Method for isolating proteins and sugars from yeast

Abstract

A method for lysing yeast, in particular brewer's yeast, in order to isolate proteins and sugars, comprising the steps of: a) providing a yeast suspension, in particular a brewer's yeast suspension; b) physically lysing the yeast in the yeast suspension; c) microfiltering the lysed yeast suspension from step b); d) ultrafiltration of the microfiltration permeate from step c), whereby a phase having proteins as the main solid component is separated as the ultrafiltration retentate; e) treating the microfiltration retentate from step c) with a protease under basic conditions and subsequently separating the phase having mannan as the main solid component, in particular by filtration and / or centrifugation; f) successively treating the phase retained in step e) first under basic conditions and then under acidic conditions, followed by separation, in particular by filtration and / or centrifugation, of the phase having glucan as the main solid component.

Description

[Technical field]

[0001] The present invention relates to a method for lysing yeast, in particular brewer's yeast, for isolating proteins and sugars. [Background technology]

[0002] Yeasts are single-celled fungi that grow by budding or division and are traditionally of great technological importance. For example, yeasts are used in the production of beer, wine, spirits, food and therapeutic substances. Basically, there are different types of yeast, the so-called baker's yeast or brewer's yeast (Saccharomyces cerevisiae) being one of the most commonly used yeasts. Therefore, considerable amounts of yeast-containing, especially brewer's yeast-containing by-products are concomitantly generated.

[0003] In particular, since the yeast cell walls contain a series of technologically useful biomolecules such as proteins, glucans, mannans, chitin and lipids, yeast-containing wastes are processed to obtain these materials, whereupon the yeast cell walls are partially or completely dissolved to release the substances of interest.

[0004] For example, EP1990419A1 (Tex-a-tec AG) describes a method for isolating glucans, proteins, mannans and lipids from yeast, in which the yeast is treated in successive steps with ultrasound at elevated temperature.

[0005] EP2272876A1 (Angel Yeast Co., Ltd.) discloses a method for extracting glucan and mannan from the cell walls of microorganisms, such as yeast 8440. The method includes the steps of: a) treating the cells of the microorganism with alkaline protease and mannanase; b) separating the mixture from step a) into a heavy phase and a light phase; c) drying the heavy phase obtained from step b) to obtain a glucan preparation; and d) drying the light phase obtained from step b) to obtain a mannan preparation. The glucan and mannan preparations are intended for therapeutic use.

[0006] WO2010 / 070207A1 (Glykos Finland Oy) describes a method for producing an immunostimulant composition containing a saccharide fraction, which comprises hydrolyzing yeast cells to obtain a soluble fraction. The saccharide fraction thus obtained can be added as a food or beverage ingredient or can be used as a pharmaceutical.

[0007] However, the previously known methods for isolating technically exploitable biomolecules from yeast are not entirely convincing, especially in large-scale approaches: for example, the ratio between yield and effort (especially with regard to energy and time requirements) is insufficient in many methods, which seriously hampers economical production.

[0008] Other methods are directed to selectively obtaining individual components (e.g., glucans or mannans) while leaving other components (e.g., proteins) unconsidered, which is a disadvantage in terms of the most complete utilization of yeast-containing by-products.

[0009] Thus, there remains a need for improved solutions that at least partially or completely overcome the above-mentioned shortcomings. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] EP1990419A1 [Patent Document 2] EP2272876A1 [Patent Document 3] WO2010 / 070207A1 Summary of the Invention [Problem to be solved by the invention]

[0011] The object of the present invention is to provide an improved method for obtaining biomolecules from yeast cells, in particular from brewer's yeast cells.In particular, it should be possible to isolate a variety of different biomolecules, particularly preferably proteins, glucans and mannans, with the highest possible yield and purity at the same time.The method should furthermore allow the biomolecules to be obtained as efficiently as possible in a large-scale industrial approach.Here, it is desirable that the method can be carried out with the lowest possible energy and time consumption. [Means for solving the problem]

[0012] The solution to said problem is defined by the features of claim 1. The essence of the invention is therefore a method for lysing yeast, in particular brewer's yeast, in order to isolate proteins and sugars, which comprises the following steps: a) providing a yeast suspension, in particular a brewer's yeast suspension; b) physically lysing the yeast in the yeast suspension; c) microfiltering the lysed yeast suspension from step b); d) ultrafiltration of the microfiltration permeate from step c), whereby a phase having proteins as the main solid component is separated as the ultrafiltration retentate; e) treating the microfiltration retentate from step c) with a protease under basic conditions and subsequently separating the phase having mannan as the main solid component, in particular by filtration and / or centrifugation; f) successively treating the phase retained in step e) first under basic conditions and then under acidic conditions, followed by separation of the phase having glucan as the main solid component, in particular by filtration and / or centrifugation.

[0013] Surprisingly, it has been shown that the combination of physical, chemical and biochemical processing steps according to the invention allows good yields with relatively low energy requirements and relatively short time requirements. This applies to all products, i.e. proteins, glucans and mannans, and yields of up to about 40% for each of these substances can be achieved with high purity.

[0014] Moreover, the method is suitable for a large-scale approach: tens of thousands of tons of yeast suspension per year, or thousands of kilograms of yeast suspension per hour, can be processed without problems.

[0015] "Glucan" is an oligosaccharide or polysaccharide composed of D-glucose molecules linked together by glycosidic bonds. Glucans as present in yeast cells are typically β-glucans having β-glycosidic bonds, particularly β-1,3-glucans.

[0016] "Mannan" is a polysaccharide that is a polymer of the sugar mannose. Mannans present in yeast typically have an α(1-6)-linked backbone and α(1-2)- and α(1-3)-linked side chains that average about two sugar units in length. "Microfiltration" refers to the process for filtration through filter media, especially membranes, with pore sizes ≧0.1 μm. In contrast, the pore sizes in ultrafiltration are less than 0.1 μm.

[0017] Unless specifically stated otherwise, each "solid ingredient" refers to the dry matter of the respective ingredient. The term "major solid ingredient" refers to the ingredient that is solid and has the greatest proportion by weight.

[0018] The yeast suspension provided in step a) is in particular a yeast suspension that has been stored and / or pasteurized at a temperature between 0 and 15° C., preferably between 2 and 12° C., in particular between 3 and 7° C. or between 3 and 5° C. This ensures that the suspension contains as few undesirable by-products as possible.

[0019] In principle, however, the yeast suspension provided in step a) may also be a non-pasteurized yeast suspension that has been stored at another temperature, in particular at room temperature.

[0020] The yeast suspension is in particular an aqueous yeast suspension, in which water is present as the continuous liquid phase of the yeast suspension.

[0021] The yeast suspension is in particular a brewer's yeast suspension, which preferably contains spent brewer's yeast. Such a brewer's yeast suspension arises, for example, as a by-product in beer production and can be used directly in the process according to the invention.

[0022] The yeast suspension preferably has a solids content of 5 to 30% by weight, in particular 7 to 20% by weight, based on the total weight of the yeast suspension. This has been found to be particularly suitable for the method according to the invention. Optionally, the solids content of the yeast suspension can be adjusted to the desired range using a liquid, in particular water.

[0023] The yeast suspension in step a) has in particular a temperature of 0 to 15° C., preferably 2 to 12° C., in particular 3 to 7° C. or 5° C. This ensures that the composition of the suspension is not further altered, e.g. by active yeast cells, or that the formation of undesired by-products is reduced.

[0024] Further preferably, step b) and optionally also steps c) and d) are carried out at a temperature of 0 to 15° C., preferably 2 to 12° C., in particular 3 to 7° C. or 5° C. This further reduces the formation of undesired by-products.

[0025] In principle, however, the yeast suspension in steps a), b), c) and / or d) may also have another temperature, e.g. room temperature.

[0026] According to an advantageous embodiment, the yeast suspension provided is subjected to a washing process prior to step b), in which at least a part of the liquid phase of the yeast suspension, in particular at least 50% by weight, in particular at least 60% by weight, of the liquid phase of the yeast suspension is replaced by a further liquid or a replacement liquid, in particular water. The washing process is preferably carried out in a centrifuge or drum filter.

[0027] If necessary, a washing process can reduce the proportion of undesired components dissolved in the liquid, thereby improving the purity and quality of the material to be isolated, however, the washing process is optional and can be omitted.

[0028] The physical dissolution in step b) is carried out in particular by means of an electric field, in a homogenizer or in a grinding media mill, in particular in a ball mill.

[0029] The homogenizer is in particular a high-pressure homogenizer, in particular operated at a pressure of more than 1000 bar, in particular more than 1200 bar.

[0030] In grinding media mills, the grinding material and freely movable grinding media, in particular balls, are circulated in a process chamber, whereby collisions occur between the grinding media, the walls of the process chamber and the grinding material, whereby the reduction of the grinding material occurs, in particular by breakage.

[0031] Lysis by grinding media mills, especially ball mills, has now proven to be particularly advantageous, since the protein to be isolated can be released gently, but nevertheless effectively. This is particularly true for brewer's yeast suspensions. However, for special applications, other methods for physical lysis can also be used.

[0032] The physical dissolution by means of an electric field in step b) can in particular be carried out by means of pulsed electric fields (also called in English PEF or "Pulsed Electric Fields"), this dissolution technique itself being otherwise known to the skilled person.

[0033] When treating with pulsed electric field, yeast suspension is placed between two electrodes and treated with pulsed electric field. Pulsed electric field is characterized by high electric field strength and short duration. There, reversible or irreversible pore formation occurs in cell membrane, so that intracellular components can be released and / or extracellular substances can enter into the cell.

[0034] It has now been found that in particular the following parameters are suitable for controlling the lysis of yeast in the yeast suspension: electric field strength [kV / cm], input temperature of the yeast suspension [°C] and specific energy input [kJ / L]. The energy required for the process (kilojoules per liter of yeast suspension) is understood as energy input. Furthermore, the energy input can be adjusted via the frequency [Hz] of the pulses, which in turn is controlled automatically depending on the flow rate and in known devices.

[0035] The electric field strength is preferably from 1 to 30 kV / cm, particularly from 5 to 24 kV / cm.

[0036] The specific energy input is preferably between 1 and 180 kJ / L, in particular between 3 and 120 kJ / L.

[0037] The temperature at which the yeast suspension is added is in particular 0 to 15°C, preferably 2 to 12°C, in particular 3 to 7°C or 5°C.

[0038] Such parameters allow for efficient lysis of the yeast in the yeast suspension.

[0039] Devices for performing physical cell lysis using pulsed electric fields are commercially available from various suppliers, such as the PEF Pilot Dual device (Elea-Technology; Germany).

[0040] Preferably, the treatment with a pulsed electric field is followed by a mechanical post-treatment, which is therefore preferably carried out between steps b) and c).

[0041] The mechanical post-treatment can be carried out in particular by means of mixers, preferably mixers with high shear forces. Rotor-stator mixers, in particular in-line rotor-stator mixers, are particularly suitable. In in-line rotor-stator mixers, the rotor and stator are typically housed in a housing with an inlet at one end and an outlet at the other end. The yeast suspension to be treated can then be passed through the mixer in a continuous flow.

[0042] The shear rate during mechanical post-treatment with a mixer is selected in particular so that yeast cells are further lysed. This allows intracellular components (proteins) that may not yet be completely released after application of a pulsed electric field to be released. The high shear forces further "squeeze" the already cleaved yeast cells, allowing intracellular components, especially proteins, to leak out of the cells.

[0043] The microfiltration in step c) is preferably carried out using a filter medium with a pore size of 0.1 to 0.5 μm, preferably 0.1 to 0.2 μm, in particular 0.1 μm, which has been found to be particularly favorable in order to achieve, after physical lysis, a relative enrichment of the protein to be isolated in the permeate, based on the solids content, and at the same time an enrichment of biomass residues, such as cell fragments and fats, in the retentate.

[0044] In particular, the microfiltration in step c) is carried out as diafiltration, in which a replacement liquid, in particular water, is supplied, preferably continuously, on the feed side, in other words, in diafiltration, the liquid phase of the lysed yeast suspension, which is preferably discharged as permeate, is at least partially, in particular completely, continuously replaced on the feed side by a replacement liquid, in particular water.

[0045] The lysed yeast suspension is in particular circulated from the feed vessel. By controlling the flow after the filter medium, the transmembrane pressure, which acts as the driving force in diafiltration, can be adjusted.

[0046] The diafiltration is preferably carried out in a closed circulation so that the discharge of the permeate leads to a continuous flow of the displacement solvent, in particular so that the circulation volume during the diafiltration remains constant.

[0047] In particular, the microfiltration in step c) is preferably carried out such that the proportion of protein in the microfiltration permeate from step c) is preferably at least 50% by weight, more preferably 50-80% by weight, in particular 60-70% by weight, based on the total weight of solids in the permeate.

[0048] The total proportion of solids in the permeate of the microfiltration in step c) is preferably 1-10% by weight, in particular 3-7% by weight, based on the total weight of the permeate.

[0049] Further preferably, the microfiltration in step c) is carried out such that the proportion of proteins in the microfiltration retentate from step c) is less than 50 wt.-%, preferably less than 40 wt.-%, in particular less than 35 wt.-%, based on the total weight of solids in the retentate.

[0050] The total proportion of solids in the retentate of the microfiltration in step c) is preferably 20-50% by weight, in particular 25-30% by weight, based on the total weight of the retentate.

[0051] According to a further possible embodiment, the lysed yeast suspension is treated with an oxidizing agent, in particular ozone (O3) and / or hydrogen peroxide (H2O2), before the microfiltration step c). This makes it possible in particular to prevent chemical reactions that lead to undesirable by-products and / or altered tastes. However, the treatment with an oxidizing agent is optional.

[0052] Additionally, the lysed yeast suspension can be enzymatically treated prior to the microfiltration step c), which can increase the efficiency and yield of the process if desired, although the enzymatic treatment prior to the microfiltration step c) is optional.

[0053] The ultrafiltration in step d) is preferably carried out using a filter medium with a pore size of less than 90 nm and / or an exclusion limit (nominal molecular weight cut-off) of 1-100 kDa, in particular 5-100 kDa, preferably 10-50 kDa, in particular 15-25 kDa, especially 20 kDa. The exclusion limit is defined as the minimum molecular weight of molecules that are 90% retained by the filter medium, in particular a membrane.

[0054] In particular, the ultrafiltration in step d) is carried out as diafiltration, in which a replacement liquid, in particular water, is supplied, preferably continuously, on the feed side. In other words, in the diafiltration in step d), the liquid phase of the permeate of the microfiltration from step c), which is discharged as permeate in the diafiltration, is preferably at least partially, in particular completely, continuously replaced, on the feed side, by a replacement liquid, in particular water.

[0055] The permeate of the microfiltration from step c), which forms the feed in step d), is in particular circulated from the feed vessel. By controlling the flow after the filter medium, the transmembrane pressure, which acts as the driving force for the diafiltration in step d), can again be adjusted.

[0056] The diafiltration in step d) is preferably carried out in a closed circulation, such that the discharge of the permeate results in a continuous flow of displacement liquid, in particular so that the circulation volume during the diafiltration remains constant.

[0057] Ultrafiltration and diafiltration in step d) allows for a targeted increase in the relative solids proportion of protein in the retentate.

[0058] Preferably, the ultrafiltration in step d) is carried out in such a way that the proportion of protein in the phase separated in step d) having protein as the main solid component is at least 75% by weight, in particular at least 85% by weight, preferably at least 90% by weight, based on the weight of the solids.

[0059] The total proportion of solids in the phase separated in step d) is preferably 0.5-7% by weight, in particular 1-3% by weight, based on the total weight of the phase separated in step d).

[0060] In particular, both the microfiltration in step c) and the ultrafiltration in step d) are carried out as diafiltration.

[0061] Particularly preferably, the phase separated in step d) with the protein as the main solid component is dried after step d), in particular spray-dried. This allows the protein to be obtained as a powdery product with high purity. Apparatuses and methods suitable for spray-drying are known per se to those skilled in the art.

[0062] Particularly preferably, before spray drying, the solid content, in particular the protein content, is concentrated, in particular to a solid content of at least 50% by weight, in particular at least 60% by weight, preferably at least 70% by weight.Concentration is especially carried out in an evaporator, which partially evaporates the liquid phase.The apparatus and methods suitable for concentration are known per se to those skilled in the art.

[0063] The phase separated in step d) having the protein as the main solid component can optionally be treated after step d) with an oxidizing agent, in particular ozone and / or hydrogen peroxide, and / or contacted with an adsorbent material, for example activated carbon. This can in particular improve the quality of the protein product. For example, undesirable discolourations and / or flavours can be chemically decomposed and / or removed.

[0064] If spray drying is performed, treatment with an oxidizing agent and / or contact with an adsorbent material preferably occurs prior to spray drying, and, if performed, prior to concentration.

[0065] The treatment with the oxidizing agent is particularly preferably carried out using a membrane contactor. In the membrane contactor, the fluids in the mass exchange are passed through, separated from one another by a porous membrane. In this case, the fluids are the phase with the protein as the main solid component, separated in step d), and the oxidizing agent. In the membrane contactor, the oxidizing agent passes through the membrane to reach the phase with the protein as the main solid component, separated in step d). It has now been found that the use of a membrane contactor is particularly advantageous and efficient compared to other contactors. This is in particular because the pressure loss and the energy requirement in the membrane contactor are relatively low, while the oxidizing agent can still exert its effect very effectively.

[0066] In step e) a protease is used. A protease is an enzyme that is capable of hydrolytically degrading proteins. Proteases are also called proteolytic enzymes.

[0067] Preferably, in step e), the protease is used in a proportion of 0.0001 to 10% by weight, in particular 0.001 to 5% by weight, in particular 0.01 to 1% by weight or 0.05 to 0.5% by weight, based on the solids content of the microfiltration retentate from step c).

[0068] The protease is preferably an alkaline protease, in particular a serine protease, particularly preferably a subtilisin.

[0069] Serine proteases are a subfamily of proteases that have the amino acid serine in their active center.

[0070] Subtilisin-type proteases (subtilases, subtilopeptidases, EC 3.4.21.62) are classified as serine proteases based on the catalytically active amino acid. They are naturally produced and secreted by microorganisms, especially Bacillus species. They act as nonspecific endopeptidases, i.e., they hydrolyze any acid-amide bond in the interior of a peptide or protein. Their pH optimum is often in the distinctly alkaline range.

[0071] Proteases, and in particular the specific proteases mentioned, have been found to be extremely advantageous in the process of the invention, in particular because they allow selective release of mannans, which can be separated in the form of a liquid phase having mannans as the main solid component.

[0072] In particular, the method is carried out without the addition of mannanase or in the absence of mannanase, which is an enzyme that degrades mannans.

[0073] The treatment in step e) is preferably carried out at a pH value of 7.5 to 13, preferably 8 to 11, in particular 8.5 to 10.5, and / or at a temperature of 40 to 80° C., preferably 50 to 70° C., in particular 55 to 65° C. The pH value is preferably adjusted to the protease used.

[0074] In particular, in step e), the pH value is adjusted to 0.1%, preferably with a pK of less than 4.75, in particular less than 1. B pK is adjusted by adding a base having a B The value is the negative of the base constant K B It is particularly preferably an inorganic base, in particular NaOH. Such bases have been found to be particularly suitable.

[0075] The base is preferably used in diluted form (for example 30 to 60% by weight in water).

[0076] The treatment in step e) lasts in particular for 10 min to 18 h, in particular for 3 to 12 h, which allows the yield to be optimized.

[0077] In a further embodiment, in step e) a pasty fraction with a small amount of liquid can be processed, which allows the enzymatic step to be optimized in terms of yield, depending on the composition of the yeast suspension and the reaction procedure, respectively.

[0078] In step f), the held phase is preferably treated for a first time period, preferably 1 to 5 hours, at a pH value of 7.5 to 13, preferably 8 to 11, in particular 8.5 to 10.5, and / or at a temperature of 40 to 95° C., preferably 65 to 85° C., in particular 75 to 85° C. Subsequently, the held phase is preferably treated for a second time period, preferably 0.5 to 2 hours, at a pH value of 2 to 6.5, preferably 3 to 5, in particular 3.5 to 4.5, and / or at a temperature of 50 to 95° C., preferably 80 to 95° C., in particular 80 to 90° C.

[0079] The "phase retained in step e)" referred to in step f) is in particular to be understood as the phase which is retained after separation of the phase having mannan as the main solid component in step e). In particular, the "phase retained in step e)" therefore corresponds to the retentate from step c) from which the phase having mannan as the main solid component has been separated.

[0080] If the separation of the phase having mannan as the main solid component is carried out in step e), for example by filtration, the "phase retained in step e)" referred to in step f) is in particular the retentate from step e), in which case the phase having mannan as the main solid component forms the permeate, which is separated in step e).

[0081] Particularly preferably, in step f), the pH value in the first period of time is selected to correspond to the pH value during the treatment in step e).

[0082] More preferably, in step f), the temperature in the first period of time is higher than the temperature during treatment in step e).

[0083] Preferably, in step f), the temperature in the first period of time is lower than the temperature in the second period of time and / or the first period of time is longer than the second period of time.

[0084] In step f), if necessary, the pH value is adjusted by adding a base, preferably NaOH, and / or by adding an acid, preferably H2SO4.

[0085] Here, the base is preferably a base as described above in step e), particularly preferably the base in step f) is the same base as in step e).

[0086] The acid has in particular a pK of less than 4.75, in particular less than 1. S It is an acid that has a pK value. S The value is negative, the acid constant K S Particularly preferably it is a mineral acid, in particular H2SO4.

[0087] The acid is preferably used in diluted form (for example 30-60% by weight in water).

[0088] These aforementioned measures, especially in combination, have been found to be particularly advantageous in terms of process efficiency.

[0089] The separation of the phase having mannan as the main solid component in step e) is preferably carried out by filtration, in particular by combined microfiltration and ultrafiltration.Similarly, the separation of the phase having glucan as the main solid component in step f) is preferably carried out by filtration, in particular by combined microfiltration and ultrafiltration.

[0090] The phase retained after separation of the mannan-bearing phase in step e) is further treated in step f) specifically as the "retained phase".

[0091] Here, microfiltration is preferably carried out using a filter medium having a pore size of 0.1 to 0.5 μm, in particular 0.1 to 0.2 μm, preferably 0.1 μm, respectively. Ultrafiltration is preferably carried out using a filter medium having a pore size of less than 90 nm and / or an exclusion limit (nominal molecular weight cut-off) of 1 to 100 kDa, in particular 5 to 100 kDa, preferably 10 to 50 kDa, in particular 15 to 25 kDa, especially 20 kDa, respectively.

[0092] The microfiltration in step e) and / or the ultrafiltration in step f) is preferably carried out as diafiltration, in particular in the same way as described in steps c) and d).

[0093] Here, in step e), in particular the phase having mannans as the main solid component is discharged in the form of a permeate of the microfiltration and fed to an ultrafiltration in order to increase the relative proportion of mannans in the retentate of the ultrafiltration.

[0094] In step f), in particular the phase having glucan as the main solid component is discharged in the form of a permeate of the microfiltration and fed to an ultrafiltration in order to increase the relative proportion of glucan in the retentate of the ultrafiltration.

[0095] In the diafiltration in steps e) and / or f), liquid is circulated, in particular from the reaction vessel in which steps e) and / or f) are carried out, and replacement liquid is preferably continuously supplied on the feed side.

[0096] According to a further advantageous embodiment, the separation in steps e) and / or f) is carried out by means of a drum filter and / or a centrifuge, in particular in step f).

[0097] Thus, in a particular embodiment, the separation in step e) is carried out by combined microfiltration and ultrafiltration, as described above, and the separation in step f) is carried out by means of a drum filter and / or a centrifuge, in particular by means of a centrifuge.

[0098] Preferably, the phase separated in step e) having mannan as the main solid component is dried, in particular spray-dried, after step e), and / or the phase separated in step f) having glucan as the main solid component is dried, in particular spray-dried, after step f), whereby both mannan and glucan can be obtained as a powder product with high purity.

[0099] Preferably, prior to spray drying, the solids, in particular the mannan and / or glucan content, are concentrated, in particular using an evaporator.

[0100] The phase separated in step e) having mannans as the main solid component can optionally be treated after step e) with an oxidizing agent, in particular ozone and / or hydrogen peroxide, and / or contacted with an adsorbent material, for example activated carbon.

[0101] Similarly, the phase separated in step f) having glucan as the main solid component can optionally be treated after step f) with an oxidizing agent, in particular ozone and / or hydrogen peroxide, and / or contacted with an adsorbent material, for example activated carbon.

[0102] As with protein separation, this can improve the quality of the mannan and / or glucan products.

[0103] If spray drying is performed after steps e) and / or f), treatment with the oxidizing agent and / or contact with the adsorbent material is preferably performed before spray drying, and insofar as it is performed, it is performed before concentration.

[0104] The treatment with the oxidizing agent is particularly preferably carried out using a membrane contactor, the advantages of which have been described above in connection with protein streams.

[0105] Further advantageous embodiments and feature combinations of the invention will become apparent from the entirety of the following detailed description and claims. [Brief description of the drawings]

[0106] 1 and 2, used for the illustration of the examples, show a process flow diagram of the method according to the invention for the lysis of brewer's yeast for the purpose of isolating proteins (P), mannans (M) and glucans (G).

[0107] Basically, the same parts are given the same reference numerals in each figure.

[0108] Methods for carrying out the invention Figures 1 and 2 show the process flow diagram of the method according to the invention for the lysis of brewer's yeast for the purpose of isolating proteins (P), mannans (M) and glucans (G).

[0109] In a first step 100, a cooled aqueous brewer's yeast suspension BS (temperature=5° C.; solids concentration=15% by weight) is delivered in a tank truck 1 for temporary storage at 5° C. in a storage tank 2 in a next step 101.

[0110] Furthermore, the brewer's yeast suspension BS is subjected to a washing process 102 in the centrifuge 3. There, the liquid phase of the brewer's yeast suspension BS is replaced by water W, which is fed to the feed tank 4.

[0111] From the feed tank 4, the washed brewer's yeast suspension is then led to a ball mill 5, where the brewer's yeast cells in the suspension are physically lysed in a subsequent process step 103. The lysed brewer's yeast suspension is subsequently subjected to a microfiltration step 104. The microfiltration is carried out as diafiltration using a filter medium with a pore size of 0.1 μm. There, the lysed brewer's yeast suspension is circulated from the feed vessel 6 by means of a pump 7 through a microfilter 8, with water W being continuously supplied as replacement liquid on the feed side. The transmembrane pressure, which can be controlled by the outflow of the permeate, is about 1.5 bar. The proportion of proteins in the permeate p1 of the microfiltration is, for example, 60-70% by weight, based on the total weight of solids in the permeate p1.

[0112] The permeate p1 of the microfiltration step 104 is then subjected to an ultrafiltration step 105. The ultrafiltration is carried out as a diafiltration using a filter medium with an exclusion limit (nominal molecular weight cut-off) of 20 kDa. There, the permeate p1 is circulated from a further feed vessel 9 by means of a pump 10 through an ultrafilter 11, with water W being continuously supplied as displacement liquid on the feed side. The transmembrane pressure, which can be controlled by the outflow of the permeate, is about 2 bar. The ultrafiltration step 105 increases the relative solids fraction of the protein in the retentate r2, so that the solids fraction of the protein is about 90% by weight, based on the total solids in the retained retentate r2.

[0113] The permeate p2 of the ultrafiltration step 105 is discharged as wastewater (which can be reprocessed if necessary).

[0114] The retentate r2 of the ultrafiltration step 105, having the protein as the main solid component, is subjected to a drying process 106, in which the retentate r2, after passing through an evaporator 12, is fed to a spray-drying device 13, from which the protein is obtained as a product P in powder form.

[0115] The protein yield over the entire process is about 38% by weight, based on the content of available protein in the brewer's yeast suspension BS.

[0116] As shown in Figure 2, the retentate r1 of the microfiltration process 104 is further treated enzymatically and chemically in sequence. Figure 2 continues from the part marked with an asterisk (*) in Figure 1.

[0117] In the proteolytic digestion step 107, the retentate r1 is treated with a protease E in the form of 0.2% by weight (based on the solids content of the retentate r1) of subtilisin, for example for 8 hours, in a reactor 14. The treatment is carried out at a pH value of about 9.5, which is adjusted and kept constant during the treatment with the protease by adding a base B in the form of a 45% aqueous solution of NaOH.

[0118] The proteolytic digestion selectively releases mannans, which are subsequently separated by microfiltration and downstream ultrafiltration in the form of an aqueous phase with mannans as the main solid component. Both microfiltration and ultrafiltration are carried out as diafiltration. During microfiltration, the liquid to be filtered is circulated from the reactor 14 through a microfiltration filter 16 (pore size: 0.1 μm) using pump 15, with replacement liquid being continuously supplied on the feed side. The permeate of the microfiltration is subsequently fed to the feed vessel 17 of the ultrafiltration stage, from where the liquid is circulated through an ultrafilter 19 (exclusion limit: 20 kDa) using pump 18. Ultrafiltration increases the relative solids proportion of mannans in the retentate of the ultrafiltration stage, so that the solids proportion of mannans is about 65% by weight, based on the total solids in the retained retentate. The permeate of the ultrafiltration stage is discharged as wastewater (which can be reprocessed if necessary).

[0119] The retentate of the ultrafiltration stage, having mannans as the main solid component, is subjected to a drying process 110, the retentate passing through an evaporator 20 before being fed to a spray drying apparatus 21, from which the mannans are obtained as product M in powder form.

[0120] The mannan yield over the entire process is about 39% by weight, based on the content of available mannan in the brewer's yeast suspension BS.

[0121] The phase retained in the reactor 14 is then heated to a temperature of about 80° C. and subjected to a basic extraction 108 at a pH value of 9.5, for a first period of about 3 hours. The pH value is kept constant by controlled addition of dilute NaOH.

[0122] Subsequently, the temperature is increased again to about 85° C. and the pH value is reduced to a value of 4 by adding 50% H2SO4 in water. The phase retained in the reactor is then subjected to an acid extraction 109 for a period of 1 hour.

[0123] After the basic and acidic extractions, the liquid phase, mainly composed of glucans as well as mannans, is separated by diafiltration through two filter stages (microfilter 16 and ultrafilter 18), thereby achieving a solids content of glucans of about 72% by weight, based on the total solids in the retentate of the diafiltration stage.

[0124] The retentate of the ultrafiltration stage, having glucan as the main solid component, is likewise subjected to a drying process 110, the retentate passing through an evaporator 20 before being fed to a spray drying apparatus 21, from which the glucan is obtained as product G in powder form.

[0125] The glucan yield over the entire process is about 42% by weight, based on the content of available glucan in the brewer's yeast suspension BS.

[0126] The invention is not, however, limited to the embodiment shown, which can therefore be modified as desired within the framework of the invention.

[0127] For example, instead of the brewer's yeast suspension, another yeast suspension can be used, for example. Likewise, non-unavoidable process steps, such as the washing process 102, can be omitted. Furthermore, cell lysis can be performed by pulsed electric fields instead of using a ball mill 5. It is also possible, inter alia, to separate the mannans and / or glucans using drum filters or centrifuges instead of using the described filter stages (microfilter 16 and ultrafilter 18).

[0128] Furthermore, a membrane contactor MK or MK' can optionally be provided before the evaporator 12 and / or before the evaporator 20, by which the corresponding phase can be treated with an oxidizing agent, for example ozone and / or hydrogen peroxide, before spray drying.

[0129] In summary, it is shown that a new and particularly advantageous method for lysing yeast, in particular brewer's yeast, for the isolation of proteins and sugars has been provided, which is particularly suitable for a large-scale approach.

Claims

1. Steps below: a) providing a yeast suspension, in particular a brewer's yeast suspension; b) physically lysing the yeast in the yeast suspension; c) microfiltering the lysed yeast suspension from step b); d) ultrafiltration of the microfiltration permeate from step c), whereby a phase having proteins as the main solid component is separated as the ultrafiltration retentate; e) treating the microfiltration retentate from step c) with a protease under basic conditions and subsequently separating the phase having mannan as the main solid component, in particular by filtration and / or centrifugation; f) successively treating the phase retained in step e) first under basic conditions and then under acidic conditions, followed by separation, in particular by filtration and / or centrifugation, of the phase having glucan as the main solid component; 2. A method for lysing yeast, in particular brewer's yeast, in order to isolate proteins and sugars, comprising:

2. 2. The method according to claim 1, wherein the yeast suspension provided in step a) has a proportion of solids of 5 to 30% by weight, in particular 7 to 20% by weight, based on the total weight of the yeast suspension.

3. The method according to claim 1, wherein the yeast suspension provided in step a) has a temperature of 0 to 15°C, preferably a temperature of 2 to 12°C, in particular a temperature of 3 to 7°C or 5°C.

4. 2. The method according to claim 1, wherein the yeast suspension is subjected to a washing process prior to step b), in which at least a part of the liquid phase of the yeast suspension, in particular at least 50% by weight, in particular at least 60% by weight of the liquid phase of the yeast suspension, is replaced by a further liquid, in particular water.

5. 2. The method according to claim 1, wherein the physical dissolution in step b) is carried out in a homogenizer or a grinding media mill, in particular a ball mill.

6. The method according to claim 1 , wherein the physical dissolution in step b) is carried out by means of a pulsed electric field.

7. The method according to claim 6, wherein the electric field strength is between 1 and 30 kV / cm, in particular between 5 and 24 kV / cm, and / or the specific energy input is between 1 and 180 kJ / L, in particular between 3 and 120 kJ / L.

8. 2. The method according to claim 1, wherein the microfiltration in step c) is carried out using a filter medium having a pore size of 0.1 to 0.5 μm, preferably 0.1 to 0.2 μm, in particular 0.1 μm, and the ultrafiltration in step d) is carried out using a filter medium having a pore size of less than 90 nm and / or an exclusion limit of 1 to 100 kDa, in particular 5 to 100 kDa, preferably 10 to 50 kDa, in particular 15 to 25 kDa, in particular 20 kDa.

9. 2. The process according to claim 1, wherein the microfiltration in step c) and / or the ultrafiltration in step d) is carried out as diafiltration, with water being preferably continuously supplied on the feed side.

10. 2. The method according to claim 1, wherein the phase having proteins as main solid components separated in step d) is dried after step d), in particular spray-dried.

11. 2. The process according to claim 1, wherein in step e) a protease is used in a proportion of 0.0001 to 10% by weight, in particular 0.001 to 5% by weight, in particular 0.01 to 1% by weight or 0.05 to 0.5% by weight, based on the solids content of the microfiltration retentate from step c), said protease being preferably a subtilisin, and said treatment in step e) is carried out at a pH value of 7.5 to 13, preferably 8 to 11, in particular 8.5 to 10.5 and / or at a temperature of 40 to 80° C., preferably 50 to 70° C., in particular 55 to 65° C.

12. The method according to claim 1, wherein the treatment with the protease in step e) lasts from 10 min to 18 h, in particular from 3 to 12 h.

13. 2. The method of claim 1, wherein in step f) the retained phase is treated for a first period of time, preferably a first period of 1 to 5 hours, at a pH value of 7.5 to 13, preferably 8 to 11, in particular 8.5 to 10.5, and at a temperature of 40 to 95°C, preferably 65 to 85°C, in particular 75 to 85°C, and then for a second period of time, preferably a second period of 0.5 to 2 hours, at a pH value of 2 to 6.5, preferably 3 to 5, in particular 3.5 to 4.5, and at a temperature of 50 to 95°C, preferably 80 to 95°C, in particular 80 to 90°C.

14. 14. The method according to claim 13, wherein in step f) the pH value in the first period corresponds to the pH value during the treatment in step e), and wherein in step f) the temperature in the first period is lower than the temperature in the second period and / or the first period is longer than the second period.

15. 2. The method according to claim 1, wherein the separation of the phase having mannan as the main solid component in step e) and / or the separation of the phase having glucan as the main solid component in step f) is carried out by filtration, in particular by combined microfiltration and ultrafiltration.

16. 16. The method according to claim 15, wherein the microfiltration is carried out using a filter medium having a pore size of 0.1 to 0.5 μm, in particular 0.1 to 0.2 μm, preferably 0.1 μm, and the ultrafiltration is carried out using a filter medium having a pore size of less than 90 nm and / or an exclusion limit of 1 to 100 kDa, in particular 5 to 100 kDa, preferably 10 to 50 kDa, in particular 15 to 25 kDa, in particular 20 kDa.

17. 16. The method according to claim 15, wherein the microfiltration in step e) and / or the ultrafiltration in step f) are carried out as diafiltration.

18. 2. The method according to claim 1, wherein the phase separated in step e) having mannan as the main solid component is dried, in particular spray-dried, after step e) and / or the phase separated by step f) having glucan as the main solid component is dried, in particular spray-dried, after step f).

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