Animal-free composite protein hydrolysate and method for preparing the same

Abstract

The present application relates to a kind of animal-source-free complex protein hydrolysate and its preparation method, the weight percentage of the peptide segment with molecular weight less than or equal to 1000Da in the animal-source-free complex protein hydrolysate is greater than or equal to 90% in the total peptide segment of the animal-source-free complex protein hydrolysate, the weight percentage of protein is greater than or equal to 65%, the animal-source-free complex protein hydrolysate is prepared by the method comprising the raw material containing plant protein and yeast is enzymolysis.The animal-source-free complex protein hydrolysate of the present application has the natural attribute of animal-source-free, is rich in small molecule polypeptide and contains a certain amount of free amino acid, nucleotide and other components, while having the characteristics of not strictly dependent on exogenous protease, processing flexibility, product plasticity, wide application field, etc., can be used in cell culture, microbial fermentation, animal nutrition, plant nutrition and other fields.

Description

Technical Field

[0001] This invention relates to the field of protein hydrolysate technology, and in particular to an animal-free composite protein hydrolysate and its preparation method. Background Technology

[0002] Animal-free complex protein hydrolysates are protein breakdown products that do not contain any animal-derived components. Their proteins originate from non-animal raw materials such as plants and microorganisms, and are processed through biotechnology such as enzymatic hydrolysis. These hydrolysates have broad application prospects in the food, pharmaceutical, and agricultural fields, especially in the biopharmaceutical sector, where they can replace traditional animal-derived protein hydrolysates, avoiding risks such as animal pathogenicity and cultural taboos.

[0003] Traditional animal-free protein hydrolysates are obtained by enzymatic hydrolysis of plant or microbial proteins with the addition of exogenous proteases. Due to the differences in properties among different types of proteins, a single protein raw material is often used for hydrolysis during processing to obtain pure protein hydrolysates. These hydrolysates are then further compounded as needed to obtain complex protein hydrolysates.

[0004] Chinese patent CN113832094A discloses a serum-free and animal-derived basal culture medium for insect cells, comprising a basic component, a hydrolysate component, and a lipid component. The basic component includes inorganic salts, amino acids and sugars, vitamins, trace elements, and organic acids. The hydrolysate component includes yeast hydrolysate and plant protein hydrolysate. The lipid component includes linoleic acid, cholesterol, and tocopherol. This invention's serum-free and animal-derived basal culture medium for insect cells contains no animal-derived components, uses inexpensive raw materials, and has a simple process, making it suitable for large-scale industrial production. However, this method directly uses a combination of yeast hydrolysate and plant protein hydrolysate, lacking the sufficient degradation of plant proteins by yeast endogenous enzymes to form amino acids and small peptides. It also lacks the reaction products of the plant and yeast raw materials during processing, such as the conversion of oligosaccharides during the early stage of yeast autolysis.

[0005] Chinese patent CN118516435A discloses a method for preparing a mixed plant protein hydrolysate, comprising the following steps: S1. Preparing pea protein using pea protein powder as raw material; S2. Preparing black bean protein using black soybeans as raw material; S3. Preparing corn protein using corn starch as raw material; S4. Mixing the pea protein, black bean protein, and corn protein, adding alkaline protease, and performing a first hydrolysis to obtain a first hydrolysate; S5. Adding papain to the first hydrolysate and performing a second hydrolysis to obtain a second hydrolysate; S6. Adding a complex protease to the second hydrolysate and performing a third hydrolysis to obtain a mixed plant protein hydrolysate. In step 4, the alkaline protease has a mass concentration of 6%, and during the first hydrolysis, the substrate mass concentration is 7%, the pH is 8, and the temperature is 40℃; in step 5, the papain mass concentration is 9%, and during the second hydrolysis, the substrate mass concentration is 5%, the pH is 8.5, and the temperature is 50℃. This invention prepares a mixed plant protein hydrolysate through three different hydrolysis processes. This mixed plant protein hydrolysate has a protective effect against stem cell damage caused by fatty degeneration and can improve the release of liver-related transaminases, thereby reducing hepatic lipid metabolism. Simultaneously, this mixed plant protein hydrolysate also helps reduce intracellular lipid peroxidation and oxidative stress, thus achieving the prevention and treatment of non-alcoholic fatty liver disease (NAFLD) and effectively intervening in its development. However, the preparation of this mixed plant protein hydrolysate requires the addition of a large amount of specific exogenous proteases, increasing the production cost of plant proteins and limiting their application. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an animal-free complex protein hydrolysate, which is rich in small molecule polypeptides and a certain amount of free amino acids and nucleotides, and has the nutritional components of both plant protein and yeast, making it nutritionally comprehensive and easily absorbed.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] Technical Solution 1: An animal-free complex protein hydrolysate, wherein peptides with a molecular weight of less than or equal to 1000 Da account for more than or equal to 90% of the total peptides in the animal-free complex protein hydrolysate, and the protein content in the animal-free complex protein hydrolysate is more than or equal to 61% by weight. The animal-free complex protein hydrolysate is prepared by a method including enzymatic hydrolysis of raw materials containing plant protein and yeast.

[0009] Technical Solution 2: According to the animal-free complex protein hydrolysate described in Technical Solution 1, wherein peptides with a molecular weight less than or equal to 1000 Da account for 90-98% of the total peptides in the animal-free complex protein hydrolysate by weight; and / or,

[0010] The animal-free complex protein hydrolysate contains 65-90% protein by weight; and / or,

[0011] The free amino acid content in the animal-free complex protein hydrolysate is 15-40 wt%; and / or,

[0012] In the animal-free complex protein hydrolysate, peptides with a molecular weight greater than or equal to 2000 Da account for 0.5-3% of the total peptides in the animal-free complex protein hydrolysate by weight; peptides with a molecular weight greater than 1000 Da and less than 2000 Da account for 1.5-5% of the total peptides in the animal-free complex protein hydrolysate by weight; peptides with a molecular weight greater than 400 Da and less than or equal to 1000 Da account for 14-27% of the total peptides in the animal-free complex protein hydrolysate by weight; and peptides with a molecular weight less than or equal to 400 Da account for 65-85% of the total peptides in the animal-free complex protein hydrolysate by weight.

[0013] Technical Solution 3: The animal-free complex protein hydrolysate according to Technical Solution 1 or 2, wherein the animal-free complex protein hydrolysate is further prepared by adding an exogenous enzyme to a raw material containing plant protein and yeast for enzymatic hydrolysis, wherein the exogenous enzyme includes an exogenous protease.

[0014] Technical Solution 4: The animal-free complex protein hydrolysate according to Technical Solution 3, wherein the exogenous enzyme further includes one or two selected from exogenous amylase and exogenous nuclease.

[0015] Technical Solution 5: A method for preparing the animal-free complex protein hydrolysate according to any one of Technical Solutions 1-4, characterized in that it includes the following steps:

[0016] (1) Prepare a dispersion by adding water to plant protein and yeast, and heat it to 40-60℃;

[0017] (2) Enzymatic hydrolysis was carried out at 40-60℃ to obtain the enzymatic hydrolysate;

[0018] (3) The enzyme hydrolysate obtained in step (2) is inactivated and separated to obtain a complex protein hydrolysate without animal origin.

[0019] Technical Solution 6: According to the preparation method described in Technical Solution 5, step (1) further includes the step of adding exogenous enzyme after heating to 40-60℃, wherein the exogenous enzyme includes exogenous protease.

[0020] Technical Solution 7: According to the preparation method described in Technical Solution 6, the exogenous protease is selected from one or more of the group consisting of papain, bromelain, alkaline protease, neutral protease, acidic protease and flavor protease.

[0021] Technical Solution 8: The preparation method according to Technical Solution 6 or 7, wherein the amount of exogenous protease added is 0.1-5 wt% based on the dry weight of plant protein and yeast.

[0022] Technical Solution 9: The preparation method according to any one of Technical Solutions 6-8, wherein the exogenous enzyme further includes the step of exogenous amylase and / or exogenous nuclease, wherein the amount of exogenous amylase and / or exogenous nuclease added is 0.1-1 wt% based on the dry weight of plant protein and yeast.

[0023] Technical Solution 10: The preparation method according to any one of Technical Solutions 5-9, wherein the enzymatic hydrolysis pH in step (2) is 3-10, and / or the enzymatic hydrolysis time is 3-48h.

[0024] Technical Solution 11: The preparation method according to any one of Technical Solutions 5-10, wherein the weight ratio of the plant protein to the yeast is 1-99:99-1.

[0025] Technical Solution 12: The preparation method according to any one of Technical Solutions 5-11, wherein the plant protein is selected from one or more of the group consisting of soybean protein, wheat protein, pea protein, rice protein, potato protein, peanut protein and cottonseed protein.

[0026] Technical Solution 13: The preparation method according to any one of Technical Solutions 5-12, wherein the yeast is active dry yeast or active wet yeast.

[0027] Technical Solution 14: The preparation method according to any one of Technical Solutions 5-13, wherein the enzyme inactivation in step (3) is carried out by heating to 65-95℃ and holding for 5-60 minutes.

[0028] Technical Solution 15: The preparation method according to any one of Technical Solutions 5-14, wherein the separation in step (3) is selected from one or more of centrifugal separation, plate and frame filtration, ceramic membrane microfiltration and organic membrane ultrafiltration.

[0029] Technical Solution 16: The preparation method according to any one of technical solutions 5-15, wherein the preparation method further includes the steps of concentrating and / or drying the animal-free complex protein hydrolysate.

[0030] Technical Solution 17: The preparation method according to Technical Solution 16, wherein the concentration is selected from triple-effect evaporation or nanofiltration membrane concentration, and / or the drying is selected from spray drying, vacuum belt drying, vacuum microwave drying or vacuum freeze drying.

[0031] Technical Solution 18: The application of the animal-free complex protein hydrolysate prepared by any one of Technical Solutions 1-5 or any one of Technical Solutions 6-17 in the fields of animal cell culture, microbial fermentation, animal nutrition or plant nutrition.

[0032] The beneficial effects of this invention are:

[0033] The animal-free complex protein hydrolysate of this invention has the natural property of being free of animal origin, is rich in small molecule peptides and contains a certain amount of free amino acids, nucleotides and other components, and has the characteristics of not being strictly dependent on exogenous proteases, having high processing flexibility, strong product plasticity and wide application fields. It can be used in cell culture, microbial fermentation, animal nutrition, plant nutrition and other fields.

[0034] Information on strain preservation

[0035] 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. This strain has been described in the patent publication text with publication number CN108220175A.

[0036] The *Wickerhamomyces anomalus* C1.7 strain used in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on December 11, 2017, with accession number CCTCC NO: M2017782. This strain has been described in the patent publication text with publication number CN110959853A.

[0037] The Pichia pastoris (Cyberlindnera fabianii) C1.8 used in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on December 11, 2017, with accession number CCTCCNO:M2017780. This strain has been described in the patent publication text with publication number CN110959853A.

[0038] The Kluyveromyces marxianus AMCC 30634 strain used in this application was deposited at the China Center for Type Culture Collection on February 27, 2023, with accession number CCTCC No: M 2023217. This strain has been described in the patent publication text with publication number CN118956622A. Attached Figure Description

[0039] Figure 1 This is a growth curve of Escherichia coli in Experiment Example 1;

[0040] Figure 2 This is a growth curve of Bacillus subtilis in Experiment Example 1;

[0041] Figure 3 This is a growth curve of Corynebacterium glutamicum in Experiment Example 1;

[0042] Figure 4 This is a growth curve of Enterococcus faecalis in Experiment Example 1;

[0043] Figure 5 The graph shows the sensitivity test results of *Coprinus rhizophilus* in Experiment Example 2.

[0044] From left to right, the protein hydrolysate obtained by mixing the protein hydrolysate obtained in Comparative Example 1 and the protein hydrolysate obtained in Comparative Example 2 in equal mass ratios, the protein hydrolysate obtained in Example 1, and the protein hydrolysate obtained in Example 2 are respectively.

[0045] Figure 6 The graph shows the sensitivity test results of Bacillus subtilis in Experiment Example 2. From left to right, the graph shows the protein hydrolysate obtained by mixing the protein hydrolysate obtained in Comparative Example 1 and Comparative Example 2 in equal mass ratios, the protein hydrolysate obtained in Example 1, and the protein hydrolysate obtained in Example 2.

[0046] Figure 7 The graph shows the sensitivity test results of Bacillus subtilis in Experiment Example 2. From left to right, the graph shows the protein hydrolysate obtained by mixing the protein hydrolysate obtained in Comparative Example 1 and Comparative Example 2 in equal mass ratios, the protein hydrolysate obtained in Example 1, and the protein hydrolysate obtained in Example 2.

[0047] Figure 8 The image shows the results of animal cell culture testing in Experiment Example 3. Detailed Implementation

[0048] 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.

[0049] The term "yeast" refers to yeast obtained by culturing yeast strains, which can be in the form of dry yeast or wet yeast.

[0050] Yeast strains can be cultured by any suitable method. Methods for culturing yeast are known in the art, and those skilled in the art know how to optimize the culture conditions for each strain according to its properties, obtaining yeast by proliferating yeast strains in a culture medium.

[0051] Therefore, for example, on an industrial scale, yeast that can be used in the context of this invention can be obtained by a method including the following steps:

[0052] (1) Yeast strains are cultured in the culture medium in several stages to obtain the proliferation of the initial yeast cells; and

[0053] (2) The resulting yeast cells are separated by centrifugation to obtain a liquid yeast extract containing 10-30% yeast dry matter.

[0054] The yeast obtained in this way is live yeast. Live yeast or active yeast means a group of yeast cells whose metabolism is active.

[0055] In this application, the live yeast can be in the form of dry yeast or wet yeast. Dry yeast is characterized by a low moisture content and typically contains more than 90% yeast dry matter, preferably 93-96%. One advantage of dry yeast is its long shelf life. Therefore, the method for producing yeast cells can further include drying the yeast cells to obtain dry yeast. Drying can be performed by freeze drying, fluidized bed drying, drum drying, or spray drying.

[0056] In a first aspect, in one specific embodiment of this application, the present invention provides an animal-free complex protein hydrolysate, wherein peptides with a molecular weight of less than or equal to 1000 Da account for more than or equal to 90% of the total peptides in the animal-free complex protein hydrolysate, and the protein content in the animal-free complex protein hydrolysate is more than or equal to 61%, and the animal-free complex protein hydrolysate is prepared by a method comprising enzymatic hydrolysis of raw materials containing plant protein and yeast.

[0057] Plant proteins are mostly derived from plant fruits, and their structures or some components possess properties that resist protease hydrolysis. However, when hydrolyzed by exogenous enzymes, they suffer from low hydrolysis levels, low yields, and drawbacks such as large amounts of protease required and high costs. Yeast, on the other hand, contains a rich protease system with diverse and abundant protease types. Therefore, the inventors of this application, through dedicated research, discovered that by activating and releasing endogenous yeast proteases, plant proteins can be enzymatically hydrolyzed simultaneously with yeast autolysis. This method offers advantages such as multiple cleavage sites, high protein yield of the hydrolysate, and abundance of small peptides (peptides with a molecular weight of 1000 Da or less) and free amino acids.

[0058] In some embodiments of this application, the total nitrogen content in the animal-free composite protein hydrolysate is greater than or equal to 8 wt%, preferably 8-15 wt%, and more preferably 9-14 wt%. In some embodiments, the total nitrogen content in the animal-free composite protein hydrolysate can be 9-14 wt%, 9.9-14 wt%, 10.6-14 wt%, 10.7-14 wt%, 11-14 wt%, 11.6-14 wt%, 11.7-14 wt%, 12-14 wt%, 12.2-14 wt%, 13.4-14 wt%, 13.8-14 wt%, or 13.9-14 wt%. In some embodiments, the total nitrogen content in the animal-free complex protein hydrolysate can be 9 wt%, 9.9 wt%, 10.6 wt%, 10.7 wt%, 11.6 wt%, 11.7 wt%, 12 wt%, 12.2 wt%, 13.4 wt%, 13.8 wt%, or 13.9 wt%, 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.

[0059] In some embodiments of this application, the amino acid nitrogen content in the animal-free composite protein hydrolysate is greater than or equal to 1.2 wt%, preferably 1.2-8 wt%, and more preferably 2-6 wt%. In some embodiments, the amino acid nitrogen content in the animal-free composite protein hydrolysate can be 2.5-6 wt%, 2.9-6 wt%, 3-6 wt%, 4-6 wt%, 4.1-6 wt%, 4.9-6 wt%, or 5.3-6 wt%. In some embodiments, the amino acid nitrogen content in the animal-free composite protein hydrolysate can be 2.5 wt%, 2.9 wt%, 3 wt%, 4 wt%, 4.1 wt%, 4.9 wt%, 5.3 wt%, or 6 wt%, or fall within a numerical 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.

[0060] In some embodiments of this application, the nucleotide content in the animal-free composite protein hydrolysate is 0.2-1.5 wt%, preferably 0.2-1.2 wt%, and more preferably 0.2-0.5%.

[0061] In some embodiments of this application, the protein weight percentage in the animal-free composite protein hydrolysate is greater than or equal to 61 wt%, preferably 65 wt%, and more preferably 65-90 wt%. In some embodiments, the protein content in the animal-free composite protein hydrolysate can be 61-90 wt%, 65-90 wt%, 66.3-90 wt%, 66.9-90 wt%, 70-90 wt%, 72-90 wt%, 72.5-90 wt%, 73-90 wt%, 73.1-90 wt%, 75-90 wt%, 76-90 wt%, 76.3-90 wt%, 80-90 wt%, 83-90 wt%, 86.3-90 wt%, or 86.9-90 wt%. In some embodiments, the protein content in the animal-free complex protein hydrolysate may be 66.3 wt%, 66.9 wt%, 70 wt%, 72 wt%, 72.5 wt%, 73 wt%, 73.1 wt%, 75 wt%, 76 wt%, 76.3 wt%, 80 wt%, 83 wt%, 86.3 wt%, or 86.9 wt%, 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 may be combined with any other range.

[0062] In some preferred embodiments of this application, the protein hydrolysate contains 9.9-13.9% total nitrogen, and / or 2.5-6% amino acid nitrogen, and / or 0.2-1.2% nucleotides, and / or 15-37.2% free amino acids, and / or 61.9-86.9% protein, and / or 92.6-97.5% small molecule peptides (≤1000 Da) by mass percentage of total peptides in the animal-free complex protein hydrolysate, and / or 50.1-90.4% yield of hydrolysate product, and / or 17.7-56.2% degree of hydrolysis.

[0063] In some embodiments of this application, the weight percentage of peptides with a molecular weight greater than or equal to 2000 Da in the animal-free complex protein hydrolysate is 0.5-3%; the weight percentage of peptides with a molecular weight greater than 1000 Da and less than 2000 Da is 1.5-5%; the weight percentage of peptides with a molecular weight greater than 400 Da and less than or equal to 1000 Da is 14-27%; and the weight percentage of peptides with a molecular weight less than or equal to 400 Da is 65-85%.

[0064] In some embodiments of this application, the weight percentage of peptides with a molecular weight of 400 Da or less in the animal-free complex protein hydrolysate is 69-85%, preferably 70-85%, and more preferably 75-85% of the total peptides in the animal-free complex protein hydrolysate. In some embodiments, the weight percentage of peptides with a molecular weight of 400 Da or less in the animal-free complex protein hydrolysate can be 69-85%, 70-85%, 74-85%, 75-85%, 76-85%, 80-85%, 81-85%, 82-85%, or 83-85%. In some embodiments, the weight percentage of peptides with a molecular weight of 400 Da or less in the animal-free complex protein hydrolysate may be 69.5%, 74.0%, 74.1%, 75.7%, 76.7%, 80.5%, 80.6%, 81.3%, 82.9%, or 83.1% of the total peptides in the animal-free complex protein hydrolysate, or may 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 may be combined with any other range.

[0065] In some embodiments of this application, in the animal-free complex protein hydrolysate, peptides of 400 Da or less account for 69.5-83.1% of the total peptides in the animal-free complex protein hydrolysate by mass, peptides of 400-1000 Da account for 14.1-26.1% of the total peptides in the animal-free complex protein hydrolysate by mass, peptides of 1000-2000 Da account for 1.5-4.7% of the total peptides in the animal-free complex protein hydrolysate by mass, and peptides of ≥2000 Da account for 0.9-2.8% of the total peptides in the animal-free complex protein hydrolysate by mass.

[0066] In some embodiments of this application, the weight percentage of peptides with a molecular weight of 1000 Da or less in the animal-free complex protein hydrolysate is 90-98%, preferably 92-98% of the total peptides in the animal-free complex protein hydrolysate. In some embodiments, the weight percentage of peptides with a molecular weight of 1000 Da or less in the animal-free complex protein hydrolysate can be 90-98%, 92-98%, 93-98%, 94-98%, 95-98%, 96-98%, or 97-98%. In some embodiments, the weight percentage of peptides with a molecular weight of 1000 Da or less in the animal-free complex protein hydrolysate can be 92.6%, 93%, 95%, 95.6%, 96.6%, 97%, 97.1%, 97.2%, or 97.5%, or falls within a range defined by any two of the above specific values ​​as endpoints. It should be understood that, in specific implementations, any of the above ranges can be combined with any other ranges.

[0067] In some embodiments of this application, the free amino acid content in the animal-free composite protein hydrolysate is 15-40 wt%, preferably 20-40 wt%. In some embodiments, the free amino acid content in the animal-free composite protein hydrolysate can be 15-40 wt%, 15.4-40 wt%, 23.1-40 wt%, 24.6-40 wt%, 25.6-40 wt%, 26.7-40 wt%, 27.7-40 wt%, 29-40 wt%, 34.8-40 wt%, or 37.2-40 wt%. In some embodiments, the free amino acid content in the animal-free composite protein hydrolysate can be 15 wt%, 15.4 wt%, 23.1 wt%, 24.6 wt%, 25.6 wt%, 26.7 wt%, 27.7 wt%, 29 wt%, 34.8 wt%, or 37.2 wt%, or fall within a numerical range defined by any two of the above specific values ​​as endpoints. It should be understood that, in specific implementations, any of the above ranges can be combined with any other ranges.

[0068] Specifically, in some embodiments of this application, the free amino acids in the animal-free complex protein hydrolysate, by weight percentage, include aspartic acid 0.3-1.9%, threonine 0.9-2.0%, serine 0.8-2.0%, glutamic acid 0.7-5.1%, glycine 0.2-1.2%, alanine 0.8-4.3%, valine 1.1-3%, methionine 0.3-2%, isoleucine 1.1-3%, leucine 2-5%, tyrosine 1.1-3.5%, phenylalanine 1.1-3.5%, lysine 0.5-2.5%, histidine 0.4-1.0%, arginine 1.1-5%, and proline 0.2-1.0%. In some embodiments, the free amino acids in the animal-free complex protein hydrolysate include aspartic acid 0.3-1.9%, threonine 1.6-2.0%, serine 0.8-2.0%, glutamic acid 0.7-5.1%, glycine 0.2-1.2%, alanine 0.8-4.3%, valine 2.6-3%, methionine 0.3-2%, isoleucine 1.9-3%, leucine 3-5%, tyrosine 2-3.5%, phenylalanine 2-3.5%, lysine 1.9-2.5%, histidine 0.4-1.0%, arginine 1.8-5%, and proline 0.2-1.0%.

[0069] In some embodiments of this application, the animal-free complex protein hydrolysate is further prepared by adding an exogenous enzyme to a raw material containing plant protein and yeast for enzymatic hydrolysis, wherein the exogenous enzyme includes an exogenous protease. Further, the exogenous enzyme includes one or both selected from exogenous amylase and exogenous nuclease.

[0070] In some embodiments of this application, the amount of exogenous protease added is 0.1-5% based on the dry weight of plant protein and yeast. Further, the amount of exogenous amylase and / or exogenous nuclease added is 0.1-1 wt% based on the dry weight of plant protein and yeast.

[0071] It should be noted that in this application, exogenous enzymes refer to enzymes added artificially during the hydrolysis of plant proteins and yeast. Exogenous proteases refer to proteases added artificially during the hydrolysis of plant proteins and yeast. Exogenous amylases or exogenous nucleases refer to amylases or nucleases added artificially during the hydrolysis of plant proteins and yeast. The animal-free complex protein hydrolysate of this application can also be obtained without adding exogenous enzymes, utilizing only endogenous enzymes produced by yeast autolysis. In this application, after the dispersion of plant protein and yeast is heated to 40-60℃, the yeast gradually becomes inactive, and simultaneously undergoes autolysis and cell wall disruption, releasing cell contents into the dispersion. The endogenous proteases in the yeast cells then hydrolyze the plant protein, finally yielding an animal-free complex protein hydrolysate containing both plant protein and yeast nutrients.

[0072] In some embodiments of this application, the exogenous protease is selected from one or more of papain, bromelain, alkaline protease, neutral protease, acidic protease, and flavor protease.

[0073] To enrich the small peptides and free amino acids in the complex protein hydrolysate, in some embodiments of this application, the exogenous protease includes papain. In some embodiments, the amount of papain added is 1-3% based on the dry weight of the plant protein and yeast.

[0074] In some embodiments of this application, the animal-free complex protein hydrolysate is prepared by a method comprising the following steps: preparing a dispersion of plant protein and yeast, enzymatically hydrolyzing it at 40-60°C, and then obtaining the animal-free protein hydrolysate by enzyme inactivation and separation.

[0075] In this application, "animal-free" in animal-free complex protein hydrolysate means that it does not contain any animal-derived components.

[0076] It is understood that in this application, the dispersion of plant protein and yeast is an aqueous dispersion.

[0077] In some embodiments of this application, the plant protein is selected from one or more of soybean protein, wheat protein, pea protein, rice protein, potato protein, peanut protein, and cottonseed protein. To further enrich the small peptides and free amino acids in the complex protein hydrolysate, in some embodiments, the plant protein includes soybean protein and / or rice protein.

[0078] In some embodiments of this application, the yeast is active dry yeast or active wet yeast.

[0079] In some embodiments of this application, the yeast is selected from one or more of Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Kluyveromyces martensii.

[0080] In some embodiments of this application, the weight ratio of the plant protein to the yeast can be 1:99-99:1. To enrich the small peptides and free amino acids in the complex protein hydrolysate, in some embodiments, the weight ratio of the plant protein to the yeast is 1-50:50-99.

[0081] In some embodiments of this application, the weight ratio of the plant protein to the yeast can be 1-80:20-99, 1-70:30-99, 1-60:40-99, 1-50:50-99, 1-40:60-99, 1-30:70-99, 1-20:80-99, 10-90:10-90, 20-90:10-80, 20-80:20-80, 30-70:30-70, or 40-60:60:40. In some embodiments, the weight ratio of the plant protein to the yeast can be 1:99, 20:80, 30:70, 40:60, or 50:50, 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.

[0082] On the other hand, this application provides a method for preparing the above-mentioned animal-free complex protein hydrolysate, comprising the following steps:

[0083] (1) Prepare a dispersion of plant protein and yeast, and raise the temperature to 40-60℃;

[0084] (2) Enzymatic hydrolysis was carried out at 40-60℃ to obtain the enzymatic hydrolysate;

[0085] (3) The enzyme hydrolysate obtained in step (2) is inactivated and separated to obtain a complex protein hydrolysate without animal origin.

[0086] In some embodiments of this application, a method for preparing the above-mentioned animal-free complex protein hydrolysate is provided, comprising the following steps:

[0087] (1) Prepare a dispersion of plant protein and yeast;

[0088] (2) Heat to 40-60℃ and add exogenous enzymes to perform enzymatic hydrolysis to obtain enzymatic hydrolysate, wherein the exogenous enzymes include exogenous proteases;

[0089] (3) The enzyme hydrolysate obtained in step (2) is inactivated and separated to obtain a complex protein hydrolysate without animal origin.

[0090] In some embodiments of this application, in order to improve the yield of the animal-free complex protein hydrolysate in the above preparation method, the weight ratio of the plant protein to the yeast is 1-99:99-1, preferably 20-90:10-80, and more preferably 30-90:10-70.

[0091] In some embodiments, the weight ratio of the plant protein to the yeast can be 1-99:99-1, 1-90:10-90, 1-80:20-99, 1-70:30-99, 1-60:40-99, 1-50:50-99, 1-40:60-99, 1-30:70-99, 1-20:80-99, 10-90:10-90, 20-90:10-80, 20-80:20-80, 30-70:30-70, or 40-60:60:40. In some embodiments, the weight ratio of the plant protein to the yeast can be 1:99, 20:80, 30:70, 40:60, or 50:50, or fall within a range defined by any two of the above specific values ​​as endpoints. It should be understood that, in specific implementations, any of the above ranges can be combined with any other ranges.

[0092] In some embodiments of this application, in the above preparation method, the amount of exogenous protease added is 0.1-5% based on the dry weight of plant protein and yeast, preferably 0.5-3%, and more preferably 2-3%.

[0093] In some embodiments of this application, in the above preparation method, the exogenous protease is selected from one or more of papain, bromelain, alkaline protease, neutral protease, acidic protease and flavor protease.

[0094] In some embodiments of this application, the exogenous protease includes papain, and in some embodiments, the amount of papain added is 1-3%. In some embodiments, the exogenous protease includes an alkaline protease, and in some embodiments, the amount of papain added is 0.5-2%. In some embodiments, the exogenous protease includes a neutral protease, and in some embodiments, the amount of neutral protease added is 1-2%. In some embodiments, the exogenous protease includes an acidic protease, and in some embodiments, the amount of acidic protease added is 2-3%. In some embodiments, the exogenous protease includes a flavor protease, and in some embodiments, the amount of flavor protease added is 0.5-1.5%.

[0095] In some embodiments of this application, the above preparation method includes the addition of exogenous amylase and / or exogenous nuclease in addition to exogenous protease. In some embodiments, the amount of exogenous amylase and / or exogenous nuclease added is 0.1-1 wt%, preferably 0.1-0.5 wt%.

[0096] The enzyme activities of the various enzymes used in this application can be as follows: papain activity can be 500,000-700,000 U / g, bromelain activity can be 500,000-700,000 U / g, alkaline protease activity can be 100,000-300,000 U / g, neutral protease activity can be 50,000-150,000 U / g, acidic protease activity can be 40,000-60,000 U / g, flavor protease activity can be 20,000-40,000 U / g, amylase activity can be 100,000-300,000 U / g, and nuclease activity can be 50,000-150,000 U / g.

[0097] In some embodiments of this application, in the above preparation method, the plant protein is selected from one or more of soybean protein, wheat protein, pea protein, rice protein, potato protein, peanut protein and cottonseed protein.

[0098] In some embodiments of this application, the yeast in the above preparation method is active dry yeast or active wet yeast. In some embodiments, the yeast is selected from one or more of Saccharomyces cerevisiae, Candida albicans, and Kluyveromyces oryzae.

[0099] In some embodiments of this application, the active dry yeast of this application may be prepared by a method comprising the following steps:

[0100] a. Seed culture: The strains preserved on slant culture were subjected to primary and secondary seed culture;

[0101] b. Seed enrichment: The secondary seed culture medium was centrifuged and washed to enrich yeast seeds;

[0102] c. The yeast seed is dried to obtain active dry yeast.

[0103] In some embodiments, the primary seed culture includes the following steps: inoculating the inoculum into a primary culture medium and culturing it at 28-32°C and pH 5.0-6.0 for 15-25 hours. In some embodiments, the primary culture medium comprises, by weight percentage, 1-2% yeast extract, 2-3% peptone, and 2-3% glucose.

[0104] 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 5.0-6.0 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.

[0105] In some embodiments of this application, the enzymatic hydrolysis temperature in the above preparation method is 40-60℃. In some embodiments, the enzymatic hydrolysis temperature can be 40-55℃, 40-50℃, or 50-55℃.

[0106] In some embodiments of this application, the enzymatic hydrolysis pH in the above preparation method is 3-10. In some embodiments, the enzymatic hydrolysis pH can be 3-9, 3-8, 3-7, 3-6, 3-5, 5-10, 6-10, 7-10, 8-10, or 9-10. In some embodiments, the enzymatic hydrolysis time can be 3-48h, 6-48h, 12-48h, 18-48h, 24-48h, or 36-48h.

[0107] In some embodiments of this application, the enzyme inactivation in the above preparation method is carried out by heating to 65-95°C and holding for 5-60 minutes.

[0108] In some embodiments of this application, the separation is selected from one or more of centrifugal separation, plate and frame filtration, ceramic membrane microfiltration, and organic membrane ultrafiltration.

[0109] In some embodiments of this application, the preparation method further includes the steps of concentrating and / or drying the animal-free complex protein hydrolysate.

[0110] In some embodiments of this application, the concentration is selected from triple-effect evaporation or nanofiltration membrane concentration; preferably, the nanofiltration membrane is a polysulfone nanofiltration membrane, preferably with a molecular weight cutoff of 200-500 Da.

[0111] In some embodiments of this application, the drying is selected from one of spray drying, vacuum belt drying, vacuum microwave drying, or vacuum freeze drying.

[0112] In some embodiments of this application, the yield of the animal-free complex protein hydrolysate in the above preparation method is 50-91%, preferably 60-91%, and more preferably 62-91%.

[0113] In some embodiments of this application, the degree of hydrolysis of the animal-free complex protein hydrolysate in the above preparation method is 17-60%, preferably 30-60%, more preferably 40-60%, and even more preferably 50-60%.

[0114] Thirdly, this application also provides animal-free complex protein hydrolysates prepared by the above method.

[0115] Fourthly, the present invention also provides the application of the above-mentioned animal-free complex protein hydrolysate or the animal-free complex protein hydrolysate prepared by the above method in the fields of animal cell culture, microbial fermentation, animal nutrition or plant nutrition.

[0116] The beneficial effects of the present invention will be further illustrated below through specific embodiments.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] The sources of the reagents and instruments used in the following examples are shown in Table 1.

[0121]

[0122]

[0123] The indicators in the examples and comparative examples were tested according to the following testing methods, and the results are shown in Tables 2-5.

[0124] (1) Determination of total nitrogen

[0125] The Kjeldahl method for nitrogen determination was adopted as described in section 6.4 of the national standard GB / T 23530-2009.

[0126] Take a sample (equivalent to 30-440 mg of total nitrogen), add 20 mL of concentrated sulfuric acid for digestion under the action of 5 g of mixed catalyst a (potassium sulfate and copper sulfate from wastewater mixed in a ratio of 97:3) and 2.5 g of catalyst b (selenium powder and potassium sulfate mixed in a ratio of 0.1:100); then distill, absorb the ammonia product with boric acid; then titrate with 0.1 mol / L hydrochloric acid, read the data, and calculate the total nitrogen content.

[0127] (2) Determination of amino acid nitrogen

[0128] The determination method for amino acid nitrogen as described in section 6.5 of the national standard GB / T 23530-2009 was adopted:

[0129] Take 5g of sample, dilute it, and titrate it with 0.5mol / L sodium hydroxide solution to pH 8.2, maintaining this titration for 1 min. Slowly add 10mL of 36% formaldehyde solution, which reacts with the non-dissociated amino group in the neutral amino acid to generate monohydroxymethyl and dihydroxymethyl inducers. This reaction proceeds completely and quantitatively. The hydrogen ions released at this point are titrated with the aforementioned sodium hydroxide solution, and the amino acid nitrogen content is calculated based on the amount of alkali consumed.

[0130] (3) Calculation of yield:

[0131] Yield = Dry weight of hydrolysate / (Total dry weight of plant protein + yeast) * 100%.

[0132] (4) Calculation of degree of hydrolysis:

[0133] Degree of hydrolysis = the ratio of amino acid nitrogen to total nitrogen.

[0134] (5) Determination of ash content:

[0135] The determination shall be carried out according to the method specified in Method I of GB 5009.4.

[0136] (6) Determination of nucleotides:

[0137] The determination shall be performed according to the method in Appendix H of GB / T 20886.2.

[0138] (7) Determination of the content and composition of free amino acids:

[0139] Free amino acids in the sample were analyzed using a fully automated amino acid analyzer (Hitachi L-8900). The specific steps are as follows: Take 0.5-1g of sample (accurate to 0.001g) and place it in a 50mL volumetric flask. Add 20mL of sulfosalicylic acid and sonicate until fully dissolved. Make up to 50mL and mix well. Accurately pipette 1mL into a 25mL volumetric flask, add citrate buffer (pH 2.2) and make up to 25mL. Mix well and pass through a 0.45μm membrane for analysis.

[0140] (8) Determination of peptide molecular weight distribution:

[0141] Principle: High-performance gel filtration chromatography (GPC) is used for determination. A porous packing material is used as the stationary phase, and the components are separated based on their relative molecular mass differences. Detection is performed under ultraviolet absorption at 220 nm. Dedicated data processing software (GPC software) for determining the relative molecular mass distribution of gel chromatography is used to process the chromatogram and data, calculating the relative molecular mass and distribution range of the peptides.

[0142] 8.1 Reagents:

[0143] a) The water used in the experiment shall meet the specifications for Class II water in GB / T 6682, and all reagents used shall be of analytical grade unless otherwise specified.

[0144] b) Acetonitrile was of chromatographic grade;

[0145] c) Standards used for the relative molecular mass distribution calibration curve;

[0146] insulin;

[0147] Bacitracin;

[0148] Glycine-glycine-tyrosine-arginine;

[0149] Glycine-glycine-glycine.

[0150] 8.2 Instruments and equipment.

[0151] a) High performance liquid chromatograph: equipped with an ultraviolet detector and a chromatography workstation or integrator containing GPC data processing software.

[0152] b) Mobile phase vacuum filtration and degassing device.

[0153] c) Electronic balance: scale division value 0.0001g.

[0154] 8.3 Operating Procedures

[0155] Chromatographic conditions and system suitability experiment (reference conditions)

[0156] Chromatographic column: TSK gel G2000SWXL 300 mm × 7.8 mm (inner diameter) or other similar gel columns suitable for the determination of proteins and peptides.

[0157] Mobile phase: Acetonitrile: Water: Trifluoroacetic acid = 20:80:0.1 (volume ratio).

[0158] Detection wavelength: 220nm.

[0159] Flow rate: 0.5 mL / min.

[0160] Detection time: 30 minutes.

[0161] Injection volume: 20 μL.

[0162] Column temperature: room temperature.

[0163] To ensure that the chromatographic system meets the detection requirements, it is stipulated that under the above chromatographic conditions, the column efficiency of gel chromatography, i.e., the theoretical plate number (N), calculated based on the standard (glycine-glycine-glycine) peak, shall not be less than 10,000.

[0164] 8.4 Preparation of relative molecular mass standard curve

[0165] Different molecular weight standard solutions with a mass concentration of 1 mg / mL were prepared using the mobile phase. After mixing in a certain proportion, the solutions were filtered through an organic membrane with a pore size of 0.2 μm-0.5 μm and then injected to obtain chromatograms of the standards. The molecular weight calibration curve and its equation were obtained by plotting the logarithm of the molecular weight against the retention time or by performing linear regression.

[0166] 8.5 Sample Preparation

[0167] Accurately weigh 10 mg of sample into a 10 mL volumetric flask, add a small amount of mobile phase, sonicate for 10 min to fully dissolve and mix the sample, dilute to the mark with mobile phase, filter through an organic membrane with a pore size of 0.2 μm-0.5 μm, and analyze the filtrate under the chromatographic conditions described above.

[0168] 8.6 Calculation of relative molecular mass distribution

[0169] After analyzing the sample solution prepared in 8.5 under the chromatographic conditions in 8.3, the chromatographic data of the sample were substituted into the calibration curve (relative molecular mass standard curve) using GPC data processing software for calculation, thus obtaining the relative molecular mass and its distribution range of the sample. The distribution of molecular mass of different peptide phases can be calculated using the peak area normalization method, as follows:

[0170]

[0171]

[0172] In the formula:

[0173] X i —The percentage by weight of peptides with relative molecular masses ≤400 Da, 400-1000 Da, 1000-2000 Da, or ≥2000 Da in the total peptides, %

[0174] A i —The sum of peak areas of peptides with different relative molecular masses (where i represents peptides with relative molecular masses ≤400Da, 400-1000Da, 1000-2000Da, or ≥2000Da).

[0175] The calculation result is rounded to one decimal place.

[0176] 8.7 Repeatability

[0177] The absolute difference between two independent measurements obtained under repeatability conditions shall not exceed 15% of the arithmetic mean of the two measurements.

[0178] (9) Protein determination

[0179] The determination was performed according to Method I in GB 5009.5-2016 National Food Safety Standard - Determination of Protein in Food.

[0180] Example 1

[0181] Preparation of animal-free complex protein hydrolysates

[0182] (1) Preparation of active dry brewing yeast FX-2

[0183] a. Seed culture: For primary seed culture, one loopful of Saccharomyces cerevisiae FX-2 was inoculated from the slant of the preserved Saccharomyces cerevisiae strain 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 28°C and 200 rpm for 18 h.

[0184] The primary culture medium, by weight percentage, consists of: 1% yeast extract, 2% peptone, 2% glucose, and the remainder is water, with its pH controlled at 5.5; the secondary culture medium, by weight percentage, consists of: 2% glucose, 2% yeast extract, 0.1% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, and the remainder is water, with its pH controlled at 5.5.

[0185] b. Seed enrichment: Centrifuge, wash three times with deionized water with low calcium and magnesium ion content to enrich the brewer's yeast seeds until the yeast wet weight is 220g / L.

[0186] c. The Saccharomyces cerevisiae seeds were dried to obtain active dry Saccharomyces cerevisiae FX-2.

[0187] (2) Enzymatic hydrolysis: Weigh 500g of soybean protein and 500g of active dry brewer's yeast FX-2, add water to prepare a dispersion with a dry matter concentration of 10%, heat to 55℃, adjust pH to 7.0, add 2wt% papain to the dispersion based on the dry matter weight of soybean protein and active dry brewer's yeast FX-2, and hydrolyze at 55℃ for 36h; after the hydrolysis is completed, heat to 95℃ to inactivate the enzyme for 5min.

[0188] (3) Separation: The enzymatic hydrolysate is separated using a disc centrifuge, and the clear liquid is collected.

[0189] (4) Concentration and drying: The separated clear liquid is evaporated by triple effect to obtain concentrated liquid, and the concentrated liquid is spray dried to obtain dried product of animal-free complex protein hydrolysate.

[0190] Example 2

[0191] Preparation of animal-free complex protein hydrolysates

[0192] (1) Enzymatic hydrolysis: Weigh 500g of soybean protein and 500g of active dry brewer's yeast FX-2 prepared in Example 1, add water to prepare a 10% dispersion, heat to 55℃, adjust pH to 7.0, and enzymatically hydrolyze for 36h under these conditions; after the enzymatic hydrolysis is completed, heat to 95℃ to inactivate the enzyme for 5min.

[0193] (2) Separation: The enzymatic hydrolysate is separated using a disc centrifuge, and the clear liquid is collected.

[0194] (3) Concentration and drying: The separated clear liquid is evaporated by triple effect to obtain concentrated liquid, and the concentrated liquid is spray dried to obtain dried product of animal-free complex protein hydrolysate.

[0195] Example 3

[0196] Preparation of animal-free complex protein hydrolysates

[0197] (1) Enzymatic hydrolysis: Weigh 700g of pea protein and 300g of active dry brewer's yeast FX-2 prepared in Example 1, add water to prepare a 15% dispersion, heat to 50℃, adjust pH to 9.0, add 2wt% alkaline protease and 0.1wt% amylase based on the dry matter weight of pea protein and active dry brewer's yeast FX-2, and hydrolyze for 6h; after the hydrolysis is completed, heat to 85℃ to inactivate the enzyme for 10min.

[0198] (2) Separation: The enzymatic hydrolysate was microfiltered using a ceramic membrane (membrane pore size of 1 μm), and the microfiltered solution was then ultrafiltered using an organic membrane (membrane pore size of 10 nm) to obtain the ultrafiltered solution.

[0199] (3) Concentration and drying: The ultrafiltrate is concentrated by passing it through a polysulfone nanofiltration membrane (with a molecular weight cutoff of 300 Da) to obtain a concentrated solution. The concentrated solution is then freeze-dried under vacuum to obtain a dried product of animal-free composite protein hydrolysate.

[0200] Example 4

[0201] Preparation of animal-free complex protein hydrolysates

[0202] (1) Enzymatic hydrolysis: Weigh 900g of wheat protein and 100g of active dry brewer's yeast FX-2 prepared in Example 1, add water to prepare a 10% dispersion, heat to 50℃, adjust pH to 8.0, add 2wt% alkaline protease and 1wt% neutral protease respectively based on the dry matter weight of wheat protein and active dry brewer's yeast FX-2, and hydrolyze for 18h; after the hydrolysis is completed, heat to 75℃ to inactivate enzyme for 30min.

[0203] (2) Separation: The enzymatic hydrolysate was filtered by plate and frame filter press to obtain the filtered clear liquid.

[0204] (3) Concentration and drying: The filtered liquid is evaporated through triple-effect evaporation to obtain a concentrated liquid. The concentrated liquid is then sprayed to obtain a dried product of animal-free complex protein hydrolysate.

[0205] Example 5

[0206] Preparation of animal-free complex protein hydrolysates

[0207] (1) Enzymatic hydrolysis: Weigh 990g of rice protein and 10g of active dry brewer's yeast FX-2 prepared in Example 1, add water to prepare a 10% dispersion, heat to 60℃, adjust pH to 7.0, add 2wt% papain and 0.1wt% nuclease according to the dry matter weight of soybean protein and active dry brewer's yeast FX-2, respectively, and hydrolyze for 3h; after the hydrolysis is completed, heat to 95℃ to inactivate enzyme for 5min.

[0208] (2) Separation: The enzyme hydrolysate is centrifuged to obtain a clear liquid.

[0209] (3) Concentration and drying: The centrifuged clear liquid is evaporated by triple effect to obtain concentrated liquid, and the concentrated liquid is dried by vacuum belt drying to obtain the dried product of animal-free complex protein hydrolysate.

[0210] Example 6

[0211] Preparation of animal-free complex protein hydrolysates

[0212] (1) Enzymatic hydrolysis: Weigh 10g of potato protein and 990g of active dry brewer's yeast FX-2 prepared in Example 1, add water to prepare a 10% dispersion, heat to 40℃, adjust pH to 6.0, and enzymatically hydrolyze for 12h under these conditions; after the enzymatic hydrolysis is completed, heat to 75℃ to inactivate the enzyme for 30min.

[0213] (2) Separation: The enzymatic hydrolysate is microfiltered through a ceramic membrane (pore size 1um) to obtain a clear microfiltrate.

[0214] (3) Concentration and drying: The microfiltrate is concentrated by passing it through a polysulfone nanofiltration membrane (with a molecular weight cutoff of 300 Da). The concentrated solution is then dried by vacuum microwave drying to obtain the dried product of animal-free composite protein hydrolysate.

[0215] Example 7

[0216] Preparation of animal-free complex protein hydrolysates

[0217] (1) Preparation of active dry Candida albicans C1.7

[0218] a. Seed culture: For primary seed culture, one loopful of Candida cerevisiae C1.7 cells was inoculated from a slant culture of Candida cerevisiae 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 28°C and 200 rpm for 18 h.

[0219] The primary culture medium, by weight percentage, consists of: 1% yeast extract, 2% peptone, 2% glucose, and the remainder is water, with its pH controlled at 5.5; the secondary culture medium, by weight percentage, consists of: 2% glucose, 2% yeast extract, 0.1% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, and the remainder is water, with its pH controlled at 5.5.

[0220] b. Seed enrichment: Centrifuge, wash three times with deionized water with low calcium and magnesium ion content, and enrich to obtain Candida albicans.

[0221] c. The Candida seeds were dried to obtain active dry Candida C1.7.

[0222] (2) Enzymatic hydrolysis: Weigh 200g of corn protein and 800g of active dry Candida albicans C1.7, add water to prepare a 10% dispersion, heat to 40℃, adjust pH to 3.0, add 3wt% acidic protease based on the dry matter weight of soybean protein and active dry Candida albicans C1.7, and hydrolyze for 48h; after the hydrolysis is completed, heat to 65℃ to inactivate the enzyme for 60min.

[0223] (3) Separation: The enzymatic hydrolysate is centrifuged, and the centrifuged clear liquid is then filtered through a plate and frame filter to obtain the filtered clear liquid.

[0224] (4) Concentration and drying: The filtered liquid is concentrated under reduced pressure to obtain a concentrated liquid, and the concentrated liquid is spray-dried to obtain a dried product of animal-free complex protein hydrolysate.

[0225] Example 8

[0226] Preparation of animal-free complex protein hydrolysates

[0227] (1) Preparation of active dry Pichia pastoris C1.8

[0228] a. Seed culture: Primary seed culture: One loopful of Pichia pastoris (Saccharomyces cerevisiae) C1.8 was inoculated from a Pichia pastoris slant into 200 mL of primary culture medium and cultured at 30°C and 220 rpm for 18 h. Secondary seed culture: 10% of the primary culture seed was inoculated into 1 L of secondary culture medium and cultured at 28°C and 200 rpm for 18 h. The primary culture medium, by weight percentage, consisted of: 1% yeast extract, 2% peptone, 2% glucose, and the remainder was water, with the pH controlled at 5.5. The secondary culture medium, by weight percentage, consisted of: 2% glucose, 2% yeast extract, 0.1% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, and the remainder was water, with the pH controlled at 5.5.

[0229] b. Seed enrichment: Centrifuge, wash three times with deionized water with low calcium and magnesium ion content, and enrich to obtain Pichia pastoris.

[0230] c. Pichia pastoris seeds were dried to obtain active dry Pichia pastoris C1.8.

[0231] (2) Enzymatic hydrolysis: Weigh 800g of cottonseed protein and 200g of active dry Pichia pastoris C1.8, add water to prepare a 10% dispersion, heat to 45℃, adjust pH to 6.0, add 2wt% neutral protease and 1wt% flavor protease based on the dry matter weight of soybean protein and active dry Pichia pastoris C1.8, and hydrolyze for 6h; after the hydrolysis is completed, heat to 65℃ to inactivate the enzyme for 60min.

[0232] (3) Separation: The enzyme hydrolysate is centrifuged to obtain a clear liquid.

[0233] (4) Concentration and drying: The centrifuged clear liquid is concentrated under reduced pressure to obtain a concentrated liquid, and the concentrated liquid is spray-dried to obtain a dried product of animal-free complex protein hydrolysate.

[0234] Example 9

[0235] Preparation of animal-free complex protein hydrolysates

[0236] (1) Enzymatic hydrolysis: Weigh 200g rice protein, 200g pea protein, 200g potato protein and 400g active dry brewer's yeast FX-2 prepared in Example 1, add water to prepare a 10% dispersion, heat to 40℃, adjust pH to 5.0, add 1wt% neutral protease and 1wt% flavor protease according to the dry matter weight of rice protein, pea protein, potato protein and active dry brewer's yeast FX-2 respectively, and hydrolyze for 24h; after the hydrolysis is completed, heat to 65℃ to inactivate enzyme for 60min.

[0237] (2) The enzymatic hydrolysate is centrifuged, the centrifuged clear liquid is microfiltered through a ceramic membrane, and the microfiltered clear liquid is then ultrafiltered through an organic membrane to obtain the ultrafiltered clear liquid.

[0238] (3) Concentration and drying: The ultrafiltrate is concentrated by passing it through a polysulfone nanofiltration membrane (with a molecular weight cutoff of 300 Da) to obtain a concentrated solution. The concentrated solution is then freeze-dried under vacuum to obtain a dried product of animal-free composite protein hydrolysate.

[0239] Example 10

[0240] Preparation of animal-free complex protein hydrolysates

[0241] (1) Preparation of active dried Kluyveromyces martensii AMCC 30634

[0242] a. Seed culture: Primary seed culture: One loopful of Kluyveromyces marxianus AMCC30634 was inoculated from a Kluyveromyces marxianus slant culture into 200 mL of primary culture medium and cultured at 30°C and 220 rpm for 18 h. Secondary seed culture: 10% of the primary culture seed was inoculated into 1 L of secondary culture medium and cultured at 28°C and 200 rpm for 18 h. The primary culture medium, by weight percentage, consisted of: 1% yeast extract, 2% peptone, 2% glucose, and the remainder was water, with a pH controlled at 5.5. The secondary culture medium, by weight percentage, consisted of: 2% glucose, 2% yeast extract, 0.1% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, and the remainder was water, with a pH controlled at 5.5.

[0243] b. Seed enrichment: Centrifuge, wash three times with deionized water with low calcium and magnesium ion content, and enrich to obtain Kluyveromyces martensii.

[0244] c. The Kluyveromyces seeds were dried to obtain active dried Kluyveromyces AMCC 30634.

[0245] (2) Enzymatic hydrolysis: Weigh 100g soybean protein, 100g wheat protein, 100g corn protein and 700g active dry Kluyveromyces macrocarpa AMCC 30634, add water to prepare a 10% dispersion, heat to 55℃, adjust pH to 10.0, add 0.5wt% alkaline protease based on the dry matter weight of soybean protein, wheat protein, corn protein and Kluyveromyces macrocarpa AMCC 30634, and hydrolyze for 18h; after the hydrolysis is completed, heat to 95℃ to inactivate the enzyme for 5min.

[0246] (3) Separation: The enzymatic hydrolysate is filtered by plate and frame filter, and the filtered liquid is filtered by ceramic membrane microfiltration to obtain microfiltrate.

[0247] (4) Concentration and drying: The microfiltrate is evaporated by triple evaporation to obtain a concentrated solution, which is then spray-dried to obtain a dried product of animal-free complex protein hydrolysate.

[0248] Comparative Example 1

[0249] (1) Enzymatic hydrolysis: Weigh 1000g of soybean protein, add water to prepare a 10% dispersion, heat to 55℃, adjust pH to 7.0, add 2wt% papain to the dispersion based on the dry weight of soybean protein, and hydrolyze at 55℃ for 36h; after the hydrolysis is completed, heat to 95℃ for 5min to inactivate the enzyme.

[0250] (2) Separation: The enzymatic hydrolysate is separated using a disc centrifuge, and the clear liquid is collected.

[0251] (3) Concentration and drying: The separated clear liquid is evaporated by triple evaporation to obtain a concentrated liquid, and the concentrated liquid is spray dried to obtain a dried product.

[0252] Comparative Example 2

[0253] (1) Enzymatic hydrolysis: Weigh 1000g of active dry brewer's yeast FX-2 prepared in Example 1, add water to prepare a 10% dispersion, heat to 55℃, adjust pH to 7.0, and react at 55℃ for 36h; after the reaction is completed, heat to 95℃ to inactivate enzymes for 5min.

[0254] (2) Separation: The enzymatic hydrolysate is separated by centrifugation to obtain the centrifuged clear liquid.

[0255] (3) Concentration and drying: The centrifuged clear liquid is evaporated through triple-effect evaporation to obtain a concentrated liquid, and the concentrated liquid is spray-dried to obtain a dried product.

[0256] Specifically, the results of various component determinations of the protein hydrolysates prepared in Examples 1-10 and Comparative Examples 1-2 are shown in Tables 2-5 below.

[0257]

[0258]

[0259]

[0260]

[0261] As shown in Table 2, the protein hydrolysates obtained in Examples 1-10 of this application have a total nitrogen content of 9.9-13.9%, an amino acid nitrogen content of 2.5-6%, a nucleotide content of 0.2-1.2%, a free amino acid content of 15-37.2%, a protein content of 61.9-86.9%, and a small molecule peptide (≤1000 Da) accounting for 92.6-97.5% of the total peptides in the animal-free complex protein hydrolysate. The yield of the hydrolysate product is 50.1-90.4%, and the degree of hydrolysis is 17.7-56.2%.

[0262] Compared to Example 1, Comparative Example 1 used only plant protein for enzymatic hydrolysis, and the content of free amino acids, nucleotides, and the percentage of small peptides in the total peptides of the animal-free complex protein hydrolysate were all lower than in Example 1 and also lower than in other examples of this application; the yield of the hydrolysate product was 45%, lower than in Example 1 and also lower than in other examples of this application; the degree of hydrolysis of Comparative Example 1 was 22.6%, much lower than in Example 1. Compared to Example 1, Comparative Example 2 used only Saccharomyces cerevisiae autolysis, and the content of free amino acids in the protein hydrolysate obtained was also significantly lower than in Example 1. It should be explained here that in Examples 1, 2-4, 6-7, and 9-10 of the present invention (except Examples 5 and 8), the protein hydrolysates obtained after hydrolysis with exogenous proteases using a combination of plant protein and yeast had a content of less than 400 Da that was less than or equal to that in Comparative Example 2 (which used only yeast and did not add plant protein). This is because yeast itself has endogenous proteases, which undergo autolysis at higher temperatures (40-60°C), degrading large molecules into smaller molecules. Since yeast endogenous enzymes are a set of proteases evolved specifically for yeast's own proteins, their enzymatic hydrolysis efficiency is high. Therefore, even without the use of exogenous proteases, hydrolysates rich in small peptides and amino acids can be formed. Consequently, the degree of hydrolysis in Examples 2-10 is also less than that in Comparative Example 2. Only the degree of hydrolysis in Example 1 is greater than that in Comparative Example 2. This is because Example 1 has two advantages over other examples. The characteristics are as follows: First, the raw material is soy protein, whose amino acid composition is very similar to that of yeast protein, which may explain why yeast endogenous enzymes can efficiently cleave soy protein. Second, an exogenous protease is added. The addition of exogenous enzymes can not only hydrolyze proteins but also help to break down yeast cell walls, allowing yeast endogenous enzymes to be released more fully, bind to and hydrolyze soy protein. Therefore, Example 1 achieved a very high degree of hydrolysis. Except for Examples 3, 4, and 6, the protein content of the protein hydrolysates obtained in Examples 1-2, 5, 7-10 is lower than that in Comparative Example 2. This is because the protein content is mainly related to the protein content of the raw materials. The protein content of soybean, corn, and rice protein raw materials is between 48-60%, which is lower than the 65% of yeast. The protein content of wheat and pea protein can reach more than 85%, so the prepared hydrolysates have a higher protein content.

[0263] In this application, the physicochemical indicators are only reference indicators for measuring the degree of hydrolysis and nutrient composition of the hydrolysate. For microorganisms and animal cells, the cultivation and application effect is the key indicator for evaluating the quality of the hydrolysate. Hydrolysates with the same degree of hydrolysis will have significant differences in application depending on the yeast source and specific process parameters. The core inventive point of this invention is that by mixing yeast and plant peptone for autolysis / enzymatic hydrolysis, firstly, the amount of exogenous enzyme added can be reduced, thus lowering production costs; secondly, a composite hydrolysate with better application effect than a combination of yeast extract and plant peptone powder can be obtained. The latter is the most important inventive point of this invention, which is further verified in Experimental Examples 1 and 2.

[0264] As shown in Table 3, in the protein hydrolysates prepared in Examples 1-10 of this application, peptides with a mass percentage of 400 Da or less account for 69.5-83.1% of the total peptides in the animal-free complex protein hydrolysates, peptides with a mass percentage of 400-1000 Da account for 14.1-26.1%, peptides with a mass percentage of 1000-2000 Da account for 1.5-4.7%, and peptides with a mass percentage of ≥2000 Da account for 0.9-2.8%. Compared to Example 1, in the protein hydrolysate obtained by enzymatic hydrolysis of plant protein in Comparative Example 1, peptides with a molecular weight of 400 Da or less accounted for 49.1% of the total peptides in the animal-free complex protein hydrolysate, peptides with a molecular weight of 400-1000 Da accounted for 20.1%, peptides with a molecular weight of 1000-2000 Da accounted for 16.6%, and peptides with a molecular weight of 2000 Da or more accounted for 14.2%. It can be seen that the proportion of peptides with a molecular weight greater than 1000 Da in Comparative Example 1 is relatively large.

[0265] As shown in Tables 4 and 5, this application contains a rich variety of amino acids, each with a certain content. Specifically, in the protein hydrolysates obtained in Examples 1-10 of this application, the content of aspartic acid is 0.3-1.9%, the content of threonine is 0.9-1.9%, the content of serine is 0.8-2%, the content of glutamic acid is 0.7-5.1%, the content of glycine is 0.2-1.2%, the content of alanine is 0.8-4.3%, and the content of valine is... The content of aspartic acid is 1.1-2.9%, methionine is 0.3-1.8%, isoleucine is 1.1-2.8%, leucine is 2.2-4.7%, tyrosine is 1.1-3.2%, phenylalanine is 1.1-2.6%, lysine is 0.5-2.4%, histidine is 0.4-0.8%, arginine is 1.1-4.8%, and proline is 0.2-0.7%. Compared with Example 1, the protein hydrolysate prepared in Comparative Example 1 contains only 0.1% aspartic acid, 0.4% threonine, 0.4% serine, 0.1% glycine, 0.3% alanine, 0.4% arginine, and 0.1% proline.

[0266] Experimental Example 1: Industrial Microbial Culture Test

[0267] The cell culture effects of the protein hydrolysates prepared in Examples 1, 2, 1, and 2 were evaluated using Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and Enterococcus faecalis, respectively.

[0268] Experimental method: Weigh 10g of hydrolysate, 0.4g of glucose, and 1.13g of M9 nutrients to prepare 1L of culture medium, dispense into 10ml test tubes, and sterilize. After sterilization, inoculate the bacterial suspension at a ratio of 1%, and culture with shaking using a Bioscreen. Detect the OD600 value at different culture times.

[0269] The hydrolysates were the protein hydrolysates prepared in Example 1, Example 2, Comparative Example 1, and Comparative Example 2, respectively; the bacterial suspensions were prepared using Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and Enterococcus faecalis, respectively.

[0270] The culture results of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and Enterococcus faecalis are as follows: Figures 1-4 As shown.

[0271] Depend on Figures 1-4It can be seen that, in the culture of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and Enterococcus faecalis, the protein hydrolysates prepared in Example 1 and Example 2 have significantly better growth-promoting effects than the protein hydrolysates prepared in Comparative Example 1 and Comparative Example 2.

[0272] Experiment Example 2: Quality Control Bacterial Sensitivity Test Experiment

[0273] The protein hydrolysate prepared in Example 1, the protein hydrolysate prepared in Example 2, and the protein hydrolysate obtained by mixing equal proportions of the protein hydrolysates prepared in Comparative Examples 1 and 2 were evaluated using sensitivity tests against Rhizoctonia solani, Bacillus subtilis, and Staphylococcus aureus.

[0274] Experimental method: Weigh 20g of hydrolysate, 5g of sodium chloride, and 12g of agar to prepare 1L of culture medium. After sterilization, pour the medium onto plates. Dilute the bacterial activation solution 10... -8 ~10 -9 Take 100 μL of the diluent, spread it evenly on a plate, and observe the colonies after 24 hours of incubation.

[0275] The hydrolysates were the protein hydrolysates prepared in Example 1, the protein hydrolysates prepared in Example 2, and the protein hydrolysates obtained by mixing the protein hydrolysates of Comparative Example 1 and Comparative Example 2 in equal mass ratios. The bacterial strains used in the experiments were Rhizotrophus cocci, Bacillus subtilis, and Staphylococcus aureus.

[0276] Colony culture results of Rhizotrophic Cochlea, Bacillus subtilis and Staphylococcus aureus are as follows Figures 5-7 As shown. Among them, Figure 5 The results of sensitivity testing for *Coprinus rhizogenes* in Experimental Example 2 are shown below. From left to right, the protein hydrolysate obtained by mixing the protein hydrolysate prepared in Comparative Example 1 and Comparative Example 2 in equal mass ratios is shown below. The protein hydrolysate obtained in Example 1 is shown below. The protein hydrolysate obtained in Example 2 is shown below. Figure 6 The results of sensitivity testing of Bacillus subtilis in Experiment Example 2 are shown below. From left to right, the protein hydrolysate obtained by mixing the protein hydrolysate prepared in Comparative Example 1 and the protein hydrolysate prepared in Comparative Example 2 in equal mass ratios is shown below. The protein hydrolysate obtained in Example 1 is shown below. The protein hydrolysate obtained in Example 2 is shown below. Figure 7 The results of the sensitivity test of Bacillus subtilis in Experiment Example 2 are shown below. From left to right, the protein hydrolysate obtained by mixing the protein hydrolysate obtained in Comparative Example 1 and Comparative Example 2 in equal mass ratios is shown below, as are the protein hydrolysate obtained in Example 1 and Example 2.

[0277] Depend on Figures 5-7The results show that, for sensitivity tests of *Rhizoctonia solani*, *Bacillus subtilis*, and *Staphylococcus aureus*, the growth-promoting effects of the protein hydrolysates from Examples 1 and 2 are superior to those obtained by mixing equal mass ratios of Comparative Examples 1 and 2. This indicates that the integrated mixed enzymatic hydrolysis and autolysis is superior to the pure product combination. Therefore, the protein hydrolysates of this application have significant application potential in detection media.

[0278] Experiment Example 3: Animal Cell Culture Test

[0279] BHK21 cells, ST cells, PK15 cells, SP9 cells, MDCK cells, and H5 cells were used for cell culture to evaluate the effects of the protein hydrolysates prepared in Example 1, Example 2, Comparative Example 1, and Comparative Example 2 on animal cell culture.

[0280] Experimental Methods: 2 g of hydrolysate was added to each liter of DMEM / F12 basal medium, thoroughly dissolved, filtered, and then inoculated into cells for 48 h of culture. At the end of the culture, 100 μL of cell suspension was mixed thoroughly with 100 μL of trypan blue staining solution. The mixture was then transferred to a cell counting chamber, and cell counts were performed using a cell counter. Three cell counts were taken, and the average value was calculated to obtain the viable cell density (VCD).

[0281] The hydrolysates were the protein hydrolysates prepared in Example 1, Example 2, Comparative Example 1, and Comparative Example 2, respectively; BHK21 cells, ST cells, PK15 cells, SP9 cells, MDCK cells, and H5 cells were used for the experiments.

[0282] The culture results of BHK21 cells, ST cells, PK15 cells, SP9 cells, MDCK cells, and H5 cells are as follows: Figure 8 As shown in the figure, the viable cell density results for each cell are, from left to right, the viable cell density of the protein hydrolysate cultured cells prepared in Comparative Example 1, the viable cell density of the protein hydrolysate cultured cells prepared in Comparative Example 2, the viable cell density of the protein hydrolysate cultured cells prepared in Example 1, and the viable cell density of the protein hydrolysate cultured cells prepared in Example 2.

[0283] Depend on Figure 8 It can be seen that the growth-promoting effects of Examples 1 and 2 on the 6 cell lines were significantly better than those of Comparative Examples 1 and 2. The average viable cell density of Example 1 was 19.5% higher than that of Comparative Example 1 and 9.6% higher than that of Comparative Example 2.

[0284] 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 an animal-free complex protein hydrolysate, characterized in that, Includes the following steps: (1) Combine soybean protein and brewer's yeast ( Saccharomyces cerevisiae FX-2 is mixed in a weight ratio of 30-70:30-70 and then water is added to prepare a dispersion. The brewer's yeast FX-2 is active dry brewer's yeast FX-2 or active wet brewer's yeast FX-2. The preservation number of the brewer's yeast FX-2 is CCTCC NO: M2016418. (2) The dispersion obtained in step (1) is hydrolyzed by papain at 40-60℃ to obtain an enzymatic hydrolysate, wherein the amount of papain added is 2-5 wt% based on the dry weight of soybean protein and brewer's yeast FX-2, and the enzymatic hydrolysis pH is 3-10. (3) The enzyme hydrolysate obtained in step (2) is inactivated and separated to obtain an animal-free complex protein hydrolysate, wherein the weight percentage of peptides with a molecular weight of less than or equal to 1000 Da in the animal-free complex protein hydrolysate is greater than or equal to 90% of the total peptides in the animal-free complex protein hydrolysate, and the weight percentage of protein in the animal-free complex protein hydrolysate is greater than or equal to 61%.

2. The preparation method according to claim 1, characterized in that, In the animal-free complex protein hydrolysate, peptides with a molecular weight of 1000 Da or less account for 90-98% of the total peptides in the animal-free complex protein hydrolysate; and / or, The animal-free complex protein hydrolysate contains 65-90% protein by weight; and / or, The free amino acid content in the animal-free complex protein hydrolysate is 15-40 wt%; and / or, In the animal-free complex protein hydrolysate, peptides with a molecular weight greater than or equal to 2000 Da account for 0.5-3% of the total peptides in the animal-free complex protein hydrolysate by weight; peptides with a molecular weight greater than 1000 Da and less than 2000 Da account for 1.5-5% of the total peptides in the animal-free complex protein hydrolysate by weight; peptides with a molecular weight greater than 400 Da and less than or equal to 1000 Da account for 14-27% of the total peptides in the animal-free complex protein hydrolysate by weight; and peptides with a molecular weight less than or equal to 400 Da account for 65-85% of the total peptides in the animal-free complex protein hydrolysate by weight.

3. The preparation method according to claim 1, characterized in that, In step (2), the enzymatic hydrolysis also includes the addition of exogenous amylase and / or exogenous nuclease, wherein the amount of exogenous amylase and / or exogenous nuclease added is 0.1-1 wt% based on the dry weight of soybean protein and brewer's yeast FX-2.

4. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis time in step (2) is 3-48 hours.

5. The preparation method according to claim 1, characterized in that, The enzyme inactivation in step (3) is achieved by heating to 65-95℃ and holding for 5-60 minutes.

6. The preparation method according to claim 1, characterized in that, The separation process in step (3) is selected from one or more of centrifugal separation, plate and frame filtration, ceramic membrane microfiltration and organic membrane ultrafiltration.

7. The preparation method according to any one of claims 1-6, characterized in that, The preparation method further includes the steps of concentrating and / or drying the animal-free complex protein hydrolysate.

8. The preparation method according to claim 7, characterized in that, The concentration is selected from triple-effect evaporation or nanofiltration membrane concentration, and / or the drying is selected from spray drying, vacuum belt drying, vacuum microwave drying or vacuum freeze drying.

9. The application of the animal-free complex protein hydrolysate prepared by the method according to any one of claims 1-8 in the fields of animal cell culture, microbial fermentation, animal nutrition or plant nutrition.

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