A low-nucleic acid yeast protein, its preparation method and application

Abstract

This invention relates to the field of microbial application technology, and particularly to a low-nucleic acid yeast protein, its preparation method, and its application. The preparation method of the low-nucleic acid yeast protein includes the following steps: (1) inactivating high-protein yeast; (2) enzymatically hydrolyzing the inactivated yeast with neutral protease, glucanase, mannanase, and a first nuclease, and separating the heavy phase after enzyme inactivation to obtain crude yeast protein; (3) homogenizing the crude yeast protein under high pressure; (4) enzymatically hydrolyzing the homogenized crude yeast protein with a second nuclease and lipase, and separating the heavy phase after enzyme inactivation to obtain low-nucleic acid yeast protein. This invention uses high-protein yeast as raw material, which undergoes inactivation, complex enzyme hydrolysis, high-pressure homogenization, and secondary enzymatic hydrolysis to obtain a yeast protein with a nucleic acid content of less than 1.0%. It has a neutral and pure taste and can be widely used in nutritional health and food fields such as protein supplementation and protein replacement.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, and in particular to a low-nucleic acid yeast protein, its preparation method, and its application. Background Technology

[0002] Dietary protein is one of the most important nutrients for humans, playing a crucial role in building and repairing body tissues, transporting various substances, maintaining nervous system function, and providing energy. Therefore, adequate intake of high-quality protein is essential for good health. Finding sustainable alternative proteins has become a hot topic in the market. Yeast, the most widely used single-celled fungus, is an excellent source of high-quality complete protein. Yeast protein not only has good nutritional value but also excellent functional properties, such as muscle building and immune regulation, thus showing broad application prospects in food, medicine, and animal feed.

[0003] The key factors affecting the application effect of extracting protein from yeast are: (1) the protein content needs to be increased as much as possible to meet the protein content requirements; (2) the residual yeast nucleic acid in yeast protein needs to 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 yeast protein, while reducing the nucleic acid content in yeast protein, thereby improving the taste and flavor of yeast protein.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] Technical Solution 1: A method for preparing low-nucleic acid yeast protein, comprising the following steps:

[0007] (1) Inactivate the high-protein yeast;

[0008] (2) Neutral protease, glucanase, mannanase and nuclease were used to enzymatically hydrolyze the inactivated yeast, and the crude yeast protein was obtained by separating the heavy phase after enzyme inactivation.

[0009] (3) The crude yeast protein was subjected to high-pressure homogenization;

[0010] (4) Enzymatic hydrolysis of crude yeast protein treated by high pressure homogenization was performed using nuclease and lipase. After enzyme inactivation, the heavy phase was separated to obtain low-nucleic acid yeast protein.

[0011] Technical Solution 2: According to the preparation method described in Technical Solution 1, the protein content of the high-protein yeast is 45-75%.

[0012] Technical Solution 3: According to the preparation method described in Technical Solution 1 or 2, wherein the inactivation treatment in step (1) includes preparing a dispersion of high-protein yeast and heating it to 50-99°C for heat treatment, preferably at 90-99°C, more preferably at 95-98°C.

[0013] Technical Solution 4: According to the preparation method described in Technical Solution 3, wherein the pH of the heat treatment in step (1) is 5.0-9.0, preferably 8.0-9.0, and more preferably 8.5-9.0;

[0014] And / or, the heat treatment time is 0.5-2 hours;

[0015] And / or, the concentration of the dispersion is 5-20 wt%.

[0016] Technical Solution 5: The preparation method according to any one of Technical Solutions 1-4, wherein, based on the mass of high-protein yeast, the amount of neutral protease added in step (2) is 0.5-2‰, the amount of dextranase added is 3-6‰, the amount of mannanase added is 1-3‰, and the amount of nuclease added is 1-3‰.

[0017] Technical Solution 6: According to the preparation method described in Technical Solution 5, the amount of neutral protease added is 1.2-2.0‰, preferably 1.4-1.6‰; and / or,

[0018] The amount of dextranase added is 3-5‰, preferably 4-5‰; and / or,

[0019] The amount of mannanase added is 2-3‰; and / or,

[0020] The amount of nuclease added is 2-3‰.

[0021] Technical Solution 7: The preparation method according to any one of Technical Solutions 1-6, wherein the enzyme activity of the neutral protease is 50,000-150,000 u / g; and / or,

[0022] The activity of the dextranase is 200-400 u / ml; and / or,

[0023] The mannanase activity is 150,000-250,000 u / g; and / or,

[0024] The enzyme activity of the nuclease is 400,000-600,000 u / g.

[0025] Technical Solution 8: The preparation method according to any one of Technical Solutions 1-7, wherein the temperature of enzymatic hydrolysis in step (2) is 40-70℃, preferably 50-70℃, and more preferably 60-70℃;

[0026] And / or, the pH of the enzymatic hydrolysis is 4.0-8.0, preferably 6.0-8.0, more preferably 7.0-8.0;

[0027] And / or, the enzymatic hydrolysis time is 5-20 h, preferably 12-20 h.

[0028] 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;

[0029] Preferably, the high-pressure homogenization treatment time is 1-5 hours, more preferably 1-3 hours, and even more preferably 1-2 hours;

[0030] More preferably, the high-pressure homogenization temperature is 50-70℃.

[0031] Technical Solution 10: The preparation method according to any one of Technical Solutions 1-9, wherein step (3) before high pressure homogenization includes the step of preparing crude yeast protein into a dispersion.

[0032] Technical Solution 11: The preparation method according to Technical Solution 10, wherein the concentration of the yeast protein dispersion is 5-20 wt%.

[0033] 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 nuclease added in step (4) is 1-3‰, and the amount of lipase added is 3-5‰.

[0034] Technical Solution 13: According to the preparation method described in Technical Solution 12, the amount of nuclease added is 2-3‰, and / or the amount of lipase added is 3-4‰.

[0035] Technical Solution 14: The preparation method according to any one of Technical Solutions 1-13, wherein the enzyme activity of the nuclease is 400,000-600,000 u / g, and / or the enzyme activity of the lipase is 4,000-6,000 u / g.

[0036] 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℃, preferably 50-70℃, and more preferably 65-70℃;

[0037] Preferably, the enzymatic hydrolysis pH is 4.0-8.0, more preferably 5.0-7.0, and even more preferably 6.5-7.0;

[0038] More preferably, the enzymatic hydrolysis time is 1-10 hours, and more preferably 3-5 hours.

[0039] Technical Solution 16: The preparation method according to any one of Technical Solutions 1-15, wherein the enzyme inactivation in steps (2) and (4) both include the step of heating the enzyme hydrolysate to 75-85℃ and keeping it at that temperature for 0.5-2h.

[0040] Technical Solution 17: The preparation method according to any one of Technical Solutions 1-16, wherein the preparation method further includes the steps of preparing the heavy phase obtained in step (4) into a dispersion and drying it.

[0041] Technical Solution 18: According to the preparation method described in Technical Solution 17, the concentration of the dispersion of the heavy phase obtained in step (4) is 20-30 wt%.

[0042] Technical Solution 19: The preparation method according to Technical Solution 17 or 18, wherein the drying is spray drying.

[0043] Technical Solution 20: A low-nucleic acid yeast protein, prepared by any one of the preparation methods in Technical Solutions 1-19 above; wherein the low-nucleic acid yeast protein has a protein content of greater than or equal to 70%; and / or,

[0044] The low-nucleic acid yeast protein has a nucleic acid content of less than or equal to 1%, and / or,

[0045] The low-nucleic acid yeast protein has a fat content of less than or equal to 6%.

[0046] 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,

[0047] The low-nucleic acid yeast protein has a nucleic acid content of 0.1-0.8%, preferably 0.1-0.5%; and / or,

[0048] The low-nucleic acid yeast protein has a fat content of 2-6%, preferably 3-5%.

[0049] Technical Solution 22: Application of the low-nucleic acid yeast protein described in Technical Solution 19 or 20 in the food industry.

[0050] Technical Solution 23: The application according to Technical Solution 22, wherein the application is the use of the low nucleic acid yeast protein in the field of protein supplementation or protein substitution.

[0051] The beneficial effects of this invention are:

[0052] This invention uses high-protein yeast as raw material, which undergoes inactivation, compound enzyme hydrolysis, high-pressure homogenization, and secondary enzymatic hydrolysis to obtain a low-nucleic acid yeast protein with a protein content of over 70% and a nucleic acid content of less than 1.0%. It has a neutral and pure taste and can be widely used in the fields of nutrition, health, and food, such as protein supplementation and protein replacement.

[0053] Strain preservation information:

[0054] The *Saccharomyces cerevisiae* FX-2 used in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on August 1, 2016, with accession number CCTCC NO: M2016418. The deposit address is: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China; telephone: (027)-68752319. This strain has been described in the patent publication text with publication number CN108220175A. Detailed Implementation

[0055] To make the objectives, 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 some embodiments of the present invention, but not all embodiments. In conjunction with the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0056] In this application, the terms "first aspect," "second aspect," "third aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0057] In this document, terms such as "preferred," "better," and "good" are merely descriptions of implementation methods or embodiments that achieve better results, and should be understood as not constituting a limitation on the scope of protection of this application. If multiple "preferred" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "preferred" term shall be independent.

[0058] In this application, unless otherwise specified, the features or solutions corresponding to "and / or" include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. Any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. For example, "M and / or N" represents the group consisting of M, N, and "a combination of M and N". "Containing M and / or N" can mean "containing M, containing N, and containing both M and N", or "containing M, containing N, or containing both M and N", and can be appropriately understood according to the context.

[0059] In a first aspect, according to one specific embodiment of this application, this application provides a method for preparing low-nucleic acid yeast protein, characterized by comprising the following steps:

[0060] (1) Inactivate the high-protein yeast;

[0061] (2) Neutral protease, glucanase, mannanase and primary nuclease were used to enzymatically hydrolyze the cell-wall-breaking yeast, and the crude yeast protein was obtained by separating the heavy phase after enzyme inactivation.

[0062] (3) The crude yeast protein was subjected to high-pressure homogenization;

[0063] (4) The crude yeast protein after high-pressure homogenization was enzymatically hydrolyzed using a second nuclease and a lipase. After enzyme inactivation, the heavy phase was separated to obtain low-nucleic acid yeast protein.

[0064] This application employs a method of inactivating high-protein yeast, causing yeast denaturation, inactivation of endogenous enzymes, and permeability of the cell membrane, thereby releasing soluble components and reducing the production of odorous substances such as amino acids due to yeast autolysis. Enzymatic hydrolysis ruptures yeast cells, breaking down the cell wall into soluble dextran and mannooligosaccharides. Subsequent separation removes these soluble dextran and mannooligosaccharides, increasing protein content and facilitating nucleic acid release. The crude yeast protein solution obtained from the first enzymatic hydrolysis is then subjected to high-pressure homogenization to further open the protein structure, exposing residual nucleic acids and fats. Further enzymatic hydrolysis with nucleases and lipases further reduces nucleic acids and removes fats.

[0065] In some embodiments of this application, the inactivation treatment in step (1) includes preparing a dispersion of high-protein yeast and heating it to 50-99°C for heat treatment, preferably for 0.5-2 hours.

[0066] In some embodiments of this 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 beneficial to the release of yeast endogenous substances, especially yeast nucleic acids, and can reduce the nucleic acid content in yeast protein, thereby avoiding excessive uric acid levels in the human body after consumption. In some embodiments of this 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.

[0067] In some embodiments of this application, the heat treatment temperature can be 50-70℃, 90-99℃, or 95-98℃. Higher heat treatment temperatures are more conducive to yeast autolysis and the release of nucleic acid substances. This invention, through secondary enzymatic hydrolysis and homogenization, can produce yeast protein with low nucleic acid content, almost no yeast flavor, and no gritty texture even at lower temperatures.

[0068] In some embodiments of this application, based on the mass of high-protein yeast, the amount of neutral protease added in step (2) is 0.5-2‰, the amount of dextranase added is 3-6‰, the amount of mannanase added is 1-3‰, and the amount of the first nuclease added is 1-3‰.

[0069] In some embodiments, the amount of neutral protease added in step (2), based on the mass of high-protein yeast, 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 neutral protease added in step (2) can be 0.5‰, 1‰, 1.2‰, 1.4‰, 1.6‰, 1.8‰, or 2‰, or within a range defined by 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 range.

[0070] In some embodiments, the amount of 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 high-protein yeast. In some embodiments, the amount of glucanase added in step (2) can be 3‰, 4‰, 5‰, or 6‰, or within a range defined by 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 range.

[0071] 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 high-protein yeast. In some embodiments, the amount of mannanase added in step (2) can be 1‰, 2‰, or 3‰, or fall within a range defined by 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 range.

[0072] In some embodiments, based on the mass of high-protein yeast, the amount of the first nuclease added in step (2) can be 1-3‰, 1-2‰, or 2-3‰. In some embodiments, the amount of nuclease added in step (2) can be 1‰, 2‰, or 3‰, or fall within a range defined by 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 range.

[0073] In some embodiments of this application, the temperature for enzymatic hydrolysis in step (2) is 40-70°C, preferably 50-70°C, and more preferably 60-70°C. In some embodiments, the pH for enzymatic hydrolysis is 4.0-8.0, preferably 6.0-8.0, and more preferably 7.0-8.0. In some embodiments, the hydrolysis time is 5-20 hours, preferably 10-20 hours, and more preferably 12-20 hours.

[0074] In some embodiments of this application, step (3) before high-pressure homogenization includes the step of preparing crude yeast protein into a dispersion, preferably with a concentration of 5-20 wt%.

[0075] In some embodiments of this 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.

[0076] In some embodiments of this application, the amount of nuclease added in step (4) is 1-3‰ and the amount of lipase added is 3-6‰, based on the dry weight of crude yeast protein.

[0077] 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 the crude yeast protein. In some embodiments, the amount of the second nuclease added in step (4) can be 1‰, 2‰, or 3‰, or fall within a range defined by 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 range.

[0078] 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 range defined by 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 range.

[0079] The enzyme activity ranges of the various enzymes used in this invention can be as follows: the neutral protease in step (2) has an activity of 50,000-150,000 u / g; the dextranase in step (2) has an activity of 200-400 u / ml; the mannanase in step (2) has an activity of 150,000-250,000 u / g; and the first nuclease in step (2) has an activity of 400,000-600,000 u / g. The second nuclease in step (4) has an activity of 400,000-600,000 u / g; and the lipase in step (4) has an activity of 4,000-6,000 u / g.

[0080] In some embodiments of this 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 1-10 hours, preferably 3-5 hours. In some embodiments of this application, the enzyme inactivation in steps (2) and (4) both include the step of heating the enzymatic hydrolysate to 75-85°C and holding it at that temperature for 0.5-2 hours.

[0081] In some embodiments of this application, the preparation method further includes the steps of preparing the heavy phase obtained in step (4) into a dispersion and drying it; preferably, the concentration of the dispersion is 20-30 wt%, and more preferably, the drying is spray drying.

[0082] Secondly, this application also provides a low-nucleic acid yeast protein prepared by the above-described method. In some embodiments, the protein content of the low-nucleic acid yeast protein is greater than or equal to 70%, preferably 75-95%. In some embodiments, the nucleic acid content is less than or equal to 1%, ranging from 0.1% to 0.8%. In some embodiments, the fat content is less than or equal to 6%.

[0083] In some embodiments of this application, the nucleic acid content in the low-nucleic acid yeast protein can 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 can 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%.

[0084] In some embodiments of this application, the protein content in the low-nucleic acid yeast protein can 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 can be 70-95%, 75-95%, 80-95%, 85-95%, 87-95%, 89-95%, or 91-95%.

[0085] In some embodiments of this application, the fat content in 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%.

[0086] It should be noted that the term "high-protein yeast" in this application refers to yeast with a protein content of 45% or more obtained by culturing yeast strains. For example, the high-protein yeast of this application can be obtained by batch fermentation or continuous fermentation of yeast strains using one or more of sugarcane molasses, hydrolyzed sugar or beet molasses as fermentation carbon source, and its protein content can be 45-75%, preferably 65-75%.

[0087] Specifically, in the method for preparing low-nucleic acid yeast protein in this application, there are no particular restrictions on the type of yeast strain used to obtain high-protein yeast. For example, it can be commercially available or obtained by any method, such as Saccharomyces cerevisiae, Kluyveromyces, Pichia pastoris, or Candida albicans. Preferably, it is Saccharomyces cerevisiae, specifically Saccharomyces cerevisiae FX-2, which was deposited at the China Center for Type Culture Collection (CCTCC) on August 1, 2016, with accession number CCTCC NO: M2016418.

[0088] Furthermore, there are no particular restrictions on the culture conditions required to obtain the high-protein yeast in this application, as long as the protein content can reach 45% or higher. For example, it can be obtained by the method disclosed in Chinese Patent CN108220175A. Specifically, the high-protein yeast in this application can be obtained by the following steps:

[0089] b. Seed culture: The strains preserved on the slant were subjected to primary and secondary seed cultures;

[0090] c. Seed enrichment: Centrifugation and washing were performed to enrich the Saccharomyces cerevisiae seeds;

[0091] d. Fermentation culture: The brewing yeast seed from step c is inoculated into a fermenter containing water and a phosphorus source for fermentation culture. The aeration ratio in the fermenter is 1.6-2.5 VVM, and the dissolved oxygen volume is 45-70%. The physiological state of the cells is monitored online in real time, and the feed rate is adjusted according to the real-time monitored physiological parameters, including carbon dioxide release rate (CER), oxygen uptake rate (OUR), and respiratory quotient (RQ). The fermentation control process does not require the addition of inorganic salts, acids, alkalis, and trace elements other than feed.

[0092] e. After fermentation is complete, separate and wash the yeast cells, and collect them.

[0093] In step d, the feed added during the fermentation control process consists of molasses and a nitrogen source.

[0094] In some embodiments, the method for preparing high-protein yeast according to this application further includes:

[0095] a. Pretreatment of fermentation raw materials: Molasses is removed from residue and sediment, and then separated and prepared into molasses with a total sugar content of 25-35%;

[0096] In some embodiments, the pretreatment of fermentation feedstock also includes the steps of preparing a nitrogen source solution with water to a concentration of 20-25% and sterilizing the treated molasses and nitrogen source solution.

[0097] In some embodiments, the primary seed culture includes the following steps: inoculating the inoculum into a primary culture medium and culturing it for 15-25 hours at 28-32°C and pH 4.0-5.5. In some embodiments, the primary culture medium comprises, by weight percentage, 1-2% yeast extract, 2-3% peptone, and 2-3% glucose.

[0098] In some embodiments, the secondary seed culture includes the following steps: inoculating the seeds from the primary culture into a secondary culture medium and culturing them at 28-32°C and pH 4.0-5.5 for 15-25 hours. In some embodiments, the secondary culture medium comprises, by weight percentage, 2-3% glucose, 2-3% yeast extract, 0.1-0.2% potassium dihydrogen phosphate, and 0.1-0.2% dipotassium hydrogen phosphate.

[0099] In some embodiments, the fermentation culture in step d is carried out at a temperature of 25-35°C, for a fermentation time of 10-20 hours, and at a pH of 4.0-7.0. In some embodiments, the amount of phosphorus source added is 0.1-0.5% by weight of the fermentation bottom water.

[0100] Thirdly, this application also provides the application of the above-mentioned low-nucleic acid yeast protein in the food field, preferably in the application of protein supplementation and protein replacement.

[0101] The beneficial effects of the present invention will be further illustrated below through specific embodiments.

[0102] All raw materials or reagents used in this invention are purchased from mainstream manufacturers on the market. Those without specified manufacturers or concentrations are all analytical grade raw materials or reagents that can be obtained routinely. There are no special restrictions as long as they can achieve the expected effect.

[0103] Unless otherwise specified in this embodiment, the techniques or conditions described in the literature in this field or in accordance with the product manual shall apply.

[0104] The present invention will now be described in more detail with reference to examples and comparative examples, but the 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.

[0105] The sources of the reagents and instruments used in the following examples are shown in Table 1.

[0106]

[0107] Example 1

[0108] 1. Preparation of high-protein yeast,

[0109] The preparation steps for high-protein yeast include:

[0110] a. Fermentation raw material processing: Molasses is removed from the sediment and separated to obtain molasses supernatant, which is adjusted to a 30% mass concentration to be used as a carbon source. The processed molasses is sterilized at 121℃ for 20 minutes. Ammonia water is diluted with sterile water to obtain a nitrogen source with a nitrogen concentration of 20%.

[0111] b. Seed culture: For primary seed culture, one loopful of yeast cells (Saccharomyces cerevisiae FX-2) was inoculated from the slant culture into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 h. For secondary seed culture, 10% of the primary culture seed was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 h. 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, 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 maintained between 4.0 and 5.5.

[0112] c. Seed enrichment: Centrifuge, wash three times with deionized water with low calcium and magnesium ion content, and enrich to obtain brewer's yeast seeds with a yeast wet weight of 400 g / L.

[0113] d. Fermentation culture: Add 45L of fermentation bottom water and 0.2% monoammonium phosphate (by weight of fermentation bottom water) to a 100L fermenter and sterilize at 121℃ for 20min. Inoculate the seed enriched in step c into the fermenter at a volume inoculation rate of 10%. Control the temperature at 30℃, ferment at atmospheric pressure, and the aeration ratio at 1.667VVM. Adjust the rotation speed to control the dissolved oxygen volume at 50%. During fermentation, physiological parameters RQ, CER, and OUR were monitored online in real time using a biological exhaust gas analyzer. The online parameter RQ value was kept within a small fluctuation range of 1.0-1.2. When RQ was greater than 1.2, the flow rate of molasses and ammonia was reduced simultaneously to decrease the RQ value. When RQ was less than 1.0, the flow rate of molasses and ammonia was increased simultaneously to increase the RQ value. Throughout the entire regulation process, the flow rate of molasses was controlled within the range of 10-50 mL / min, the flow rate of nitrogen source was controlled within the range of 1-5 mL / min, and the pH was controlled within the range of 4.2-6.5 throughout the fermentation process.

[0114] e. Fermentation broth separation: After 12 hours of fermentation, the culture was centrifuged at 5000 rpm and 15°C for 10 minutes using a Beckman centrifuge to harvest the bacterial phase. The cells were then washed with deionized water, and this washing process was repeated once to harvest the high-protein yeast cells. The protein content of the high-protein yeast was measured to be 65.3%.

[0115] 2. Preparation of low-nucleic acid yeast protein

[0116] (1) High-protein yeast solution preparation: Take 200g (dry) of the high-protein yeast cells obtained by fermentation in step (1) and prepare a 2000g solution with deionized water so that the yeast mass fraction is 10%. Adjust the pH to 6.2 with sodium hydroxide solution and inactivate at 75℃ for 60 minutes, keeping the mixture stirred throughout the process.

[0117] (2) Cool the yeast emulsion obtained in step (1) to 60℃, adjust the pH to 5.7 with citric acid, and add 1‰ neutral protease, 5‰ dextranase, 2‰ mannanase, and 2‰ nuclease based on the mass of high-protein yeast. Perform enzymatic hydrolysis for 15 hours, keeping the mixture stirred throughout the process. Then, raise the temperature to 80℃ to inactivate the enzymes for 30 minutes, centrifuge at 5000 r / min for 10 minutes, and remove the supernatant. Prepare a crude yeast protein dispersion with deionized water at a concentration of 18 wt%.

[0118] (3) The crude yeast protein dispersion was subjected to high-pressure homogenization, with the temperature controlled at 60℃, the pressure at 50MPa, and the homogenization time at 5 minutes.

[0119] (4) After homogenization, keep 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 let it act for 5 hours while stirring throughout the process; then raise the temperature to 80°C to inactivate the enzyme for 30 minutes, centrifuge at 5000r / min for 10 minutes, remove the supernatant, and use deionized water to prepare a yeast protein solution with a concentration of 25wt%.

[0120] (5) Spray dry the yeast protein solution to make its moisture content <5%, 100% pass rate through a 100-mesh sieve, inlet air temperature 100-190℃, and outlet air temperature 50-120℃.

[0121] Example 2

[0122] 1. Preparation of high-protein yeast

[0123] The preparation steps of high-protein yeast include:

[0124] a. Fermentation raw material processing: Molasses is removed from the sediment and separated to obtain molasses supernatant, which is adjusted to a 30% mass concentration to be used as a carbon source. The processed molasses is sterilized at 121℃ for 20 minutes. Ammonia water is diluted with sterile water to obtain a nitrogen source with a nitrogen concentration of 20%.

[0125] b. Seed culture: For primary seed culture, one loopful of yeast cells (Saccharomyces cerevisiae FX-2) was inoculated from the slant culture into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 h. For secondary seed culture, 10% of the primary culture seed was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 h. 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, 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 maintained between 4.0 and 5.5.

[0126] c. Seed enrichment: Centrifuge, wash three times with deionized water with low calcium and magnesium ion content, and enrich to obtain brewer's yeast seeds with a yeast wet weight of 400 g / L.

[0127] d. Fermentation culture: Add 45L of fermentation bottom water and 1 / 1000 of monoammonium phosphate (by weight of fermentation bottom water) to a 100L fermenter and sterilize at 121℃ for 20min. Inoculate the seed enriched in step c into the fermenter at a volume inoculation rate of 10%. Control the temperature at 28℃, ferment at atmospheric pressure, and the aeration ratio at 1.6VVM. Adjust the rotation speed to control the dissolved oxygen volume at 45%. During fermentation, physiological parameters RQ, CER, and OUR were monitored online in real time using a biological exhaust gas analyzer. The online parameter RQ value was kept within a small fluctuation range of 1.0-1.2. When RQ was greater than 1.2, the flow rate of molasses and ammonia was reduced simultaneously to decrease the RQ value. When RQ was less than 1.0, the flow rate of molasses and ammonia was increased simultaneously to increase the RQ value. Throughout the entire control process, the flow rate of molasses was controlled within the range of 10-40 mL / min, the flow rate of nitrogen source was controlled within the range of 1-3 mL / min, and the pH was controlled within the range of 4.2-6.0 throughout the fermentation process.

[0128] e. Fermentation broth separation: After 16 hours of fermentation, the culture was centrifuged at 5000 rpm and 15°C for 10 minutes using a Beckman centrifuge to harvest the bacterial phase. The cells were then washed with deionized water, and this washing process was repeated once to harvest the high-protein yeast cells. The protein content of the high-protein yeast was measured to be 51.6%.

[0129] 2. Preparation of low-nucleic acid yeast protein

[0130] The experiment was conducted according to the method in Example 1, the difference being the different process parameters. The process parameters for Example 2 are shown in Table 2, and the rest are the same as those for Example 1.

[0131] Example 3

[0132] 1. Preparation of high-protein yeast

[0133] The preparation steps of high-protein yeast include:

[0134] a. Fermentation raw material processing: Molasses is removed from the sediment and separated to obtain molasses supernatant, which is adjusted to a 30% mass concentration to be used as a carbon source. The processed molasses is sterilized at 121℃ for 20 minutes. Ammonia water is diluted with sterile water to obtain a nitrogen source with a nitrogen concentration of 20%.

[0135] b. Seed Culture: For primary seed culture, one loopful of yeast cells (Saccharomyces cerevisiae FX-2) was inoculated from the slant culture into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 h. For secondary seed culture, 10% of the primary culture seed was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 h. 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, 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 maintained between 4.0 and 5.5.

[0136] c. Seed enrichment: Centrifuge, wash three times with deionized water with low calcium and magnesium ion content, and enrich to obtain brewer's yeast seeds with a yeast wet weight of 400 g / L.

[0137] d. Fermentation culture: Add 45L of fermentation bottom water and 1 / 1000 of monoammonium phosphate (by weight of fermentation bottom water) to a 100L fermenter and sterilize at 121℃ for 20min. Inoculate the enriched seeds into the fermenter at a volume inoculation rate of 10%. Control the temperature at 29℃, ferment at atmospheric pressure, and the aeration ratio at 1.63VVM. Adjust the rotation speed to control the dissolved oxygen volume at 46%. During fermentation, physiological parameters RQ, CER, and OUR were monitored online in real time using a biological exhaust gas analyzer. The online parameter RQ value was kept within a small fluctuation range of 1.0-1.2. When RQ was greater than 1.2, the flow rate of molasses and ammonia was reduced simultaneously to decrease the RQ value. When RQ was less than 1.0, the flow rate of molasses and ammonia was increased simultaneously to increase the RQ value. Throughout the entire control process, the flow rate of molasses was controlled within the range of 10-40 mL / min, the flow rate of nitrogen source was controlled within the range of 1-3 mL / min, and the pH was controlled within the range of 4.2-6.0 throughout the fermentation process.

[0138] e. Fermentation broth separation: After 15 hours of fermentation, the culture was centrifuged at 5000 rpm and 15°C for 10 minutes using a Beckman centrifuge to harvest the bacterial phase. The cells were then washed with deionized water, and this washing process was repeated once to harvest the high-protein yeast cells. The protein content of the high-protein yeast was measured to be 55.8%.

[0139] 2. Preparation of low-nucleic acid yeast protein

[0140] The experiment was conducted according to the method in Example 1, the difference being the different process parameters. The process parameters of Example 3 are shown in Table 2, and the rest are the same as those of Example 1.

[0141] Example 4

[0142] 1. Preparation of high-protein yeast

[0143] The preparation steps of high-protein yeast include:

[0144] a. Fermentation raw material processing: Molasses is removed from the sediment and separated to obtain molasses supernatant, which is adjusted to a 30% mass concentration to be used as a carbon source. The processed molasses is sterilized at 121℃ for 20 minutes. Ammonia water is diluted with sterile water to obtain a nitrogen source with a nitrogen concentration of 20%.

[0145] b. Seed Culture: For primary seed culture, one loopful of yeast cells (Saccharomyces cerevisiae FX-2) was inoculated from the slant culture into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 h. For secondary seed culture, 10% of the primary culture seed was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 h. 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, 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 maintained between 4.0 and 5.5.

[0146] c. Seed enrichment: Centrifuge, wash three times with deionized water with low calcium and magnesium ion content, and enrich to obtain brewer's yeast seeds with a yeast wet weight of 400 g / L.

[0147] d. Fermentation culture: Add 45L of fermentation bottom water and 0.2% monoammonium phosphate (by weight of fermentation bottom water) to a 100L fermenter and sterilize at 121℃ for 20min. Inoculate the enriched seeds into the fermenter at a volume inoculation rate of 10%. Control the temperature at 30℃, ferment at atmospheric pressure, and the aeration ratio at 1.65VVM. Adjust the rotation speed to control the dissolved oxygen volume at 50%. During fermentation, physiological parameters RQ, CER, and OUR were monitored online in real time using a biological exhaust gas analyzer. The online parameter RQ value was kept within a small fluctuation range of 1.0-1.2. When RQ was greater than 1.2, the flow rate of molasses and ammonia was reduced simultaneously to decrease the RQ value. When RQ was less than 1.0, the flow rate of molasses and ammonia was increased simultaneously to increase the RQ value. Throughout the entire control process, the flow rate of molasses was controlled within the range of 10-50 mL / min, the flow rate of nitrogen source was controlled within the range of 1-4 mL / min, and the pH was controlled within the range of 4.2-6.5 throughout the fermentation process.

[0148] e. Fermentation broth separation: After 13 hours of fermentation, the culture was centrifuged at 5000 rpm and 15°C for 10 minutes using a Beckman centrifuge to harvest the bacterial phase. The cells were then washed with deionized water, and this washing process was repeated once to harvest the high-protein yeast cells. The protein content of the high-protein yeast was measured to be 60.3%.

[0149] 2. Preparation of low-nucleic acid yeast protein

[0150] The experiment was conducted according to the method in Example 1, the difference being the different process parameters. The process parameters of Example 4 are shown in Table 2, and the rest are the same as those of Example 1.

[0151] Example 5

[0152] 1. Preparation of high-protein yeast

[0153] The preparation steps of high-protein yeast include:

[0154] a. Fermentation raw material processing: Molasses is removed from the sediment and separated to obtain molasses supernatant, which is adjusted to a 30% mass concentration to be used as a carbon source. The processed molasses is sterilized at 121℃ for 20 minutes. Ammonia water is diluted with sterile water to obtain a nitrogen source with a nitrogen concentration of 20%.

[0155] b. Seed Culture: For primary seed culture, one loopful of yeast cells (Saccharomyces cerevisiae FX-2) was inoculated from the slant culture into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 h. For secondary seed culture, 10% of the primary culture seed was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 h. 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, 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 maintained between 4.0 and 5.5.

[0156] c. Seed enrichment: Centrifuge, wash three times with deionized water with low calcium and magnesium ion content, and enrich to obtain brewer's yeast seeds with a yeast wet weight of 400 g / L.

[0157] d. Fermentation culture: 45L of fermentation bottom water and 0.2% monoammonium phosphate (by weight of fermentation bottom water) were placed in a 100L fermenter and sterilized at 121℃ for 20min. The enriched seeds were inoculated into the fermenter at a volume inoculation rate of 10%. The temperature was controlled at 31℃, and the fermentation was carried out at atmospheric pressure with an aeration ratio of 1.7VVM. The dissolved oxygen volume was controlled at 52% by adjusting the rotation speed. During fermentation, physiological parameters RQ, CER, and OUR were monitored online in real time using a biological exhaust gas analyzer. The online parameter RQ value was kept within a small fluctuation range of 1.0-1.2. When RQ was greater than 1.2, the flow rate of molasses and ammonia was reduced simultaneously to decrease the RQ value. When RQ was less than 1.0, the flow rate of molasses and ammonia was increased simultaneously to increase the RQ value. Throughout the entire control process, the flow rate of molasses was controlled within the range of 10-50 mL / min, the flow rate of nitrogen source was controlled within the range of 1-5 mL / min, and the pH was controlled within the range of 4.2-6.8 throughout the fermentation process.

[0158] e. Fermentation broth separation: After 12 hours of fermentation, the culture was centrifuged at 5000 rpm and 15°C for 10 minutes using a Beckman centrifuge to harvest the bacterial phase. The microbial phase was then washed with deionized water, and this washing process was repeated once to harvest the high-protein yeast cells. The protein content of the high-protein yeast was measured to be 66.2%.

[0159] 2. Preparation of low-nucleic acid yeast protein

[0160] The experiment was conducted according to the method in Example 1, the difference being the different process parameters. The process parameters of Example 5 are shown in Table 2, and the rest are the same as those of Example 1.

[0161] Example 6

[0162] 1. Preparation of high-protein yeast

[0163] The preparation steps of high-protein yeast include:

[0164] a. Fermentation raw material processing: Molasses is removed from the sediment and separated to obtain molasses supernatant, which is adjusted to a 30% mass concentration to be used as a carbon source. The processed molasses is sterilized at 121℃ for 20 minutes. Ammonia water is diluted with sterile water to obtain a nitrogen source with a nitrogen concentration of 20%.

[0165] b. Seed Culture: For primary seed culture, one loopful of yeast cells (Saccharomyces cerevisiae FX-2) was inoculated from the slant culture into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 h. For secondary seed culture, 10% of the primary culture seed was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 h. 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, 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 maintained between 4.0 and 5.5.

[0166] c. Seed enrichment: Centrifuge, wash three times with deionized water with low calcium and magnesium ion content, and enrich to obtain brewer's yeast seeds with a yeast wet weight of 400 g / L.

[0167] d. Fermentation culture: Add 45L of fermentation bottom water and 0.3% monoammonium phosphate (by weight of fermentation bottom water) to a 100L fermenter and sterilize at 121℃ for 20min. Inoculate the enriched seeds into the fermenter at a volume inoculation rate of 10%. Control the temperature at 32℃, ferment at atmospheric pressure, and the aeration ratio at 1.8VVM. Adjust the rotation speed to control the dissolved oxygen volume at 60%. During fermentation, physiological parameters RQ, CER, and OUR were monitored online in real time using a biological exhaust gas analyzer. The online parameter RQ value was kept within a small fluctuation range of 1.0-1.2. When RQ was greater than 1.2, the flow rate of molasses and ammonia was reduced simultaneously to decrease the RQ value. When RQ was less than 1.0, the flow rate of molasses and ammonia was increased simultaneously to increase the RQ value. Throughout the entire control process, the flow rate of molasses was controlled within the range of 10-50 mL / min, the flow rate of nitrogen source was controlled within the range of 1-5 mL / min, and the pH was controlled within the range of 4.2-6.8 throughout the fermentation process.

[0168] e. Fermentation broth separation: After 12 hours of fermentation, the culture was centrifuged at 6000 rpm and 15°C for 20 minutes using a Beckman centrifuge to harvest the bacterial phase. The cells were then washed with deionized water, and this washing process was repeated once to harvest the high-protein yeast cells. The protein content of the high-protein yeast was measured to be 69.8%.

[0169] 2. Preparation of low-nucleic acid yeast protein

[0170] The experiment was conducted according to the method in Example 1, the difference being the different process parameters. The process parameters of Example 6 are shown in Table 2, and the rest are the same as those of Example 1.

[0171] Example 7

[0172] 1. Preparation of high-protein yeast

[0173] The preparation steps of high-protein yeast include:

[0174] a. Fermentation raw material processing: Molasses is removed from the sediment and separated to obtain molasses supernatant, which is adjusted to a 30% mass concentration to be used as a carbon source. The processed molasses is sterilized at 121℃ for 20 minutes. Ammonia water is diluted with sterile water to obtain a nitrogen source with a nitrogen concentration of 20%.

[0175] b. Seed Culture: For primary seed culture, one loopful of yeast cells (Saccharomyces cerevisiae FX-2) was inoculated from the slant culture into 200 ml of primary culture medium and cultured at 30°C and 220 rpm for 18 h. For secondary seed culture, 10% of the primary culture seed was inoculated into 1 L of secondary culture medium and cultured at 30°C and 200 rpm for 18 h. 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, 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 maintained between 4.0 and 5.5.

[0176] c. Seed enrichment: Centrifuge, wash three times with deionized water with low calcium and magnesium ion content, and enrich to obtain brewer's yeast seeds with a yeast wet weight of 400 g / L.

[0177] d. Fermentation Culture: 45L of fermentation bottom water and 0.3% monoammonium phosphate were added to a 100L fermenter and sterilized at 121℃ for 20min. The enriched seed was inoculated into the fermenter at a 10% volumetric inoculum rate. The temperature was controlled at 33℃, fermentation was carried out at atmospheric pressure, and the aeration ratio was 2.0 VVM. The dissolved oxygen volume was controlled at 70% by adjusting the rotation speed. During fermentation, the physiological parameters RQ, CER, and OUR were monitored online in real time using a biological exhaust gas analyzer. The online parameter RQ value was kept within a small fluctuation range of 1.0-1.2. When RQ was greater than 1.2, the flow rate of molasses and ammonia was simultaneously reduced to decrease the RQ value. When RQ was less than 1.0, the flow rate of molasses and ammonia was simultaneously increased to increase the RQ value. Throughout the entire control process, the flow rate of molasses was controlled within the range of 10-50 mL / min, the flow rate of nitrogen source was controlled within the range of 1-5 mL / min, and the pH was controlled within the range of 4.2-6.8.

[0178] e. Fermentation broth separation: After 11 hours of fermentation, the bacterial culture was harvested by centrifugation at 5000 rpm and 15°C for 10 minutes using a Beckman centrifuge. The culture was then washed with deionized water, and this washing process was repeated once to harvest the bacterial cells. The protein content of the high-protein yeast was measured to be 73.7%.

[0179] 2. Preparation of low-nucleic acid yeast protein

[0180] The experiment was conducted according to the method in Example 1, the difference being the different process parameters. The process parameters of Example 7 are shown in Table 2, and the rest are the same as those of Example 1.

[0181]

[0182]

[0183] Comparative Example 1

[0184] The difference from Example 1 is that step (2) does not contain neutral protease, but only adds 5‰ of dextranase, 2‰ of mannanase and 2‰ of nuclease for enzymatic hydrolysis.

[0185] Comparative Example 2

[0186] The difference from Example 1 is that step (2) does not contain nuclease, but only adds 1‰ of neutral protease, 5‰ of dextranase and 2‰ of mannanase for enzymatic hydrolysis.

[0187] Comparative Example 3

[0188] The difference from Example 1 is that step (2) does not contain nuclease and neutral protease, but only adds 5‰ glucanase and 2‰ mannanase for enzymatic hydrolysis.

[0189] Comparative Example 4

[0190] The difference from Example 1 is that high-pressure homogenization is not used; instead, the crude yeast protein dispersion obtained in step (2) is directly hydrolyzed by nucleases and lipases.

[0191] Comparative Example 5

[0192] The difference from Example 1 is that instead of using nucleases and lipases to enzymatically hydrolyze the homogenized yeast protein, the homogenized yeast protein is directly spray-dried.

[0193] Comparative Example 6

[0194] (1) High-protein yeast solution preparation: 200g of high-protein yeast with a protein content of 65.3% (dry weight) was mixed with deionized water to prepare a solution of 2000g (the yeast milk was prepared in the same way 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 cell wall was broken at 75°C for 60 minutes, with stirring throughout the process.

[0195] (2) Cool the cell wall broken yeast emulsion obtained in step (1) to 60℃, adjust the pH to 5.7 with citric acid, add 5‰ glucanase, 2‰ mannanase and 5‰ lipase based on the mass of high protein yeast, and perform enzymatic hydrolysis for 15h, keeping the mixture stirred throughout the process; then raise the temperature to 80℃ to inactivate the enzymes for 30 minutes, centrifuge at 5000r / min for 10 minutes, and remove the supernatant. Prepare a crude yeast protein dispersion with deionized water at a concentration of 18wt%.

[0196] (5) Spray dry the crude yeast protein solution to make its moisture content <5%, 100% pass rate through a 100-mesh sieve, and set the inlet and outlet air temperatures.

[0197] The protein, fat, and nucleic acid contents in yeast were determined using the following methods. The results are shown in Table 3.

[0198] 1) Determination of protein content

[0199] The protein content was determined according to GB 5009.5-2016, the method for determination of protein in food. The specific operation is as follows: take 1g of sample, add 20mL of concentrated sulfuric acid for digestion under the action of mixed catalyst (0.4g copper sulfate pentahydrate + 6g potassium sulfate), then distill, absorb the nitrogen of the product with boric acid, and titrate with 0.1mol / L hydrochloric acid. Read the data, calculate the nitrogen content, and use N (nitrogen content) × 6.25 as the protein coefficient to calculate the protein content.

[0200] 2) Determination of fat content

[0201] The fat content was determined according to GB 5009.6-2016, the method for determining fat in food. The specific procedure is as follows: weigh 2-5g of sample and place it in a filter paper tube. Place the filter paper tube in a Soxhlet extractor and extract with ether or petroleum ether. Then, recover the ether or petroleum ether in the receiving bottle, dry it, weigh it, and calculate the fat content.

[0202] 3) The nucleic acid content was determined according to the detection method for nucleic acids in yeast, as follows:

[0203] A. Weigh approximately 0.500-0.800 g (accurate to 0.001 g) of yeast protein sample into a centrifuge tube. Accurately add 8 mL of 0.25 mol / L perchloric acid solution cooled at 4°C to the centrifuge tube and vortex until homogeneous. Immediately place the centrifuge tube in a 4°C water bath for 15 minutes without shaking. Then centrifuge at 4000 rpm for 10 minutes, and discard the supernatant. Add 5 mL of 0.5 mol / L perchloric acid solution to the precipitate and vortex until homogeneous. Place the centrifuge tube in a 70°C water bath for 15 minutes, shaking every 3-4 minutes. Centrifuge at 4000 rpm for 10 minutes, collect 1 mL of the supernatant, and dilute to 100 mL with distilled water, mixing well.

[0204] B. Preparation of RNA Standard Solution: Accurately weigh approximately 10.5 mg (accurate to 0.1 mg) of RNA standard, add 5 mL of 0.5 mol / L perchloric acid solution, and vortex to mix. Place the centrifuge tube in a 70℃ water bath and incubate for 15 min, vortexing every 3-4 min. Centrifuge at 4000 rpm for 10 min, and take 1 mL of the supernatant. Dilute to 100 mL with distilled water and mix well. Accurately pipette 3.5 mL, 4 mL, 5 mL, 6 mL, 7.5 mL, and 10 mL of the RNA standard solution into 10 mL brown volumetric flasks, respectively, and dilute to the mark with water to obtain a series of working RNA standard solutions with 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. The above-mentioned RNA standard series working solutions were added to a UV spectrophotometer, with distilled water as a blank, and the absorbance was measured at a wavelength of 260 nm. A standard curve was plotted with absorbance on the ordinate and the concentration of the standard series working solutions on the abscissa, and the linear regression equation was calculated.

[0205] 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 take the average of the two measurements. Note: The sample absorbance should be controlled between 0.2 and 0.8, which can be controlled by weighing the sample or by dilution factor.

[0206] Specifically, the test results of various indicators in low-nucleic acid yeast protein are shown in Table 3 below.

[0207]

[0208] As shown in Table 3, the yeast proteins obtained in Examples 1-7 have a protein content of 75.3-91.2%, all above 70%; a fat content of 3.1-5.8%, all below 6%; and a nucleic acid content of 0.28-0.76%, all below 0.8%. In particular, the yeast proteins obtained in Examples 6-7 have a protein content of 89.8-91.2%, a nucleic acid content of only 0.28-0.31%, and a fat content as low as 3.1-3.5%.

[0209] Compared to Example 1, Comparative Example 3 did not add neutral protease and nuclease during the first enzymatic hydrolysis, and the resulting yeast protein contained 1.98% nucleic acid. Comparative Example 1 did not add neutral protease during the first enzymatic hydrolysis, and the resulting yeast protein contained 1.68% nucleic acid. Comparative Example 2 did not add nuclease during the first enzymatic hydrolysis, and the resulting yeast protein contained 2.32% nucleic acid. In contrast, Example 1 used both neutral protease and nuclease, along with glucanase and mannooligosaccharase, for enzymatic hydrolysis, and the resulting yeast protein contained 0.51% nucleic acid. Therefore, it can be seen that the synergistic effect of neutral protease and nuclease significantly reduces the nucleic acid content in yeast protein.

[0210] Compared to Example 1, Comparative Example 4 did not undergo high-pressure homogenization, and the nucleic acid content in its yeast protein was 2.58%, significantly higher than that of Example 1. Compared to Example 1, Comparative Example 5 did not undergo secondary enzymatic hydrolysis, and the nucleic acid content in its yeast protein was also 2.58%, significantly higher than that of Example 1. Furthermore, the nucleic acid content in the yeast protein obtained in Comparative Example 6 was 2.95%, also significantly higher than that of the examples.

[0211] Experimental Example

[0212] 1. Sensory analysis of the texture and taste of yeast protein

[0213] Sensory evaluation was used to assess the flavor and texture of yeast protein. Nine sensory evaluators were selected to conduct sensory assessments. The assessment process was as follows: 2g of low-nucleic acid yeast protein was weighed and added to 98mL of water to prepare a 2% solution. Then, the subjects rated the taste and texture, with each item ranging from 0 to 6 points. The evaluation criteria are shown in Table 4, and the results are shown in Table 5.

[0214]

[0215]

[0216] As shown in Table 5, the sensory evaluation results indicate that the yeast proteins in Examples 1-7 have acceptable odor and taste, and none of them have a gritty feel. Among them, Examples 1-2, 4, and 6-7 have almost no yeast odor, weak yeast taste, neutral taste, and no gritty feel.

[0217] Compared with Example 1, no neutral protease and nuclease were added during the first enzymatic hydrolysis of Comparative Example 3, no neutral protease was added during the first enzymatic hydrolysis of Comparative Example 1, and no nuclease was added during the first enzymatic hydrolysis of Comparative Example 2. The yeast protein obtained by Comparative Examples 1-3 had a worse odor, taste and smoothness than that of Example 1.

[0218] Compared to Example 1, Comparative Example 4 did not undergo high-pressure homogenization, and Comparative Example 5 did not undergo secondary enzymatic hydrolysis. The yeast proteins obtained in Comparative Examples 4 and 5 had inferior odor, taste, and smoothness compared to Example 1. Furthermore, the yeast proteins obtained in Comparative Example 6 also had inferior odor, taste, and smoothness compared to the examples.

[0219] 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 intended to limit it. 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 to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a low nucleic acid yeast protein, characterized by, The method comprises the following steps: (1) inactivating high-protein yeast, wherein the protein content of the high-protein yeast is 45-75%, and the inactivation treatment comprises preparing a dispersion of the high-protein yeast and heating to 50-99°C for heat treatment, wherein the pH of the heat treatment is 5.0-9.0, the time of the heat treatment is 0.5-2h, and the concentration of the dispersion is 5-20wt%; (2) enzymatically treating the yeast after the inactivation treatment in step (1) with neutral protease, glucanase, mannanase and first nuclease, separating and taking the heavy phase after enzyme inactivation to obtain crude yeast protein, wherein the addition amount of the neutral protease is 0.5-2‰, the addition amount of the glucanase is 3-6‰, the addition amount of the mannanase is 1-3‰, and the addition amount of the first nuclease is 1-3‰, based on the mass of the high-protein yeast, wherein the enzymolysis temperature is 40-70°C, the enzymolysis pH is 4.0-8.0, and the enzymolysis time is 5-20h; (3) high-pressure homogenization treatment of the crude yeast protein, wherein the pressure of the high-pressure homogenization treatment is 50-200MPa, the time of the high-pressure homogenization treatment is 1-5min, and the temperature of the high-pressure homogenization treatment is 50-70°C; (4) enzymatically treating the crude yeast protein after the high-pressure homogenization treatment with second nuclease and lipase, separating and taking the heavy phase after enzyme inactivation to obtain low-nucleic-acid yeast protein, wherein the addition amount of the second nuclease is 1-3‰, and the addition amount of the lipase is 3-5‰, based on the dry weight of the crude yeast protein, wherein the enzymolysis temperature is 40-70°C, the enzymolysis pH is 4.0-8.0, and the enzymolysis time is 1-10h.

2. The production method according to claim 1, characterized by, The addition amount of the neutral protease is 1.2-2.0‰; and / or, The addition amount of the glucanase is 3-5‰; and / or, The addition amount of the mannanase is 2-3‰; and / or, The addition amount of the first nuclease is 2-3‰.

3. The production method according to claim 1, characterized by, The enzyme activity of the neutral protease is 50000-150000u / g; and / or, The enzyme activity of the glucanase is 200-400u / ml; and / or, The enzyme activity of the mannanase is 150000-250000u / g; and / or, The enzyme activity of the first nuclease is 400000-600000u / g.

4. The method of claim 1, wherein, Step (3) comprises the step of preparing a crude yeast protein dispersion before the high-pressure homogenization treatment.

5. The preparation method according to claim 4, characterized in that, The concentration of the crude yeast protein dispersion is 5-20wt%.

6. The method of claim 1, wherein, The addition amount of the second nuclease is 2-3‰, and / or the addition amount of the lipase is 3-4‰, based on the dry weight of the crude yeast protein.

7. The preparation method according to claim 1, characterized in that, The enzyme activity of the second nuclease is 400000-600000u / g, and / or the enzyme activity of the lipase is 4000-6000u / g.

8. The method of claim 1, wherein, The enzyme inactivation in steps (2) and (4) both comprises the step of heating the enzymolysis solution to 75-85°C for 0.5-2h.

9. The method of claim 1, wherein, The preparation method further comprises the steps of preparing a dispersion of the heavy phase obtained in step (4) and drying.

10. The method of claim 9, wherein, The concentration of the separated dispersion liquid of the heavy phase obtained in step (4) is 20-30 wt%.

11. The preparation method according to claim 9, characterized in that, The drying is spray drying.

12. A low nucleic acid yeast protein, characterized in that, The low nucleic acid yeast protein is prepared by the preparation method in any one of claims 1-11, wherein the protein content in the low nucleic acid yeast protein is 85-95%, the nucleic acid content in the low nucleic acid yeast protein is 0.1-0.5%, and the fat content in the low nucleic acid yeast protein is 3-5%.

13. Use of the low nucleic acid yeast protein in claim 12 in the preparation of a protein supplement or a protein substitute for the food field.

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