereafter is passed over a microfiltration unit, the permeability of the ultra- filtration membranes used in the ultrafiltration 2 separation unit being 80 to 140 1./h.m ' the inlet pressure being 0.15 to 0.4 14Pa and the outlet pressure C being 0.1 to 0.3 MPa, between the membranes and the filter lead-off plates there is arranged a supporting. fabric, the solution first being concentrated by the' ultrafiltration separation unit to about one tenth, whereafter the concentrate is diluted with water in the ratio of 1:1 and diafiltered in circulation until the starting volume of concentrate is again achieved, whereafter the purified invertase concentrate solution obtained is passed over the microfiltration separation unit, the pore diameter of the microfiltration material used being about 0.2 pm. and the inlet pressure being not greater than 0.3 MPa and the filter material, as in the case of the ultrafiltration separation unit, being provided with a supporting fabric, whereafter the invertase concentrate solution obtained is stabilised, standardised and packed in known manner.

T.

2. Process according to claim 1, wherein the yeast suspension used is adjusted to a temperature of 10 to 12'C.

3. Process according to claim 1 or 2, wherein the invertase solution obtained is stabilised by mixing with glycerol in a ratio of 1:1.

4. Process according to claim 1 for the preparation of invertase, substantially as hereinbeford described and exemplified.

5. Invertase, whenever prepared by the process according to any of claims 1. to 4.

Published 1988 at The Patent Office, State House, 66M High Holborn, London WC1R 47-r..-r-trther copies may be obtained from The Patent Office, Salles Branch.. S. Mary Cray, Orpington.,'er-- BR5 3RD Printed by Multiplex techniques ltd. St Mary Crky. Kent. Con. 1187.