yeast proteins

Abstract

The present invention relates to a process for obtaining a yeast protein comprising the following steps: a) providing a yeast cream; b) exposing the yeast cream to heat plasmolysis at a temperature of 70 to 95°C for 30 seconds to 4 hours, preferably 1 minute to 3 hours, more preferably 40 minutes to 2 hours; b') separating the insoluble fraction and the soluble fraction; c) subjecting the insoluble fraction to the activity of at least one ribonuclease and one glucanase, sequentially or simultaneously, at a temperature of 40 to 65°C, preferably 60°C, for 8 to 24 hours, preferably 18 hours; d) separating the insoluble fraction from the soluble fraction; wherein the insoluble fraction collected in step d) has no taste, a nucleotide content of less than 3% and a true protein content of at least 72%. Step b') is optional. In this case, the whole composition obtained after heat plasmolysis of the yeast cream is subjected to the enzymatic activity.

Description

[0001] This application is a divisional application of patent application No. 201980028345.9 (International Application No.: PCT / EP2019 / 060750), filed on April 26, 2019, entitled "Yeast Protein". Technical Field

[0002] This invention relates to the field of proteins derived from microorganisms, more specifically from yeast, which can be widely used for the nutrition, health and well-being of humans and animals. Background Technology

[0003] Proteins represent the main components of human and animal tissues. From a nutritional point of view, proteins are hydrolyzed into peptides and amino acids by proteases and peptidases. Amino acids provide the body with essential elements such as nitrogen, carbohydrates, and sulfur. The body cannot fix inorganic nitrogen to incorporate it into organic molecules; therefore, nitrogen is provided by amino acids. Sulfur-containing amino acids provide important sulfur for metabolism. Amino acids are also necessary elements for the synthesis of proteins, peptides, and physiologically essential low-molecular-weight substances such as glutathione, creatine, dopamine, serotonin, etc. Therefore, the demand for protein in human and animal nutrition is expanding with the growth of the world's population. Consequently, there is a need to find alternative and / or alternative protein sources for animal protein. Proteins have been recovered from plants (grains, legumes) or insects. Obtaining microbial-derived proteins relies on fermentation mechanisms that have been around for centuries. Microbial proteins can be components of whole cells or cell walls, or isolated proteins. One drawback of microbial-derived proteins used in human food is the nucleic acid content of the isolates. This content must be low because one of the end products of nucleic acid metabolism is uric acid, which the human body lacks the enzyme uricase needed to metabolize, and therefore cannot degrade. Other drawbacks include the extraction yield or purity of proteins obtained by isolating from microorganisms.

[0004] Cells, such as yeast cells, after lysis, typically recover proteins in the "noble" stage, the soluble stage (which corresponds to yeast extract and contains compounds with a strong flavor). However, the taste may be undesirable depending on the intended application of the obtained protein extract.

[0005] Three patents from the same team relate to the acquisition of concentrated protein extracts.

[0006] US Patent 3867555 discloses a method for obtaining a protein extract with low nucleic acid content. Cells are mechanically ruptured (high pressure, sonication) or chemically ruptured (autolysis, exogenous enzymes), allowing proteins to remain in the soluble fraction. However, these proteins are mixed with hydrolysates, i.e., amino acids, peptides, and polysaccharides or monosaccharides. After centrifugation to remove the insoluble fraction, the protein is precipitated, treated with alkali to hydrolyze the nucleic acids, and then removed by filtration. However, the overall recovery rate of the "unhydrolyzed" protein is very low.

[0007] Patent US3887431 discloses the use of endogenous nucleases found in the soluble fraction after yeast lysis for the degradation of nucleic acids. A vacuum concentration step can yield a concentrated protein extract and a product with virtually no taste or odor.

[0008] US3991215 discloses a method of applying ultra-high temperature heating to a soluble cytoplasmic fraction after cell rupture, which allows for the production of a protein-rich extract containing very little nucleic acid.

[0009] Finally, several directions can be followed to improve methods for isolating proteins from microorganisms in order to obtain protein extracts with better purity, better yield, and / or that can be used directly for nutrition or as food supplements. Summary of the Invention

[0010] Contrary to conventional techniques, the inventors have developed a method for isolating microbial proteins from the insoluble fraction obtained after microbial lysis. This novel method enables the production of concentrated, non-hydrolyzed protein extracts with low nucleic acid content, mild odor and taste, and high yield.

[0011] The method of the present invention begins with cell lysis, preferably via thermoplasmolysis, which leads to the inactivation of microbial enzymes and their release into a matrix of free amino acids, including glutamate. These enzymes are then subjected to enzymes of the type of dextranase and ribonuclease, followed by separation, after which the insoluble fraction represents the target product having a true protein content of at least 72%. In another embodiment of the invention, a first separation is performed after the thermoplasmolysis stage, and only the insoluble fraction containing proteins, polysaccharides, and RNA is subjected to dextranase and ribonuclease, followed by a second separation to retain the protein-rich insoluble fraction.

[0012] The obtained protein extract was tasteless and odorless, with low nucleic acid content.

[0013] Protein extracts that still contain lipids derived from the membrane can be defatted using methods known to those skilled in the art, such as extraction with supercritical CO2 using solvents of the hexane or ethanol type, or by treatment with lipase or phospholipase followed by separation from the dissolved phase.

[0014] The obtained proteins can be used directly in nutrition, food supplements, and other applications.

[0015] definition

[0016] Microorganisms are living organisms invisible to the naked eye but visible under a microscope. In this invention, the microorganisms are preferably bacteria (prokaryotic microorganisms) or yeast (eukaryotic microorganisms). Yeast, expressed in singular or plural form, is a general term for eukaryotic microorganisms that can cause fermentation of organic matter. In the context of yeast, the genera *Saccharomyces*, *Candida*, *Pichia*, and *Kluyveromyces* are mentioned in a non-exhaustive manner.

[0017] Yeast extract refers to a yeast suspension obtained after propagation in a container and centrifugation (the centrifugation separates the suspension from the surrounding liquid (also known as the slurry (must))). Propagation can also be referred to as cultivation, or more broadly, fermentation.

[0018] Proteins are large molecules composed of sequences of amino acids linked together by peptide bonds. They are the main building blocks of all plant and animal cells. They account for up to 50% of the dry weight of organisms and 15% to 20% of our daily energy intake. Dietary and body proteins are the main sources of nitrogen and amino acids, which have several major metabolic functions: they are substrates for protein synthesis, precursors to important nitrogenous compounds in the body (nucleic acids, nitric oxide, glutathione, etc.), and substrates for energy metabolism.

[0019] Typically, in agri-food products, protein content is considered to be nitrogen level multiplied by 6.25 (the coefficient 6.25 originates from the average nitrogen content of the protein, i.e., 100 / 6.25, or 16%). True protein refers to a protein content closer to reality, corrected for biases caused by non-protein nitrogen (such as nitrogen in nucleic acids). Therefore, as a formula, true protein content can be total nitrogen minus nitrogen from nucleic acids and ammonia nitrogen multiplied by 6.25. Alternatively, true protein content can be evaluated by measuring total amino acids. Intact natural proteins refer to microbial proteins in the state found in living microorganisms. Denatured natural proteins, through expansion, folding, or coagulation, possess potentially modified spatial conformations. Proteins can also be partially or completely hydrolyzed.

[0020] Plasmolysis refers to the impermeability of microorganisms, particularly yeast, or the rupture of their compartments. The membrane loses water after permeation. Attached Figure Description

[0021] Figure 1 Two main embodiments of the method of the present invention are shown. Figure 1 A illustrates a method that eliminates the separation step between plasmolysis and enzymatic hydrolysis. Figure 1 B illustrates a method for separating the plasmolysis step and enzymatically hydrolyzing the insoluble fraction derived from that separation.

[0022] Figure 2 Protein profiles are provided for protein extracts derived from the methods of the present invention and for so-called conventional protein extracts obtained by known methods for extracting proteins from soluble fractions obtained through plasmolysis steps. Detailed Implementation

[0023] The method of this invention can be applied to any type of yeast, and more specifically, to any yeast used for human or animal nutrition. From an industrial perspective, the method of this invention is applied to yeast extract as defined above, i.e., a suspension of yeast. Preferably, the yeast is selected from yeasts of the genera *Saccharomyces*, *Pichia*, *Candida*, *Kluyveromyces*, *Yarrowia*, or *Wickerhamomyces*. More preferably, the yeast is selected from *Saccharomyces cerevisiae*, *Pichia jadinii* (also known as *Candida utilis*), *Pichia pastoris*, *Kluyveromyces lactis*, *Kluyveromyces marxianus*, *Yarrowia lipolytica*, *Wickerhamomyces anomalus*, etc. These yeasts contain 6% to 11% nitrogen. Nitrogen is measured using the Kjeldahl method, known to those skilled in the art. As described above, the true protein content is extrapolated from this nitrogen determination using a multiplier of 6.25.

[0024] Yeast is cultured according to methods known to the technician. A procedure is described in Gerald Reed and TilakW. Nagodawithana, “Yeast Technology” (ISBN 0-442-31892-8), pages 284-293. The culture (also called biomass) is collected by centrifugation or filtration and can be washed. A yeast extract is obtained.

[0025] The cells are then mechanically or chemically ruptured using known methods, such as high-pressure homogenization, mechanical grinding, ultrasonic disintegration, repeated freeze-thaw cycles, osmotic shock, or enzymatic lysis. In a preferred embodiment of the invention, the yeast is subjected to thermoplasmolysis at a temperature of 70 to 95°C, depending on the yeast type. The temperature is preferably between 80 and 90°C. The temperature and / or lysis time are adjusted within ranges conceivable to those skilled in the art, for example, to inactivate the cells and / or enzymes in this step due to their resistance. The time ranges from 30 seconds to 3 hours, up to 4 hours, more preferably 1 minute, 5 minutes, 10 minutes, 30 minutes, 45 minutes, 1 hour, 1 hour and 30 minutes, or 2 hours, up to 3 hours, or even 4 hours. This plasmolysis step denatures the yeast, inactivates endogenous enzymes, and permeates the membrane, thereby releasing the soluble fraction. The soluble fraction mainly comprises free amino acids, small peptides, and minerals, and among these amino acids, some have a flavor, such as glutamic acid.

[0026] In a first embodiment of the invention, the whole, derived from a plasmolysis step and comprising a soluble and an insoluble portion, is subjected to one or more enzymatic activities. The purpose of this step is to dissolve the maximum amount of non-protein components without damaging the protein. Ribonuclease activity (EC 3.1.4.1) and dextranase activity (EC 3.2.1) are preferably used. The enzyme activities can be performed sequentially or simultaneously. The ribonuclease activity converts RNA into 5' nucleotides and dissolves the 5' nucleotides, thereby allowing them to enter the soluble portion. Several ribonucleases can be used, such as a mixture of endonucleases and exonucleases. Optionally, deaminases can be used to convert AMP to IMP to restore the flavor-enhancing properties of the soluble portion. The action of one or more dextranases dissolves the cell wall polysaccharides into soluble oligosaccharides. The time and temperature are adjusted according to the function of the enzymes used according to their instructions. The temperature is between 40 and 65°C, preferably 60°C. The time is 8 to 24 hours, preferably 16 to 24 hours, more preferably 18 hours. This enzymatic step first yields a new soluble fraction containing nucleotides, polysaccharides (approximately 60% of the total carbohydrates), and a small proportion of amino acids, followed by an insoluble fraction that is primarily composed of proteins.

[0027] The final step is to separate the soluble and insoluble portions.

[0028] In a second embodiment of the invention, the soluble and insoluble portions derived from the plasmolysis step are separated. The soluble portion is set aside, removed, and recycled as yeast extract. The insoluble portion includes the shell (or wall), polymers, polysaccharides, RNA, and proteins due to heat coagulation. This insoluble portion is retained. It is subjected to enzymatic activity of ribonuclease and dextranase as described above according to a similar incubation protocol (temperature, time), and the soluble and insoluble portions are separated. The final soluble portion is set aside and used.

[0029] The final insoluble fraction from enzymatic digestion contains 70% or more "true" protein, preferably 72% or more, 80%, and up to 85%. Typically, yeast contains 50% to 55% "true" protein. Using the method of the present invention, many elements can be removed, resulting in a product with a high protein concentration. Optionally, the amount of lipids in the insoluble fraction can be reduced by the action of a solvent (ethanol, hexane), by supercritical CO2, or by the action of lipases or phospholipases. A true protein purity of over 80% can then be achieved.

[0030] In one embodiment of the invention, the respective actions of ribonuclease and dextranase are performed sequentially in any order. In a preferred embodiment of the invention, ribonuclease and dextranase activities are applied simultaneously.

[0031] The obtained product can be freeze-dried, dried using methods known to those skilled in the art (e.g., by spray drying or vacuum drying), and stored while preserving its quality and properties.

[0032] Therefore, in one embodiment, the present invention relates to a method for obtaining yeast protein, comprising the following steps:

[0033] a) Provide yeast paste;

[0034] b) Expose the yeast extract to thermal plasmolysis at a temperature of 70 to 95°C for 1 minute to 3 hours, preferably 40 minutes to 2 hours;

[0035] c) Subject the whole to the activity of at least one ribonuclease and one dextranase sequentially or simultaneously at a temperature of 40 to 65°C for 8 to 24 hours;

[0036] d) Separate the insoluble and soluble portions.

[0037] The insoluble fraction collected in step d) is tasteless, contains less than 3% nucleotides, and contains at least 72% true protein.

[0038] In another embodiment, the present invention relates to a method for obtaining yeast protein, comprising the following steps:

[0039] a) Provide yeast paste;

[0040] b) Expose the yeast extract to thermal plasmolysis at a temperature of 70 to 95°C for 1 minute to 3 hours, preferably 40 minutes to 2 hours;

[0041] b') Separate the insoluble and soluble portions;

[0042] c) Subject the insoluble portion to the activity of at least one ribonuclease and one dextranase sequentially or simultaneously at a temperature of 40 to 65°C for 8 to 24 hours;

[0043] d) Separate the insoluble and soluble portions.

[0044] The insoluble fraction collected in step d) is tasteless, contains less than 3% nucleotides, and contains at least 72% true protein.

[0045] In the remainder of this specification, the insoluble fraction collected in step d) may also be referred to as "the (yeast) protein extract of the present invention". Conversely, the yeast protein extract obtained from the soluble fraction obtained from plasmolysis according to known prior art methods may be referred to as "conventional protein extract".

[0046] In a preferred embodiment of the invention, the yeast extract in step a) is derived from yeast fermentation, wherein the yeast is selected from *Saccharomyces cerevisiae*, *Pichia* (preferably *Pichia jadinii*), *Kluyveromyces* (preferably *Kluyveromyces marxianus* or *Kluyveromyces lactis*), *Yarrowia* (preferably *Yarrowia lipolytica*), or *Wickerhamomyces* (preferably *Wickerhamomyces anomalus*). Advantageously, the yeast is selected from *Saccharomyces cerevisiae*, *Pichia jadinii*, or *Kluyveromyces marxianus*. The preferred yeast is *Saccharomyces cerevisiae*.

[0047] In a preferred embodiment of the present invention, the thermoplastic-mass separation step a) is carried out at a temperature between 80 and 90°C.

[0048] In a preferred embodiment of the invention, both dextranase and ribonuclease activities are applied simultaneously.

[0049] Alternatively, deaminase activity can also be applied.

[0050] Advantageously, the yeast extract used in step a) contains selenium-enriched yeast. The selenium-enriched yeast can be cultured according to methods known to those skilled in the art, such as those described in application EP 1478732. The selenium content in the yeast culture can then reach 3000 ppm or higher.

[0051] The product obtained in the insoluble fraction has a specific protein nitrogen content of 11.5% dry matter or higher, i.e., 72% true protein (using the conversion factor of 6.25 given above). Typically, the maximum nitrogen content of yeast is 11%, which yields 69% potential protein. Yeast also contains non-protein nitrogenous substances (nucleic acids, RNA, DNA). When nucleic acid nitrogen is subtracted from total nitrogen, yeast contains only 50% to 55% protein. Therefore, the minimum protein content of 72% obtained in the final insoluble fraction is characteristic of the method of the present invention. In this insoluble fraction, the total nucleotide content is 0.8% to 1.5%, and at most 3%, the total carbohydrate content is 1% to 8% (anthrone determination known to those skilled in the art), the dextran content is 0.2% to 4%, the mannan content is 0.2% to 4%, the lipid content is 7% to 15%, and the mineral content is 1% to 7%.

[0052] Interestingly, the yeast protein extract obtained by the method of the present invention differs from prior art extracts (i.e., protein extracts obtained from the soluble fraction after cell lysis and concentration steps). The yeast protein extract obtained by the method of the present invention has a protein content of at least 72% and a nucleotide content of less than 3%.

[0053] For this purpose, the total lipids in the protein extract derived from *Saccharomyces cerevisiae* were determined by gravimetric analysis after acid hydrolysis and extraction with hexane on a Soxhlet extractor. The lipid levels were systematically higher than 7%. Therefore, the lipid content of the insoluble fraction collected in step d) of the extraction method of the present invention was also higher than 7%. In contrast, the lipid content in "conventional" yeast protein extracts is less than 1%. Lipid content can vary depending on the yeast species or strain. Typically, for industrial products, the starting microorganism is indicated in the technical data sheet. Those skilled in the art will be able to extrapolate and infer the species / lipid content combination in the protein extract.

[0054] For the yeast protein extract of the present invention that has undergone a defatting process, the lipid content may be insufficient to distinguish between the two sources. Therefore, a comparative analysis was completed by evaluating the protein solubility profiles of the protein extracts. The solubility percentage was determined by the following formula:

[0055]

[0056] Wherein, N% represents the percentage of nitrogen determined by the Kjeldahl method.

[0057] The protein extract of this invention has a solubility of less than 3.5% in water.

[0058] In contrast, "conventional" yeast protein extracts are considered soluble, containing only 5% to 10% insoluble elements.

[0059] For the same purpose, molecular weight spectra of the yeast protein extract of the present invention and those of prior art protein extracts were compared. The yeast protein extract obtained by the present invention did not exhibit low molecular weight peaks. Most protein spectra were distributed around 40-45 kDa. The smallest compound found was approximately 500 Da, corresponding to small peptides. In contrast, the spectra of "conventional" yeast proteins showed multiple molecular weight peaks, as well as low molecular weights in non-negligible amounts. These spectra can be extrapolated and the ratios measured.

[0060]

[0061] (Use standard methods for the determination of amino acids in food, such as the official method of Commission Regulation (EC) No. 152 / 2009). For the yeast protein extract of the present invention, this ratio is close to 1 and is always higher than 0.9, while for “conventional” yeast protein extracts, this ratio can be between 0.30 and 0.85.

[0062] The product obtained by the method of this invention is distinguished from known protein extracts by its microbial origin (in other words, non-plant and non-animal), its high protein content, low nucleic acid content, and the presence of cell membrane lipids (if defatting treatment has not been applied). Furthermore, it is tasteless. Its microbial origin also has the advantage of being derived from raw materials known not to cause allergies.

[0063] The main advantage of yeast protein lies in its nutritional quality. Therefore, the product obtained using the method of this invention has a major advantage as a protein source: a quality expressed as 1 by a PDCAAS score (protein digestibility-corrected amino acid score), meaning its quality is similar to that of reference animal proteins (e.g., casein and ovalbumin). Assessment of protein quality can determine its ability to meet metabolic requirements. Consequently, the protein extract obtained by the method of this invention can be used in any application related to nutrition and food or sports supplements. The applications cited herein are not intended to be exhaustive.

[0064] Within the scope of human nutrition, health, and well-being, the protein extract can be used for weight management, as a supplement for athletes or the elderly, as a food supplement, or in the form of high-protein bars or beverages. For example, the protein extract can be used as a non-animal protein source in vegetarian diets, such as in shakes, hamburgers, chicken nuggets, plant-based deli meats, wonton fillings, meatballs, oatmeal, and pasta seasonings. Similarly, protein-rich breads or bread-making products can use the protein extract obtained by the methods of this invention. As previously mentioned, an advantage of these protein extracts is that they are tasteless, bitter, or odorless, which are characteristic of some contemporary plant-based or algal proteins. In human health, the protein extract can be used in infant nutrition or clinical nutrition, i.e., oral or enteral nutrition to correct nutritional imbalances. Advantageously, when protein extracts are obtained from selenium-enriched yeast extract, they can be used as a food supplement to stimulate immunity and / or enhance the quality of skin, hair, and / or nails.

[0065] Finally, the protein extract can be used as a protein supply in animal feed. Advantageously, when the protein extract is obtained from selenium-rich yeast extract, animals will benefit from a combined supply of protein and selenium, which is bioavailable because it is integrated into the protein in the form of selenomethionine.

[0066] Therefore, the present invention also relates to the use of a yeast protein extract having a true protein content of at least 72%, obtained according to any embodiment of the method of the present invention, as a food supplement for weight control in older adults or athletes; to the use of the protein extract as a non-animal protein source in beverages, bread-making products, or plant-based processed meats; to the use of the protein extract for oral or enteral clinical nutrition; or to the use of the protein extract for animal nutrition. In other words, in one embodiment, the present invention relates to a method for food supplementation for human and / or animal nutrition, comprising the following steps:

[0067] - To obtain a yeast protein extract by implementing any embodiment of the method for obtaining yeast protein according to the present invention; and

[0068] - The protein extract was administered to humans as a food supplement for weight management in older adults or athletes, and / or to animals as a protein intake.

[0069] As previously mentioned, the final soluble fraction of the method of the present invention can also be used. It contains a high total carbohydrate content, comparable to that of sweet juice. The total carbohydrate content is between 45% and 70%. These carbohydrates are present in the form of dextran (25% to 40%) and mannan (25% to 35%), thus making this soluble fraction suitable for immunostimulation, particularly for animal health. Depending on the type of dextranase used, free glucose may also be released. When AMP is converted to IMP by deaminase, this soluble fraction is rich in total nucleotides, especially 5'-GMP and 5'-IMP. Therefore, it can be used as a flavor enhancer. When AMP is not converted to IMP, the product can be used to mask bitterness or off-flavors, as described in patent application FR1762074. Its composition also serves as a good growth substrate for various microorganisms, especially bacteria.

[0070] The following examples illustrate the invention. They should not be construed as limiting the scope of the invention.

[0071] Example

[0072] Example 1: Preparation of concentrated protein extract (>75%) from Saccharomyces cerevisiae

[0073] Saccharomyces cerevisiae fermentation was carried out under conditions that resulted in a high nitrogen content of approximately 10% dry matter nitrogen in the yeast. The fermentation slurry was centrifuged to obtain a yeast extract containing 16-18% dry matter yeast, which was then washed.

[0074] In the laboratory, 3 kg of this nitrogen-rich, washed paste was subjected to thermoplethysmography: the paste was heated to a temperature between 70 and 95°C using an exchanger, and then incubated in a beaker immersed in a hot water bath. The paste was stirred and maintained at this temperature in the beaker for 2 hours. The temperature of the reaction medium was then lowered to 60°C. A portion was transferred to a 500 mL Erlenmeyer flask. The Erlenmeyer flask was immersed in a hot water bath adjusted to 60°C. The following doses of enzymes were added: 0.2% (g / 100g paste) of a mixture of two dextranases and 0.1% of a mixture of endonuclease and exonuclease. The mixture was incubated with stirring for 18 hours. After incubation, the mixture was centrifuged. The soluble fraction containing free nucleotides, polysaccharides, and a small amount of amino acids was allowed to stand. The insoluble fraction was a yeast protein extract. After washing three times, its composition was as follows: 13.0% nitrogen, 3% total nucleotides, and 4.7% total carbohydrates, i.e., 77% true protein.

[0075] Example 2: A concentrated protein extract (75%) was prepared from Saccharomyces cerevisiae according to a variation of Method 1.

[0076] In a variation of the method described in Example 1, after thermoplasmolysis, the insoluble fraction is separated from the supernatant containing free amino acids by centrifugation. The insoluble fraction is absorbed into tap water of an equal volume to the discarded supernatant, and then centrifuged again. This operation is performed a total of 3 times. A final recovery of 2 kg of 16% dry matter insoluble fraction, containing protein, polysaccharide, and nucleic acid material, is achieved. A portion is transferred to a 500 mL Erlenmeyer flask. The flask is immersed in a hot water bath at 60°C, and the remaining steps of the method are the same as those described in the previous examples.

[0077] The final protein extract had the following composition: 12.2% nitrogen, 1.4% total nucleotides, and 2.9% total carbohydrates, which is 75% true protein.

[0078] Example 3: Preparation of concentrated protein extract (>80%) from Saccharomyces cerevisiae

[0079] Following the scheme described in Example 2, the insoluble fraction of Saccharomyces cerevisiae containing protein, polysaccharide and nucleic acid materials is prepared by means of thermoplasmolysis, centrifugation and washing.

[0080] The dry extract of this fraction was adjusted to 14%, and the pH was adjusted to the optimal pH for the enzyme. 200 g of this fraction was incubated for 18 hours in a conical flask immersed in a 60°C hot water bath with stirring, in the presence of the following enzyme dosage: 0.2% dextranase purified liquid preparation and a mixture of 0.1% endoribonuclease and exonuclease. The insoluble fraction was collected by centrifugation and washed three times. The obtained protein extract contained 13.6% nitrogen, 2.4% total nucleotides, and 4.2% total carbohydrates, representing 83% true protein.

[0081] Example 4: Rapid preparation of concentrated protein extract (75%) from Saccharomyces cerevisiae

[0082] In a variation of the method described in Example 2, the incubation time can be shortened to 8 hours. The resulting protein extract then contains 12.5% ​​nitrogen, 3% total nucleotides, and 4.4% total carbohydrates, which is 75% true protein.

[0083] Example 5: Preparation of concentrated protein extract (80%) from Saccharomyces cerevisiae

[0084] In a variation of the method described in Example 2, the protein extract was dried, then extracted with five times its volume of ethanol, washed, drained, and dried under vacuum again. The resulting protein extract contained 13.3% nitrogen, 2% total nucleotides, and 3.1% total carbohydrates, representing 81% true protein.

[0085] Example 6: Preparation of concentrated protein extract from Pichia pastoris

[0086] Fermentation of *Pichia pastoris* was carried out under conditions that resulted in a nitrogen content of 9% dry matter in the obtained yeast. The fermentation slurry was centrifuged to obtain a yeast extract of 11-13% dry matter, and the extract was washed.

[0087] In the laboratory, following the same procedures as with the Saccharomyces cerevisiae strain, 3 kg of this nitrogen-rich paste was subjected to thermoplasmolysis at 70–95 °C for 2 hours. The insoluble fraction was collected by centrifugation and washed three times.

[0088] Adjust the dry extract of this fraction to 11-13% and adjust the pH to the optimal pH for the enzyme. Incubate 200g of this fraction for 18 hours in a conical flask immersed in a 60°C hot water bath with stirring, in the presence of the following enzyme dosage: 0.2% dextranase purified liquid preparation and a mixture of 0.1% endoribonuclease and exonuclease. Centrifuge to collect the insoluble fraction and wash three times. The resulting protein extract then contains 11.7% nitrogen, 1.4% total nucleotides, and 13.3% total carbohydrates, i.e., 72% true protein.

[0089] Example 7: Preparation of concentrated protein extract from Kluyveromyces martensii.

[0090] Kluyveromyces martensii fermentation was carried out under conditions that brought the nitrogen content of the yeast to 8% dry matter. The fermentation slurry was centrifuged to obtain a yeast extract, which was then washed. The extract was subjected to thermoplethysmosis as described above, followed by centrifugation, washing of the insoluble fraction, and incubation with an enzyme mixture containing dextranase, endonuclease, and exonuclease. The extract was then centrifuged again and washed to remove the insoluble fraction containing protein.

[0091] The obtained protein extract contained 11.8% nitrogen, 1.3% total nucleotides and 8.8% total carbohydrates, which is 72% true protein.

[0092] Example 8: The total lipids in the “insoluble” protein extract obtained by the method of the present invention were determined and compared with the lipid content of a “conventional” yeast protein extract obtained from the soluble portion of yeast after cell lysis according to the prior art.

[0093] The method used is a gravimetric analysis following acid hydrolysis and extraction with hexane on a Soxhlet extractor. This method is described in EC Regulation 152 / 2009. The results are given in Table 1 below. From three batches of protein extracts obtained according to the present invention, these results indicate a lipid content of approximately 10-11 g per 100 g of extract. In contrast, the lipid content of prior art yeast protein extracts obtained from the soluble fraction of lysed yeast is shown to be less than 1 g per 100 g of extract.

[0094]

[0095] Example 9: Determination of protein solubility curves

[0096] For the sample obtained from batch 1 of the protein extract according to the invention (see Example 8), protein solubility profiles were determined in aqueous solution at three different pH values ​​and using a final protein concentration of 2% (w / v). After a dissolution time of 60 minutes with stirring at ambient temperature, the supernatant was collected by centrifugation, and the total nitrogen content in the supernatant was determined by the Kjeldahl method according to standard NF EN ISO 5983-2. For each pH value, the solubility percentage was determined by the following formula:

[0097]

[0098] The result was less than 3.5%.

[0099] Example 10: Determination of the molecular weight profiles of the protein extract of the present invention and the "conventional" protein extract.

[0100] As described above, "protein extract of the present invention" refers to a yeast protein extract obtained from the insoluble portion of lysed yeast cells. Conversely, "conventional yeast protein extract" refers to yeast protein obtained by extraction from the soluble portion of lysed yeast cells. Extraction is performed using methods known to those skilled in the art.

[0101] As shown in Example 9, the protein extract of the present invention is insoluble in aqueous conditions. Specific conditions for dissolution and analysis by size exclusion chromatography have been developed. The procedure is as follows ( Figure 2 Method SEC1 in the middle:

[0102] Dissolve 10 mg of sample in 5 mL of mobile phase (stirring at 90 °C for 48 hours).

[0103] -Analysis at 40℃ PL gel column, 20 μm, MIXED-A (2000 to 40000)

[0104] The solution obtained at 000 g / mol (PS equivalent).

[0105] Mobile phase: DMSO / LiCl 0.25M.

[0106] RI (refractive index) and ultraviolet (280nm) detectors.

[0107] Methods for “conventional” yeast protein extracts ( Figure 2 Method SEC2) is excerpted from an OIV monograph (International Organisation of Vine and Wine):

[0108] - Dissolve the sample in water;

[0109] - Analyzed on a SUPERDEX 200 10 / 300GL column (10,000 to 600,000 Da, globular protein equivalent; 1,000 to 100,000 g / mol, dextran equivalent).

[0110] Mobile phase: phosphate buffer + 0.25M NaCl, pH 7.2

[0111] UV detection at 214, 260 and 280 nm.

[0112] exist Figure 2 Each provides its own protein map.

[0113] The protein extract spectrum of the yeast of the present invention is distributed at approximately 45 kDa and does not exhibit low molecular weight peaks (range 2-10 kDa). In contrast, the protein extract spectrum of "conventional" yeast is significantly different: it shows two distinct peaks in the low molecular weight range of 2-10 kDa, and overall, the peaks in the spectrum are more distinct and broader.

[0114] The ratio can also be determined using the official method for the determination of total and free amino acids given in EU Regulation 152 / 2009:

[0115]

[0116] For the yeast protein extract of the present invention (for the three batches of lipid determination in Example 8), the ratio is very close to 1 (or 100%) and is always higher than 0.9 (or 90%). For the applicant's "conventional" yeast protein extract and commercially available extracts, it is typically 0.30 to 0.85 (30% to 85%).

[0117] Example 11: Preparation of concentrated protein extract (>75%) from selenium-enriched Saccharomyces cerevisiae culture.

[0118] A batch of selenium-enriched yeast extract was subjected to the protein extraction method described in Example 1 or Example 2. The selenium content in the yeast extract was approximately 3200 ppm.

[0119] The obtained protein extract contained 13.5% total nitrogen and 76.7% true protein. The total selenium content was 4,750 ppm (mg Se / kg, dry weight). The selenomethionine content was 84% ​​(equivalent Se / Se tot). In comparison, selenized yeast... The selenomethionine content of 3000 (a bioavailable organic selenium source) is 63%.

Claims

1. A method for obtaining a yeast protein extract, comprising the following steps: a) Provide yeast extract; b) Expose the yeast extract to thermal plasmolysis at a temperature of 70 to 95°C for 1 minute to 4 hours; b') Separates the insoluble and soluble portions; c) Subject the insoluble portion to the activity of at least one ribonuclease and at least one dextranase sequentially or simultaneously at a temperature of 40 to 65°C for 8 to 24 hours; d) Separate the insoluble portion from the soluble portion, wherein the insoluble portion is the yeast protein extract; The insoluble fraction collected in step d) is tasteless, contains less than 3% nucleotides, and has a true protein content of at least 72%. The yeast extract refers to a yeast suspension obtained after propagation in a container and centrifugation.

2. The method according to claim 1, wherein, In step b), the yeast extract is exposed to thermal plasmolysis at a temperature of 70 to 95°C for 1 minute to 3 hours.

3. The method according to claim 1, wherein, In step b), the yeast extract is exposed to thermal plasmolysis at a temperature of 70 to 95°C for 40 minutes to 2 hours.

4. The method according to claim 1, wherein, In step c), the insoluble portion is subjected to the activity of at least one ribonuclease and at least one dextranase sequentially or simultaneously at a temperature of 60°C for 18 hours.

5. The method according to claim 1, wherein, Deaminase activity is also applied in step c).

6. The method according to claim 1, wherein, The yeast is selected from the genus *Gynostemma*. Saccharomyces Pichia pastoris ( ) Pichia ), Candida genus ( Candida Kluyveromyces ( ) Kluyveromyces ), Yersinia ( Yarrowia ) or Wickham yeast ( Wickerhamomyces ).

7. The method according to claim 1, wherein, The yeast is selected from brewer's yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae ), Pichia pastoris ( Pichia jadinii ) or Max Kluyveromycin ( Kluyveromyces marxianus ).

8. The method according to any one of claims 1 to 7, wherein, Simultaneously use the enzyme used in step c).

9. The method according to any one of claims 1 to 7, wherein, The lipid content of the insoluble fraction collected in step d) is higher than 7%.

10. The method according to any one of claims 1 to 7, wherein, The insoluble fraction collected in step d) was treated with a solvent to remove lipids and increase the true protein content to 80%.

11. The method according to any one of claims 1 to 7, wherein, The insoluble fraction collected in step d) was treated with ethanol or supercritical CO2 to remove lipids and increase the true protein content to 80%.

12. The method according to any one of claims 1 to 7, wherein, The yeast paste used in step a) contains selenium-enriched yeast.

13. Use in human nutrition of a yeast protein extract having a true protein content of at least 72%, obtained by the method of any one of claims 1 to 12.

14. The use according to claim 13, wherein it is used as a food supplement for weight control in the elderly or in athletes.

15. The use according to claim 13, wherein it is used as a supply of non-animal protein in beverages, bread-making products or plant-based processed meats.

16. The use according to claim 13, for oral or non-therapeutic enteral clinical nutrition.

17. Use in animal nutrition of a yeast protein extract having a true protein content of at least 72%, obtained by the method of any one of claims 1 to 12.

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