YEAST PROTEINS

Description

[0001] The present invention relates to the field of proteins derived from microorganisms and more precisely from yeasts, which can be used widely for the nutrition, well-being and health of humans and animals. BIBLIOGRAPHY

[0002] Proteins are the main component of human and animal tissues. From a nutritional point of view, proteins are hydrolyzed by proteases and peptidases into peptides and amino acids. Amino acids provide essential elements for the body such as nitrogen, carbohydrates and sulfur. The body does not fix mineral nitrogen to incorporate it into organic molecules; amino acids therefore provide nitrogen. Sulfur-containing amino acids provide sulfur, which is important for metabolism. Amino acids are also essential elements for the synthesis of proteins, peptides and physiologically essential low-molecular substances such as glutathione, creatine, dopamine, serotonin, etc. Therefore, the need for protein for human and animal nutrition is growing with the growth of the world's population.Consequently, it is necessary to find additional and / or alternative sources of protein to animal proteins. Proteins from plants (cereals, legumes) or insects are already valued. Obtaining proteins of microbial origin uses fermentation mechanisms known for centuries. Microbial proteins are then either components of the whole cell or the wall, or isolated proteins. One of the disadvantages of proteins of microbial origin for human consumption is the level of nucleic acids in the isolates. This level must be low because one of the end products of nucleic acid metabolism is uric acid, which the human body, lacking uricase, the enzyme necessary for its catabolism, does not degrade. Other disadvantages are, for example, the extraction yield and / or purity of proteins obtained by isolation from microorganisms.

[0003] After cell lysis, for example of yeast cells, proteins are conventionally recovered in the "noble" phase, i.e. the soluble phase, which corresponds to yeast extracts and contains compounds rich in taste. However, taste can be a disadvantage, depending on the intended application of the protein extract obtained. Three patents from the same team deal with obtaining protein-concentrated extracts. Patent US3867555 discloses a process for obtaining protein extracts with a low level of nucleic acids. The cells are disrupted mechanically (high pressure, ultrasound) or chemically (autolysis, external enzymes) so that the proteins remain in the soluble fraction. These proteins are, however, mixed with the result of hydrolysis, i.e. amino acids, peptides and polysaccharides or simple sugars.After centrifugation to separate and discard the insoluble fraction, the proteins are precipitated and an alkaline treatment allows the hydrolysis of the nucleic acids which are then removed by filtration. The overall recovery yield of "non-hydrolyzed" proteins is however low.

[0004] US patent 3887431 discloses the use of endogenous nuclease present in the soluble fraction after yeast lysis to degrade nucleic acids. A vacuum concentration step makes it possible to obtain both a concentrated protein extract and a product with low taste and odor.

[0005] Patent US3991215 discloses a process which, after cell disruption, applies an ultra-high temperature treatment to the soluble cytoplasmic fraction which makes it possible to obtain a product enriched in proteins, with few nucleic acids.

[0006] Finally, there are several directions for improving the processes for isolating proteins from microorganisms, with the aim of obtaining better purity, better yields and / or protein extracts that can be directly used in nutrition or as food supplements. Documents FR 2 207 985 A1 and US 4 080 260 A describe processes for obtaining protein extracts from a soluble fraction. SUMMARY OF THE INVENTION

[0007] Contrary to common techniques, the inventors have developed a method for isolating microorganism proteins from the insoluble fraction obtained after lysis of said microorganisms. This new method makes it possible to obtain, with good yield, concentrated extracts of non-hydrolyzed proteins, low in nucleic acids, odor and taste.

[0008] The method according to the invention begins with a lysis of the cells, preferably by thermal plasmolysis, which results in an inactivation of the microorganism enzymes and the release into the medium of free amino acids, including glutamic acid. The whole is then subjected to a glucanase-type enzyme and a ribonuclease-type enzyme and then subjected to a separation at the end of which the insoluble fraction represents the product of interest, the true protein content of which is at least 72%. According to another embodiment of the invention, a first separation is carried out after the thermal plasmolysis phase and only the insoluble fraction, which comprises the proteins, the polysaccharides and the RNA, is subjected to the action of a glucanase and a ribonuclease and then separated again to preserve the insoluble fraction rich in proteins.

[0009] The protein extract obtained is odorless and tasteless, low in nucleic acids. The protein extract which still contains lipids from the membrane can be delipidated by methods known to those skilled in the art such as extraction with a solvent such as hexane or ethanol, with supercritical CO2 or treatment with lipases or phospholipases followed by separation of the solubilized phase.

[0010] The proteins thus obtained can be used directly for applications in nutrition, food supplements, etc. DEFINITIONS

[0011] By microorganism is meant a living organism invisible to the naked eye and visible under a microscope. In the context of the present invention, the microorganisms are preferably bacteria (prokaryotic microorganisms) or yeasts (eukaryotic microorganisms). Yeast, in the singular or plural, is a generic term designating eukaryotic microorganisms capable of causing fermentation of organic matter. Among yeasts, the following genera will be mentioned, but not limited to: Saccharomyces, Candida, Pichia, Kluyveromyces.

[0012] Yeast cream refers to the yeast suspension obtained after multiplication in a vat, and after centrifugation which separates said suspension from the ambient liquid also called must. Multiplication can also be called culture or, by extension, fermentation. Proteins are macromolecules made up of a chain of amino acids linked by peptide bonds. They are among the main basic constituents of all animal and plant cells. They represent up to 50% of the dry weight of a living being and constitute 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 of important nitrogen compounds in the body (nucleic acids, nitric oxide, glutathione, etc.) and substrates for energy metabolism.

[0013] Generally speaking, in the food industry, the protein content is equated to the nitrogen content multiplied by 6.25 (the coefficient 6.25 comes from the average nitrogen content of a protein which is 100 / 6.25 or 16%). By true proteins, we mean a protein content closer to reality, corrected for the bias induced by non-protein nitrogen, for example, nucleic acid nitrogen. Thus, in formula form, the true protein content can be equated to total nitrogen less nucleic acid nitrogen and ammoniacal nitrogen, multiplied by 6.25. Alternatively, the true protein content can be assessed by measuring total amino acids. Integral native proteins are the proteins of the microorganism that are in the state in which they are found in the living microorganism. By extension, denatured native proteins have a potentially modified spatial conformation, by folding or coagulation.Proteins can also be partially or completely hydrolyzed.

[0014] Plasmolysis refers to a breakdown of the membrane seal of the microorganism, preferably yeast, or of its compartmentalized appearance. Water is lost due to membrane permeabilization. DETAILED DESCRIPTION OF THE INVENTION AND EMBODIMENTS

[0015] There Figure 1 represents the two main embodiments of the method according to the invention. The Figure 1A illustrates the process in which there is no separation step between plasmolysis and enzymatic hydrolysis. The Figure 1B illustrates the process in which a separation is carried out after the plasmolysis step and in which enzymatic hydrolysis is then carried out on the insoluble fraction resulting from this separation. Figure 2presents the respective protein profiles of a protein extract resulting from a process according to the invention, and of a so-called conventional protein extract resulting from a known process for extracting proteins from the soluble fraction obtained after the plasmolysis step.

[0016] The method according to the invention applies to any type of yeast, more specifically any yeast with a history of consumption in human or animal nutrition. From an industrial point of view, the method according to the invention is carried out on a yeast cream as defined above, that is to say on a suspension of yeasts. Preferably, the yeasts are chosen from yeasts of the genus Saccharomyces, Pichia, Candida, Kluyveromyces, Yarrowia Or Wickerhamomyces. Preferably, the yeasts are chosen from Saccharomyces cerevisiae, Pichia jadinii (also called Candida utilis), Pichia pastoris, Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica, Wickerhamomyces anomalus etc.These yeasts comprise between 6 and 11% nitrogen. Nitrogen is measured by the Kjeldahl method known to those skilled in the art. As indicated above, the true protein level is extrapolated from this nitrogen measurement using the multiplier coefficient 6.25.

[0017] Yeast culture is carried out according to methods known to those skilled in the art. A protocol is described in the reference book "Yeast technology" by Gerald Reed and Tilak W. Nagodawithana (ISBN 0-442-31892-8) on pages 284 to 293. Said culture, also called biomass, is harvested by centrifugation or filtration and can be washed. A yeast cream is obtained.

[0018] The cells are then subjected to mechanical or chemical disruption using known methods such as high-pressure homogenization, mechanical grinding, ultrasonic disintegration, repeated freeze-thaw cycles, osmotic shock or enzymatic lysis. According to a preferred embodiment of the invention, the yeasts are subjected to thermal plasmolysis, at a temperature between 70 and 95°C adapted according to the type of yeast. The temperature is preferably between 80 and 90°C. Those skilled in the art will be able to adjust the temperature and / or the duration of the lysis, for example depending on the resistance of the cells and / or the enzymes that they wish to inactivate during this step. The duration will be between 30 seconds and 3 hours, up to 4 hours, preferably 1 min., 5 min., 10 min., 30 min., 45 min., 1 hour, 1 hour 30 minutes, or 2 hours, up to 3 or even 4 hours.This plasmolysis step allows denaturation of the yeast, inactivation of endogenous enzymes and permeabilization of the membrane which allows the release of a soluble fraction. The soluble fraction mainly includes free amino acids, small peptides, and minerals and among these amino acids, some which have an impact on the taste such as glutamic acid.

[0019] In a first embodiment of the invention, the assembly resulting from the plasmolysis step and containing the soluble fraction and the insoluble fraction is then subjected to one or more enzymatic activities. The objective of this step is to solubilize the maximum number of constituents that are not proteins, without attacking the proteins. Preferably, a ribonuclease activity (EC 3.1.4.1) and a glucanase activity (EC 3.2.1) will be used. The enzymatic activities can be carried out sequentially or simultaneously. The ribonuclease activity will transform the RNA into 5' nucleotides and solubilize it, thus passing it into the soluble fraction. It is possible to use several ribonucleases, and for example a mixture of endo- and exo-nucleases. Optionally, a deaminase can be used to transform AMP into IMP, in order to enhance the flavor-enhancing power of the soluble fraction.The action of one or more glucanases will solubilize the wall polysaccharides into soluble oligosaccharides. The time and temperature will be adjusted according to the specifications of the enzymes used. The temperature will be between 40 and 65°C, preferably 60°C. The duration will be between 8 and 24 hours, preferably between 16 and 24 hours, more preferably 18 hours. This enzymatic step makes it possible to obtain, on the one hand, a new soluble fraction which includes nucleotides, polysaccharides (approximately 60% of total carbohydrates) and a small proportion of amino acids, and on the other hand an insoluble fraction which essentially includes proteins.

[0020] The last step is a separation of the soluble fraction and the insoluble fraction.

[0021] In a second embodiment of the invention, the soluble and insoluble fractions resulting from the plasmolysis step are separated. The soluble fraction will be set aside, eliminated and can be used as yeast extract. The insoluble fraction includes the bark (or walls), polymers, polysaccharides, RNA and proteins coagulated by heat. This insoluble fraction is retained. It is then subjected to the ribonuclease and glucanase enzymatic activities as indicated above, according to a similar protocol of incubation (temperature, time) and separation of the soluble and insoluble fractions. The final soluble fraction can be set aside and used.

[0022] The final insoluble fraction, resulting from enzymatic digestion, has a "true" protein content greater than or equal to 70%, preferably greater than or equal to 72%, 80% and up to 85%. Generally, yeast comprises between 50 and 55% of "true" proteins. The process according to the invention makes it possible to eliminate numerous elements and to obtain a product with a higher protein concentration. Optionally, the quantity of lipids in this insoluble fraction can be reduced, either by the action of solvents (ethanol, hexane), or by supercritical CO 2, or by the action of a lipase or a phospholipase. True protein purities of more than 80% can then be obtained.

[0023] According to one embodiment of the invention, the respective actions of the ribonucleases and glucanases are implemented sequentially, without importance of the order. According to a preferred embodiment of the invention, the ribonuclease and glucanase activities are applied at the same time.

[0024] The product thus obtained can be freeze-dried, dried by a method known to those skilled in the art, for example by atomization or under vacuum, and preserved while retaining its properties and quality.

[0025] Thus, according to one embodiment, the invention relates to a method for obtaining yeast proteins comprising the following steps: a) providing a yeast cream; b) exposing this yeast cream to thermal plasmolysis at a temperature between 70 and 95°C, for a period of between 1 minute and 3 hours, preferably between 40 minutes and 2 hours; c) subjecting the whole to the activity of at least one ribonuclease and one glucanase, sequentially or simultaneously, at a temperature between 40 and 65°C for a period of between 8 and 24 hours; d) separating the insoluble fraction and the soluble fraction, wherein the insoluble fraction collected in step d) is tasteless, has a nucleotide content of less than 3% and a true protein content of at least 72%.

[0026] According to another embodiment, the invention relates to a method for obtaining yeast proteins comprising the following steps: a) providing a yeast cream; b) exposing this yeast cream to thermal plasmolysis at a temperature between 70 and 95°C, for a period of between 1 minute and 3 hours, preferably between 40 minutes and two 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 between 40 and 65°C for a period of between 8 and 24 hours; d) separating the insoluble fraction and the soluble fraction, wherein the insoluble fraction collected in step d) is tasteless, has a nucleotide content of less than 3% and a true protein content of at least 72%.

[0027] In the remainder of this description, the insoluble fraction collected in step d) may also be called “protein extract (from yeast) according to the invention”. On the contrary, a yeast protein extract obtained, according to a process known from the prior art, from the soluble fraction resulting from plasmolysis, may be called “classic protein extract”.

[0028] According to a preferred embodiment of the invention, the yeast cream of step a) is obtained from the fermentation of yeasts chosen from Saccharomyces cerevisiae, Pichia, preferentially Pichia jadinii, Kluyveromyces, preferentially Kluyveromyces marxianus Or Kluyveromyces lactis, Yarrowia, preferentially Yarrowia lipolytica, Or Wickerhamomyces, preferentially Wickerhamomyces anomalus. Advantageously, the yeast is chosen from Saccharomyces cerevisiae, Pichia jadinii Or Kluyveromyces marxianus. The preferred yeast is Saccharomyces cerevisiae.

[0029] According to a preferred embodiment of the invention, step a) of thermal plasmolysis is carried out at a temperature between 80 and 90°C.

[0030] According to a preferred embodiment of the invention, the glucanase and ribonuclease activities are applied simultaneously.

[0031] Optionally, deaminase activity can also be applied.

[0032] Advantageously, the yeast cream used in step a) contains a selenium-enriched yeast. A selenium-enriched yeast culture can be carried out according to a method known to those skilled in the art, for example that described in application EP 1478732. The selenium level in the yeast culture can then reach 3,000 ppm or even more.

[0033] The product obtained in the insoluble fraction has an exclusively protein nitrogen content greater than or equal to 11.5% on dry matter, i.e. 72% of true proteins (using the conversion coefficient of 6.25 indicated above). Normally, yeast has a maximum nitrogen content of 11% nitrogen, which gives 69% of potential proteins. Yeast also contains non-protein nitrogenous material (RNA / DNA nucleic acids). By subtracting the nucleic nitrogen from the total nitrogen, a yeast contains only 50 to 55% of proteins. Thus, the minimum level of 72% of proteins obtained in the final insoluble fraction is characteristic of the process according to the invention.In this same insoluble fraction, the level of total nucleotides is between 0.8 and 1.5%, up to 3%, the level of total carbohydrates is 1 to 8% (anthrone assay according to a method known to those skilled in the art), the respective levels of glucans are 0.2 to 4%, of mannans 0.2 to 4%, of lipids 7 to 15%, of mineral matter 1 to 7%.

[0034] It may be interesting to distinguish a yeast protein extract obtained by the process according to the invention from an extract according to the prior art, namely a protein extract obtained from the soluble fraction after lysis of the cells and having undergone a concentration step, which would then have a protein level of at least 72% and possibly a nucleotide level of less than 3%.

[0035] For this purpose, total lipids were measured on protein extracts from Saccharomyces cerevisiae,by a gravimetric method after acid hydrolysis and extraction with hexane using a Soxhlet apparatus. The lipid levels are systematically greater than 7%. Thus, the insoluble fraction collected in step d) of an extraction process according to the invention also has a lipid level greater than 7%. In comparison, the lipid level is less than 1% on a “classic” yeast protein extract. The lipid level may be different depending on the species or strain of yeast. Generally speaking, for an industrial product, the starting microorganism is indicated in the technical data sheet. Those skilled in the art will therefore be able to extrapolate and deduce a species / lipid level combination in the protein extract.

[0036] In the case of a yeast protein extract according to the invention that has been subjected to a delipidation step, the lipid content may not be sufficient to distinguish the two origins. An evaluation of the protein solubility profile of the protein extract can then complete the comparative analysis. The percentage of solubility is determined according to the following equation: % s o l u b i l i t é = % N total dans le surnageant % N initial dans le milieu réactionnel where %N denotes the percentage of nitrogen determined by the Kjeldahl method.

[0037] The solubility of a protein extract according to the invention is less than 3.5% in water.

[0038] In comparison, a "classic" yeast protein extract is considered soluble and comprises only between 5 and 10% insoluble elements.

[0039] For the same purpose, the molecular weight profiles of a yeast protein extract according to the invention and a protein extract according to the prior art were compared. A yeast protein extract obtained according to the invention does not exhibit a low molecular weight peak. The bulk of the protein profile is distributed around 40-45 kDa. The smallest compounds are at around 500 Da, which corresponds to small peptides. In comparison, the profile of a "classic" yeast protein extract exhibits several molecular weight peaks and a significant amount at the low molecular weight level. It is then possible to extrapolate from these profiles and measure the ratio acides aminés totaux − acides aminés libres acides aminés totaux (amino acids measured according to a classic method for measuring amino acids in foods, for example the official method from EC Regulation EU152 / 2009). This ratio is close to 1, always greater than 0.9 for a yeast protein extract according to the invention, whereas it can be between 0.30 and 0.85 for “classic” yeast protein extracts.

[0040] The product obtained by the process according to the invention is distinguished from known protein extracts by its microbial origin, in other words non-plant and non-animal, its high protein content, its low nucleic acid content, the lipids of the cell membrane still present if no delipidating treatment is applied. It is also tasteless. Its microbial origin also has the advantage that it comes from a raw material that is not known to be an allergen.

[0041] Yeast proteins have a major advantage in their nutritional quality. Thus, the product obtained by the process according to the invention is a source of protein which has a major advantage: its quality, represented by the PDCAAS score (Protein Digestibility Corrected AminoAcids Score) which is equal to 1, i.e. similar to that of reference animal proteins such as casein and ovalbumin. The evaluation of the quality of proteins makes it possible to determine their capacity to satisfy metabolic needs. Consequently, any application related to nutrition and food supplements or sports supplements can use a protein extract obtained by the process according to the invention. The applications cited here do not claim to be exhaustive.

[0042] In the context of nutrition, health and human well-being, such a protein extract can be used for weight control, as a supplement for athletes or for the elderly, in the form of a food supplement or a high-protein bar or drink. For example, such a protein extract can be used as a non-animal protein source for vegan diets, for example in milkshakes, burgers, nuggets, vegetable charcuterie, ravioli stuffing, meatballs, oat flakes, and pasta seasoning preparations. Similarly, protein-rich breads or bakery products can use the protein extracts obtained by the process according to the invention. As indicated above, these protein extracts have the advantage of not imparting any taste, bitterness, or unpleasant notes (off-notes), characteristic of certain vegetable or algae proteins today.In human health, such a protein extract can be used in infant nutrition or clinical nutrition, i.e., in oral or enteral nutrition to compensate for nutritional imbalance. Advantageously, when the protein extract is obtained from a yeast cream enriched with selenium, it can be used as a food supplement stimulating immunity and / or strengthening the quality of the skin, hair and / or nails.

[0043] Finally, such a protein extract can be used for protein intake in animal feed. Advantageously, when the protein extract is obtained from a yeast cream enriched with selenium, the animal will benefit from a combined intake of proteins and selenium, said selenium being bioavailable because it is integrated into the proteins, in the form of selenomethionine.

[0044] Thus, the invention also relates to the use of a yeast protein extract having a true protein content of at least 72%, obtained according to any one of the embodiments of the method according to the invention, as a food supplement for weight control, for the elderly or for athletes, to the use of such a protein extract as a non-animal protein supply in drinks, bakery products or vegetable charcuterie, to the use of such a protein extract in clinical, oral or enteral nutrition, or to the use of such a protein extract in animal nutrition. In other words, according to one embodiment, the invention relates to a method of food supplementation in human nutrition, and / or in animal nutrition comprising the steps consisting of: obtaining a yeast protein extract by implementing any of the embodiments of the method for obtaining yeast proteins according to the invention; and administering said protein extract respectively to a human individual as a food supplement for weight control, for the elderly or for athletes, and / or to an animal as a protein intake.

[0045] As mentioned above, the final soluble fraction of the process according to the invention can also be set aside and recovered. Due to its richness in total carbohydrates, it can be compared to a sweet juice. The total carbohydrate content is between 45 and 70%. These carbohydrates are in the form of glucans (25 to 40%) and mannans (25 to 35%), which allows this soluble fraction to be used for immunostimulation, particularly for animal health. Depending on the type of glucanase used, free glucose can also be released. This soluble fraction is rich in total nucleotides, more precisely in 5'-GMP and 5'-IMP when AMP is transformed into IMP by a deaminase. Thus, 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-notes, as disclosed in patent application FR1762074.Its composition also makes it a good growth substrate for various microorganisms, especially bacteria.

[0046] The examples below may illustrate the present invention. They cannot be considered as limiting the scope of the invention. EXAMPLES Example 1: Preparation of a concentrated protein extract (>75%) from Saccharomyces cerevisiae

[0047] Carry out a fermentation of Saccharomyces cerevisiae under conditions that allow a high nitrogen content of the yeast to be achieved, approximately 10% nitrogen on dry matter. Use centrifugal separation on the must from this fermentation to obtain a yeast cream with 16-18% dry matter-yeast then wash the cream.

[0048] In the laboratory, carry out a thermal plasmolysis of 3 kg of this nitrogen-rich washed cream: raise the temperature of the cream to a chosen temperature between 70 and 95°C using an exchanger then incubate the 3 kg of cream in a beaker immersed in a water bath. Leave the cream stirring and maintain the temperature of the cream in the beaker for 2 hours. Then lower the temperature of the reaction medium to 60°C. Transfer a fraction into a 500 mL Erlenmeyer flask. Immerse the Erlenmeyer flask in a water bath whose temperature is regulated at 60°C. Add the following doses of enzymes: 0.2% (g per 100g of cream) of a mix of two glucanases and 0.1% of a mix of endo and exo-ribonucleases. Incubate for 18 hours with stirring. At the end of this incubation, centrifuge. Discard the soluble fraction, which contains free nucleotides, polysaccharides, and a small proportion of amino acids. The insoluble fraction is the yeast protein extract.Its composition, measured after 3 washes, is as follows: 13.0% nitrogen, 3% total nucleotides and 4.7% total carbohydrates, or 77% true protein. Example 2: Preparation of a concentrated protein extract (75%) from Saccharomyces cerevisiae according to a variant of method 1.

[0049] In a variation of the process presented in Example 1, separate the insoluble fraction from the supernatant comprising the free amino acids by centrifugation at the end of the thermal plasmolysis. The insoluble fraction is then taken up in a volume of tap water equivalent to the volume of supernatant discarded and then centrifuged again. This operation is carried out 3 times in total. Finally, recover 2 kg of insoluble fraction at 16% dry matter, containing the proteins, polysaccharides and nucleic material. Transfer a fraction into a 500 mL Erlenmeyer flask. Immerse the Erlenmeyer flask in a water bath whose temperature is regulated at 60°C and apply the rest of the process as described in the previous example.

[0050] The composition of the final protein extract obtained is as follows: 12.2% nitrogen, 1.4% total nucleotides and 2.9% total carbohydrates, or 75% true protein. Example 3: Preparation of a concentrated protein extract (>80%) from Saccharomyces cerevisiae

[0051] Prepare, using the steps of thermal plasmolysis, centrifugal separation and then washing, the insoluble fraction of Saccharomyces cerevisiae containing proteins, polysaccharides and nucleic material according to the protocol described in example 2.

[0052] Adjust the dry extract of this fraction to 14% and the pH to the optimum pH of the enzymes. Incubate with stirring 200 g of this fraction in an Erlenmeyer flask immersed in a water bath at 60°C for 18 hours in the presence of the following enzyme doses: 0.2% of a purified liquid preparation of glucanase and 0.1% of a mix of endo and exo-ribonucleases. Recover the insoluble fraction by centrifugation and wash 3 times. The protein extract thus obtained then comprises 13.6% nitrogen, 2.4% total nucleotides and 4.2% total carbohydrates, i.e. 83% true proteins. Example 4: Rapid preparation of a concentrated protein extract (75%) from Saccharomyces cerevisiae

[0053] In a variant of the method presented in Example 2, the incubation time can be reduced to 8 hours. The protein extract obtained then comprises 12.5% ​​nitrogen, 3% total nucleotides and 4.4% total carbohydrates, i.e. 75% true proteins. Example 5: Preparation of a concentrated protein extract (80%) from Saccharomyces cerevisiae

[0054] In a variation of the process presented in Example 2, the protein extract is dried before being extracted with five volumes of ethanol, washed, then drained and dried again under vacuum. The protein extract obtained then comprises 13.3% nitrogen, 2% total nucleotides and 3.1% total carbohydrates, or 81% true protein. Example 6: Preparation of a concentrated protein extract from Pichia jadinii

[0055] Carry out a fermentation of Pichia jadiniiunder conditions allowing a nitrogen content of the yeast to be reached of around 9% on dry matter. Use centrifugal separation on the must from this fermentation in order to obtain a yeast cream with 11-13% dry matter then wash the cream.

[0056] In the laboratory, carry out thermal plasmolysis of 3 kg of this nitrogen-rich washed cream at a temperature between 70 and 95°C, for 2 hours according to the same protocol as that applied to the strain Saccharomyces cerevisiae. Recover the insoluble fraction by centrifugation and then wash it 3 times.

[0057] Adjust the dry extract of this fraction to 11-13% and the pH to the optimum pH of the enzymes. Incubate with stirring 200 g of this fraction in an Erlenmeyer flask immersed in a water bath at 60°C for 18 hours in the presence of the following enzyme doses: 0.2% of a purified liquid preparation of glucanase and 0.1% of a mix of endo and exo-ribonucleases. Recover the insoluble fraction by centrifugation and wash 3 times. The protein extract thus obtained then comprises 11.7% nitrogen, 1.4% total nucleotides and 13.3% total carbohydrates, i.e. 72% true proteins. Example 7: Preparation of a concentrated protein extract from Kluyveromyces marxianus.

[0058] Carry out a fermentation of Kluyveromyces marxianusunder conditions that allow a nitrogen content of the yeast to be reached of around 8% on dry matter. Use centrifugal separation on the must from this fermentation to obtain a yeast cream and then wash the cream. Carry out thermal plasmolysis, centrifuge, wash the insoluble fraction and incubate with a mix of enzymes containing glucans and endo and exo-ribonucleases according to the protocol described previously. Centrifuge again then wash the insoluble fraction containing the proteins.

[0059] The protein extract thus obtained comprises 11.8% nitrogen, 1.3% total nucleotides and 8.8% total carbohydrates, i.e. 72% true protein. Example 8: determination of total lipids in an “insoluble” protein extract obtained by a method according to the invention and comparison with the lipid level of a “classic” yeast protein extract, extracted from the soluble part of the yeasts after cell lysis according to the prior art.

[0060] The method used is a gravimetric method after acid hydrolysis and hexane extraction using a Soxhlet apparatus. This method is described in EC Regulation No. 152 / 2009. The results are presented in Table 1 below. From three batches of protein extract obtained according to the invention, these results indicate a lipid content of around 10 to 11 g per 100 g of extract. In comparison, a yeast protein extract according to the prior art, obtained from the soluble fraction of lysed yeasts will have a lipid content of less than 1 g per 100 g of extract. Extrait proteique selon l'invention (sur fraction insoluble) Protein extract according to the prior art (on soluble fraction) lot 1 lot 2 lot 3 Total fat (g / 100g) 11,2 10,8 11,1 <1 Example 9: Determination of the protein solubility profile.

[0061] On a sample from batch 1 of protein extract according to the invention (see example 8), the protein solubility profile was determined at three distinct pH values ​​in aqueous solution and using a final protein concentration of 2% (m / v). After 60 minutes of solubilization with stirring at room temperature, the supernatant is recovered by centrifugation and the total nitrogen content in the supernatant is determined by the Kjeldahl method according to standard NF EN ISO 5983-2. For each pH value, the percentage solubility is determined according to the following equation: % s o l u b i l i t é = % N total dans le surnageant % N initial dans le milieu réactionnel

[0062] The result obtained is less than 3.5%.

[0063] Example 10: determination of the molecular weight profiles of a protein extract according to the invention and a “classic” protein extract respectively.

[0064] As in all of the above, “protein extract according to the invention” designates a yeast protein extract obtained from the insoluble fraction of lysed yeast cells. On the contrary , a “classical” yeast protein extract” means yeast proteins obtained by extraction from the soluble fraction of lysed yeast cells. Extraction according to methods known to those skilled in the art.

[0065] As indicated in Example 9, the protein extract according to the invention is not soluble in aqueous conditions. Specific conditions for solubilization and analysis by size exclusion chromatography have been developed. The protocol is as follows (SEC1 method on the Figure 2 ) : solubilization of 10 mg of the sample in 5 mL of the mobile phase (with stirring for 48 hours at 90°C), analysis of the solution obtained on an Agilent ®< PLgel 20 µm MIXED-A column (2,000 to 40,000,000 g / mol, PS equivalent) at 40°C.

[0066] Mobile phase: DMSO / LiCI 0.25 M.

[0067] RI (refractive index) and UV (280 nm) detectors.

[0068] The method (SEC2 method on the Figure 2 ) used for the “classic” yeast protein extract comes from the OIV (International Wine Organization) monograph: solubilization of the sample in water; analysis on SUPERDEX 200 10 / 300 GL column (10,000 to 600,000 Da, globular protein equivalent; 1,000 to 100,000 g / mol, dextran equivalent).

[0069] Mobile phase: phosphate buffer + 0.25 M NaCl, pH 7.2

[0070] UV detection at 214, 260 and 280 nm.

[0071] The respective protein profiles are presented on the Figure 2.

[0072] The profile of the yeast protein extract according to the invention is distributed around 45 kDa and does not show a low molecular weight peak (range 2-10 kDa). In comparison, the profile of a "classical" yeast protein extract is significantly different; it shows two clear peaks in the low molecular weight range, 2-10 kDa and overall a profile with sharper and more distributed peaks.

[0073] The determination of total amino acids and free amino acids according to the official method from EC Regulation EU152 / 2009 also makes it possible to determine the ratio acides aminés totaux − acides aminés libres acides aminés totaux

[0074] This is very close to 1 (or 100%) and always greater than 0.9 (or 90%) on a yeast protein extract according to the invention (determination on the 3 batches whose lipids were measured in example 8). It is typically between 0.30 and 0.85 (30 to 85%) on “classic” yeast protein extracts from the Applicant, and commercially available extracts. Example 11: Preparation of a concentrated protein extract (>75%) from a culture of Saccharomyces cerevisiae enriched with selenium.

[0075] A batch of selenium-enriched yeast cream was subjected to the protein extraction process as described in Example 1 or Example 2. The selenium level in this yeast cream was close to 3,200 ppm.

[0076] The total nitrogen content in the protein extract obtained was 13.5%, the true protein content was 76.7%. The total selenium content was 4,750 ppm (mg Se / kg dry). The selenomethionine content was 84% ​​(Eq Se / Se tot). In comparison, the selenium yeast SelSaf ®< 3000, a source of bioavailable organic selenium, has a selenomethionine content of 63%.

Claims

1. A method for obtaining yeast proteins, comprising the following steps: a) providing a yeast cream; b) exposing this yeast cream to thermal plasmolysis at a temperature of between 70 and 95 °C for a time of between 30 seconds and 4 hours, preferably between 1 minute and 3 hours, preferably between 40 minutes and 2 hours; c) subjecting the whole to the activity of at least one ribonuclease and one glucanase, sequentially or simultaneously, at a temperature of between 40 and 65 °C, preferably 60°C for a time of between 8 and 24h, preferably 18h; d) separating the insoluble fraction and soluble fraction; wherein the insoluble fraction collected at step d) is taste-free, has a nucleotide content of less than 3 % and true protein content of at least 72 %.

2. A method for obtaining yeast proteins comprising the following steps: a) providing a yeast cream; b) exposing this yeast cream to thermal plasmolysis at a temperature of between 70 and 95 °C for a time of between 30 seconds and 4 hours, preferably between 1 minute and 3 hours, more preferably between 40 minutes and 2 hours; b') separating the insoluble fraction and 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 between 40 and 65 °C, preferably 60° C for a time of between 8 and 24h, preferably 18h; d) separating the insoluble fraction and soluble fraction; wherein the insoluble fraction collected at step d) is taste-free, has a nucleotide content of less 3 % and true protein content of at least 72 %.

3. The method according to claim 1 or 2, wherein deaminase activity is also applied at step c).

4. The method according to one of claims 1 to 3, wherein the yeasts are selected from among the species Saccharomyces, Pichia, Candida, Kluyveromyces, Yarrowia, Wickerhamomyces.

5. The method according to claim 4, wherein the yeasts are selected from among Saccharomyces cerevisiae, Pichia jadinii, Kluyveromyces marxianus.

6. The method according to one of claims 1 to 5, wherein the enzymes used at step c) are used simultaneously.

7. The method according to one of claims 1 to 6, wherein the insoluble fraction collected at step d) also has a lipid content higher than 7 %.

8. The method according to one of claims 1 to 7, wherein the insoluble fraction collected at step d) is treated with ethanol, a solvent or supercritical CO2 to remove the lipids and increase the true protein content to 80 %.

9. The method according to one of claims 1 to 8, wherein the yeast cream used at step a) contains a selenium-enriched yeast.

10. Use of a yeast protein extract having a true protein content of at least 72 %, obtained with the method of one of claims 1 to 9, in human nutrition.

11. The use according to claim 10 as a food supplement for weight control, for the elderly, or for sportspersons.

12. The use according to claim 10 as supply of non-animal protein in beverages, breadmaking products or plant-based deli meats.

13. The use according to claim 10 in oral or enteral clinical nutrition.

14. Use of a yeast protein extract having a true protein content of at least 72 %, obtained with the method of one of claims 1 to 9, in animal nutrition.