CS225684B1 - Method of the native fraction liberation from the monocell microorganism by means of mechanical desintegration - Google Patents

Description

The invention relates to a process for the release of native fractions from unicellular microorganisms by mechanical disintegration in order to isolate individual fractions or mixtures thereof.

E.g. in the preparation of food protein concentrates from micro - organisms, it is necessary to:

to release the intracellular protein by disrupting the cell wall, either in a mechanical, biochemical or chemical manner. The effect of cellulolytic enzymes or hydrolysis in a strongly acidic or alkaline environment is exploited, which however has a destructive effect on the native protein. Alkaline hydrolysis allows, for example, the formation of toxic compound amino acids (lysinoalanine) or racemized amino acids, whereby the protein value is substantially reduced from the food point of view.

For enzymatic cell disruption, the necessary lytic enzymes or enzyme mixture must be prepared in advance; the process itself is thus time-consuming and thus disadvantageous, since the isolation of individual enzymes is expensive and proteases which can break down proteins are also present / Vršanská M., Bielý P. und Krátký Z .: Zechr. Allg. Mikr. 17, 465 (1977); Funatsu M., Aizono O.H. and Shinioda T.s. Agr. Biol. Chem. 42, 1975, 1978]. Although several processes have been patented in the field of enzyme degradation of cell walls in the last decade, this process has not been used on an industrial scale yet. No. 1, 281,618 (1972), Japanese Patent No. 56,682 (1978)).

The use of mechanical cell wall disruption techniques makes it possible to obtain a suitable protein

235 684

225 684 for food purposes (aut. Certificate ČSSR Nr. 161299). In addition, wastes in such production can be used to isolate other biologically active cell components such as ergosterol.

Of the mechanical methods of cell disruption, extrusion, ballotin disintegration and disruption by rapid decompression seem to be the most acceptable method.

Extrusive disintegration is based on forcing the cell suspension from the high pressure region through the micro-slit of the desintegration device to the normal pressure region. The main factors determining cell disintegration are - hydrostatic pressure value, its gradient and rate of pressure change.

Of the disintegration devices utilizing this method, gaulin (USA) hydraulic high-pressure homogenizers are best known (Gaulin Teohnioal Bulletin, 1968, 7, Hetherington PJ et al. Trans. Chem. Engrs., 1971, 49, 142). ). The process of cell disruption in extrusive homogenizers is characterized by high working pressure values (50 to 80 MPa) and relatively low productivity (50 to 200 l.hr · ^ ·).

In order to obtain a high degree of cell disruption (80-90%) it is necessary to repeat the operating cycle several times, resulting in cell fragments of uneven size and difficulty in separating the solid and liquid phases of the obtained suspension. complete inactivation of the intracelllular components. A modified high-pressure pump (Brookman J.S., Davies M. Biotechnol. Bioeng. 1973, 15, 693) works on a similar principle, with a slurry productivity of 15 liters / hour at an operating pressure of up to 310 MPa.

With the use of ballotin disintegrators, any desired degree of cell disruption can be achieved on an industrial scale with continuous technology, but with a corresponding increase in material processing time. The disadvantages of this method are the local overheating of the treated substance, which necessarily requires intensive cooling. The repeated crushing of already obtained cell fragments with the formation of too small particles (from units to hundredths of a micrometer) further complicates the following solid and liquid phase separation. All this together, ie increased water consumption for electric power cooling, longer biomass processing time adds cost to the process and reflects the hay price of the product.

Also known is a method and apparatus of continuous disintegration using the decompression effect (aut. USSR Nr. 477742/1973 / »aut. USSR Nr. 492118/1973 / $ aut. USSR Nr. 602550/1974 /» aut. USSR No. 602551 (1975), U.S. Patent No. 4084757 (Franc, No. 7624795), whose function is based on the principle of rapid decompression. The suspension of microorganisms is saturated with a pressurized gas (nitrogen, air, carbon dioxide), which must not react with intracellular components and also must not inhibit enzymes, particularly endophenic nuclease useful for degrading nucleic acids in the mictobial protein. The cells are also saturated with gas until the partial pressure is equalized from both sides of the cell wall, whereupon the suspension is suddenly brought to atmospheric pressure. The result is a gas pressure gradient in the cell directed to the outside. Since the rate of gas leakage from the cell is higher than the rate of diffusion through the cell wall, the wall ruptures.

Disintegration by rapid decompression takes place in a device based on com3

225 684 gas pressurizer, high-pressure micro-organism suspension pump and two high-pressure cylinders for saturation and suspension transfer. The indisputable advantage of decompression disintegration lies in the fact that the cells resp. the cell walls remain whole after disintegration or show only a small degree of crushing and can therefore be easily separated by separation from protoplasm.

The described procedure does not lead to inactivation of intracellular enzymes and allows the chemical components of the cell to be isolated in the native state. The energy consumption for decompression disintegration is low and is 0.06 kWh. kg \ while consuming 0.2 to 0.4 kWh.kg for extrusion or ballotin cell crushing \

However, the decompression disintegration process alone does not provide a sufficiently high degree of release of the intracellular components to the external environment and since it is substantially influenced by the pH of the treated suspension, the process needs to be supplemented by extraction of the biomass of disintegrated cells.

This is documented in Tab. No. 1, where the pH of the yeast suspension of Sacharomyces cerevisiae is given as a function of the release of water-soluble proteins into the cell supernatant from cells disrupted by the decompression method. For comparison, values for one and six times disintegration are given.

The amount of water-soluble protein in the intact cell is 20% of the total protein content (N χ 6.25).

Table 1.

Influence of pH on the degree of extraction of water-soluble protein from yeast cells of Sakaromyces cerevisiae, disrupted by decompression.

pH value Degree of water soluble protein extraction in%

jednonásob. disintegration six times. disintegration 4,5 8.0 ^5l -1 7.0 12.0 68.3 9.0 18.1 96.5 11.0 24.9 124.0

As can be seen from the table, virtually all of the water-soluble protein is released in ** from disintegrated cells only at pH = 9.0. The degree of extraction at pH = 11.0, in excess of 100%, can be explained by the additional dissolution of the alkali soluble protein fractions. The destructive effect of decompression on the cell wall takes hundredths of a second. Thereafter, the cell is no longer mechanically stressed, so that only a portion of the protoplasm containing the protein flows out of it. Upon repeated decompression, another part of the protein passes into the intercellular space, the amount of which depends on the pH of the environment and the number of disintegrations. However, the cell wall is no longer disturbed. Thus, there is a proportionality between the amount of protein released from the cell and the number of individual disintegration operations as well as the pH value of the suspension.

The disadvantage of this method is the low protein yield from suspension disintegration at pH

225,684 at about 7.0, possibly requiring higher pH for faster release or extraction of proteins from disrupted cells, and the fact that the active nuclease needed for nucleic acid degradation is released to a lesser extent than by disintegration with ballotin disintegrators.

These disadvantages are overcome by the subject-matter of the invention, wherein the microbial suspension at a concentration of 1 to 20% by weight at a pH of 4.5 to 9.5 and at a temperature of 0 to 70 ° C is first subjected to disintegration by decompression at an operating overpressure of 3 to 30 MPa. after which the pre-crushed suspension is treated by mechanical crushing and the released native fractions are isolated by a generally known method.

It is an object of the invention to isolate intracellular chemical components in the native state of the biomass of microorganisms in less time and at lower energy consumption in high yield and to obtain a suspension containing sufficiently large cell fragments whose isolation on standard separators does not cause problems.

This is achieved by the use of combined mechanical disintegration, where the suspension of microorganism cells is disintegrated one or more times by decompression at 0 to 70 ° C, pH = 4.5 to 9.5, a working pressure in the system of 3 to 30 MPa, and then processed in a ballotin disintegrator containing ballotins (the bulk volume of the ballotins is preferably in a ratio of 1.7 sl to the volume of the suspension).

They can be subjected to yeast disintegration, for example of the genus Sacharomyces cerevisiae, Candida torulopsis, Rhodotorula, Ovidium, Pichia, Hansenula, bacteria of the genus Megaterium, Medtanomonas, Eacherichia coli, molds and algae.

The proposed method consists of the following!

The isolation of native fractions from unicellular microorganisms is carried out from their aqueous suspension with a cell concentration of 1 to 20% by weight, at a temperature of 0 to 70 ° C and a pH = 4.5 to 9.5. The suspension is treated one or more times in a periodic or continuous disintegrator. The working gas used is air, nitrogen or any other chemically inert gas relative to the cells. Compressed air is introduced into the suspension at a pressure of 3 to 30 MPa, maintained at a pressure of 15 to 60 sec ·, and transferred to the barometric pressure range. The decompression disintegration of the microorganism cells takes place.

Further, the suspension is processed in a ballotin disintegrator comprising ballotins with a diameter of 0.40 to 0.60 mm, preferably at a ratio of the bulk volume of the dry ballotin to the volume of the cell suspension of 1.7 and 1. Within 5 to 10 minutes. the disintegration becomes a solution of 80 to 95% of the water-soluble cell fractions in the native state, which are isolated and purified by known methods.

Below are examples of using a disintegration method to isolate native protein from microorganism cells of the invention.

Example 1

The aqueous suspension of Sakaromyces cerevisiae yeast with a cell concentration of 9.8% by weight at 20 ° C and pH = 8.4 was treated once in a laboratory decompression disintegrator with periodic action, consisting of a cylindrical chamber - high pressure transducer with a working volume of 35 ml. shut-off valve, inclined-expander a

225 684 collection containers. The slurry was introduced into the chamber, saturated with pressurized nitrogen under a positive pressure of 9-10 MPa, and after 25 sec. Gas saturation was shot through a shut-off valve into the cyclone-expander. The disintegrated cell suspension was collected in a collection vessel and further processed in a single-disc ballotin vertical disintegrator with a disc speed of 180 m-sec. ¹ and glass halotines of 0.40-0.56 mm diameter; the volume ratio of the bulk powder suspension volume in the disintegrator was 1: 1.7. The disintegration efficiency was evaluated according to the rate of release of the water-soluble protein into the intercellular environment.

For comparison, an experiment was conducted in which the biomass was processed only in a ballotin disintegrator.

The graph in FIG. 1 shows the results of disintegration on a ballotin mill versus time according to the process of the invention. Curve # 1 shows the combination crushing process of the present invention with a single crush, Curve # 2 with two crushes and Curve # 3 without crushing.

(VRB = water-soluble protein and ACS = absolute yeast dry matter).

As is apparent from FIG. 1, in the preliminary decompression disintegration experiment, virtually all of the water-soluble protein goes into solution within 5 minutes of ballotin disintegration; For ballotin disintegration without pre-decompression processing, 24 min is required to release the same amount of protein. Thus, pre-decompression disintegration makes it possible to reduce the time of ballotin disintegration to 21%,

Example 2

The experiment was carried out in the same suspension and under the same conditions as in Example 1, but the pre-decompression disintegration was performed in two phrases. The time course of the experiment and the yield are expressed by curve 2 in Fig. 1,

Example 3

The aqueous suspension of Sacharomyces cerevisiae with a cell concentration of 15% by weight at 50 ° C and pH 7.5 was treated once in a decompression disintegrator at a pressure of 19 to 20 MPa. The other conditions are the same as in Example 1. The protein recovery over the same time period is 16% per yeast dry matter (as in Example 1).

Example 4

Instead of the yeast suspension of Sacharomyces cerevisiae, a suspension of Métharibmonas was subjected to disintegration. The disintegration conditions were the same as in Example 1. The protein recovery for the same time period was 19% per dry bacteria.

The method of disintegrating the unicellular microorganisms of the present invention can be used where efficient separation of the solid and liquid phases of the disrupted cell suspension in standard centrifuges or separators is required or isolated in the native state of intracellular chemical components that deactivate at higher temperature or too acidic. alkaline environment. The method can be used, for example, in obtaining food protein substances from hiomass of unicellular microorganisms when, simultaneously with proteins, it is necessary to isolate intracellular nucleases in the active state, used to reduce the nucleic acid content of the obtained product.

The use of the proposed method shows that the time is processed. cells in the ballotin disinte225 684 grator is shortened 4-5 times if the suspension has been pre-subjected to decompression disintegration. In doing so, the degree of heating of the suspension is significantly reduced, which contributes to increasing the productivity of the ballotin disintegrator, saving cooling water and energy.

In the case of isolation of any thermolabile fractions of cellular content, their yield increases and retains activity. The use of the method according to the invention makes it possible to isolate cellular enzymes in the active state, e.g. intracellular nuclease and further used to disrupt the nucleic acids found in the cells of microorganisms.

Claims (2)

Process for releasing native fractions from unicellular microorganisms by mechanical disintegration, characterized in that the microbial suspension at a concentration of 1-20% by weight, at a pH of 4.5 to 9.5 and at a temperature of 0 to 70 ° C, is first subjected to disintegration by working decompression. at a pressure of from 3 to 30 MPa, after which the pre-crushed suspension is treated by mechanical crushing and the released native fractions are isolated by a generally known method. 2. A method according to claim 1, wherein the microbial suspension is decompressed at least twice to disintegrate.