FR2607148A1 - BIOTECHNICAL PROCESS FOR PREPARING ENZYME INVERTASE - Google Patents

Abstract

THE PRESENT INVENTION CONCERNS A BIOTECHNICAL PROCESS FOR PREPARING INVERTASE ENZYME FROM BAKER YEAST, PREFERABLY USED IN THE FOOD INDUSTRY. WITHIN THE FRAMEWORK OF THE PRESENT INVENTION, THE YEAST CELLS ARE MECHANICALLY DISAGREGLED FROM A BAKERY YEAST SUSPENSION USING A TREE MILL. THE SEPARATION OF THE CELLULAR DEBRIS FROM THE SOLUTION CONTAINING INVERTASE IS THEN CARRIED OUT. THE SOLUTION IS SUBJECT TO CLARIFICATION USING A LAMINATED FILTER. PURIFICATION, CONCENTRATION AND STERILIZATION ARE PERFORMED ON A COMBINED ULTRA AND MICROFILTRATION PLANT, OR FIRST THE PURIFICATION AND CONCENTRATION BY MEANS OF ULTRAFILTRATION IN THE CYCLE USING ULTRAFILTRATION, THEN STERILIZATION IS PERFORMED THANKS TO MICROFILTRATION.

Description

The present invention relates to a biotechnical process for the preparation of

  the invertase enzyme from baker's yeast, which is used

  preferably in the food industry.

  A large number of processes are known

  of preparation of invertase from yeasts.

  Essentially these processes are characterized

  by the following operating steps: - Disintegration of the yeast cells to release the invertase - Separation of the invertase from the components of the cell

  - Concentration and purification of the solution

holding the invertase

- Stabilization of invertase.

  We thus know p. ex. carry out cell destruction with organic solvents, e.g.

  ex. with toluene. After the end of the plasmolysis /

  tolyse a separation of undissolved yeast cells, p. ex. by pressure filtration. After a corresponding adjustment of the value

  the pH of the autolysate is obtained by obtaining the inversion

  tase via a precipitation of acetone

  to perform several times. After an extra step

  separation and dissolution,

  removes solvent residues the solution of invertase obtained p. ex. by means of a vacuum evaporator

  and concentrate. Then stabilized with glycerol.

  (Waldschmidt - Leitz, E. and Balls, A.K. in Handbuch der Lebensmittelchemie Vol II / 2nd part, Editions

  Spriniger, Berlin 1935, pp 734-738).

  The disadvantages of this process are that, from time to time,

  toxic toluene persist in the solution of inversion

  tase, in that the disintegration of the cells is long, which increases the danger of contamination, and in that the yields and activities obtained

  are low, the energy expenditure being relatively

high level.

  The additional treatment of the autolysate obtained by the addition of toluene by means of papain

  and subsequent measures such as ad-

  Sorption on hydrated alumina or calcium phosphate, elution with lactate solution, water or sucrose solution, precipitation with alcohol or a solution of picrate, dialysis and other purification operations born

  not lead to a significant increase in the effectiveness of

  city of the process (Hartmeier, W. GORDIAN, 78, 320-

324 (1978)).

  Another known method has been described in

the patent of RDA N 1401.

  After the usual yeast culture, the active substance (hexosidases) is enriched in the yeast mass. This is followed by a concentration by separation followed by the addition of liberating substances such as green malt or diastatic active ingredients including cytase. Cell destruction is done by

  ultrasound action on the mass of yeast in rotation.

  After the liberation of the bodies contained in the yeast, the separation of the invertase solution from cell debris and other solid components is carried out by a separation technique. The purification of the cellular juice is carried out using a resin

weakly acidic exchange.

  The disadvantage of this process lies especially in the fact

  the importance of the expenditure of industrial technology

  trielle and the complicated mode of realization related to it as well as the great energy expenditure and the relatively long time passed to the liberation of the bodies contained in the yeast with the too low yield or activity which one can reach for

  the resulting invertase makes it unprofitable.

  Also known is a method for obtaining soluble invertase from yeast as described

in German Patent No. 26,626.

  This process is essentially characterized by the fact that plasmolysis and autolysis are carried out by the action of cellulases, proteases and lipases on the yeast cells. As starting yeast, the freeze-dried yeast is used, which is then

  resuspension. Once the plasmolysis /

  the separation of the cell fragments takes place

  then the filtrate is separated into the yeast extract and invertase by means of a

  ultrafiltration. Then a stabilization

  tion or standardization of invertase using a solution of glycerol or sorbitol. It is also revealed in this process that the energy expenditure is relatively important. The disintegration times are as before high (8-20 h), which can lead to contamination and thus to loss of activity. In addition there is in the use of lyophilization of yeast and the use of a high temperature attached thereto (up to 240 C) a danger of inactivation

partial of the enzyme.

  In the East German patents N

  110 425 and 116 135 are described methods for obtaining

  yeast extract, yeast glycan, lower yeast protein isolates

in nucleic acids.

  The yeast cells (baker's yeast or baker's yeast) are then broken down in a known manner p. ex. by homogenization under pressure, trituration in a sand or colloid mill, spraying with sound waves, repeated freeze-dew cycles, lytic enzymes, etc. A heat treatment is then carried out to separate the nucleic acid from the protein. The insoluble protein product fraction is then separated, not described in more detail, from the soluble fraction. Obtaining yeast extract, yeast glycan and yeast protein isolate with reduced nucleic acid content is carried out by centrifugation,

  concentration under vacuum and drying.

  These methods are used firstly to obtain yeast protein isolates with reduced nucleic acid content, yeast glycans.

and yeast extracts.

  The stages of the process are certainly close to those which are applied in obtaining invertase, but it is not found in

  inventions no indication as to a possible

  transposition of teaching recom-

mandated to obtain invertase.

  In particular, the high-pressure homogenization method preferably recommended for cell destruction and the recommended thermal treatment process give rise to doubts.

  as for an effective preparation of invertase.

  German Patent No. 2639129 discloses enzyme separation methods in which the enzymes are solubilized by disintegrating p. ex. of yeast cells and / or treatment with detergents and dispersed with the debris of cells or cells between different phases of a multi-phase aqueous system containing at least one high molecular weight body among the groups formed by polyalcohols, polyethers, polyesters, polyvinylpyrrolidones and polysaccharides

  optionally substituted and at least one inorganic salt

  nique, or containing at least two bodies with high molecular weight, then the phases are separated from each other p. ex. by precipitation, ultrafiltration, dialysis, gel permeation, adsorption, exchange

ions or electrophoresis.

  In the recommended teaching, it is certainly said that one can obtain any enzymes, but concretely one only refers to pullulanases or maltases. The results achieved with the recommended process in terms of activities and yields compared to expenditure on resources

  necessary, however, can not satisfy.

  The object of the invention is to develop an energy-efficient biotech process for the preparation of invertase of activity and purity raised from baker's yeast, in which no organic or other solvents are used. chemicals and / or enzymes and short preparation times are achieved. Furthermore, the method, by the use of corresponding devices, must be easy to control and uncomplicated, and must be able to be implemented continuously, and one must simultaneously obtain a high yield of concentrated invertase and be able to

  further treat the residual or sub-products

products that appear.

  The cells are continuously broken

  yeast of an active yeast suspension of bou-

  change with a dry yeast substance (HTS) of approximately 10% and a temperature of 8-15 C in

  a mill with a tree, and glass microbeads presented

  diameter of 0.5-1.2 mm and the speed of

  the stirring shaft is 2500-400 rpm.

  In a subsequent separator, the macroscopic separation between debris is carried out continuously.

  cell and solution containing invertase.

  In order to avoid losses of enzyme yield, the introduction of the suspension before desliming (Entschlammung) is stopped and the rotor is washed for about 5 minutes with water, the emptying space being emptied only when the water

  clear when the separator flows.

  Continuous clarification filtration,

  which comes next, from the solution containing

  vertase, is carried out on a stratified filter, the filter layers having a permeability of 425-475 l / h.m2. Then there is the clear side of the filter layers of the filter paper, the filter layers being moistened before

  start of filtration for about 20 minutes.

  The pressure difference between the inlet and the outlet of the filter must not be greater than 200-250 kPa

during filtration.

  The laminated filter is mounted so that in addition to the filtration process a closed circuit is possible, so that the occurrence of an overpressure which can result in damaging

  filtration plates. The rest of the solution of

  vertase to be clarified, remaining in the mode of

  conduct of the cycle is again subject to a separate

  ration. Purification, concentration and sterilization of the invertase solution are carried out

  on a combined installation of ultra- and micro-

  filtration. The ultrafiltration separation unit is arranged with respect to the microfiltration separation unit so that the solution is

  first purified and concentrated on the separating unit

  ultrafiltration and then sterilization

  passing through the microfiltration separation unit. The permeability of the UF membranes used in the ultrafiltration separation unit is 80-140 l / h.m2, the inlet pressure being approximately 0.15-0.4 and the outlet pressure at about 0.1-0.3 MPa. To increase the mechanical stability of the flat membranes, a support fabric, for example a textile, is introduced between the membranes and the bypass plates of the filtration.

nonwoven.

  The invertase solution is concentrated

  at about 1/10 by the separation unit of the ultra-

  filtration. To continue the purification of the invertase solution concentrate, a diafiltration is carried out

  addition to the separation unit of the ultra-

  filtration. The concentrate is then diluted with water in a ratio of 1: 1 and diafiltered in the cycle until the volume is again reached.

starting point of concentrate.

  After this ultra- and diafiltration, the purified invertase concentrate solution is introduced into the microfiltration separation unit for separation of the denatured proteins.

  contained in the solution as well as micro-organisms

  nisms. The pore diameter of the microbial material

  filtration used amounts to 0.2 μm, and one does not

  must not exceed an inlet pressure of 0.3 MPa.

  To increase the mechanical stability of the filter material used, a support fabric is used,

  as it says in the separation unit of the ultra-

  filtration. With the subsequent stabilization, known per se, the solution is adjusted to an activity of about

  1500 U.ml-1 and makes it suitable for preservation.

  The process according to the invention is particularly suitable

  specifically to prepare invertase preparations of high specific activity (400-800 U / mg) for which analytical purposes are of particular interest, in that diafiltration

  is repeated 3 to 8 times as described above.

above.

  The yield of stable invertase solution

  glycerol obtained according to the procedure mentioned above is 75% relative to

  active baker's yeast used.

EXEMPLARY EMBODIMENT

  The exemplary embodiment is explained using

  of a figure in which the reference signs represent

feel the following.

  120 kg of active baker's yeast (HTS 30-33%) stored at 24 C with 240 L of water are placed in a mixing and equilibrium temperature container 1. After about 30 minutes a suspension is homogeneous yeast of HTS 10-15%, which is stored at a temperature between

and 12 C.

  The cooled suspension is then added via a dosing pump 2 of a

  shaft mill operating continuously 3.

  The disintegration of the yeast cells is then carried out in a milling vessel of about 3 liters cooled with water, which is filled with about 2400 g of 0.8 to 1 mm glass microbeads. The speed of the stirring shaft is approximately

  3000 turns / rainet the flow of the suspension at 16 1 / h.

  The disaggregated yeast suspension flows through the upper flow device of the grinding vessel into a mixing vessel and equilibrates the temperature 4. By means of a metering pump 5 is introduced the suspension in a separator 6. On

  separates here most of the cellular debris

  with the solution containing invertase.

  From the product flow of the separator 6 flows

  phase largely free of cellular debris

  in the intermediate container 7. The rate of introduction of the suspension to be separated amounts to

  at about 50 1 / h. The separator 6 is here to disarm

  sea every time after about 3/4 of an hour. In order to avoid greater losses of enzyme yield, the introduction of the yeast suspension is stopped before desliming and the rotor is washed with water until clear water comes out of the water. product. We then introduce the water

adjustment and empty the sludge.

  From the intermediate container 7 the solution containing the invertase is added using a dosing pump 8 to a laminate filter 9

  to carry out clarification by filtration.

  The clarification is carried out by the use of KP-10A type filter layers (40 x 40 cm) with a permeability of approximately 450 l / hm. In order to increase the quality of the filtration, the filter layers

  Niederschlag 291 filter paper (40 x 40 cm).

  Before the beginning of the filtration, the filter layers introduced are humidified for about 20 minutes. The filtration rate is set at about -100 l / h. The maximum pressure difference between the inlet and the outlet of the filtration must not be

  here be greater than 200 or depending on the case 250 kPa.

  The laminated filter 9 is here mounted so that in addition to the filtration process,

  average traffic, so that

  overpressure, which may lead to

  early destruction of the filter material.

  The residue, remaining in the circulation mode, of the solution containing invertase to be clarified is again conveyed to the separator.

  6 after the end of the clarification by filtration.

  - The solution to be treated is transferred further into an intermediate container 10. The steps

  subsequent purification, concentration and sterility

  the solution containing invertase

  made in a special installation of ultra-

combined microfiltration 11.

  The plate package on a support is used with a filtration area of 5 m2 for ultrafiltration and 4 m2 for microfiltration. For the ultrafiltration separation unit 12, UF membranes are used

  a permeability of 80-140 l / h.m2 of CAM-type

  UF-120-CP (VEB Zellstoff- und Zellwollewerke Witten-

  bank). This type provides a selectivity over invertase of over 99%. To increase the mechanical stability is also available between the membrane and the filter bypass plate

  a nonwoven textile support fabric.

  Through the microfiltration unit 13 is stretched a microfilter for traces of cores made of polyester (ZfK AdW Rossendorf) having a

  pore diameter of 0.2 μm. To increase the

  this filtration material is reinforced

a non-woven fabric.

  From the intermediate container 10 the invertase-containing solution is pumped by means of a metering pump 14 through a double-tube cooler 15 into the ultrafiltration unit 12. The solution is conducted according to the principle of

  distribution on the filter plates, where the

  molecules with a molecular weight greater than 150 000 are retained. The permeate flow rate here is about 80-140 l / h at a starting pressure of 0.1-0.3 MPa. The invertase activity in the permeate product is located below

0.5%.

  The solution obtained after clarification by filtration is concentrated by ultrafiltration to 1 / 10th of its volume. The concentrate obtained after the ultrafiltration is then diluted with water in

  a ratio of 1: 1 and diafiltrate it for purification.

  Subsequent charging in the cycle, through the ultrafiltration separation unit 12, until the starting volume of the concentrate is reached again. By blocking the ultrafiltration cycle, the ultra- or diafiltered concentrate which is in the intermediate container 10 is transported via a metering pump 16 through the refrigerant 15 to the unit.

microfiltration 13.

  There the denatured proteins contained in the concentrate as well as the microorganisms which affect the quality of the product, are separated. The active filtration surface mentioned above is set at a flow rate of about 20 l / h, the inlet pressure

  not to be greater than 0.3 MPa.

  The purified invertase solution is introduced

  concentrated and sterilized in a container

  mixing and balancing the temperature

  17 and mixed with glycerol in a 1: 1 ratio and stabilized therein. The yield of the stabilized invertase solution is kg (75% relative to baker's yeast)

active used).

Claims (1)

CLAIM   A biotechnological process for the preparation of invertase from active baker's yeast, in which active baker's yeast is suspended with a dry yeast substance (HTS) of 30-33% with the aid of water at a rate of HTS of about 10% and at a temperature of 10-12 C, characterized in that the yeast cells of this suspension are broken in a tree mill known per se 3 using glass microbeads having a diameter from 0.5 to 1.2 mm and a stirring shaft speed of 2500-4000 rpm / rmin continuously, and then in a separator known per se 6 a solution containing invertase, o before the desliming is stopped the flow of the suspension and the rotor is washed with water until clear water appears in the flow of the product, then in that one submits the solution to a clarification by   filtration in itself known by means of a strati-   9, where the filter layers have a   meandering 425-475 1 / h.m2, the clear sides of the filter layers are further supported with filter paper and before the beginning of clarification by filtration these filter layers are moistened for about 20 minutes with the difference in pressure between the inlet and outlet of the filter during filtration is not greater than 200-250 kPa, the laminated filter is mounted so that   of the filtration process operation in.   cycle is possible and the residue of the invertase solution to be clarified remains separated again and the solution is then   to a combined installation of ultra- and micro-   filtration 11 so that the solution goes through   firstly the separation unit of ultrafiltration   12, is subjected in the cycle to diafiltration,   then taken to the microfilter separation unit   13, the permeability of the UF membranes employed in the ultrafiltration separation unit is 80-140 l / h.m2, the inlet pressure   amounted to 0.15-0.4 and the outlet pressure to 0.1-   0.3 MPa, a support fabric, for example non-woven fabric, is arranged between membranes and filtration bypass plates, the solution is first concentrated by the ultrafiltration separation unit 12 to about 1 / 10, then the concentrate   is diluted with water in a 1: 1 ratio and di-   filtered in the cycle until the concentrate start volume is reached again, and then the purified invertase concentrate solution is   conducted through the separation unit of the micro-   filtration 13, where the pore diameter of the microfiltration material used is 0.2 μm and the inlet pressure is not more than 0.3 MPa and the filter material is provided in the separation unit ultrafiltration 12 of a fabric   reinforcement, then the concentrate concentrate solution   vertase is stabilized, standardized and crafted in known manner.