Functional yeast protein concentrate

Abstract

The present invention relates to a method for preparing a yeast protein concentrate, comprising lysing yeast cells in a suspension adjusted to a specific pH before lysis, then subjecting the soluble fraction obtained from the lysis to filtration to reduce the content of molecules less than 30 kDa, and optionally drying the solution obtained from the filtration. The present invention also relates to a yeast protein concentrate obtainable by the method of the present invention. The yeast protein concentrate contains a large amount of proteins that are still folded and can aggregate upon heating to form a solid protein matrix. Furthermore, the yeast protein concentrate of the present invention has a mild taste and is therefore particularly suitable for use in the preparation of food products such as meat substitute products.

Description

Technical Field

[0001] The present invention relates to a method for preparing a yeast protein concentrate, which comprises lysing yeast cells in a suspension adjusted to a specific pH before lysis, and then subjecting the soluble fraction obtained from the lysis to filtration to reduce the content of molecules less than 30 kDa, and optionally drying the solution obtained by filtration. The present invention further relates to a yeast protein concentrate obtainable by the method of the present invention. The yeast protein concentrate contains a large amount of proteins that can aggregate and form a solid protein matrix when heated because they are still folded. Furthermore, the yeast protein concentrate of the present invention has a mild taste and is therefore particularly suitable for use in the preparation of foods such as meat substitute products.

Background Art

[0002] The World Health Organization (WHO) has assumed that the world population will increase by one-third by 2050, thereby posing a major problem for the food and agricultural industries. As a result of population growth, world meat production is predicted to double from current levels, having a profound impact on the environment. Already today, the production of livestock feed consumes approximately one-third of the world's total land area. The increasing demand in agricultural areas leads to further destruction of tropical rainforests and massive emissions of greenhouse gases.

[0003] Therefore, there is a great need for foods that can be produced in a resource- and climate-friendly way. Specifically, meat production involves a significant waste of resources, so the development of meat substitute products is an important factor in coping with the increasing world population. Several different meat substitute products based on plant proteins have been developed. However, the production of proteins from plants such as soybeans generally requires cultivating the plants on arable land and is therefore generally cumbersome and time-consuming. Therefore, an alternative source of protein that can be used in the manufacture of foods such as meat substitute products is still needed.

[0004] The present invention is based on the finding that a protein concentrate obtained from yeast can form an extraordinary solid protein gel having a specific texture and high consistency. Since yeast cells require neither complex nutrients nor growth conditions, they are a particularly advantageous starting material for producing a protein-enriched composition that can be further used for preparing foods, especially meat substitute products. The protein concentrate of the present invention is non-GMO, gluten-free, suitable for vegetarian and vegan diets, and can thereby meet the requirements of a wide range of consumers.

Summary of the Invention

[0005] In one aspect, the present invention is based on the insight that the hardness of a gel produced by heating a yeast protein concentrate can be adjusted by adjusting the conditions applied during the production of the protein concentrate. In particular, it has been found that the selection of an appropriate pH during the production of the concentrate strongly affects the gelling behavior of the final protein concentrate.

[0006] The protein concentrate produced by the method of the present invention can form a stable protein gel having a preferred consistency similar to that of firm egg white. The present invention also provides a novel protein concentrate that can be used for preparing a stable protein gel. The protein concentrate contains a large amount of protein that essentially maintains its structural integrity. This is achieved by preparing the concentrate under conditions that avoid protein unfolding. For example, the method is preferably carried out at a temperature low enough to avoid protein unfolding and at the same time reduce the protease activity in the lysed yeast cell suspension.

[0007] The concentrate of the present invention can be a liquid or a dry powder. When the concentrate is provided in the form of a dry powder, it must be reconstituted with water to prepare the gel. When water is added to the powder, the protein is solubilized and heating to 50 °C or higher forms a gel-like protein matrix. Since the taste of the yeast protein concentrate is mild, it can be mixed with different aromas to produce foods with different tastes.

[0008] Thus, in a first aspect, the present invention is a method for preparing a yeast protein concentrate comprising folded yeast protein molecules, comprising: (a) preparing a suspension comprising yeast cells; (b) adjusting the pH of the suspension to a value between 6.5 and 8.5; (c) lysing the yeast cells by mechanical means, preferably at a temperature below 40 °C; (d) subjecting the soluble fraction of the lysate to filtration to reduce the content of molecules less than 30 kDa; (e) optionally, drying the solution obtained by filtration in step (d) to obtain a protein concentrate powder and relates to a method comprising.

[0009] In the first step of the above method, a suspension comprising yeast cells is prepared. In a simple embodiment, the suspension of yeast cells can be a fraction of the cell-containing culture medium used for culturing the yeast cells. This cell-containing culture medium can be used directly as the suspension in the sense of step (a) of the above method. Culture media and methods for growing yeast are well known in the art. Suitable media include, for example, YPD medium from Sigma Aldrich (Taufkirchen, Germany).

[0010] In another embodiment, the suspension used in step (a) is an aqueous suspension of yeast cells. For this purpose, the yeast cells are recovered by growing the yeast cells to a certain density and then separating the cells from the culture medium. For example, the cell-containing medium is subjected to centrifugation or separation to sediment the cells. Optionally, the cells can be washed with water or a suitable buffer to remove the medium components. Thereafter, the cell pellet is resuspended in water or a suitable buffer. In that case, the yeast cells resuspended in water or buffer become the yeast cell-containing suspension in step (a) of the above method.

[0011] Preferably, the cell suspension prepared in step (a) of the above method has a volume of at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 5000, or at least 10000 liters. Preferably, the suspension is an aqueous suspension.

[0012] It is preferable that the yeast cell suspension prepared in step (a) is adjusted to have a dry matter content of about 4 to 20%, preferably about 5 to 16%, even more preferably about 6 to 14%, for example 12%. The dry matter percentage cited herein refers to the weight % based on the total weight of the suspension. The dry matter content of the suspension can be determined according to standard procedures using a commercially available device, for example, a Moisture Analyzer (Mettler-Toledo GmbH, Giessen, Germany). Once the dry matter content of the starting suspension is determined, this suspension can be adjusted to a predetermined value by diluting or concentrating the suspension.

[0013] The type of yeast used for preparing the protein concentrate of the present invention is not particularly limited. Yeast cells that can be used in the method of the present invention include, for example, yeasts belonging to the genus Saccharomyces, such as S. cerevisiae, S. chevalieri, S. boulardii, S. bayanus, S. italicus, S. delbrueckii, S. rosei, S. microellipsodes, S. carlsbergensis, S. bisporus, S. fermentati, S. rouxii, or S. uvarum; yeasts belonging to the genus Schizosaccharomyces, such as S. japonicus, S. kambucha, S. octosporus, or S. pombe; yeasts belonging to the genus Hansenula, such as H. wingei, H. arni, H. henricii, H. americana, H. canadensis, H. capsulata, or H. polymorpha; yeasts belonging to the genus Candida, such as C. albicans, C. utilis, C. boidinii, C. stellatoidea, C. famata, C. tropicalis, C. glabrata, or C. parapsilosis; yeasts belonging to the genus Pichia, such as P. pastoris, P. kluyveri, P. polymorpha, P. barkeri, P. cactophila, P.cactophila), P. rhodanensis, P. cecembensis, P. cephalocereana, P. eremophilia, P. fermentans, or P. kudriavzevii; yeasts belonging to the genus Kluyveromyces, such as K. marxianus; and yeasts belonging to the genus Torulopsis, such as T. bovina or T. glabrata.

[0014] In a particularly preferred embodiment, the method of the present invention uses a suspension containing yeast cells derived from the genus Saccharomyces, more preferably from S. cerevisiae.

[0015] In another particularly preferred embodiment of the present invention, the cells are washed with an alkaline buffer such as sodium hydroxide buffer. Such a washing step is useful for reducing the strong taste of the yeast suspension and thus helps to achieve a protein concentrate with a mild taste. If the method of the present invention includes a washing step with an alkaline buffer, at least one subsequent washing step with water is included in the method to ensure removal of the residual alkaline buffer. For example, when washing a yeast suspension with a certain volume of alkaline buffer, it is then washed with twice the volume of water before lysis.

[0016] Before lysing the cells, in step (b) of the above method, adjust the pH of the suspension to a value between 6.5 and 8.5. Standard basic and acidic reagents such as HCl and NaOH can be used to adjust the pH. It has been found that a pH in the range of 6.5 to 8.5 is surprisingly useful for improving the gelling behavior of the final protein concentrate. In a preferred embodiment, the pH of the yeast cell suspension is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5. A pH of 7.0 to 8.0 or 7.2 to 7.8, for example 7.5, is particularly preferred.

[0017] In another preferred embodiment of the present invention, a ribonuclease (RNase) enzyme is added to the suspension immediately before cell lysis. It has been found that the addition of the RNase enzyme significantly improves the separation of soluble substances from insoluble substances after lysis. RNase enzymes can be obtained from various manufacturers. Suitable enzymes may include PureLink RNase A from Thermo Fisher Scientific (Bremen, Germany) or RNase A from Sigma Aldrich (Taufkirchen, Germany). Those skilled in the art will have no problem determining the optimal amount of RNase enzyme to add to the yeast cell suspension. The appropriate amount of RNase enzyme is usually in the range of about 0.1% to 1% per dry weight.

[0018] After adjusting the pH of the cell suspension, lyse the yeast cells in step (c) of the above method to release the yeast proteins. Yeast species such as Saccharomyces cerevisiae have a thick cell wall that determines their shape and protects their interior. The yeast cell wall consists mainly of β-glucan, mannoprotein and chitin in a covalently bonded matrix and constitutes about 1 / 3 of the total yeast dry weight. Yeast cells have a lipid bilayer under the cell wall. To release proteins from inside the cell, it is necessary to break both of these protective barriers.

[0019] According to the method of the present invention, since the use of other enzymes such as proteases or glucanases commonly used for yeast cell lysis requires high temperatures, cell lysis is carried out mechanically. As a result, rapid degradation of proteins is brought about. In this step, it is particularly important to control the temperature. High temperatures induce protein denaturation, which means that the protein can no longer form a stable network even when heated, so it must be ensured that the temperature does not exceed 40°C. Furthermore, yeast cells contain a variety of proteases that can hydrolyze proteins into amino acids. When the yeast cells are disrupted, the proteases are released and can degrade other protein molecules released by the yeast cells. Since the protein concentrate prepared by the method of the present invention is intended for use in human food, the use of protease blockers such as EDTA or PMSF should be avoided because these chemicals are not safe for human consumption. Therefore, according to the present invention, it is preferable to carry out cell lysis at a low temperature in order to reduce the activity of proteases.

[0020] It is preferable to carry out steps (a) to (d) of the method of the present invention at a temperature of less than 40°C, more preferably less than 30°C, and even more preferably less than 20°C. In order to avoid unwanted degradation of proteins by proteases released from yeast cells, a temperature of less than 20°C, for example 15°C or 10°C, is particularly preferred.

[0021] According to a preferred embodiment, yeast cell lysis is achieved by the use of a bead mill. On the one hand, this ensures that the proteins remain intact during cell rupture. On the other hand, the use of a bead mill does not require additives that could impair the food-grade quality of the resulting protein concentrate. A bead mill typically comprises a chamber filled with beads that are moved by a set of impellers. When the cell-containing suspension passes through the chamber, the cells are disrupted upon collision with the beads. The efficiency of cell lysis can be adjusted by conventional means, for example, by changing the flow rate that determines how fast the yeast suspension passes through the chamber of the bead mill. Usually, a low flow rate results in a high lysis efficiency because the cells have a longer time to collide with the grinding beads. Depending on the volume of the chamber of the bead mill, a flow rate of at least 10 kg / hour, for example at least 20 kg / hour, at least 50 kg / hour, or at least 100 kg / hour can be selected. During operation, it is particularly preferred to cool the bead mill to a temperature below 20 °C, for example 18 °C, 16 °C, 14 °C, 12 °C or 10 °C. Bead mills are provided by various manufacturers, for example, the Dyno®-Mill Multi Lab Wab manufactured by Willy A. Bachofen AG (Muttenz, Switzerland).

[0022] Another factor that directly affects the efficiency of dissolution is the bead material and size. The beads can be made of various materials, such as glass, ceramic, or plastic. Particularly good results have been achieved with zirconium oxide beads, such as yttria-stabilized zirconium oxide beads. Furthermore, these beads are significantly more durable compared to glass beads. The beads used to disrupt yeast cells, such as zirconium oxide beads, can typically have various sizes in the range of 0.2 - 2.0 mm. It has been found that a bead size of 0.25 - 0.35 mm gives particularly good results for disrupting yeast cells. Therefore, a bead size of 0.25 - 0.35 mm is particularly preferred. A bead filling amount of 30 - 80%, for example, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% can be used. According to the present invention, a bead filling amount of 50 - 60% is particularly preferred.

[0023] Another way to affect the efficiency of cell disruption is to adapt the impeller structure. The impeller usually has plastic fins for moving the beads. For improved cell disruption, for example, it is possible to provide an accelerator on the rotor. The accelerator is designed to make the beads collide more frequently. The impeller rotation speed is another parameter that can be adjusted to improve cell disruption. A rotor speed of 1 - 20 m / second, for example, about 1 m / second, about 2 m / second, about 3 m / second, about 4 m / second, about 5 m / second, about 6 m / second, about 7 m / second, about 8 m / second, about 9 m / second, about 10 m / second, about 11 m / second, about 12 m / second, about 13 m / second, about 14 m / second, or about 15 m / second can be used. Those skilled in the art will easily be able to find the optimal parameters for disrupting yeast suspensions in a bead mill by ordinary experiments.

[0024] Yet another method for mechanically lysing yeast cells uses high-pressure homogenization (HPH). High-pressure homogenization is a method commonly used in the pharmaceutical, chemical, and food industries to stabilize emulsions and the like. However, it can also be used to disrupt bacteria and yeast in order to extract intracellular products from the cells. During HPH, the cells to be disrupted pass through a narrow slit under high pressure. As the yeast cells pass through this narrow slit, the flow rate increases sharply and the pressure drops suddenly. The resulting shear force causes cell disruption.

[0025] HPH devices for use in the method of the present invention can be obtained from various manufacturers. For example, the EmulsiFlex-C3 from Avestin Europe GmbH (Mannheim, Germany) or the 1000 / 2000 homogenizer from SPX Flow Technology Germany GmbH (Maels, Germany) can be used. The yeast suspension can be passed through the HPH device one or several times. Passing the suspension through the device several times increases the efficiency of cell rupture. The pressure of the device can be set to a pressure of at least 1000 bar. Preferably, the pressure is at least 1100 bar, at least 1200 bar, at least 1300 bar, at least 1400 bar, at least 1500 bar, at least 1600 bar, at least 1700 bar, at least 1800 bar, at least 1900 bar, or at least 2000 bar or more.

[0026] After disrupting the cells as described above, the pH of the lysate can approach a value of 6.0 or less. It is preferable to readjust the pH of the lysate to a value of 6.5 - 8.5. For example, the pH of the lysate is readjusted to about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5. A pH of 7.0 - 8.0 or 7.2 - 7.8, for example 7.5, is particularly preferred.

[0027] After disrupting the yeast cells, among the numerous other components, the soluble fraction containing the folded yeast proteins is preferably separated from the insoluble fraction. The insoluble fraction mainly contains cell wall components, organelles, and undissolved cells. The separation of the soluble fraction from the insoluble fraction can be carried out by various methods including centrifugation, filtration, and other methods. In a simple embodiment, the lysate containing the disrupted cells, diluted or undiluted, is subjected to centrifugation at, for example, 2000 - 25000 g to sediment the insoluble substances. The supernatant containing the soluble cell components released from the interior of the cell and containing the folded proteins is obtained and preferably cooled to below 20 °C until further use.

[0028] Alternatively, the separation of the soluble fraction and the insoluble fraction is carried out by standard filtration techniques using a filtration material that retains the insoluble substances. For example, dead-end filtration using a filter with a pore size of 0.2 - 15 μm can be performed. Cross-flow filtration or tangential flow filtration is also possible to avoid early clogging of the filter. The molecular weight cut-off (MWCO) should be greater than 300 kDa to allow all proteins to pass through.

[0029] In step (d) of the above method, the soluble fraction of the lysate is subjected to a filtration step that selectively reduces the content of molecules less than 30 kDa. Preferably, the content of molecules less than 30 kDa is reduced by at least 25%, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the content of these molecules in the soluble fraction of the lysate obtained in step (c) of the above method.

[0030] The molecules removed by the filtration step are preferably less than 20 kDa, more preferably less than 10 kDa, and even more preferably less than 5 kDa.

[0031] Filtration methods suitable for removing molecules less than 30 kDa are known and include, for example, activated carbon filtration, ultrafiltration, or nanofiltration. Activated carbon filtration is a separation method based on the adsorption of molecules onto the surface of carbon particles and the capture of small molecules in the depressions and pores of the activated carbon. For example, a fluid can be passed through a filtration bed of activated carbon molecules, and the organic small molecules are bound by the carbon particles in the filtration bed. Alternatively, activated carbon can be directly added to the fluid and incubated for a certain period of time with or without mixing, and then removed from the carbon.

[0032] Alternatively or additionally, ultrafiltration and / or nanofiltration can be used to remove molecules less than 30 kDa, preferably less than 20 kDa, more preferably less than 10 kDa, and even more preferably less than 5 kDa. These filtration techniques use membranes that act as physical barriers with pores of a specific size. Both filtration techniques can be applied as dead-end filtration or tangential flow filtration. The membrane is designed to allow only particles with a diameter smaller than the pore size to pass through. During filtration, the initial solution is separated into two different fractions: the fraction that passes through the membrane (permeate) and the fraction that is retained by the membrane (retentate). The filters used during ultrafiltration are typically defined by a molecular weight cut-off (MWCO) in the range of 0.1 to 1000 kDa. In one embodiment, ultrafiltration is performed in step (d) of the above method using a membrane having a molecular weight cut-off (MWCO) of 30 kDa or less, preferably 20 kDa or less, 10 kDa or less, and most preferably 5 kDa or less.

[0033] Optionally, the protein solution obtained after filtration is sterilized or pasteurized. It is preferred to sterilize or pasteurize the protein solution without heating the product, for example, by UV sterilization or the like. Alternatively, sterilization or pasteurization can be performed by subjecting the protein solution to a very short ultra-high temperature treatment. For example, the protein solution can be heated at a temperature of 120 to 150 °C for 3 to 5 seconds.

[0034] The solution obtained after the filtration step (d) is a liquid protein concentrate in the meaning of the present invention. The liquid concentrate can be used to prepare a gel. For this purpose, an aliquot of the concentrate is heated to a temperature of 50 °C or higher. The high temperature unfolds the proteins in the concentrate and causes them to associate with each other, thereby forming a gel.

[0035] The solution obtained after the filtration step (d) can also be dried, thereby obtaining a protein concentrate powder. Since the proteins in the powder are no longer susceptible to degradation by endogenous or exogenous proteases, the powder has a significantly improved shelf life. The degradation of yeast proteins would result in a decrease in the gelling properties of the protein concentrate. A further advantage is that the powder is protected from the growth of microbial contaminants that can affect food safety. According to the present invention, it is preferred to carry out the drying of the solution obtained from filtration by freeze-drying or spray-drying.

[0036] The principle of spray-drying is based on the dispersion of the solution into fine droplets that are introduced into a stream of hot air. The solvent evaporates from the substrate droplets, resulting in the remaining dry product clusters. In this case, it is important to select temperature conditions that do not cause protein denaturation. Spray-drying is usually carried out at a temperature higher than the melting temperature of the protein, but this does not mean that the protein unfolds completely during spray-drying. Protein denaturation is not an instantaneous event but a process with many transition steps. Therefore, by limiting the period of high temperature during spray-drying, the denaturation of yeast proteins can be avoided. Standard spray-drying devices such as the Mini Spray Dryer B-290 manufactured by Buchi Labortechnik GmbH (Essen, Germany) or the Mobile Minor (trademark) Spray Dryer manufactured by GEA (Berlin, Germany) can be used.

[0037] Freeze-drying or lyophilization is a method of removing water from a product to extend its shelf life. Freeze-drying involves freezing the product, reducing the pressure, and applying heat to sublimate the frozen water in the material. Various methods can be applied to freeze the product. For example, freezing can be achieved by using a standard freezer or a cooling bath. By cooling the product below its triple point, sublimation is guaranteed to occur upon heating. Freezing is carried out rapidly to prevent the formation of large crystals that can damage the structure of the product being dried. When the frozen water sublimates, approximately 95% of the water in the product is removed. Most materials can be dried to a residual moisture content of 1 - 5%. Standard freeze-drying equipment such as the Lyovac (trademark) device manufactured by GEA (Berlin, Germany), the Gamma 2-20 Freeze Dryer LCM-1 manufactured by Christ (Osterode am Harz, Germany), or the Christ Martin (trademark) Alpha 1-2 Lyophilisator manufactured by Fisher Scientific GmbH (Schwerte, Germany) can be used.

[0038] In another aspect, the present invention provides a yeast protein concentrate obtainable by the above method.

[0039] In yet another aspect, the present invention provides a yeast protein concentrate in liquid or powder form, comprising a mixture of non-folded proteins and folded proteins, wherein the folded proteins contained in the protein concentrate unfold in the temperature range of 45°C to 83°C. The presence of the folded proteins and their respective unfolding temperatures can be analyzed by standard equipment for measuring protein stability and functionality, such as Tycho NT.6 (NanoTemper Technologies GmbH, Munich). Tycho NT.6 quantifies protein unfolding at increasing temperatures by spectrophotometric measurement of absorbance at 330 nm and 350 nm. The change in this ratio corresponds to protein unfolding. When the sample contains a complex mixture of various proteins, there is no distinct melting temperature, but there is a melting range.

[0040] According to a preferred embodiment of the present invention, at least 40% of the proteins contained in the concentrate have an apparent size of more than 5 kDa. Preferably, at least 50%, at least 60% or at least 70% of the proteins contained in the protein concentrate have an apparent size of more than 5 kDa.

[0041] According to another preferred embodiment of the present invention, at least 40% of the proteins contained in the yeast protein concentrate have an apparent size of more than 10 kDa. Preferably, at least 50%, at least 60% or at least 70% of the proteins contained in the protein concentrate have an apparent size of more than 10 kDa.

[0042] According to another preferred embodiment of the present invention, at least 40% of the proteins contained in the yeast protein concentrate have an apparent size of more than 20 kDa. Preferably, at least 50% or at least 60% of the proteins contained in the protein concentrate have an apparent size of more than 20 kDa.

[0043] According to another preferred embodiment of the present invention, at least 40% of the proteins contained in the yeast protein concentrate have an apparent size greater than 30 kDa. Preferably, at least 50% or at least 60% of the proteins contained in the protein concentrate have an apparent size greater than 30 kDa.

[0044] According to another preferred embodiment of the present invention, at least 30% of the proteins contained in the yeast protein concentrate have an apparent size greater than 60 kDa. Preferably, at least 40% of the proteins contained in the protein concentrate have an apparent size greater than 60 kDa.

[0045] According to another preferred embodiment of the present invention, at least 20% of the proteins contained in the yeast protein concentrate have an apparent size greater than 150 kDa. Preferably, at least 25% of the proteins contained in the protein concentrate have an apparent size greater than 150 kDa.

[0046] According to the present invention, it is more preferable that the yeast protein concentrate of the present invention contains a compound that is not a protein of less than 45%. More preferably, the content of non-protein compounds in the protein concentrate is less than 40%, less than 35%, or less than 30%.

[0047] Furthermore, the protein concentrate of the present invention has particularly small amounts of free amino acids and dipeptides or tripeptides. Preferably, the total amount of free amino acids, dipeptides and tripeptides is less than 20%, more preferably less than 18%.

[0048] The protein concentrate of the present invention preferably has an RNA content of less than 15% (w / w), more preferably less than 14%, less than 13%, less than 12%, or less than 11%. The amount of free nucleotides is preferably less than 5% (w / w), more preferably less than 4%, less than 3%, or less than 2%.

[0049] In yet another aspect, the present invention provides a yeast concentrate in the form of a liquid or a powder, which contains at least 55% (w / w), preferably at least 60% (w / w), at least 65% (w / w), or at least 70% (w / w) of total crude protein per dry matter, and at least 40% of the protein in the concentrate has a size greater than 60 kDa.

[0050] Here, the total crude protein content is preferably determined by measuring the nitrogen content of the sample by the generally used Kjeldahl method and multiplying the result by a conversion factor of 6.25. In other words, the total crude protein content is determined by the following formula: Protein = Kjeldahl nitrogen content × 6.25. In this context, the term "total crude protein" indicates that nitrogen-containing compounds other than proteins in the sample, such as urea or free amino acids, may contribute to this value to some extent. The Kjeldahl method is widely known in the prior art as the most common procedure for determining the total crude protein content in a sample. Fully automated devices for determining the crude protein content by the Kjeldahl method are sold by several manufacturers, for example, the Kjeltec™ 8400 device (Foss GmbH, Hamburg, Germany).

[0051] Here, the concentrate of the present invention contains at least 55% (w / w) of total crude protein per dry matter, which means that 1.0 g of the concentrate contains at least 0.55 g of total crude protein on a dry matter basis. Dry matter refers to the weight of a completely dried compound. Preferably, the total crude protein is measured using the Kjeltec™ 8400 device.

[0052] In a concentrate containing at least 55% (w / w) of total crude protein per dry matter, at least 40% of the protein has an apparent size greater than 60 kDa. In other words, the concentrate of the present invention contains a large amount of larger proteins. The apparent size of the protein in the concentrate can be measured by conventional methods such as size exclusion chromatography (SEC).

[0053] The yeast protein concentrate according to the present invention preferably contains β-glucan of 30% (w / w) or less, preferably 25% (w / w) or less, more preferably 20% (w / w) or less on a dry matter basis. In other words, 1.0 g of the concentrate contains at most 0.3 g of β-glucan on a dry matter basis, preferably less than that.

[0054] In the protein concentrate containing at least 55% (w / w) of total crude protein on a dry matter basis, preferably at least 40% (w / w), more preferably at least 45% (w / w) or at least 50% (w / w) of the total dry matter is soluble crude protein. The amount of soluble crude protein in a given sample can be determined by centrifuging the sample to remove the insoluble protein component and then measuring the crude protein content of the supernatant. The amount of soluble crude protein is preferably by the following procedure.

[0055] A 1% (wt / wt) protein concentrate solution in deionized water is prepared at 20 °C and gently stirred for 30 minutes. Foam formation must be strictly avoided. Then, 1.5 ml of the said solution is transferred to a 2 ml reaction tube, and the solution is centrifuged at 25000×g at 4 °C for 20 minutes. 1 ml of the supernatant is taken out, and the crude protein content on a dry matter basis is measured in the said supernatant by the Kjeldahl method. The measurement is carried out 3 times, and the average result obtained from this measurement reflects the soluble crude protein content of the sample on a dry matter basis.

[0056] The protein concentrate of the present invention may have a soluble crude protein content of at least 40% (w / w) on a dry matter basis as measured by the Kjeldahl method.

[0057] The yeast protein concentrate according to the invention, containing at least 40% (w / w) soluble crude protein per dry matter as shown above, particularly preferably contains a high proportion of soluble crude protein that can be precipitated by heating the concentrate. Preferably, at least 20% (w / w), more preferably at least 25% (w / w), at least 30% (w / w), at least 35% (w / w), at least 40% (w / w), at least 45% (w / w) or at least 50% (w / w) of the soluble crude protein in the concentrate can be precipitated by heating the concentrate at 90°C for 10 minutes. Preferably, the precipitation method is carried out as follows.

[0058] Prepare a 1% (wt / wt) protein concentrate solution in deionized water at 20°C and gently stir for 30 minutes. Formation of foam must be strictly avoided. Then, transfer 1.5 ml of the said solution to a 2 ml reaction tube. Incubate the tube in a water bath at 90°C for 10 minutes and then place it on ice for 10 minutes. Then, centrifuge the tube at 25000×g at 4°C for 20 minutes. Take out 1 ml of the supernatant and measure the crude protein content per dry matter in the said supernatant by the Kjeldahl method.

[0059] Perform the measurement three times, and the average result obtained from this measurement reflects the soluble crude protein content of the sample after heat treatment. Based on this value and the total soluble protein content of the sample, the proportion of soluble crude protein that can be precipitated by heat treatment at 90°C can be determined by the following formula:

Number

[0060] Those skilled in the art will have no problem in determining the proportion of crude soluble protein that can be precipitated by the above heat treatment.

[0061] In a protein concentrate containing at least 55% (w / w) total crude protein, preferably at least 40% (w / w) soluble crude protein, per dry matter, the folded proteins contained therein preferably unfold in a temperature range between 45°C and 83°C. Further, in a protein concentrate containing at least 55% (w / w) total crude protein, preferably at least 40% (w / w) soluble crude protein, per dry matter, the total amount of free amino acids, dipeptides and tripeptides contained therein is preferably less than 20%, more preferably less than 18%. Further, in a protein concentrate containing at least 55% (w / w) total crude protein, preferably at least 40% (w / w) soluble crude protein, per dry matter, preferably at least 40% of the protein has an apparent size of more than 5 kDa.

[0062] In a preferred embodiment, the yeast protein concentrate described herein is a liquid having a dry matter content of at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, or at least 5%. In another preferred embodiment, the yeast protein concentrate described herein is a powder having a dry matter content of at least 90%, at least 92%, at least 94%, or at least 96%.

[0063] In another aspect, the present invention is a method for preparing a protein gel, comprising: (a) preparing the liquid yeast protein concentrate as described above; and (b) heating the concentrate to a temperature of at least 55°C to obtain a protein gel. A method comprising the above steps is provided.

[0064] This method includes the preparation of a liquid yeast protein concentrate. To prepare the gel, the liquid concentrate or an aliquot thereof is heated to a temperature of at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, or at least 80°C. At this temperature, the folded proteins in the liquid denature and entangle to form a gel.

[0065] In yet another aspect, the present invention is a method of preparing a protein gel, comprising: (a) providing the yeast protein concentrate powder as described above; and (b) mixing the yeast protein concentrate powder with an aqueous carrier fluid; and (c) heating the mixture to a temperature of at least 55° C. to obtain a protein gel. A method starting from a dry concentrate includes mixing the concentrate powder with an aqueous carrier fluid. The aqueous carrier fluid can be water such as tap water or another suitable aqueous carrier such as a buffer solution. The reconstitution of the powder is preferably done such that a solution of at least 2% (w / w), at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% is obtained. The mixing of the yeast protein concentrate powder and the aqueous carrier fluid can be carried out with stirring, for example, by agitating.

[0066] The obtained mixture is then heated to a temperature of at least 55° C., preferably at least 60° C., at least 65° C., at least 70° C., at least 75° C., or at least 80° C. At this temperature, the folded proteins in the mixture denature and entangle to form a gel.

[0067]

[0068] In a final aspect, the present invention relates to the use of the yeast protein concentrate described herein for preparing a protein-rich food or feed product. The protein-rich food can be a dessert or a pudding. Additionally, the protein-rich food can be a meat alternative product.

[0069] The meat alternative product can include additional compounds commonly used in the manufacture of such products, such as starches derived from rice, wheat, corn, potato, sweet potato, barley or sorghum, and vegetable oils such as soybean, olive, rapeseed, palm, peanut, corn, flax, sunflower, safflower or cottonseed oil.​

Brief Description of Drawings

[0070]

Figure 1

Figure 2

Figure 3

Figure 4

Examples

[0071] The following examples are provided to illustrate the present invention. However, it should be understood that the scope of the present invention is not limited by the examples. Those skilled in the art will understand that some modifications can be made without departing from the scope of the present invention.

[0072] Example 1: Preparation method using carbon filtration Saccharomyces cerevisiae A2W5 yeast cells were recovered from a yeast cell culture and suspended in water. The dry matter content was adjusted to 14%. The suspension had a volume of 1 L. NaOH was used to adjust the pH of the suspension to pH 7.5.

[0073] Cell disruption was carried out using yttria-stabilized zirconium oxide beads with a size of 0.25 - 0.35 mm in a Dyno®-Mill Multi Lab Wab from Willy A. Bachofen AG (Muttenz, Switzerland). The flow rate was set at 7 kg / hour and the rotor speed was set at 8 m / second. The bead filling was set at 65%.

[0074] The efficiency of cell disruption was confirmed by measuring the protein extraction yield using the method of Kjeldahl, J. Fresenius, Zeitschrift f. anal. Chemie (1883) 22:366.

[0075] The pH of the lysate obtained from the bead mill was adjusted to 7.6 using NaOH. Subsequently, the lysate was subjected to centrifugation at 25,000 × g for 120 minutes in a Heraeus Multifuge X3R. The supernatant was separated from the precipitate and subjected to activated carbon filtration using an activated carbon filter cartridge (height: 5 cm, diameter: 6 cm) filled with steam-activated Norit® SX Plus.

[0076] The solution obtained by activated carbon filtration was sterilized at a temperature of 130 °C for 3 seconds and then spray-dried using a Mini Spray Dryer B-290 from Buchi Labortechnik GmbH (Esslingen, Germany) at a constant inlet temperature of 133 - 136 °C and an outlet temperature of 93 ± 2 °C.

[0077] The powder thus obtained was chemically analyzed for its free amino acid content:

Table 1

Table 2

[0078] Example 2: Preparation method using ultrafiltration The experiment described in Example 1 was repeated, except that ultrafiltration was used instead of activated carbon filtration. Ultrafiltration was performed using a polyethersulfone (PES) UF GR 81PP membrane with a MWCO of 10 kDa. Seven support plates were equipped with stop disks so that a total of 14 filters were used. The filter device was cleaned according to the manufacturer's instructions. Initially, the retentate flow rate was set at 2.3 L / min and the permeate flow rate was set at 0.064 L / min. After 20 minutes, the retentate was flowed at 2.2 L / min and the permeate was allowed to flow out at 0.054 L / min. The permeate was analyzed using size exclusion chromatography (SEC). The results are shown in Figure 3. It can be seen that only particles smaller than the membrane cut-off were able to pass through. This indicates that the yeast protein remained in the retentate and there was no significant protein loss. The solution obtained from ultrafiltration was sterilized and spray-dried as described in Example 1 above.

[0079] Example 3: Preparation of protein gel The powders obtained in Examples 1 and 2 were tested for their gelling properties when reconstituted in water. For this purpose, the protein powder was dissolved in tap water to obtain a 20% (w / w) solution.

[0080] Next, using a Tycho NT.6 (NanoTemper Technologies GmbH, Munich), the melting temperature range of the proteins in the protein concentrate was measured between room temperature and 95°C. Figure 4 shows that the proteins inside the product of the present invention mainly melt between 45°C and 83°C. Protein gels were evaluated by at least seven sensory panels for gel hardness and flavor profile. There was general agreement within the panel regarding the main perceptions of the samples. The taste was described as salty, sweet, and roasted. The panel did not point out a strong yeast flavor. At the same time, it was found that the gel was hard and resembled cooked egg white in terms of consistency.

[0081] Example 4: Determination of total crude protein content In Example 1, a sample of the liquid yeast protein concentrate obtained after activated carbon filtration was analyzed for the content of total crude protein per dry matter. The liquid concentrate had a dry matter concentration of 5.3% (w / w). For the digestion step, 500 mg aliquots of the sample were analyzed by the Kjeldahl method using a FOSS Kjeltec-Analyser 8400 equipped with a Kjeltec-Sampler 8420 and a Tecator-Digestor Auto equipped with a Tecator-Scrubber. A conversion factor of 6.25 was used to calculate the total crude protein content. As a result, it was measured that the total crude protein content per dry matter of the liquid yeast protein concentrate was 70.5% (w / w).

[0082] Example 5: Determination of soluble crude protein content In Example 1, a sample of the liquid yeast protein concentrate obtained after activated carbon filtration was diluted to a dry matter concentration of 1% (w / w) using deionized water. This solution was stirred at 20 °C for 30 minutes. 1.5 ml of the diluted protein solution was filled into 2 ml-Eppendorf tubes. The tubes were centrifuged at 25000×g for 20 minutes at 4 °C. After centrifugation, 1 ml of the supernatant was taken out of the tubes and subjected to the Kjeldahl protein measurement described in Example 4. As a result, it was measured that the soluble crude protein content per dry matter was 61.3% (w / w).

[0083] Example 6: Determination of proportion of heat-reactive proteins Another aliquot of the sample used in Example 5 having a dry matter concentration of 1% (w / w) was used by stirring at 20 °C for 30 minutes as described above. A 2 ml-Eppendorf tube was filled with 1.5 ml of the diluted protein solution. The tube was then heat-treated by placing it in a water bath at 90 °C for 10 minutes. Thereafter, the tube was placed on ice for 10 minutes. Thereafter, the tube was centrifuged at 25,000×g at 4 °C for 20 minutes. After centrifugation, 1 ml of the supernatant was removed from the tube and subjected to the Kjeldahl protein measurement described in Example 4. As a result, it was determined that the soluble crude protein content after heat treatment per dry matter was 32.8% (w / w). The proportion of soluble crude protein that can be precipitated by heat is determined by the following formula.

Number

[0084] When the soluble crude protein content measured in Example 5 and the soluble crude protein after heat incubation measured in Example 6 are used in the above formula, the following is obtained:

Number

[0085] Therefore, the above calculation shows that 46.5% of the soluble crude protein can be precipitated by the above heating.

Claims

1. A method for preparing a yeast protein concentrate containing a non-denatured yeast protein molecule, comprising: (a) preparing a suspension containing yeast cells; (b) adjusting the pH of the suspension to a value between 6.5 and 8.5; (c) lysing the yeast cells by mechanical means; (d) subjecting the soluble fraction of the lysate to filtration to reduce the content of molecules less than 30 kDa; (e) optionally, drying the solution obtained by filtration in step (d) to obtain a protein concentrate powder ; steps (a) to (d) are carried out at a temperature below 40°C, the protein concentrate obtained in step (d) or (e) contains at least 55% (w / w) of total crude protein per dry weight, and at least 40% of the proteins in the concentrate have an apparent size greater than 60 kDa, a method.

2. The method according to claim 1, wherein steps (a) to (d) are carried out at a temperature below 20°C.

3. The method according to any one of claims 1 to 2, wherein the suspension in step (a) has a dry matter content of 5 to 20%.

4. The method according to any one of claims 1 to 3, wherein the suspension in step (a) is washed with a basic washing buffer before cell lysis.

5. The method according to any one of claims 1 to 4, wherein the content of molecules less than 10 kDa is reduced in step (d).

6. The method according to any one of claims 1 to 5, wherein the lysis of the yeast cells in step (c) is carried out using a bead mill.

7. The method according to any one of claims 1 to 6, wherein the pH of the lysate is readjusted to 6.5 to 8.5 after step (c).

8. The method according to any one of claims 1 to 7, wherein the solution obtained after the filtration is sterilized.

9. The method according to any one of claims 1 to 8, wherein the solution obtained after the filtration is freeze-dried or spray-dried to obtain a powder.

10. A yeast protein concentrate obtainable by the method according to any one of claims 1 to 9, containing at least 55% (w / w) of total crude protein per dry weight, and at least 40% of the proteins in the concentrate having an apparent size greater than 60 kDa.

11. The yeast protein concentrate according to claim 10, containing at least 60% (w / w) of total crude protein per dry weight.

12. The yeast protein concentrate according to claim 10 or 11, comprising β-glucan of 30% (w / w) or less per dry matter.

13. The yeast protein concentrate according to any one of claims 10 to 12, comprising at least 40% (w / w) of soluble crude protein per dry matter.

14. The yeast protein concentrate according to any one of claims 10 to 13, wherein at least 20% (w / w) of the soluble crude protein in the concentrate can be precipitated by heating the concentrate at 90 °C for 10 minutes.

15. The yeast protein concentrate according to any one of claims 10 to 14, which is a liquid having a dry matter content of at least 1%.

16. The yeast protein concentrate according to any one of claims 10 to 14, which is a powder having a dry matter content of at least 90%.

17. A method for preparing a protein gel, comprising: (a) preparing a yeast protein concentrate powder according to any one of claims 10 to 16; (b) mixing the yeast protein concentrate powder with an aqueous carrier fluid; (c) heating the mixture to a temperature of at least 55 °C to obtain the protein gel. The method includes the above steps.

18. Use of the yeast protein concentrate according to any one of claims 10 to 16 in the preparation of food.

19. Use of the yeast protein concentrate according to claim 18, wherein the food is a meat substitute product or a protein-rich food / feed product.

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