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 saccharides. [Background Art]

[0002] Yeast are unicellular fungi that proliferate by budding or fission and have long been of great technical importance. For example, yeast is used in the manufacture of beer, wine, spirits, food and therapeutic substances. Basically, there are various types of yeast, so-called baker's yeast or brewer's yeast (Saccharomyces cerevisiae) is one of the most commonly used yeasts. Accordingly, considerable amounts of yeast-containing, in particular brewer's yeast-containing by-products are incidentally generated.

[0003] In particular, since yeast cell walls contain a series of technically useful biomolecules, such as proteins, glucans, mannans, chitin and lipids, yeast-containing waste is processed to obtain these substances. Therein, the yeast cell wall is partially or completely lysed to release the substances of interest.

[0004] For example, EP1990419A1 (Tex-a-tec AG) (Patent Document 1) describes a method for isolating glucan, protein, mannan and lipid from yeast, wherein the yeast is treated with ultrasound at elevated temperature in successive steps.

[0005] EP2272876A1 (Angel Yeast Co., Ltd.) (Patent Document 2) discloses a method for extracting glucans and mannans from the cell walls of microorganisms, such as yeast 8440. The method comprises the following steps: a) treating the microbial cells with an alkaline protease and a 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) (Patent Document 3) describes a method for producing an immunostimulant composition containing a sugar fraction, comprising the step of hydrolyzing yeast cells to obtain a soluble fraction. The sugar fraction thus obtained can be added as a food or beverage component, or can be used as a pharmaceutical.

[0007] However, conventional methods for isolating technically usable biomolecules from yeast are not entirely satisfactory, especially in large-scale approaches. For example, the yield-to-labor ratio (particularly with respect to energy and time requirements) is insufficient in many methods, which significantly hinders economic production.

[0008] Other methods are geared towards selectively obtaining individual components (e.g., glucans or mannans), but other components (e.g., proteins) are left unconsidered. This is disadvantageous in terms of achieving the most complete utilization of yeast-containing by-products.

[0009] Therefore, improved solutions that overcome the aforementioned shortcomings, at least partially or completely, are still needed. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] EP1990419A1 [Patent Document 2] EP2272876A1 [Patent Document 3] WO2010 / 070207A1 [Overview of the project] [Problems that the invention aims to solve]

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

[0012] The solution to the aforementioned problem is defined by the features of claim 1. Accordingly, the essence of the present invention is a method for lysing yeast, particularly brewer's yeast (Bierhefe), in order to isolate proteins and sugars, comprising the following steps: a) A step of providing a yeast suspension, particularly a brewer's yeast suspension; b) A step of physically dissolving the yeast in the yeast suspension; c) Microfiltration of the dissolved yeast suspension from step b); d) A step of ultrafiltration the permeate from step c), wherein a phase having protein as the main solid component is separated as the retained material of the ultrafiltration; e) The retained material from the microfiltration in step c) is treated with a protease under basic conditions, and subsequently the phase having mannan as the main solid component is separated, in particular by filtration and / or centrifugation; f) The phase retained in step e) is successively treated, first under basic conditions and then under acidic conditions, and subsequently the phase having glucan as the main solid component is separated, particularly by filtration and / or centrifugation.

[0013] Remarkably, the combination of physical, chemical, and biochemical processing steps according to the present invention has been shown to enable good yields with relatively low energy requirements and relatively short processing times. This applies to all products, namely proteins, glucans, and mannans, and yields of up to approximately 40% for each of these substances can be achieved with high purity.

[0014] Furthermore, the method is suitable for large-scale approaches. It can process tens of thousands of tons of yeast suspension per year, or thousands of kilograms of yeast suspension per hour, without any problems.

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

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

[0017] Unless otherwise specifically stated, the term "solid component" refers to the dry matter of each component, respectively. The term "main solid component" refers to the solid component having the largest proportion by weight.

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

[0019] However, in principle, the yeast suspension provided in step a) may also be a yeast suspension that is stored at another temperature, in particular room temperature, and is not pasteurized.

[0020] The yeast suspension is in particular an aqueous yeast suspension, wherein 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 is produced, for example, as a by-product in beer production and can be used directly in the method according to the present invention.

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

[0023] In step a), the yeast suspension has in particular a temperature of from 0 to 15°C, preferably from 2 to 12°C, in particular from 3 to 7°C or 5°C. This ensures that the composition of the suspension does not change further, for example due to active yeast cells, or that the formation of undesired by-products is reduced.

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

[0025] However, in principle, the yeast suspension in steps a), b), c) and / or d) may be at a different temperature, such as room temperature.

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

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

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

[0029] Homogenizers, especially high-pressure homogenizers, are operated at pressures exceeding 1000 bar, particularly exceeding 1200 bar.

[0030] In a grinding media mill, the material to be ground and the freely moving grinding media, especially balls, are circulated within the processing chamber. During this process, collisions occur between the grinding media, the walls of the processing chamber, and the material to be ground. As a result, the material is broken down, particularly by fracture.

[0031] Since the protein to be isolated can be released gently, but nevertheless effectively, dissolution using a grinding medium mill, particularly a ball mill, proved to be especially advantageous in this case. This is especially true for brewer's yeast suspensions. However, for specific applications, other methods of physical dissolution can also be used.

[0032] The physical dissolution using an electric field in step b) can be carried out in particular using a pulsed electric field (PEF or "Pulsed Electric Fields" in English). This dissolution technique itself is known to those skilled in the art in relation to other matters.

[0033] When treated with a pulsed electric field, the yeast suspension is placed between two electrodes and treated with the pulsed electric field. The pulsed electric field is characterized in particular by a high electric field intensity and a short duration. This causes reversible or irreversible pore formation in the cell membrane, thereby allowing intracellular components to be released and / or extracellular substances to enter the cell.

[0034] In this study, the following parameters were found to be particularly suitable for controlling the solubility of yeast in yeast suspension: electric field strength [kV / cm], the temperature at which the yeast suspension was introduced [°C], and the specific energy input [kJ / L]. The energy required for the process (kilojoules per liter of yeast suspension) is understood as the energy input. Furthermore, the energy input can be adjusted via the pulse frequency [Hz], which is controlled automatically according to the flow rate and in known devices.

[0035] Preferably, the electric field strength is 1 to 30 kV / cm, and particularly 5 to 24 kV / cm.

[0036] The specific energy input is preferably 1 to 180 kJ / L, and more particularly 3 to 120 kJ / L.

[0037] The temperature at which the yeast suspension is added is particularly 0-15°C, preferably 2-12°C, and especially 3-7°C or 5°C.

[0038] These parameters allow for effective dissolution of yeast in a 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, mechanical post-processing is performed following the pulsed electric field treatment. Therefore, this is preferably performed between steps b) and c).

[0041] Mechanical post-processing can be carried out, in particular, by a mixer, preferably one with high shear force. A rotor-stator mixer, especially an inline rotor-stator mixer, is particularly suitable. In an inline rotor-stator mixer, the rotor and stator are typically housed in a housing having an inlet at one end and an outlet at the other end. Thus, the yeast suspension to be processed can be passed through the mixer in a continuous flow.

[0042] The shear rate during mechanical post-processing using a mixer is selected to further lyse the yeast cells. This allows for the release of intracellular components (proteins) that may not have been completely released after the application of a pulsed electric field. High shear force further "squeezes" already cleaved yeast cells, causing intracellular components, especially proteins, to leak out of the cells.

[0043] The microfiltration in step c) is preferably carried out using a filter medium having a pore size of 0.1 to 0.5 μm, preferably 0.1 to 0.2 μm, and particularly 0.1 μm. This has been found to be particularly preferable in order to achieve relative enrichment of the proteins to be isolated in the permeate with respect to the solid content after physical dissolution, and at the same time enrichment of biomass residues, such as cell fragments and lipids, in the retained material.

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

[0045] The dissolved yeast suspension is circulated, in particular, from the feed container. By controlling the flow after the filter media, the intermembrane pressure acting as the driving force in diafiltration can be adjusted.

[0046] Diafiltration is preferably performed in a closed circulation so that the removal of permeate results in a continuous flow (Nachfliessen) of the replacement solvent, and in particular so that the circulating volume remains constant during diafiltration.

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

[0048] In step c), the proportion of solids in the permeate from microfiltration is preferably 1 to 10% by weight, and particularly 3 to 7% by weight, based on the total weight of the permeate.

[0049] More preferably, the microfiltration in step c) is carried out such that the proportion of protein in the retained material from the microfiltration from step c) is less than 50% by weight, preferably less than 40% by weight, and particularly less than 35% by weight, based on the total weight of solids in the retained material.

[0050] In step c), the proportion of solids in the retained material after microfiltration is preferably 20-50% by weight, and particularly 25-30% by weight, based on the total weight of the retained material.

[0051] According to further possible embodiments, the dissolved yeast suspension is treated with an oxidizing agent, particularly ozone (O3) and / or hydrogen peroxide (H2O2), prior to the microfiltration step c). This helps prevent chemical reactions that would result in undesirable byproducts and / or changes in taste. However, treatment with an oxidizing agent is optional.

[0052] Furthermore, the dissolved yeast suspension can be enzymatically treated before step c) microfiltration. This can increase the efficiency and yield of the process, if necessary. However, the enzymatic treatment before step c) microfiltration is optional.

[0053] Ultrafiltration in step d) 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 cutoff) of 1 to 100 kDa, particularly 5 to 100 kDa, preferably 10 to 50 kDa, particularly 15 to 25 kDa, and especially 20 kDa. The exclusion limit is defined as the smallest molecular weight of the molecules that are retained by 90% of the filter medium, especially the membrane.

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

[0055] The permeate from the microfiltration from step c), which forms the feed (Vorlage) in step d), is circulated, in particular, from the feed container. By controlling the flow after the filter media, the intermembrane pressure acting as the driving force for diafiltration in step d) can be readjusted.

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

[0057] Ultrafiltration and diafiltration in step d) allow for a targeted increase in the relative proportion of solids to proteins in the retained material.

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

[0059] The proportion of solid matter in the phase separated in step d) is preferably 0.5 to 7% by weight, and particularly 1 to 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 performed as diafiltration.

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

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

[0063] The phase separated in step d), which has protein as its main solid component, can optionally be treated after step d) with an oxidizing agent, particularly ozone and / or hydrogen peroxide, and / or contacted with an adsorbent material, such as activated carbon. This can particularly improve the quality of the protein product. For example, undesirable discoloration and / or flavor 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 is preferably performed before spray drying, and if done, before concentration.

[0065] Treatment with an oxidizing agent is particularly preferably carried out using a membrane contactor. In a membrane contactor, the fluids undergoing mass exchange are separated from each other by a porous membrane. In this case, the fluids are the phase having 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 and reaches the phase having protein as the main solid component, separated in step d). The use of a membrane contactor was found to be particularly advantageous and efficient compared to other contactors. This is because the pressure loss and energy requirements in a 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 can hydrolyze proteins. Proteases are also called protein-degrading enzymes.

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

[0068] The protease is preferably an alkaline protease, more preferably a serine protease, and especially preferably subtilisin.

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

[0070] Subtilisin-type proteases (subtyralases, subtyropeptidases, EC 3.4.21.62) are classified as serine proteases based on their catalytically active amino acid. They are naturally produced and secreted by microorganisms, particularly Bacillus species. They act as nonspecific endopeptidases, meaning they hydrolyze any acid-amide bonds within peptides or proteins. Their optimal pH conditions are often clearly in the alkaline range.

[0071] Proteases, and in particular the specific proteases mentioned, have been found to be extremely advantageous in the methods of the present invention. In particular, mannan can be selectively released by the protease, and the mannan can be separated in the form of a liquid phase having mannan as the main solid component.

[0072] In particular, this method is carried out without the addition of mannanase, or in the absence of mannanase. Mannanase is an enzyme that breaks down mannan.

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

[0074] In particular, in step e), the pH value is a base, preferably less than 4.75, and especially less than 1 pK. B It is adjusted by adding a base with a specific value. pK B Value, negative, base constant K B This represents the base-10 logarithm of [the given value]. Particularly preferred is an inorganic base, especially NaOH. Such bases have been found to be particularly suitable.

[0075] The base is preferably used in a diluted form (for example, at a rate of 30-60% by weight in water).

[0076] The processing in step e) lasts particularly from 10 minutes to 18 hours, and especially from 3 to 12 hours. This allows for optimization of the yield.

[0077] In further embodiments, step e) may involve processing a paste-like fraction containing a small amount of liquid. This allows for optimization of the enzyme step with respect to yield, depending on the composition of the yeast suspension and the reaction procedure.

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

[0079] The "phase retained in step e)" mentioned in step f) is understood in particular as the phase retained after the separation of the phase having mannan as the main solid component in step e). In particular, the "phase retained in step e)" corresponds to the retained material from step c), from which the phase having mannan as the main solid component was 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, then the "phase retained in step e)" referred to in step f) is, in particular, the retained material from step e). In this case, in step e), the phase having mannan as the main solid component forms a permeate, which is then separated.

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

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

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

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

[0085] Here, the base is preferably one of the bases described above in step e). Particularly preferably, the base in step f) is the same base as the base in step e).

[0086] Acids, in particular, have a pK of less than 4.75, especially less than 1. S It is an acid that has a pK value. S Value, negative, acid constant K S This represents the base-10 logarithm of [the function]. Particularly preferred is a mineral acid, especially H2SO4.

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

[0088] These aforementioned methods (especially in combination) were 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, particularly 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, particularly by combined microfiltration and ultrafiltration.

[0090] The retained phase after the separation of the mannan-containing phase in step e) is further processed in step f) as the “retained phase” in particular.

[0091] Here, microfiltration is carried out using filter media having pore sizes of preferably 0.1 to 0.5 μm, particularly 0.1 to 0.2 μm, and preferably 0.1 μm. Ultrafiltration is preferably carried out using filter media having pore sizes of less than 90 nm and / or exclusion limits (nominal molecular weight cutoffs) of 1 to 100 kDa, particularly 5 to 100 kDa, preferably 10 to 50 kDa, particularly 15 to 25 kDa, and especially 20 kDa.

[0092] Microfiltration in step e) and / or ultrafiltration in step f) are preferably carried out as diafiltration, in particular in the same manner as described in steps c) and d).

[0093] In step e), in particular, the phase having mannan as the main solid component is discharged in the form of a permeate from microfiltration and supplied to ultrafiltration, thereby increasing the relative proportion of mannan in the material retained by ultrafiltration.

[0094] In step f), in particular, the phase having glucan as the main solid component is discharged in the form of a permeate from microfiltration and supplied to ultrafiltration, thereby increasing the relative proportion of glucan in the material retained by ultrafiltration.

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

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

[0097] Accordingly, in certain embodiments, the separation in step e) is carried out by a combination of microfiltration and ultrafiltration as described above, and the separation in step f) is carried out by a drum filter and / or a centrifuge, particularly by a centrifuge.

[0098] Preferably, the phase having mannan as the main solid component, separated in step e), is dried after step e), particularly by spray drying, and / or the phase having glucan as the main solid component, separated in step f), is dried after step f), particularly by spray drying. This allows both mannan and glucan to be obtained as powdered products with high purity.

[0099] Preferably, prior to spray drying, the solid content, particularly the mannan and / or glucan content, is concentrated, especially using an evaporator.

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

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

[0102] This allows for an improvement in the quality of the mannan and / or glucan products, similar to the case of protein isolation.

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

[0104] Treatment with an oxidizing agent is particularly preferably carried out using a membrane contactor. The advantages of this are described above in relation to protein flow.

[0105] Further advantageous embodiments and combinations of features of the present invention will be revealed from the following detailed description and the claims as a whole. [Brief explanation of the drawing]

[0106] Figures 1 and 2, used to illustrate the examples, show process flow diagrams of the method according to the present invention for solubility of brewer's yeast with the aim of isolating protein (P), mannan (M), and glucan (G).

[0107] Generally, identical parts in each figure are assigned the same reference numerals.

[0108] Method for carrying out the invention Figures 1 and 2 show process flow diagrams of the method according to the present invention for solubilization of brewer's yeast with the aim of isolating protein (P), mannan (M), and glucan (G).

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

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

[0111] Next, the washed brewer's yeast suspension is led from the feed tank 4 to the ball mill 5, where the brewer's yeast cells in the suspension are physically lysed in the subsequent process step 103. The lysed brewer's yeast suspension is then subjected to a microfiltration step 104. Microfiltration is carried out as diafiltration using a filter medium with a pore size of 0.1 μm. The lysed brewer's yeast suspension is circulated from the feed container 6 through the microfilter 8 using a pump 7, and water W is continuously supplied as a replacement liquid on the feed side. The intermembrane pressure (which can be controlled by the outflow of permeate) is approximately 1.5 bar. The percentage of protein 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 from the microfiltration step 104 is then subjected to the ultrafiltration step 105. Ultrafiltration is performed as diafiltration using a filter medium having an exclusion limit (nominal molecular weight cutoff) of 20 kDa. Thereafter, the permeate p1 is circulated from another feed container 9 via a pump 10 through the ultrafilter 11, and water W is continuously supplied as the replacement liquid on the feed side. The intermembrane pressure (which can be controlled by the outflow of permeate) is approximately 2 bar. The ultrafiltration step 105 increases the relative solids ratio of protein in the retained material r2, resulting in a solids ratio of protein of approximately 90% by weight relative to the total solids in the retained material r2.

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

[0114] The retained material r2 from the ultrafiltration step 105, which has protein as its main solid component, is subjected to a drying process 106. After passing through the evaporator 12, the retained material r2 is supplied to the spray dryer 13. From there, protein is obtained as the product P in powder form.

[0115] The protein yield throughout the entire process is approximately 38% by weight, based on the available protein content in the brewer's yeast suspension (BS).

[0116] As shown in Figure 2, the retained material r1 from the microfiltration process 104 is subsequently subjected to further enzymatic and chemical treatment. Figure 2, marked with an asterisk (*), follows the sections marked with an asterisk in Figure 1.

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

[0118] Through proteolytic digestion, mannan is selectively released, which is subsequently separated in the form of an aqueous phase with mannan as the main solid component by microfiltration and downstream ultrafiltration. Both microfiltration and ultrafiltration are performed as diafiltration. During microfiltration, the liquid to be filtered is circulated from reactor 14 through microfiltration filter 16 (pore size: 0.1 μm) using pump 15, and replacement liquid is continuously supplied on the feed side. Subsequently, the permeate from microfiltration is supplied to feed container 17 for the ultrafiltration stage, from which the liquid is circulated through ultrafilter 19 (exclusion limit: 20 kDa) using pump 18. Ultrafiltration increases the relative solid content of mannan in the retained material at the ultrafiltration stage, resulting in a mannan solid content of approximately 65% ​​by weight relative to the total solid content in the retained material. The permeate from the ultrafiltration stage is discharged as wastewater (which can be retreated if necessary).

[0119] The retained material from the ultrafiltration stage, which has mannan as its main solid component, is subjected to a drying process 110. After passing through the evaporator 20, the retained material is supplied to the spray dryer 21. From there, mannan is obtained as product M in powder form.

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

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

[0122] Next, the temperature is raised again to approximately 85°C, and the pH value is lowered to 4 by adding 50% H2SO4 in the water. Then, the phase held in the reactor is subjected to acid extraction 109 for a period of 1 hour.

[0123] After basic and acidic extraction, the liquid phase, which contains glucan as the main component, is separated by diafiltration through two filtration stages (microfilter 16 and ultrafilter 18), similar to mannan. This achieves a glucan solid content of approximately 72% by weight, relative to the total solid content in the retained material at the diafiltration stage.

[0124] The retained material from the ultrafiltration stage, which has glucan as its main solid component, is similarly subjected to the drying process 110, and after passing through the evaporator 20, it is supplied to the spray dryer 21. From there, glucan is obtained as product G in powder form.

[0125] The glucan yield throughout the entire process is approximately 42% by weight, based on the available glucan content in the brewer's yeast suspension (BS).

[0126] However, the present invention is not limited to the embodiments shown. Therefore, it can be modified as appropriate within the framework of the present invention.

[0127] For example, a different yeast suspension can be used instead of the brewer's yeast suspension. Similarly, non-essential process steps, such as the washing process 102, can be omitted. Furthermore, cell lysis can be carried out by pulsed electric field instead of using the ball mill 5. In particular, it is also possible to separate mannans and / or glucans using a drum filter or centrifuge instead of using the described filtering steps (microfilter 16 and ultrafilter 18).

[0128] Furthermore, a membrane contactor MK or MK' may optionally be provided before the evaporator 12 and / or before the evaporator 20. This allows the corresponding phase to be treated with an oxidizing agent, such as ozone and / or hydrogen peroxide, before spray drying.

[0129] In summary, it is shown that a novel and particularly advantageous method for lysing yeast, especially brewer's yeast, for the isolation of proteins and sugars is provided, a method particularly suitable for large-scale approaches. While this application relates to the invention described in the claims, it may also encompass the following other embodiments. 1. Follow these steps: a) A step of providing a yeast suspension, particularly a brewer's yeast suspension; b) A step of physically dissolving the yeast in the yeast suspension; c) Microfiltration of the dissolved yeast suspension from step b); d) A step of ultrafiltration the permeate from step c), wherein a phase having protein as the main solid component is separated as the retained material of the ultrafiltration; e) The retained material from the microfiltration in step c) is treated with a protease under basic conditions, and subsequently the phase having mannan as the main solid component is separated, in particular by filtration and / or centrifugation; f) The phase retained in step e) is treated sequentially, first under basic conditions and then under acidic conditions, and subsequently the phase having glucan as the main solid component is separated, particularly by filtration and / or centrifugation. A method for dissolving yeast, particularly brewer's yeast, in order to isolate proteins and sugars, including those contained therein. 2. The method according to 1), wherein the yeast suspension provided in step a) has a solid content of 5 to 30% by weight, particularly 7 to 20% by weight, based on the total weight of the yeast suspension. 3. The method according to 1 or 2 above, wherein the yeast suspension provided in step a) has a temperature of 0 to 15°C, preferably 2 to 12°C, and particularly 3 to 7°C or 5°C. 4. The method according to any one of 1 to 3 above, wherein the yeast suspension is subjected to a washing process before step b), in which at least a portion of the liquid phase of the yeast suspension, in particular at least 50% by weight, especially at least 60% by weight, of the liquid phase of the yeast suspension is further replaced with another liquid, in particular water. 5. The method according to any one of 1 to 4 above, wherein the physical dissolution in step b) is carried out in a homogenizer or a grinding medium mill, particularly a ball mill. 6. The method according to any one of 1 to 5 above, wherein the physical dissolution in step b) is carried out by a pulsed electric field. 7. The method according to 6 above, wherein the electric field strength is 1 to 30 kV / cm, particularly 5 to 24 kV / cm, and / or the specific energy input is 1 to 180 kJ / L, particularly 3 to 120 kJ / L. 8. The method according to any one of 1 to 7 above, 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, and particularly 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, particularly 5 to 100 kDa, preferably 10 to 50 kDa, particularly 15 to 25 kDa, and particularly 20 kDa. 9. The method according to any one of 1 to 8 above, wherein the microfiltration in step c) and / or the ultrafiltration in step d) are performed as diafiltration, and water is preferably continuously supplied on the feed side. 10. The method according to any one of 1 to 9 above, wherein the phase having protein as the main solid component, separated in step d), is dried after step d), and in particular spray-dried. 11. The method according to any one of 1 to 10 above, wherein in step e), the protease is used in a proportion of 0.0001 to 10% by weight, particularly 0.001 to 5% by weight, especially 0.01 to 1% by weight or 0.05 to 0.5% by weight, based on the solid content of the retained material from the microfiltration from step c), the protease is preferably subtilisin, and the treatment in step e) is carried out at a pH value of 7.5 to 13, preferably 8 to 11, particularly 8.5 to 10.5, and / or at a temperature of 40 to 80°C, preferably 50 to 70°C, particularly 55 to 65°C. 12. The method according to any one of the above 1 to 11, wherein the treatment with protease in step e) lasts for 10 minutes to 18 hours, particularly 3 to 12 hours. 13. The method according to any one of 1 to 12 above, wherein in step f), the retained phase is treated for a first period, preferably 1 to 5 hours, at a pH value of 7.5 to 13, preferably 8 to 11, particularly 8.5 to 10.5, and at a temperature of 40 to 95°C, preferably 65 to 85°C, particularly 75 to 85°C, and then for a second period, preferably 0.5 to 2 hours, at a pH value of 2 to 6.5, preferably 3 to 5, particularly 3.5 to 4.5, and at a temperature of 50 to 95°C, preferably 80 to 95°C, particularly 80 to 90°C. 14. The method according to 13, wherein in step f), the pH value in the first period is the same as the pH value during the treatment in step e), and 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. The method according to any one of 1 to 14 above, 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, particularly combined microfiltration and ultrafiltration. 16. The method according to 15, wherein the microfiltration is carried out using a filter medium having a pore size of 0.1 to 0.5 μm, particularly 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, particularly 5 to 100 kDa, preferably 10 to 50 kDa, particularly 15 to 25 kDa, particularly 20 kDa. 17. The method according to 15 or 16 above, wherein the microfiltration in step e) and / or the ultrafiltration in step f) are performed as diafiltration. 18. The method according to any one of 1 to 17 above, wherein the phase having mannan as the main solid component, separated in step e), is dried after step e), particularly by spray drying, and / or the phase having glucan as the main solid component, separated by step f), is dried after step f), particularly by spray drying.

Claims

1. The following steps: a) Step of providing a yeast suspension; b) A step of physically dissolving the yeast in the yeast suspension; c) Microfiltration of the dissolved yeast suspension from step b); d) A step of ultrafiltration the permeate from step c), wherein a phase having protein as the main solid component is separated as the retained material of the ultrafiltration; e) Treat the retained material from the microfiltration of step c) with a protease under basic conditions, and subsequently separate the phase having mannan as the main solid component; f) The remaining phase from step e), separated from the phase having mannan as the main solid component, is treated sequentially, first under basic conditions and then under acidic conditions, and subsequently the phase having glucan as the main solid component is separated. A method for dissolving yeast and isolating proteins and sugars, including [specific components / methods].

2. The method according to claim 1, wherein a phase having mannan as the main solid component is separated by filtration and / or centrifugation, and / or a phase having glucan as the main solid component is separated by filtration and / or centrifugation.

3. The method according to claim 1, wherein the yeast suspension provided in step a) has a solid content of 5 to 30% by weight based on the total weight of the yeast suspension.

4. The method according to claim 1, wherein the yeast suspension provided in step a) has a temperature of 0 to 15°C.

5. 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 portion of the liquid phase of the yeast suspension is further replaced by another liquid.

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

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

8. The method according to claim 7, wherein the electric field strength is 1 to 30 kV / cm and / or the specific energy input is 1 to 180 kJ / L.

9. 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, 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.

10. The method according to claim 1, wherein microfiltration in step c) and / or ultrafiltration in step d) are performed as diafiltration, and water is supplied on the feed side during this process.

11. The method according to claim 10, wherein water is continuously supplied on the feed side.

12. The method according to claim 1, wherein the phase having protein as the main solid component, separated in step d), is dried after step d).

13. The method according to claim 1, wherein in step e), the protease is used in a proportion of 0.0001 to 10% by weight, based on the solid content of the retained material from the microfiltration from step c), and the treatment in step e) is carried out at a pH of 7.5 to 13 and / or at a temperature of 40 to 80°C.

14. The method according to claim 1, wherein the treatment with protease in step e) lasts for 10 minutes to 18 hours.

15. The method according to claim 1, wherein in step f), the remaining phase is treated in a first period at a pH of 7.5 to 13 and at a temperature of 40 to 95°C, and then in a second period at a pH of 2 to 6.5 and at a temperature of 50 to 95°C.

16. The method according to claim 15, wherein in step f), the pH value in the first period is the same as the pH value during the treatment in step e), and 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.

17. 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 performed by filtration.

18. The method according to claim 17, 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 performed by a combination of microfiltration and ultrafiltration.

19. The method according to claim 18, wherein the microfiltration is performed using a filter medium having a pore size of 0.1 to 0.5 μm, and the ultrafiltration is performed using a filter medium having a pore size of less than 90 nm and / or an exclusion limit of 1 to 100 kDa.

20. The method of claim 18, wherein the microfiltration in step e) and / or the ultrafiltration in step f) are performed as diafiltration.

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

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