Low-nucleic-acid yeast protein as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microorganism application, in particular to low-nucleic-acid yeast protein as well as a preparation method and application thereof. The preparation method of the low-nucleic-acid yeast protein comprises the following steps: (1) carrying out inactivation treatment on high-protein yeast; (2) carrying out enzymolysis on the inactivated yeast by adopting neutral protease, glucanase, mannase and first nuclease, and after enzyme deactivation, separating and taking a heavy phase to obtain crude yeast protein; (3) performing high-pressure homogenization treatment on the crude yeast protein; and (4) carrying out enzymolysis on the crude yeast protein subjected to high-pressure homogenization treatment by adopting second nuclease and lipase, and after enzyme deactivation, separating and taking a heavy phase to obtain the low-nucleic-acid yeast protein. The yeast protein with the nucleic acid content of 1.0% or below is obtained by taking the high-protein yeast as a raw material through inactivation, compound enzyme enzymolysis, high-pressure homogenization treatment and secondary enzymolysis, is neutral and pure in taste, and can be widely applied to the fields of nutrition, health and food such as protein supplementation and protein substitution.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, and in particular to a low-nucleic acid yeast protein and a preparation method and application thereof. Background Art

[0002] Food protein is a crucial nutrient for humans, playing a role in building and repairing tissue, transporting various substances, maintaining nervous system function, and providing energy. Therefore, adequate and high-quality protein intake is crucial for good health. The search for sustainable protein alternatives has become a hot topic in the current market. Yeast, the most widely used single-celled fungus, is an excellent source of high-quality, complete protein. Yeast protein not only offers excellent nutritional value but also possesses excellent functional properties, such as muscle building and immune regulation. Therefore, it holds broad application prospects in a variety of fields, including food, medicine, and animal feed.

[0003] The key factors affecting the effectiveness of protein extraction from yeast are: (1) the protein content must be increased as much as possible to meet the protein content requirements; (2) the yeast nucleic acid remaining in the yeast protein must be reduced as much as possible to avoid the problem of high uric acid and gout after consumption. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing low-nucleic acid yeast protein, which can increase the protein content in the yeast protein while reducing the nucleic acid content in the yeast protein, thereby improving the taste and flavor of the yeast protein.

[0005] To achieve the above object, the present invention provides the following technical solutions: Technical Solution 1: A method for preparing low-nucleic acid yeast protein, comprising the following steps: (1) Inactivation of high-protein yeast; (2) Using neutral protease, glucanase, mannanase and nuclease to enzymatically hydrolyze the inactivated yeast, and after inactivation of the enzyme, separate the heavy phase to obtain crude yeast protein; (3) High-pressure homogenization of crude yeast protein; (4) The crude yeast protein treated by high-pressure homogenization was enzymatically hydrolyzed using nuclease and lipase. After the enzymes were inactivated, the heavy phase was separated and the low nucleic acid yeast protein was obtained.

[0006] Technical Solution 2: The preparation method according to Technical Solution 1, wherein the protein content of the high-protein yeast is 45-75%.

[0007] Technical Solution 3: The preparation method according to Technical Solution 1 or 2, wherein the inactivation treatment in step (1) comprises preparing the high-protein yeast into a dispersion and heating it to 50-99°C for heat treatment, preferably the heat treatment temperature is 90-99°C, more preferably 95-98°C.

[0008] Technical Solution 4: The preparation method according to Technical Solution 3, wherein the pH of the heat treatment in step (1) is 5.0-9.0, preferably 8.0-9.0, more preferably 8.5-9.0; and / or, the heat treatment time is 0.5-2h; And / or, the concentration of the dispersion is 5-20wt%.

[0009] Technical Solution 5: The preparation method according to any one of Technical Solutions 1-4, wherein, based on the mass of the high-protein yeast, the amount of the neutral protease added in step (2) is 0.5-2‰, the amount of the glucanase added is 3-6‰, the amount of the mannanase added is 1-3‰, and the amount of the nuclease added is 1-3‰.

[0010] Technical Solution 6: The preparation method according to Technical Solution 5, wherein the amount of the neutral protease added is 1.2-2.0‰, preferably 1.4-1.6‰; and / or, The amount of glucanase added is 3-5‰, preferably 4-5‰; and / or, The added amount of the mannanase is 2-3‰; and / or, The added amount of the nuclease is 2-3‰.

[0011] Technical Solution 7: The preparation method according to any one of Technical Solutions 1-6, wherein the enzymatic activity of the neutral protease is 50,000-150,000 u / g; and / or, The enzymatic activity of the glucanase is 200-400u / ml; and / or, The mannanase has an enzyme activity of 150,000-250,000 u / g; and / or, The enzymatic activity of the nuclease is 400,000-600,000 u / g.

[0012] Technical Solution 8: The preparation method according to any one of Technical Solutions 1-7, wherein the temperature of the enzymatic hydrolysis in step (2) is 40-70°C, preferably 50-70°C, more preferably 60-70°C; and / or, the pH of the enzymatic hydrolysis is 4.0-8.0, preferably 6.0-8.0, more preferably 7.0-8.0; And / or, the enzymatic hydrolysis time is 5-20 h, preferably 12-20 h.

[0013] Technical Solution 9: The preparation method according to any one of Technical Solutions 1-8, wherein the pressure of the high-pressure homogenization treatment in step (3) is 50-200 MPa, preferably 100-200 MPa, and more preferably 150-200 MPa; Preferably, the high-pressure homogenization treatment time is 1-5 hours, preferably 1-3 hours, more preferably 1-2 hours; More preferably, the temperature of the high pressure homogenization treatment is 50-70°C.

[0014] Technical Solution 10: The preparation method according to any one of Technical Solutions 1-9, wherein step (3) comprises preparing the crude yeast protein into a dispersion before the high-pressure homogenization treatment.

[0015] Technical Solution 11: The preparation method according to Technical Solution 10, wherein the concentration of the yeast protein dispersion is 5-20wt%.

[0016] Technical Solution 12: The preparation method according to any one of Technical Solutions 1-11, wherein, based on the mass of crude yeast protein, the amount of the nuclease added in step (4) is 1-3‰, and the amount of the lipase added is 3-5‰.

[0017] Technical Solution 13: The preparation method according to Technical Solution 12, wherein the added amount of the nuclease is 2-3‰, and / or the added amount of the lipase is 3-4‰.

[0018] Technical Solution 14: The preparation method according to any one of Technical Solutions 1-13, wherein the enzymatic activity of the nuclease is 400,000-600,000 u / g, and / or the enzymatic activity of the lipase is 4,000-6,000 u / g.

[0019] Technical Solution 15: The preparation method according to any one of Technical Solutions 1-14, wherein the enzymatic hydrolysis temperature in step (4) is 40-70°C, preferably 50-70°C, and more preferably 65-70°C; Preferably, the enzymatic hydrolysis pH is 4.0-8.0, preferably 5.0-7.0, more preferably 6.5-7.0; More preferably, the enzymatic hydrolysis time is 1-10 h, preferably 3-5 h.

[0020] Technical Solution 16: The preparation method according to any one of Technical Solutions 1-15, wherein the enzyme inactivation in step (2) and step (4) both includes the step of heating the enzymatic hydrolyzate to 75-85°C and keeping it warm for 0.5-2h.

[0021] Technical Solution 17: The preparation method according to any one of Technical Solutions 1-16, wherein the preparation method further comprises the steps of preparing a dispersion of the heavy phase separated in step (4) and drying the dispersion.

[0022] Technical Solution 18: The preparation method according to Technical Solution 17, wherein the concentration of the dispersion of the heavy phase separated in step (4) is 20-30wt%.

[0023] Technical Solution 19: The preparation method according to Technical Solution 17 or 18, wherein the drying is spray drying.

[0024] Technical Solution 20: A low nucleic acid yeast protein, which is prepared by the preparation method of any one of the above technical solutions 1-19; wherein the protein content of the low nucleic acid yeast protein is greater than or equal to 70%; and / or, The nucleic acid content in the low nucleic acid yeast protein is less than or equal to 1%, and / or, The fat content in the low nucleic acid yeast protein is less than or equal to 6%.

[0025] Technical Solution 21: The low nucleic acid yeast protein according to Technical Solution 20, wherein the protein content of the low nucleic acid yeast protein is 75-95%, preferably 85-95%; and / or, The nucleic acid content of the low nucleic acid yeast protein is 0.1-0.8%, preferably 0.1-0.5%; and / or, The fat content of the low nucleic acid yeast protein is 2-6%, preferably 3-5%.

[0026] Technical Solution 22: The low nucleic acid yeast protein described in Technical Solution 19 or 20 is used in the food field.

[0027] Technical Solution 23: The application according to Technical Solution 22, wherein the application of the low nucleic acid yeast protein is in the field of protein supplementation or protein replacement.

[0028] Beneficial effects of the present invention: The present invention uses high-protein yeast as raw material and obtains a low-nucleic acid yeast protein through inactivation, complex enzyme hydrolysis, high-pressure homogenization treatment and secondary hydrolysis. The low-nucleic acid yeast protein has a protein content of more than 70% and a nucleic acid content of less than 1.0%. The taste is neutral and pure, and can be widely used in nutrition, health and food fields such as protein supplementation and protein replacement.

[0029] Strain deposit information: The yeast strain Saccharomyces cerevisiae FX-2 used in the present invention was deposited with the China Center for Type Culture Collection (CCTCC) on August 1, 2016, with the deposit number CCTCC NO: M2016418. The deposit address is: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072; Telephone: (027) 68752319. This strain is described in the patent publication number CN108220175A. DETAILED DESCRIPTION

[0030] To make the purpose, technical solutions, and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. The embodiments described below are part of the embodiments of the present invention, not all of them. In combination with the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] In this application, the terms "first," "second," "third," etc., are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first," "second," etc., are used only for non-exhaustive enumeration and description purposes and should not constitute a closed-ended limitation on quantity.

[0032] Herein, the terms "preferred," "better," and "excellent" are used solely to describe preferred implementations or examples and should not be construed as limiting the scope of protection of this application. If multiple "preferred" terms appear in a technical solution, each "preferred" term is considered independent unless otherwise specified and there are no contradictions or mutual constraints.

[0033] In this application, unless otherwise specified, the features or solutions corresponding to "and / or" include any one of two or more relevant listed items, and also include any and all combinations of the relevant listed items, wherein any and all combinations include any two relevant listed items, any more relevant listed items, or a combination of all relevant listed items. For example, "M and / or N" means a group consisting of M, N, and "a combination of M and N." Among them, "including M and / or N" can mean "including M, including N, and including M and N", and can also mean "including M, including N, or including M and N", which can be appropriately understood according to the sentence in which it is located.

[0034] In a first aspect, in a specific embodiment of the present application, the present application provides a method for preparing a low-nucleic acid yeast protein, characterized in that it comprises the following steps: (1) Inactivation of high-protein yeast; (2) enzymatically hydrolyzing the yeast cell wall by using neutral protease, glucanase, mannanase and first nuclease, inactivating the enzymes and separating the heavy phase to obtain crude yeast protein; (3) High-pressure homogenization of crude yeast protein; (4) The crude yeast protein after high-pressure homogenization is enzymatically hydrolyzed using a second nuclease and a lipase, and after inactivating the enzyme, the heavy phase is separated to obtain the low-nucleic acid yeast protein.

[0035] The present application adopts the method of inactivating high-protein yeast to denature the yeast, inactivate endogenous enzymes and make the cell membrane permeable, thereby releasing the soluble part, which can reduce the production of odor substances such as amino acids due to yeast autolysis; the yeast cells are ruptured by enzymatic hydrolysis, and the cell walls are enzymatically hydrolyzed into soluble glucans and manno-oligosaccharides, which are then removed by subsequent separation, thereby increasing the protein content and contributing to the release of nucleic acids. The crude yeast protein liquid obtained by the primary enzymatic separation is subjected to high-pressure homogenization to further open the protein structure, further exposing the residual nucleic acids and fats, and then enzymatic hydrolysis is carried out with nucleases and lipases to further reduce nucleic acids and remove fats.

[0036] In some embodiments of the present application, the inactivation treatment in step (1) comprises preparing the high-protein yeast into a dispersion and heating it to 50-99° C. for heat treatment, preferably for 0.5-2 h.

[0037] In some embodiments of the present application, the concentration of the high-protein yeast dispersion is 5-20 wt %. In some embodiments, the pH of the heat treatment is 5.0-9.0, preferably 8.0-9.0, which is conducive to the release of endogenous substances in the yeast, particularly yeast nucleic acids, and can reduce the nucleic acid content in the yeast protein, thereby preventing excessive uric acid in the human body after consumption. In some embodiments of the present application, the pH of the heat treatment can be 5.0-8.0, 5.0-7.5, 5.0-7.0, 5.0-6.5, 5.0-6.0, 8.0-9.0, or 8.5-9.0.

[0038] In some embodiments of the present application, the heat treatment temperature can be 50-70°C, 90-99°C, or 95-98°C. Higher heat treatment temperatures are more conducive to yeast autolysis and the release of nucleic acid. The present invention utilizes secondary enzymatic hydrolysis and homogenization to produce yeast protein with low nucleic acid content, virtually no yeast flavor, and no grittiness, even at lower temperatures.

[0039] In some embodiments of the present application, based on the mass of high-protein yeast, the amount of the neutral protease added in step (2) is 0.5-2‰, the amount of the glucanase added is 3-6‰, the amount of the mannanase added is 1-3‰, and the amount of the first nuclease added is 1-3‰.

[0040] In some embodiments, based on the mass of high-protein yeast, the amount of the neutral protease added in step (2) can be 0.5-2‰, 0.5-1.8‰, 0.5-1.6‰, 0.5-1.5‰, 0.5-1.4‰, 0.5-1.2‰, 0.5-1‰, 1-2‰, 1-1.5‰, 1.5-2‰, 1.4-2‰, 1.4-1.8‰ or 1.4-1.6‰. In some embodiments, the amount of the neutral protease added in step (2) can be 0.5‰, 1‰, 1.2‰, 1.4‰, 1.6‰, 1.8‰ or 2‰, or within a numerical range consisting of any two of the above specific values ​​as endpoints. It should be understood that in specific embodiments, any of the above ranges can be combined with any other ranges.

[0041] In some embodiments, the amount of the glucanase added in step (2) can be 3-6‰, 3-5‰, 3-4‰, 4-6‰, 4-5‰, or 5-6‰, based on the mass of the high-protein yeast. In some embodiments, the amount of the glucanase added in step (2) can be 3‰, 4‰, 5‰, or 6‰, or within a numerical range consisting of any two of the above specific values ​​as endpoints. It should be understood that, in specific embodiments, any of the above ranges can be combined with any other ranges.

[0042] In some embodiments, the amount of mannanase added in step (2) can be 1-3‰, 1-2‰, or 2-3‰, based on the mass of the high-protein yeast. In some embodiments, the amount of mannanase added in step (2) can be 1‰, 2‰, or 3‰, or within a numerical range consisting of any two of the above specific values ​​as endpoints. It should be understood that in specific embodiments, any of the above ranges can be combined with any other ranges.

[0043] In some embodiments, the amount of the first nuclease added in step (2) can be 1-3‰, 1-2‰, or 2-3‰, based on the mass of the high-protein yeast. In some embodiments, the amount of the nuclease added in step (2) can be 1‰, 2‰, or 3‰, or within a numerical range consisting of any two of the above specific values ​​as endpoints. It should be understood that in specific embodiments, any of the above ranges can be combined with any other ranges.

[0044] In some embodiments of the present application, the temperature of the enzymatic hydrolysis in step (2) is 40-70°C, preferably 50-70°C, and more preferably 60-70°C. In some embodiments, the enzymatic hydrolysis pH is 4.0-8.0, preferably 6.0-8.0, and more preferably 7.0-8.0. In some embodiments, the enzymatic hydrolysis time is 5-20 hours, preferably 10-20 hours, and more preferably 12-20 hours.

[0045] In some embodiments of the present application, step (3) includes preparing the crude yeast protein into a dispersion before the high-pressure homogenization treatment, and the concentration of the yeast protein dispersion is preferably 5-20 wt%.

[0046] In some embodiments of the present application, the pressure of the high-pressure homogenization treatment is 50-200 MPa, preferably 100-200 MPa, and more preferably 150-200 MPa. In some embodiments, the temperature of the high-pressure homogenization treatment is 50-70° C. In some embodiments, the time of the high-pressure homogenization treatment is 1-5 hours, preferably 1-3 hours, and more preferably 1-2 hours.

[0047] In some embodiments of the present application, based on the dry weight of crude yeast protein, the amount of the nuclease added in step (4) is 1-3‰ and the amount of the lipase added is 3-6‰.

[0048] In some embodiments, the amount of the second nuclease added in step (4) can be 1-3‰, 1-2‰, or 2-3‰, based on the dry weight of crude yeast protein. In some embodiments, the amount of the second nuclease added in step (4) can be 1‰, 2‰, or 3‰, or within a numerical range consisting of any two of the above specific values ​​as endpoints. It should be understood that in specific embodiments, any of the above ranges can be combined with any other ranges.

[0049] In some embodiments, the amount of lipase added in step (4) can be 3-6‰, 3-5‰, or 3-4‰, based on the mass of crude yeast protein. In some embodiments, the amount of nuclease added in step (4) can be 3‰, 4‰, 5‰, or 6‰, or within a numerical range consisting of any two of the above specific values ​​as endpoints. It should be understood that in specific embodiments, any of the above ranges can be combined with any other ranges.

[0050] The enzymatic activity ranges of the various enzymes used in the present invention can be as follows: the enzymatic activity of the neutral protease in step (2) can be 50,000-150,000 u / g, the enzymatic activity of the glucanase in step (2) can be 200-400 u / ml, the enzymatic activity of the mannanase in step (2) can be 150,000-250,000 u / g, the enzymatic activity of the first nuclease in step (2) can be 400,000-600,000 u / g. The enzymatic activity of the second nuclease in step (4) can be 400,000-600,000 u / g, and the enzymatic activity of the lipase in step (4) can be 4,000-6,000 u / g.

[0051] In some embodiments of the present application, the enzymatic hydrolysis temperature in step (4) is 40-70°C, preferably 50-70°C, and more preferably 65-70°C. In some embodiments, the enzymatic hydrolysis pH is 4.0-8.0, preferably 5.0-7.0, and more preferably 6.5-7.0. In some embodiments, the enzymatic hydrolysis time is 1-10h, preferably 3-5h. In some embodiments of the present application, the enzyme inactivation in step (2) and step (4) both include the step of heating the enzymatic hydrolysis solution to 75-85°C and keeping it warm for 0.5-2h.

[0052] In some embodiments of the present application, the preparation method further comprises the steps of preparing a dispersion of the heavy phase separated in step (4) and drying the dispersion; preferably, the concentration of the dispersion is 20-30 wt%, and more preferably, the drying is spray drying.

[0053] In a second aspect, the present application also provides a low-nucleic acid yeast protein produced by the above-described preparation method. In some embodiments, the low-nucleic acid yeast protein has a protein content greater than or equal to 70%, preferably 75-95%. In some embodiments, the nucleic acid content is less than or equal to 1%, preferably 0.1-0.8%. In some embodiments, the fat content is less than or equal to 6%.

[0054] In some embodiments of the present application, the nucleic acid content of the low nucleic acid yeast protein may be less than or equal to 1%, less than or equal to 0.8%, less than or equal to 0.6%, less than or equal to 0.4%, less than or equal to 0.35%, or less than or equal to 0.2%. In some embodiments, the nucleic acid content may be 0.1-1%, 0.1-0.8%, 0.1-0.7%, 0.1-0.6%, 0.1-0.5%, 0.1-0.4%, 0.1-0.35%, or 0.1-0.3%.

[0055] In some embodiments of the present application, the protein content in the low nucleic acid yeast protein may be greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 87%, greater than or equal to 89%, or greater than or equal to 91%. In some embodiments, the protein content may be 70-95%, 75-95%, 80-95%, 85-95%, 87-95%, 89-95%, or 91-95%.

[0056] In some embodiments of the present application, the fat content of the low nucleic acid yeast protein may be less than or equal to 6%, less than or equal to 4%, or less than or equal to 3.5%. In some embodiments, the fat content may be 2-6%, 2-5%, 2-4%, or 3-4%.

[0057] It should be noted that the term "high-protein yeast" in this application refers to yeast obtained by culturing a yeast strain with a protein content of greater than or equal to 45%. For example, the high-protein yeast in this application can be obtained by batch fermentation or continuous fermentation of a yeast strain using one or more of sugarcane molasses, hydrolyzed sugar or beet molasses as fermentation carbon sources, and its protein content can be 45-75%, preferably 65-75%.

[0058] Specifically, in the preparation method of low-nucleic acid yeast protein of the present application, there is no particular restriction on the type of yeast strain used to obtain high-protein yeast. For example, it can be Saccharomyces cerevisiae, Kluyveromyces, Pichia pastoris or Candida albicans purchased commercially or obtained by any method, preferably Saccharomyces cerevisiae, which can be Saccharomyces cerevisiae FX-2, wherein Saccharomyces cerevisiae FX-2 was deposited in the China Center for Type Culture Collection (CCTCC) on August 1, 2016, with the deposit number CCTCC NO: M2016418.

[0059] Furthermore, the culture conditions required for obtaining high-protein yeast in the present application are not particularly limited, as long as the protein content can reach or exceed 45%. For example, the yeast can be obtained by the method disclosed in Chinese Patent CN108220175A. Specifically, the high-protein yeast in the present application can be obtained by the following steps: b. Seed culture: Perform primary and secondary seed culture on the strains stored on the slant; c. Seed enrichment: centrifugation, washing, and enrichment to obtain Saccharomyces cerevisiae seeds; d. Fermentation: The Saccharomyces cerevisiae seeds from step c are inoculated into a fermentation tank containing water and a phosphorus source for fermentation. The fermentation tank has an aeration ratio of 1.6-2.5 VVM and a dissolved oxygen volume of 45-70%. The physiological state of the bacteria is monitored online in real time, and the feeding rate is regulated based on the real-time monitored physiological parameters, including carbon dioxide evolution rate (CER), oxygen uptake rate (OUR), and respiratory quotient (RQ). No additional inorganic salts, acids, bases, or trace elements are required during the fermentation control process, except for feeding. e. After fermentation is completed, separate and wash the yeast cells and collect them.

[0060] Wherein, the feed added during the fermentation regulation process in step d is molasses and a nitrogen source.

[0061] In some embodiments, the method for preparing high-protein yeast of the present application further comprises: a. Fermentation raw material pretreatment: molasses is precipitated and separated to prepare molasses with a total sugar content of 25-35%; In some embodiments, the pretreatment of the fermentation feedstock further comprises the steps of adding water to the nitrogen source to prepare a solution with a concentration of 20-25%; and sterilizing the treated molasses and nitrogen source solution.

[0062] In some embodiments, the primary seed culture comprises the following steps: inoculating the bacterial strain into a primary culture medium and culturing at 28-32° C. and a pH of 4.0-5.5 for 15-25 hours. In some embodiments, the primary culture medium comprises, by weight, 1-2% yeast extract, 2-3% peptone, and 2-3% glucose.

[0063] In some embodiments, the secondary seed culture comprises the following steps: inoculating the primary cultured seeds into a secondary culture medium and culturing at 28-32°C and a pH of 4.0-5.5 for 15-25 hours. In some embodiments, the secondary culture medium comprises, by weight, 2-3% glucose, 2-3% yeast extract, 0.1-0.2% potassium dihydrogen phosphate, and 0.1-0.2% dipotassium hydrogen phosphate.

[0064] In some embodiments, the fermentation temperature in step d is 25-35° C., the fermentation time is 10-20 h, and the fermentation pH is 4.0-7.0. In some embodiments, the phosphorus source is added in an amount of 0.1-0.5% by weight of the fermentation bottom water.

[0065] In a third aspect, the present application also provides the application of the low nucleic acid yeast protein in the food field, preferably in protein supplementation and protein replacement.

[0066] The beneficial effects of the present invention are further illustrated below through specific examples.

[0067] The raw materials and reagents used in the present invention are all purchased from mainstream manufacturers in the market. Those without manufacturer or concentration are all analytically pure raw materials and reagents that can be obtained routinely. As long as they can play the expected role, there are no special restrictions.

[0068] If no specific technology or conditions are specified in this embodiment, the technology or conditions described in the literature in this field or the product instructions shall be followed.

[0069] Hereinafter, the present invention will be described in more detail using Examples and Comparative Examples, but the technical scope of the present invention is not limited to these Examples. It should be noted that, unless otherwise specified, all percentages, parts, and ratios used in the present invention are based on mass.

[0070] The sources of the reagents and instruments used in the following examples are shown in Table 1.

[0071]

[0072] Example 1 1. Prepare high-protein yeast, The preparation steps of high protein yeast include: a. Fermentation Feedstock Processing: Remove the molasses residue and precipitate. Separate the molasses supernatant and adjust the concentration to 30% by mass as a carbon source. Sterilize the treated molasses at 121°C for 20 minutes. Dilute the ammonia solution with sterile water to a nitrogen source with a nitrogen concentration of 20%.

[0073] b. Seed culture: For primary seed culture, a loopful of cells (Saccharomyces cerevisiae FX-2) was inoculated from the culture storage slant into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 hours. For secondary seed culture, 10% of the volume of the secondary culture medium was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 hours. The primary culture medium consisted of 1% yeast extract, 2% peptone, and 2% glucose, with the remainder being water. The secondary culture medium consisted of 2% glucose, 2% yeast extract powder, 0.1% potassium dihydrogen phosphate, and 0.1% dipotassium hydrogen phosphate, with the remainder being water. The pH of both primary and secondary cultures was controlled between 4.0 and 5.5.

[0074] c. Seed enrichment: Centrifuge and wash three times with deionized water low in calcium and magnesium ions to obtain Saccharomyces cerevisiae seeds with a wet yeast weight of 400 g / L.

[0075] d. Fermentation: Add 45 L of fermentation bottom water and 0.2% monoammonium phosphate (based on the weight of the fermentation bottom water) to a 100 L fermentor and sterilize at 121°C for 20 min. Inoculate the fermentor with the seeds enriched in step c at a 10% volume inoculum. Fermentation was performed at 30°C under normal pressure with an aeration ratio of 1.667 VVM. The dissolved oxygen volume was controlled at 50% by volume by adjusting the rotational speed. During the fermentation process, the physiological parameters RQ, CER, and OUR were monitored online in real time by a biogas analyzer. The online parameter RQ value was maintained at a small fluctuation between 1.0 and 1.2. When RQ was greater than 1.2, the flow rates of molasses and ammonia water were simultaneously reduced to lower the RQ value. When RQ was less than 1.0, the flow rates of molasses and ammonia water were simultaneously increased to increase the RQ value. During the entire regulation process, the flow rate of molasses was controlled in the range of 10-50 mL / min, the flow rate of the nitrogen source was controlled in the range of 1-5 mL / min, and the pH of the entire fermentation process was controlled at 4.2-6.5. e. Fermentation Broth Isolation: After 12 hours of fermentation, the fermented broth was centrifuged in a Beckman centrifuge at 5000 rpm and 15°C for 10 minutes to harvest the yeast phase. The yeast was then washed with deionized water and washed again. The high-protein yeast was harvested. The protein content was measured to be 65.3%.

[0076] 2. Preparation of Low Nuclease Yeast Protein (1) High-protein yeast solution: 200 g (dry) of the high-protein yeast obtained by fermentation in step (1) was prepared with deionized water to make a 2000 g solution, so that the yeast mass fraction was 10%. The pH was adjusted to 6.2 with sodium hydroxide solution and the yeast was inactivated at 75°C for 60 minutes, with stirring maintained throughout the process.

[0077] (2) The yeast emulsion obtained in step (1) was cooled to 60°C, adjusted to pH 5.7 with citric acid, and 1‰ neutral protease, 5‰ glucanase, 2‰ mannanase, and 2‰ nuclease were added based on the mass of the high-protein yeast. The enzymatic hydrolysis was carried out for 15 hours, with stirring maintained throughout the entire process. The mixture was then heated to 80°C to inactivate the enzymes for 30 minutes, centrifuged at 5000 r / min for 10 minutes, and the supernatant was removed. The separated heavy phase was prepared into a crude yeast protein dispersion with deionized water at a concentration of 18 wt%.

[0078] (3) The crude yeast protein dispersion was subjected to high-pressure homogenization treatment, with the temperature controlled at 60°C, the pressure at 50 MPa, and the homogenization time being 5 minutes.

[0079] (4) After homogenization, maintain the temperature at 60°C, adjust the pH to 5.5 with citric acid, add 2‰ nuclease and 5‰ lipase according to the dry matter weight of the crude yeast protein dispersion, and allow to act for 5 hours. Keep stirring during the whole process; then heat to 80°C to inactivate the enzyme for 30 minutes, centrifuge at 5000 r / min for 10 minutes, remove the supernatant, and use deionized water to prepare a 25wt% yeast protein solution with the separated heavy phase.

[0080] (5) Spray dry the yeast protein solution to make its moisture content less than 5%, the passing rate of 100 mesh screen is 100%, the inlet air temperature is 100-190℃, and the outlet air temperature is 50-120℃.

[0081] Example 2 1. Preparation of high-protein yeast The preparation steps of high protein yeast include: a. Fermentation Feedstock Processing: Remove the molasses residue and precipitate. Separate the molasses supernatant and adjust the concentration to 30% by mass as a carbon source. Sterilize the treated molasses at 121°C for 20 minutes. Dilute the ammonia solution with sterile water to a nitrogen source with a nitrogen concentration of 20%.

[0082] b. Seed culture: For primary seed culture, a loopful of cells (Saccharomyces cerevisiae FX-2) was inoculated from the culture storage slant into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 hours. For secondary seed culture, 10% of the volume of the secondary culture medium was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 hours. The primary culture medium consisted of 1% yeast extract, 2% peptone, and 2% glucose, with the remainder being water. The secondary culture medium consisted of 2% glucose, 2% yeast extract powder, 0.1% potassium dihydrogen phosphate, and 0.1% dipotassium hydrogen phosphate, with the remainder being water. The pH of both primary and secondary cultures was controlled between 4.0 and 5.5.

[0083] c. Seed enrichment: Centrifuge and wash three times with deionized water low in calcium and magnesium ions to obtain Saccharomyces cerevisiae seeds with a wet yeast weight of 400 g / L.

[0084] d. Fermentation: 45 L of fermentation bottom water and 1 / 1000th of monoammonium phosphate (based on the weight of the fermentation bottom water) were added to a 100 L fermentor and sterilized at 121°C for 20 min. The seeds enriched in step c were inoculated at a 10% volume ratio into the fermentor. The temperature was maintained at 28°C, and fermentation was carried out under normal pressure with an aeration ratio of 1.6 VVM. The dissolved oxygen volume was controlled at 45% by volume by adjusting the rotational speed. During the fermentation process, the physiological parameters RQ, CER, and OUR were monitored online in real time by a biogas analyzer. The online parameter RQ value was maintained at a small fluctuation between 1.0 and 1.2. When RQ was greater than 1.2, the flow rates of molasses and ammonia water were simultaneously reduced to lower the RQ value. When RQ was less than 1.0, the flow rates of molasses and ammonia water were simultaneously increased to increase the RQ value. During the entire regulation process, the flow rate of molasses was controlled in the range of 10-40 mL / min, the flow rate of the nitrogen source was controlled in the range of 1-3 mL / min, and the pH of the entire fermentation process was controlled at 4.2-6.0. e. Fermentation Broth Isolation: After 16 hours of fermentation, the fermented broth was centrifuged in a Beckman centrifuge at 5000 rpm and 15°C for 10 minutes to harvest the yeast phase. The yeast was then washed with deionized water and washed again. The high-protein yeast was harvested. The protein content was measured to be 51.6%.

[0085] 2. Preparation of Low Nuclease Yeast Protein The experiment was carried out according to the method in Example 1, except that the process parameters were different. The process parameters of Example 2 are shown in Table 2, and the rest are the same as in Example 1.

[0086] Example 3 1. Preparation of high-protein yeast The preparation steps of high protein yeast include: a. Fermentation Feedstock Processing: Remove the molasses residue and precipitate. Separate the molasses supernatant and adjust the concentration to 30% by mass as a carbon source. Sterilize the treated molasses at 121°C for 20 minutes. Dilute the ammonia solution with sterile water to a nitrogen source with a nitrogen concentration of 20%.

[0087] b. Seed culture: For primary seed culture, a loopful of cells (Saccharomyces cerevisiae FX-2) was inoculated from the culture storage slant into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 hours. For secondary seed culture, 10% of the volume of the secondary culture medium was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 hours. The primary culture medium consisted of 1% yeast extract, 2% peptone, and 2% glucose, with the remainder being water. The secondary culture medium consisted of 2% glucose, 2% yeast extract powder, 0.1% potassium dihydrogen phosphate, and 0.1% dipotassium hydrogen phosphate, with the remainder being water. The pH of the primary and secondary cultures was controlled between 4.0 and 5.5.

[0088] c. Seed enrichment: Centrifuge and wash three times with deionized water low in calcium and magnesium ions to obtain Saccharomyces cerevisiae seeds with a wet yeast weight of 400 g / L.

[0089] d. Fermentation: Add 45 L of fermentation base water and 1 / 1000th of monoammonium phosphate (based on the weight of the fermentation base water) to a 100 L fermenter and sterilize at 121°C for 20 min. Inoculate the enriched seeds into the fermenter at a 10% volume inoculum. Maintain the temperature at 29°C, ferment under normal pressure, and maintain an aeration ratio of 1.63 VVM. Adjust the rotational speed to maintain the dissolved oxygen volume at 46%. During the fermentation process, the physiological parameters RQ, CER, and OUR were monitored online in real time by a biogas analyzer. The online parameter RQ value was maintained at a small fluctuation between 1.0 and 1.2. When RQ was greater than 1.2, the flow rates of molasses and ammonia water were simultaneously reduced to lower the RQ value. When RQ was less than 1.0, the flow rates of molasses and ammonia water were simultaneously increased to increase the RQ value. During the entire regulation process, the flow rate of molasses was controlled in the range of 10-40 mL / min, the flow rate of the nitrogen source was controlled in the range of 1-3 mL / min, and the pH of the entire fermentation process was controlled at 4.2-6.0. e. Fermentation Broth Isolation: After 15 hours of fermentation, the fermented broth was centrifuged in a Beckman centrifuge at 5000 rpm and 15°C for 10 minutes to harvest the yeast phase. The yeast was then washed with deionized water and washed again. The high-protein yeast was harvested. The protein content was measured to be 55.8%.

[0090] 2. Preparation of Low Nuclease Yeast Protein The experiment was carried out according to the method in Example 1, except that the process parameters were different. The process parameters of Example 3 were as shown in Table 2, and the rest were the same as in Example 1.

[0091] Example 4 1. Preparation of high-protein yeast The preparation steps of high protein yeast include: a. Fermentation Feedstock Processing: Remove the molasses residue and precipitate. Separate the molasses supernatant and adjust the concentration to 30% by mass as a carbon source. Sterilize the treated molasses at 121°C for 20 minutes. Dilute the ammonia solution with sterile water to a nitrogen source with a nitrogen concentration of 20%.

[0092] b. Seed culture: For primary seed culture, a loopful of cells (Saccharomyces cerevisiae FX-2) was inoculated from the culture storage slant into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 hours. For secondary seed culture, 10% of the volume of the secondary culture medium was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 hours. The primary culture medium consisted of 1% yeast extract, 2% peptone, and 2% glucose, with the remainder being water. The secondary culture medium consisted of 2% glucose, 2% yeast extract powder, 0.1% potassium dihydrogen phosphate, and 0.1% dipotassium hydrogen phosphate, with the remainder being water. The pH of the primary and secondary cultures was controlled between 4.0 and 5.5.

[0093] c. Seed enrichment: Centrifuge and wash three times with deionized water low in calcium and magnesium ions to obtain Saccharomyces cerevisiae seeds with a wet yeast weight of 400 g / L.

[0094] d. Fermentation: Add 45 L of fermentation base water and 0.2% monoammonium phosphate (based on the weight of the fermentation base water) to a 100 L fermenter and sterilize at 121°C for 20 min. Inoculate the enriched seeds into the fermenter at a 10% volume inoculum. Maintain the temperature at 30°C, ferment under normal pressure, and maintain an aeration ratio of 1.65 VVM. Adjust the rotational speed to maintain the dissolved oxygen volume at 50%. During the fermentation process, the physiological parameters RQ, CER, and OUR were monitored online in real time by a biogas analyzer. The online parameter RQ value was maintained at a small fluctuation between 1.0 and 1.2. When RQ was greater than 1.2, the flow rates of molasses and ammonia water were simultaneously reduced to lower the RQ value. When RQ was less than 1.0, the flow rates of molasses and ammonia water were simultaneously increased to increase the RQ value. During the entire regulation process, the flow rate of molasses was controlled in the range of 10-50 mL / min, the flow rate of the nitrogen source was controlled in the range of 1-4 mL / min, and the pH of the entire fermentation process was controlled at 4.2-6.5. e. Fermentation Broth Separation: After 13 hours of fermentation, the fermented broth was centrifuged in a Beckman centrifuge at 5000 rpm and 15°C for 10 minutes to harvest the yeast phase. This was then washed with deionized water, and the washing was repeated once to harvest the high-protein yeast. The protein content of the high-protein yeast was measured to be 60.3%.

[0095] 2. Preparation of Low Nuclease Yeast Protein The experiment was carried out according to the method in Example 1, except that the process parameters were different. The process parameters of Example 4 were as shown in Table 2, and the rest were the same as in Example 1.

[0096] Example 5 1. Preparation of high-protein yeast The preparation steps of high protein yeast include: a. Fermentation Feedstock Processing: Remove the molasses residue and precipitate. Separate the molasses supernatant and adjust the concentration to 30% by mass as a carbon source. Sterilize the treated molasses at 121°C for 20 minutes. Dilute the ammonia solution with sterile water to a nitrogen source with a nitrogen concentration of 20%.

[0097] b. Seed culture: For primary seed culture, a loopful of cells (Saccharomyces cerevisiae FX-2) was inoculated from the culture storage slant into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 hours. For secondary seed culture, 10% of the volume of the secondary culture medium was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 hours. The primary culture medium consisted of 1% yeast extract, 2% peptone, and 2% glucose, with the remainder being water. The secondary culture medium consisted of 2% glucose, 2% yeast extract powder, 0.1% potassium dihydrogen phosphate, and 0.1% dipotassium hydrogen phosphate, with the remainder being water. The pH of the primary and secondary cultures was controlled between 4.0 and 5.5.

[0098] c. Seed enrichment: Centrifuge and wash three times with deionized water low in calcium and magnesium ions to obtain Saccharomyces cerevisiae seeds with a wet yeast weight of 400 g / L.

[0099] d. Fermentation: Place 45 L of fermentation base water and 0.2% monoammonium phosphate (based on the weight of the fermentation base water) in a 100 L fermenter and sterilize at 121°C for 20 min. Inoculate the enriched seeds into the fermenter at a 10% volume inoculum. Maintain the temperature at 31°C, ferment under normal pressure, and maintain an aeration ratio of 1.7 VVM. Adjust the rotational speed to maintain the dissolved oxygen volume at 52%. During the fermentation process, the physiological parameters RQ, CER, and OUR were monitored online in real time by a biogas analyzer. The online parameter RQ value was maintained at a small fluctuation between 1.0 and 1.2. When RQ was greater than 1.2, the flow rates of molasses and ammonia water were simultaneously reduced to lower the RQ value. When RQ was less than 1.0, the flow rates of molasses and ammonia water were simultaneously increased to increase the RQ value. During the entire regulation process, the flow rate of molasses was controlled in the range of 10-50 mL / min, the flow rate of the nitrogen source was controlled in the range of 1-5 mL / min, and the pH of the entire fermentation process was controlled at 4.2-6.8. e. Fermentation broth separation: After 12 hours of fermentation, the fermented broth was centrifuged in a Beckman centrifuge at 5000 rpm and 15°C for 10 minutes to harvest the yeast phase. The yeast was then washed with deionized water, and the washing was repeated once to harvest the high-protein yeast. The protein content of the high-protein yeast was measured to be 66.2%.

[0100] 2. Preparation of Low Nuclease Yeast Protein The experiment was carried out according to the method in Example 1, except that the process parameters were different. The process parameters of Example 5 are shown in Table 2, and the rest are the same as in Example 1.

[0101] Example 6 1. Preparation of high-protein yeast The preparation steps of high protein yeast include: a. Fermentation Feedstock Processing: Remove the molasses residue and precipitate. Separate the molasses supernatant and adjust the concentration to 30% by mass as a carbon source. Sterilize the treated molasses at 121°C for 20 minutes. Dilute the ammonia solution with sterile water to a nitrogen source with a nitrogen concentration of 20%.

[0102] b. Seed culture: For primary seed culture, a loopful of cells (Saccharomyces cerevisiae FX-2) was inoculated from the culture storage slant into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 hours. For secondary seed culture, 10% of the volume of the secondary culture medium was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 hours. The primary culture medium consisted of 1% yeast extract, 2% peptone, and 2% glucose, with the remainder being water. The secondary culture medium consisted of 2% glucose, 2% yeast extract powder, 0.1% potassium dihydrogen phosphate, and 0.1% dipotassium hydrogen phosphate, with the remainder being water. The pH of the primary and secondary cultures was controlled between 4.0 and 5.5.

[0103] c. Seed enrichment: Centrifuge and wash three times with deionized water low in calcium and magnesium ions to obtain Saccharomyces cerevisiae seeds with a wet yeast weight of 400 g / L.

[0104] d. Fermentation: Add 45 L of fermentation base water and 0.3% monoammonium phosphate (based on the weight of the fermentation base water) to a 100 L fermenter and sterilize at 121°C for 20 min. Inoculate the enriched seeds into the fermenter at a 10% volume inoculum. Maintain the temperature at 32°C, ferment under normal pressure, and maintain an aeration ratio of 1.8 VVM. Adjust the rotational speed to maintain the dissolved oxygen volume at 60%. During the fermentation process, the physiological parameters RQ, CER, and OUR were monitored online in real time by a biogas analyzer. The online parameter RQ value was maintained at a small fluctuation between 1.0 and 1.2. When RQ was greater than 1.2, the flow rates of molasses and ammonia water were simultaneously reduced to lower the RQ value. When RQ was less than 1.0, the flow rates of molasses and ammonia water were simultaneously increased to increase the RQ value. During the entire regulation process, the flow rate of molasses was controlled in the range of 10-50 mL / min, the flow rate of the nitrogen source was controlled in the range of 1-5 mL / min, and the pH of the entire fermentation process was controlled at 4.2-6.8. e. Fermentation Broth Isolation: After 12 hours of fermentation, the fermented broth was centrifuged in a Beckman centrifuge at 6000 rpm and 15°C for 20 minutes to harvest the yeast phase. The yeast was then washed with deionized water and washed again. The high-protein yeast was harvested. The protein content was measured to be 69.8%.

[0105] 2. Preparation of Low Nuclease Yeast Protein The experiment was carried out according to the method in Example 1, except that the process parameters were different. The process parameters of Example 6 were as shown in Table 2, and the rest were the same as in Example 1.

[0106] Example 7 1. Preparation of high-protein yeast The preparation steps of high protein yeast include: a. Fermentation Feedstock Processing: Remove the molasses residue and precipitate. Separate the molasses supernatant and adjust the concentration to 30% by mass as a carbon source. Sterilize the treated molasses at 121°C for 20 minutes. Dilute the ammonia solution with sterile water to a nitrogen source with a nitrogen concentration of 20%.

[0107] b. Seed culture: For primary seed culture, a loopful of cells (Saccharomyces cerevisiae FX-2) was inoculated from the culture storage slant into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 hours. For secondary seed culture, 10% of the volume of the secondary culture medium was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 hours. The primary culture medium consisted of 1% yeast extract, 2% peptone, and 2% glucose, with the remainder being water. The secondary culture medium consisted of 2% glucose, 2% yeast extract powder, 0.1% potassium dihydrogen phosphate, and 0.1% dipotassium hydrogen phosphate, with the remainder being water. The pH of the primary and secondary cultures was controlled between 4.0 and 5.5.

[0108] c. Seed enrichment: Centrifuge and wash three times with deionized water low in calcium and magnesium ions to obtain Saccharomyces cerevisiae seeds with a wet yeast weight of 400 g / L.

[0109] d. Fermentation: 45L of fermentation bottom water and 0.3% monoammonium phosphate were placed in a 100L fermenter and sterilized at 121°C for 20 minutes. The enriched seeds were inoculated into the fermenter at a volume ratio of 10%. The temperature was controlled at 33°C, and fermentation was carried out under normal pressure with an aeration ratio of 2.0VVM. The dissolved oxygen volume was controlled at 70% by adjusting the rotational speed. During the fermentation process, the physiological parameters RQ, CER, and OUR were monitored online in real time by a biogas analyzer. The online parameter RQ value was maintained at a small fluctuation between 1.0 and 1.2. When RQ was greater than 1.2, the flow rates of molasses and ammonia water were simultaneously reduced to lower the RQ value. When RQ was less than 1.0, the flow rates of molasses and ammonia water were simultaneously increased to increase the RQ value. During the entire regulation process, the flow rate of molasses was controlled within the range of 10-50mL / min, and the flow rate of the nitrogen source was controlled within the range of 1-5mL / min. The pH of the entire fermentation process was controlled between 4.2 and 6.8. e. Fermentation Broth Separation: After 11 hours of fermentation, the fermented broth was centrifuged in a Beckman centrifuge at 5000 rpm and 15°C for 10 minutes to harvest the bacterial phase. The broth was then washed with deionized water, and the washing was repeated once to harvest the bacterial cells. The protein content of the high-protein yeast was measured to be 73.7%.

[0110] 2. Preparation of Low Nuclease Yeast Protein The experiment was carried out according to the method in Example 1, except that the process parameters were different. The process parameters of Example 7 are shown in Table 2, and the rest are the same as in Example 1.

[0111]

[0112]

[0113] Comparative Example 1 The difference from Example 1 is that step (2) does not contain neutral protease, and only 5‰ of glucanase, 2‰ of mannanase and 2‰ of nuclease are added for enzymatic hydrolysis.

[0114] Comparative Example 2 The difference from Example 1 is that step (2) does not contain nuclease, and only 1‰ neutral protease, 5‰ glucanase and 2‰ mannanase are added for enzymatic hydrolysis.

[0115] Comparative Example 3 The difference from Example 1 is that step (2) does not contain nuclease and neutral protease, and only 5‰ of meso-glucanase and 2‰ of mannanase are added for enzymatic hydrolysis.

[0116] Comparative Example 4 The difference from Example 1 is that high-pressure homogenization treatment is not used, that is, the crude yeast protein dispersion obtained in step (2) is directly subjected to nuclease and lipase enzymolysis.

[0117] Comparative Example 5 The difference from Example 1 is that nuclease and lipase are not used to enzymatically hydrolyze the homogenized yeast protein, and the homogenized yeast protein is directly spray-dried.

[0118] Comparative Example 6 (1) High-protein yeast solution: 200 g (dry basis) of high-protein yeast with a protein content of 65.3% was mixed with deionized water to make a 2000 g solution (wherein, yeast milk was prepared according to the same method as in Example 1) so that the yeast mass fraction was 10%. The pH was adjusted to 6.2 with sodium hydroxide solution and the yeast was broken at 75°C for 60 minutes, with stirring maintained throughout the process.

[0119] (2) The broken yeast emulsion obtained in step (1) was cooled to 60°C, adjusted to pH 5.7 with citric acid, and 5‰ of glucanase, 2‰ of mannanase, and 5‰ of lipase were added based on the mass of the high-protein yeast. The enzymatic hydrolysis was carried out for 15 hours, with stirring maintained throughout the entire process. The mixture was then heated to 80°C to inactivate the enzymes for 30 minutes, centrifuged at 5000 r / min for 10 minutes, and the supernatant was removed. The separated heavy phase was prepared into a crude yeast protein dispersion with deionized water at a concentration of 18 wt%.

[0120] (5) Spray dry the crude yeast protein solution to make its moisture content less than 5%, the passing rate of 100 mesh screen 100%, the inlet air temperature, and the outlet air temperature.

[0121] The protein content, fat content and nucleic acid content in yeast were determined by the following method. The results are shown in Table 3.

[0122] 1) Determination of protein content The protein content was determined according to the method for determination of protein in foods in GB 5009.5-2016. The specific operation was as follows: 1 g of sample was added to 20 mL of concentrated sulfuric acid for digestion under the action of a mixed catalyst (0.4 g of copper sulfate pentahydrate + 6 g of potassium sulfate), followed by distillation. The product nitrogen was absorbed by boric acid, and then titrated with 0.1 mol / L hydrochloric acid. The data was read and the nitrogen content was calculated. The protein coefficient was N (nitrogen content) × 6.25 to calculate the protein content.

[0123] 2) Determination of fat content The fat content was determined according to the method for determination of fat in foods in GB 5009.6-2016. The specific procedure was as follows: 2-5 g of sample was weighed and placed in a filter paper tube. The filter paper tube was placed in a Soxhlet extractor and extracted with ether or petroleum ether. The ether or petroleum ether in the receiving bottle was then recovered, dried, and weighed to calculate the fat content.

[0124] 3) Determination of nucleic acid content: The nucleic acid content was determined according to the method for detecting nucleic acid in yeast. The specific method is as follows: A. Weigh approximately 0.500-0.800 g (accurate to 0.001 g) of yeast protein sample into a centrifuge tube. Add 8 mL of 0.25 mol / L perchloric acid solution, chilled at 4°C, to the tube and vortex thoroughly. Immediately place the tube in a 4°C water bath for 15 minutes without shaking. Centrifuge at 4000 rpm for 10 minutes, aspirate and discard the supernatant. Add 5 mL of 0.5 mol / L perchloric acid solution to the precipitate and vortex to mix thoroughly. Place the tube in a 70°C water bath and keep warm for 15 minutes, vortexing every 3-4 minutes. Centrifuge at 4000 rpm for 10 minutes, aspirate 1 mL of the supernatant, dilute to 100 mL with distilled water, and mix thoroughly.

[0125] B. Preparation of RNA Standard Solution: Accurately weigh approximately 10.5 mg (accurate to 0.1 mg) of RNA standard solution, add 5 mL of 0.5 mol / L perchloric acid solution, and vortex to mix thoroughly. Place the centrifuge tube in a 70°C waterbath and incubate for 15 minutes, vortexing every 3-4 minutes. Centrifuge at 4000 rpm for 10 minutes, aspirate 1 mL of the supernatant, dilute to 100 mL with distilled water, and mix thoroughly. Accurately pipette 3.5 mL, 4 mL, 5 mL, 6 mL, 7.5 mL, and 10 mL of the RNA standard solution into a 10 mL brown volumetric flask, and dilute to the mark with water. The resulting RNA standard working solutions have concentrations of 7.0 μg / mL, 8.0 μg / mL, 10.0 μg / mL, 12.0 μg / mL, 15.0 μg / mL, and 20.0 μg / mL, respectively. Add each of the above RNA standard working solutions to a UV spectrophotometer, using distilled water as a blank, and measure the corresponding absorbance at 260 nm. Plot a standard curve with absorbance as the y-axis and the standard working solution concentration as the x-axis, and calculate the linear regression equation.

[0126] C. Rinse the cuvette with the test solution obtained in step A. Then, fill the cuvette and place it in a UV spectrophotometer. Measure the absorbance at 260 nm, using distilled water as a blank. Record the absorbance, repeat the measurement once, and average the two measurements. Note: The sample absorbance should be between 0.2 and 0.8. This can be controlled by weighing the sample or by the dilution factor.

[0127] Specifically, the test results of various indicators in the low nucleic acid yeast protein are shown in Table 3 below.

[0128]

[0129] As shown in Table 3, the yeast proteins prepared in Examples 1-7 had protein contents ranging from 75.3% to 91.2%, all exceeding 70%; fat contents ranging from 3.1% to 5.8%, all less than 6%; and nucleic acid contents ranging from 0.28% to 0.76%, all less than 0.8%. Specifically, the yeast proteins prepared in Examples 6-7 had protein contents ranging from 89.8% to 91.2%, nucleic acid contents of only 0.28% to 0.31%, and fat contents as low as 3.1% to 3.5%.

[0130] Compared to Example 1, in Comparative Example 3, where no neutral protease and nuclease were added during the first enzymatic hydrolysis, the resulting yeast protein had a nucleic acid content of 1.98%. In Comparative Example 1, where no neutral protease was added during the first enzymatic hydrolysis, the resulting yeast protein had a nucleic acid content of 1.68%. In Comparative Example 2, where no nuclease was added during the first enzymatic hydrolysis, the resulting yeast protein had a nucleic acid content of 2.32%. In Example 1, where both neutral protease and nuclease were used in conjunction with glucanase and mannooligosaccharidase for enzymatic hydrolysis, the resulting yeast protein had a nucleic acid content of 0.51%. This indicates that the synergistic effect of neutral protease and nuclease significantly reduces the nucleic acid content in yeast protein.

[0131] Compared to Example 1, Comparative Example 4, which did not undergo high-pressure homogenization, produced yeast protein with a nucleic acid content of 2.58%, significantly higher than that of Example 1. Compared to Example 1, Comparative Example 5, which did not undergo secondary enzymatic hydrolysis, produced yeast protein with a nucleic acid content of 2.58%, also significantly higher than that of Example 1. Furthermore, the nucleic acid content of the yeast protein produced in Comparative Example 6 was 2.95%, also significantly higher than that of the Examples.

[0132] Experimental example 1. Sensory analysis of yeast protein taste and flavor The flavor and mouthfeel of yeast protein were evaluated using sensory analysis. Nine sensory evaluators were selected to conduct the sensory test. The test procedure was as follows: 2g of low-nucleic acid yeast protein was weighed and added to 98mL of water to make a 2% solution. The subjects then rated the mouthfeel and flavor on a scale of 0-6 for each item. The evaluation criteria are shown in Table 4, and the results are shown in Table 5.

[0133]

[0134]

[0135] As shown in Table 5, the sensory evaluation results show that the yeast protein of Examples 1-7 had an acceptable odor and taste, and none of them had a gritty feel. Specifically, Examples 1-2, 4, and 6-7 had almost no yeast odor, a weak yeast flavor, a neutral taste, and no gritty feel.

[0136] Compared with Example 1, in Comparative Example 3, no neutral protease and nuclease were added during the first enzymatic hydrolysis, in Comparative Example 1, no neutral protease was added during the first enzymatic hydrolysis, and in Comparative Example 2, no nuclease was added during the first enzymatic hydrolysis. The smell, taste, and smoothness of the yeast proteins prepared in Comparative Examples 1-3 were all worse than those in Example 1.

[0137] Compared with Example 1, Comparative Example 4 did not undergo high-pressure homogenization, and Comparative Example 5 did not undergo secondary enzymatic hydrolysis. The odor, taste, and smoothness of the yeast protein produced in Comparative Examples 4-5 were all inferior to those in Example 1. Furthermore, the odor, taste, and smoothness of the yeast protein produced in Comparative Example 6 were also inferior to those in the Examples.

[0138] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing low-nucleic acid yeast protein, characterized in that: The steps include: (1) Inactivation of high-protein yeast; (2) enzymatically hydrolyzing the yeast inactivated in step (1) using a neutral protease, a glucanase, a mannanase, and a first nuclease, and separating the heavy phase after enzyme inactivation to obtain crude yeast protein; (3) High-pressure homogenization of crude yeast protein; (4) The crude yeast protein after high-pressure homogenization is enzymatically hydrolyzed using a second nuclease and a lipase, and after inactivating the enzyme, the heavy phase is separated to obtain the low-nucleic acid yeast protein.

2. The preparation method according to claim 1, characterized in that The protein content of the high-protein yeast is 45-75%.

3. The preparation method according to claim 1, characterized in that The inactivation treatment in step (1) comprises preparing the high-protein yeast into a dispersion liquid and heating it to 50-99° C. for heat treatment.

4. The preparation method according to claim 3, characterized in that The pH of the heat treatment is 5.0-9.0; and / or, The heat treatment time is 0.5-2h; and / or, The concentration of the dispersion is 5-20 wt %.

5. The preparation method according to claim 1, characterized in that Based on the mass of high-protein yeast, the amount of the neutral protease added in step (2) is 0.5-2‰, the amount of the glucanase added is 3-6‰, the amount of the mannanase added is 1-3‰, and the amount of the first nuclease added is 1-3‰.

6. The preparation method according to claim 5, characterized in that The amount of the neutral protease added is 1.2-2.0‰; and / or, The added amount of the dextranase is 3-5‰; and / or, The added amount of the mannanase is 2-3‰; and / or, The added amount of the first nuclease is 2-3‰.

7. The preparation method according to claim 1, characterized in that The enzymatic activity of the neutral protease is 50,000-150,000 u / g; and / or, The enzymatic activity of the glucanase is 200-400u / ml; and / or, The mannanase has an enzyme activity of 150,000-250,000 u / g; and / or, The enzyme activity of the first nuclease is 400,000-600,000 u / g.

8. The preparation method according to any one of claims 1 to 5, characterized in that The temperature of the enzymatic hydrolysis in step (2) is 40-70°C; and / or, The pH of the enzymatic hydrolysis in step (2) is 4.0-8.0; and / or, The enzymatic hydrolysis time in step (2) is 5-20h.

9. The preparation method according to any one of claims 1 to 5, characterized in that The pressure of the high-pressure homogenization treatment in step (3) is 50-200 MPa; and / or, The high pressure homogenization treatment time in step (3) is 1-5 hours; and / or, The temperature of the high pressure homogenization treatment in step (3) is 50-70°C.

10. The preparation method according to any one of claims 1 to 5, characterized in that Step (3) includes the step of preparing the crude yeast protein into a dispersion before the high-pressure homogenization treatment.

11. The preparation method according to claim 9, characterized in that The concentration of the crude yeast protein dispersion is 5-20 wt %.

12. The preparation method according to any one of claims 1 to 5, characterized in that Based on the dry weight of crude yeast protein, the amount of the second nuclease added in step (4) is 1-3‰, and the amount of the lipase added is 3-5‰.

13. The preparation method according to claim 12, characterized in that Based on the dry weight of crude yeast protein, the added amount of the second nuclease is 2-3‰, and / or the added amount of the lipase is 3-4‰.

14. The preparation method according to claim 12, characterized in that The enzymatic activity of the second nuclease is 400,000-600,000 u / g, and / or the enzymatic activity of the lipase is 4,000-6,000 u / g.

15. The preparation method according to any one of claims 1 to 5, characterized in that The enzymatic hydrolysis temperature in step (4) is 40-70°C; and / or, The enzymatic hydrolysis pH in step (4) is 4.0-8.0; and / or, The enzymatic hydrolysis time in step (4) is 1-10 h.

16. The preparation method according to any one of claims 1 to 5, characterized in that The enzyme inactivation in step (2) and step (4) both include the step of heating the enzymatic hydrolysate to 75-85° C. and keeping the temperature for 0.5-2 h.

17. The preparation method according to any one of claims 1 to 5, characterized in that The preparation method further comprises the steps of preparing a dispersion of the heavy phase separated in step (4) and drying the dispersion.

18. The preparation method according to claim 17, characterized in that The concentration of the dispersion of the heavy phase separated in step (4) is 20-30 wt%.

19. The preparation method according to claim 17, characterized in that The drying is spray drying.

20. A low nucleic acid yeast protein, characterized in that It is prepared by the preparation method according to any one of claims 1 to 19; wherein the protein content of the low nucleic acid yeast protein is greater than or equal to 70%, and / or, The nucleic acid content of the low nucleic acid yeast protein is less than or equal to 1%; and / or, The fat content in the low nucleic acid yeast protein is less than or equal to 6%.

21. The low nucleic acid yeast protein according to claim 20, characterized in that The protein content of the low nucleic acid yeast protein is 85-95%; and / or, The nucleic acid content of the low nucleic acid yeast protein is 0.1-0.5%; and / or, The fat content of the low nucleic acid yeast protein is 3-5%.

22. Use of the low nucleic acid yeast protein according to claim 20 or 21 in the food field.

23. The use according to claim 22, wherein: The application is the application of the low nucleic acid yeast protein in the field of protein supplementation or protein replacement.

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