Yeast hydrolysate as well as preparation method and application thereof
By optimizing the composition and preparation process of yeast hydrolysate, the problems of insufficient protein content, functional amino acid ratio, and nucleic acid content in existing yeast hydrolysate have been solved, thereby achieving the effects of increasing feed intake and reducing disease incidence in piglets.
Patent Information
- Application Number
- CN202511869669.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-11
- Publication Date
- 2026-04-14
AI Technical Summary
There is still room for improvement in existing yeast hydrolysates in terms of increasing piglet weight and reducing piglet disease incidence, especially in terms of protein content, functional amino acid ratio, and nucleic acid content.
A yeast hydrolysate containing a specific ratio of true protein, aromatic amino acids, and sulfur-containing amino acids, combined with disodium nucleotides, is prepared and its molecular weight distribution and particle size are optimized through specific enzymatic hydrolysis and spray drying processes to form a highly efficient feed additive.
It significantly increases feed intake in piglets, reduces the incidence of diseases in piglets, including white scours, yellow scours, red scours, and paratyphoid, increases body weight by 34%, and reduces the incidence of diseases by 57%.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of feed, specifically to a yeast hydrolysate, its preparation method, and its application. Background Technology
[0002] With the rapid development of the livestock industry, the demand for feed is increasing, especially the supply and demand imbalance of feed protein sources is becoming increasingly prominent. To solve this problem, developing new feed protein resources has become a hot topic in the industry.
[0003] Yeast hydrolysate (yeast single-cell protein hydrolysate) has superior nutritional quality compared to existing protein feed ingredients, helping to reduce the use of soybean meal and fishmeal, thereby meeting the production needs of new feed protein resources. However, existing yeast hydrolysates still need improvement in terms of increasing piglet weight and reducing piglet disease incidence.
[0004] Therefore, developing yeast hydrolysates that can help further increase the weight of piglets and reduce the incidence of diseases in piglets is a research hotspot in the field. Summary of the Invention
[0005] This invention provides a yeast hydrolysate, its preparation method, and its application, which helps to further increase feed intake and reduce the incidence of diseases in piglets.
[0006] This invention provides a yeast hydrolysate comprising 35% to 65% by mass of true protein; the yeast hydrolysate further comprises aromatic amino acids and sulfur-containing amino acids, wherein the mass ratio of the aromatic amino acids to the sulfur-containing amino acids is 2 to 5; in the yeast hydrolysate, the mass ratio of the component with a weight average molecular weight of 200 to 2000 Da to the component with a weight average molecular weight greater than 2000 Da is 4 to 10, and the mass ratio of the component with a weight average molecular weight of 200 to 2000 Da to the component with a weight average molecular weight less than 200 Da is 4 to 9; the yeast hydrolysate further comprises disodium nucleotides, including disodium inosinate, disodium guanylate, disodium cytidine, and disodium uridine, wherein the ratio of the sum of the masses of disodium inosinate and disodium guanylate, the sum of the masses of disodium cytidine and disodium uridine, and the disodium nucleotide is 1:(0.6 to 1.2):(1.6 to 2.2).
[0007] Optionally, the aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and / or, the sulfur-containing amino acids include cysteine and methionine; and / or, the mass ratio of the aromatic amino acids to the sulfur-containing amino acids is 3 to 4.
[0008] Optionally, in the yeast hydrolysate, the mass ratio of the component with a weight average molecular weight of 200-2000 Da to the component with a weight average molecular weight greater than 2000 Da is 6-8; and / or, the mass ratio of the component with a weight average molecular weight of 200-2000 Da to the component with a weight average molecular weight less than 200 Da is 5-7; and / or, the ratio of the sum of the masses of inosinate disodium and guanylate disodium to the sum of the masses of cytidine disodium and uridine disodium is 1:(0.7-0.8).
[0009] Optionally, the D of the yeast hydrolysate 50 The diameter is 0.3µm to 5.0µm; and / or, at 25°C and 101kPa, the angle of repose of the yeast hydrolysate is 25.5° to 37.6°.
[0010] This invention provides a method for preparing yeast hydrolysate as described above, comprising: 1) inoculating yeast into a liquid culture medium for fermentation, and determining the OD of the fermentation system. 600 After reaching 120-160, fermentation is terminated to obtain fermentation broth; wherein, the yeast includes one or more of Yeast lipolytica, Pichia pastoris, Saccharomyces cerevisiae, and Candida tropicalis, and the fermentation time is 16-24 hours; 2) the fermentation broth is subjected to solid-liquid separation to obtain solid material containing yeast cells; 3) the solid material is mixed with water, and the mixture is sequentially enzymatically hydrolyzed under the action of protease and nuclease, and then the protease and nuclease are inactivated to obtain enzymatic hydrolysate; 4) the enzymatic hydrolysate is spray-dried to obtain yeast hydrolysate.
[0011] Optionally, the protease includes one or more of papain, alkaline protease, neutral protease, and acidic protease; and / or, the mass ratio of the protease to the mixture is 0.1% to 0.5%; and / or, the pH for enzymatic hydrolysis of the protease is 5.0 to 7.0, preferably 6.0 to 6.5; and / or, the temperature for enzymatic hydrolysis of the protease is 45°C to 60°C, preferably 50°C to 55°C; and / or, the time for enzymatic hydrolysis of the protease is 8h to 16h, preferably 10h to 13h.
[0012] Optionally, and / or, the nuclease includes one or more of ribonuclease, deoxyribonuclease, and deaminase; and / or, the mass ratio of the nuclease to the mixture is 0.2% to 0.3%; and / or, the pH for enzymatic hydrolysis of the nuclease is 4.5 to 6.0, preferably 5.0 to 5.5; and / or, the temperature for enzymatic hydrolysis of the nuclease is 60°C to 80°C, preferably 70°C to 75°C; and / or, the time for enzymatic hydrolysis of the nuclease is 8h to 16h, preferably 10h to 13h.
[0013] Optionally, the dry weight percentage of the yeast cells in the mixture is 10% to 25%, preferably 12% to 17%; and / or, during the spray drying process, the inlet air temperature is 165 to 180°C, the outlet air temperature is 70 to 105°C, the atomization pressure is 100 to 120 bar, and the material collection temperature is 25 to 35°C.
[0014] The present invention provides a feed comprising the yeast hydrolysate as described above or the yeast hydrolysate obtained by the preparation method described above.
[0015] Optionally, the yeast hydrolysate has a mass percentage content of 0.1% to 7.5% in the feed, preferably 1.5% to 5.0%.
[0016] This invention provides a yeast hydrolysate, its preparation method, and its application. The yeast hydrolysate has a high content of true protein, and a balanced ratio of functional amino acids, functional peptides, and nucleotides, making it a high-quality feed additive. As a high-quality feed additive, yeast hydrolysate (yeast single-cell protein hydrolysate) has the characteristics of high protein content, rich nucleic acid, and high small peptide content. It plays an important role in promoting animal feed intake, improving intestinal health, and enhancing immunity, thus promoting animal growth, increasing weight, and reducing disease incidence. Detailed Implementation
[0017] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. Based on 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.
[0018] Existing feed yeast hydrolysates (yeast single-cell protein hydrolysates) have problems such as excessively high protein content, unbalanced proportion of functional peptides, unreasonable proportion of functional amino acids, low content of nucleic acid substances and imbalance of free nucleotides.
[0019] To overcome the deficiencies in the prior art, embodiments of the present invention provide a yeast hydrolysate comprising 35% to 65% by mass of true protein; the yeast hydrolysate further comprises aromatic amino acids and sulfur-containing amino acids, wherein the mass ratio of aromatic amino acids to sulfur-containing amino acids is 2 to 5; in the yeast hydrolysate, the mass ratio of components with a weight average molecular weight of 200 to 2000 Da to components with a weight average molecular weight greater than 2000 Da is 4 to 10, and the mass ratio of components with a weight average molecular weight of 200 to 2000 Da to components with a weight average molecular weight less than 200 Da is 4 to 9; the yeast hydrolysate further comprises disodium nucleotides, including disodium inosinate, disodium guanylate, disodium cytidine, and disodium uridine, wherein the ratio of the sum of the masses of disodium inosinate and disodium guanylate, the sum of the masses of disodium cytidine and disodium uridine, and disodium nucleotides is 1:(0.6 to 1.2):(1.6 to 2.2).
[0020] According to research and analysis, the yeast hydrolysate of this invention has a high content of true protein, and the mass ratio of functional amino acids such as aromatic amino acids and sulfur-containing amino acids, the molecular weight distribution of functional peptides, and the proportion of nucleotides are balanced. This makes the yeast hydrolysate a high-quality feed additive. As a high-quality feed additive, yeast hydrolysate (yeast single-cell protein hydrolysate) has the characteristics of high protein content, rich nucleic acid, and high small peptide content. It plays an important role in promoting animal feed intake, improving intestinal health, and enhancing immunity. Therefore, it can promote animal growth, increase weight, and reduce the incidence of disease.
[0021] In practical applications, the above-mentioned yeast hydrolysate can reduce the incidence of diseases in piglets, specifically reducing the incidence of piglet white scours, piglet yellow scours, piglet red scours, piglet paratyphoid, swine epidemic diarrhea, and swine transmissible gastroenteritis, and helps increase the weight of piglets.
[0022] The mass percentage of the above-mentioned true protein in yeast hydrolysate can be calculated by the following process: Completely hydrolyze the true protein to obtain 18 amino acids, including methionine, lysine, isoleucine, phenylalanine, leucine, tryptophan, threonine, serine, cysteine, tyrosine, valine, histidine, alanine, arginine, aspartic acid, glutamic acid, glycine, and proline. Calculate the percentage of the total mass of the above 18 amino acids in the yeast hydrolysate, which is the mass percentage of the true protein in the yeast hydrolysate.
[0023] For example, the mass percentage of true protein in yeast hydrolysate can be 35%, 40%, 45%, 50%, 55%, 65%, or any combination thereof.
[0024] The mass ratio of aromatic amino acids to sulfur-containing amino acids is 2 to 5, for example, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or any combination thereof, preferably 3.0 to 4.0.
[0025] Specifically, aromatic amino acids include phenylalanine, tryptophan, and tyrosine.
[0026] Specifically, sulfur-containing amino acids include cysteine and methionine.
[0027] Components with a weight-average molecular weight of 200-2000 Da are functional peptides with a weight-average molecular weight of 200-2000 Da; components with a weight-average molecular weight greater than 2000 Da are functional peptides with a weight-average molecular weight greater than 2000 Da; and components with a weight-average molecular weight less than 200 Da are functional peptides with a weight-average molecular weight less than 200 Da.
[0028] Therefore, in the gel permeation chromatogram of yeast hydrolysate, the ratio of the content of components with a weight average molecular weight of 200-2000 Da, the content of components with a weight average molecular weight greater than 2000 Da, and the content of components with a weight average molecular weight less than 200 Da represents the mass percentage of functional peptides with a weight average molecular weight of 200-2000 Da, functional peptides with a weight average molecular weight greater than 2000 Da, and functional peptides with a weight average molecular weight less than 200 Da.
[0029] The ratio of the content of components with a weight average molecular weight of 200 to 2000 Da to the content of components with a weight average molecular weight greater than 2000 Da is 4 to 10, for example, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 or any combination thereof, preferably 6.0 to 8.0, i.e., 6 to 8.
[0030] The ratio of the content of components with a weight average molecular weight of 200 to 2000 Da to the content of components with a weight average molecular weight of less than 200 Da is 4 to 9, for example, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 or any combination thereof, preferably 5.0 to 7.0, i.e., 5 to 7.
[0031] Yeast hydrolysate also includes disodium nucleotides, which refers to the free disodium salt of nucleotides.
[0032] Specifically, disodium nucleotides include four types of free disodium nucleotides, namely disodium inosinate (free disodium inosinate), disodium guanylate (free disodium guanylate), disodium cytidine (free disodium cytidine), and disodium uridine (free disodium uridine).
[0033] The ratio of the sum of the masses of disodium inosinate and disodium guanylate, the sum of the masses of disodium cytidine and disodium uridine, and the mass of disodium nucleotide is 1:(0.6~1.2):(1.6~2.2), preferably 1:(0.7~0.8):(1.7~1.8).
[0034] The content of the above-mentioned disodium nucleotide (free disodium nucleotide salt) can be determined by liquid chromatography, and then the mass ratio of the above-mentioned disodium nucleotide (free disodium nucleotide salt) can be calculated.
[0035] In addition, the particle size of yeast hydrolysate can be 0.3µm to 5.0µm, for example, 0.3µm, 0.5µm, 1.0µm, 1.5µm, 2.0µm, 2.5µm, 3.0µm, 3.5µm, 4.0µm, 4.5µm, 5.0µm or any combination thereof.
[0036] At 25°C and 101 kPa, the angle of repose of yeast hydrolysate ranges from 25.5° to 37.6°, for example, 25.5°, 26.0°, 26.5°, 27.0°, 26.5°, 26.0°, 26.0°, 26.5°, 26.0°, 26.5°, 26.0°, 26.0°, 26.5°, 26.0°, 26.5°, 26.0°, 26.0°, 26.5°, 26.0°, 26.5°, 26.0°, 26.5°, and 37.6°, which is beneficial for its application in downstream feed premixing.
[0037] This invention also provides a method for preparing yeast hydrolysate, comprising: 1) inoculating yeast into a liquid culture medium for fermentation, and determining the OD of the fermentation system. 600 Once the temperature reaches 120-160°C, fermentation is terminated to obtain a fermentation broth. The yeast strains include one or more of Yeastia lipolytica, Pichia pastoris, Saccharomyces cerevisiae, and Candida tropicalis, and the fermentation time is 16-24 hours. 2) The fermentation broth is subjected to solid-liquid separation to obtain solid material containing yeast cells. 3) The solid material is mixed with water, and the mixture is sequentially hydrolyzed by protease and nuclease. The protease and nuclease are then inactivated to obtain the hydrolysate. 4) The hydrolysate is spray-dried to obtain yeast hydrolysate.
[0038] According to research analysis, using the above-mentioned types of yeast for liquid deep culture fermentation helps to improve the properties of yeast hydrolysate, such as the content of true protein, the mass ratio of functional amino acids, the mass ratio of functional peptides, and the ratio of nucleotides. Yeast hydrolysate can be obtained by spray drying after proteolytic and nucleolytic hydrolysis of the fermented yeast cells.
[0039] The above preparation method is simple to operate, and the prepared yeast hydrolysate can be used as a high-quality feed additive, which plays an important role in promoting feed intake and growth of animals (such as piglets) and reducing the incidence of diseases.
[0040] The inventors discovered in their research that the yeast includes one or more of *Yersinia lipolytica*, *Pichia pastoris*, *Saccharomyces cerevisiae*, and *Candida tropicalis*. Fermentation with these yeasts can produce yeast hydrolysates with excellent properties, including high true protein content, high percentage of functional amino acids, high percentage of functional peptides, and high nucleotide ratio. Preferably, the yeast includes *Yersinia lipolytica*.
[0041] Understandably, using highly active yeast strains for fermentation can help improve the performance of yeast hydrolysate, including the content of true protein, the mass ratio of functional amino acids, the mass ratio of functional peptides, and the proportion of nucleotides.
[0042] The above method for preparing yeast hydrolysate also includes activating the stored yeast, specifically including the following steps:
[0043] a) Take the frozen bacteria out of the -80℃ freezer, thaw it, take 10~20μL, and then spread it evenly on yeast extract peptone glucose agar (YPD) plates and incubate in a constant temperature incubator at 28~32℃ for 36h~48h;
[0044] b) Pick a single colony from the cultured YPD plate and inoculate it into 3-5 ml of LYPD liquid medium in a test tube. Incubate in a shaker at 28-32°C for 22-26 h at a shaker speed of 200-220 rpm to obtain the first seed culture.
[0045] c) Inoculate the first seed culture into a 5L shake flask containing 1.5-2.0L of YPD liquid culture medium and incubate at 28-32℃ in a shaker for 14-18 hours at a shaker speed of 200-220 rpm to obtain the second seed culture, thus completing the activation of the yeast.
[0046] Next, the second seed culture is inoculated into the liquid culture medium in the fermenter for fermentation, thereby preparing the fermentation broth.
[0047] The above-mentioned fermenter can be a 50L fermenter, which contains a liquid culture medium with a volume of 30~40L.
[0048] Liquid culture media include carbon sources, nitrogen sources, and inorganic salts.
[0049] In some embodiments, the carbon source includes acetic acid. In particular, the acetic acid may be derived from a byproduct of agricultural processing or an industrial byproduct.
[0050] In some embodiments, the nitrogen source described above includes an organic nitrogen source, such as corn steep liquor.
[0051] In addition, the aforementioned inorganic salts include one or more of sodium dihydrogen phosphate, magnesium sulfate, potassium sulfate, and calcium hydroxide. These inorganic salts can supplement the trace elements required for yeast growth.
[0052] In practice, the liquid culture medium can be prepared according to the formula shown in Table 1, and then the pH of the liquid culture medium can be adjusted to 5.5-6.5 with ammonia water of concentration of 24-28%.
[0053] Table 1. Formulation of liquid culture medium
[0054]
[0055] The fermentation temperature can be 28~32℃.
[0056] The pH of the fermentation system can be 5.5 to 6.5.
[0057] The dissolved oxygen content of the fermentation system can be 15-55%.
[0058] Understandably, during fermentation, stirring can be carried out at a speed of 200-800 rpm, and the aeration rate can be 0.2-3 vvm. During this period, carbon source (such as acetic acid solution with a mass percentage of 40%-70%) and nitrogen source (such as ammonia water with a mass percentage of 2%-10%) need to be continuously added.
[0059] Solid-liquid separation can be carried out by centrifugation or plate and frame filtration.
[0060] For example, the fermentation broth is filtered through a plate and frame filter press, and the filter cake is collected to obtain solid material containing yeast cells. Typically, the water content of the filter cake, i.e., the water percentage by mass, is around 75%.
[0061] After solid-liquid separation, yeast cells are collected, yielding a solid material containing yeast cells. This solid material is then mixed with water, and the solid content is adjusted using water. The solid content is the percentage of yeast cells by dry weight in the mixture. Specifically, the percentage of yeast cells by mass in the mixture can be 10% to 25%, for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, or any combination thereof, preferably 12% to 17%.
[0062] The water mentioned above may include deionized water.
[0063] The mass ratio of protease to the mixture can be 0.1% to 0.5%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or any combination thereof.
[0064] The protease may include one or more of papain, alkaline protease, neutral protease, and acidic protease. Preferably, the protease may include a complex enzyme preparation consisting of at least two of the above enzymes.
[0065] Specifically, the mass ratio of papain to the mixture can be 0.1% to 0.2%, the mass ratio of alkaline protease to the mixture can be 0% to 0.1%, the mass ratio of neutral protease to the mixture can be 0% to 0.1%, and the mass ratio of acidic protease to the mixture can be 0% to 0.1%.
[0066] The pH range for enzymatic hydrolysis of the protease is 5.0 to 7.0, for example, 5.0, 5.5, 6.0, 6.5, 7.0 or any combination thereof, preferably 6.0 to 6.5.
[0067] The temperature for enzymatic hydrolysis of the protease is 45℃~60℃, for example, 45℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃ or any combination thereof, preferably 50℃~55℃.
[0068] The enzymatic hydrolysis time of the protease is 8h to 16h, for example, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h or any combination thereof, preferably 10h to 13h.
[0069] Nucleases may include one or more of ribonucleases (such as RNase A), deoxyribonucleases (such as DNase I), and deaminases. Preferably, nucleases may include complex enzyme preparations composed of at least two of the above enzymes.
[0070] The mass ratio of nuclease to the mixture can be 0.2% to 0.3%.
[0071] The mass ratio of ribonuclease (such as RNase A) to the mixture can be 0.1% to 0.2%, the mass ratio of deoxyribonuclease (such as DNase I) to the mixture can be 0.1% to 0.2%, and the mass ratio of deaminase to the mixture can be 0% to 0.1%.
[0072] The pH range for nuclease enzymatic hydrolysis is 4.5 to 6.0, for example, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 or any combination thereof, preferably 5.0 to 5.5.
[0073] The temperature for enzymatic hydrolysis of nuclease is 60℃~80℃, for example, 60℃, 62℃, 64℃, 66℃, 68℃, 70℃, 72℃, 74℃, 75℃, 77℃, 79℃, 80℃ or any combination thereof, preferably 70℃~75℃.
[0074] The enzymatic hydrolysis time of the nuclease is 8h to 16h, for example, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h or any combination thereof, preferably 10h to 13h.
[0075] The process of sequentially hydrolyzing the mixture under the action of protease and nuclease may specifically include: adjusting the pH of the mixture to 5.0-7.0, preferably 6.0-6.5, then adjusting to a suitable temperature, and then adding protease for enzymatic hydrolysis to obtain a first hydrolysate; then adjusting the pH of the first hydrolysate to 4.5-6.0, preferably 5.0-5.5, and then adjusting to a suitable temperature, and then adding nuclease for enzymatic hydrolysis to obtain a second hydrolysate; and then inactivating the protease and nuclease in the hydrolysate to obtain the enzymatic hydrolysis product.
[0076] The pH can be adjusted using dilute sulfuric acid.
[0077] The above-mentioned process of inactivating proteases and nucleases can be carried out by heating.
[0078] During the enzymatic hydrolysis process described above, stirring can be performed, such as stirring at a speed of 100~200 rpm.
[0079] During the spray drying process, the inlet air temperature is 165~180℃, the outlet air temperature is 70~105℃, the atomization pressure is 100~120 bar, and the material collection temperature is 25~35℃.
[0080] In addition, the feed rate can be 1~10 mL / min during spray drying.
[0081] After spray drying, the spray-dried product can be granulated to obtain yeast hydrolysate, which can then be used in feed.
[0082] This invention provides a feed comprising the above-described yeast hydrolysate or a yeast hydrolysate prepared according to the above-described preparation method.
[0083] Based on this yeast hydrolysate, the feed helps to further increase piglet feed intake and reduce the incidence of piglet diseases.
[0084] In some embodiments, the above-mentioned feed can increase the average daily weight of piglets by at least 34%, such as 34% to 45%, and reduce the incidence of disease by at least 57%, such as 57% to 63%.
[0085] Specifically, the mass percentage of yeast hydrolysate in the feed can be 0.1% to 7.5%, for example, 0.1%, 0.5%, 1.5%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 7.5% or any combination thereof, preferably 1.5% to 5.0%.
[0086] When the mass percentage of yeast hydrolysate in feed meets the above range, it helps to increase the weight of animals (such as piglets) and reduce the incidence of disease, while also having good economic benefits.
[0087] In addition, the above-mentioned feed may also include one or more of the following: corn, soybean meal, fish meal, soybean oil, dicalcium phosphate, limestone powder, lysine, salt, and citric acid.
[0088] In some embodiments, the composition of the feed and the mass percentage of each component in the feed are shown in Table 2.
[0089] Table 2
[0090]
[0091] This invention also provides a method for preparing the above-mentioned feed, comprising:
[0092] The feed is obtained by mixing the raw material system including yeast hydrolysate.
[0093] The above-mentioned feed preparation method is simple to operate.
[0094] Furthermore, by mixing the raw material system including yeast hydrolysate, the mixed raw materials can be made into pellets to obtain feed. This pelleted feed can be used to feed piglets.
[0095] The present invention will be further described below through specific embodiments and comparative examples. Unless otherwise specified, the reagents, materials and instruments used below are all conventional reagents, materials and instruments, all of which are commercially available, and the reagents and materials involved can also be synthesized by conventional synthetic methods.
[0096] The formulation for Yeast Extract Peptone Glucose Agar (YPD) plates is as follows: 1 wt% Yeast Extract, 2 wt% Peptone, 2 wt% Dextrose (glucose), 2 wt% agar powder, and the balance being water.
[0097] The YPD liquid culture medium formula is: 1 wt% Yeast Extract, 2 wt% Peptone, 2 wt% Dextrose (glucose), with the balance being water.
[0098] 1. Example 1
[0099] This invention provides a method for preparing yeast hydrolysate, comprising:
[0100] a) Take the frozen yeast from the -80℃ freezer, thaw it, take 20μL, and then spread it evenly on yeast extract peptone glucose agar (YPD) plates. Incubate in a 30℃ constant temperature incubator for 40h. The yeast includes Yeast lipolytica.
[0101] b) Pick a single colony from the cultured YPD plate and inoculate it into YPD liquid medium in a test tube. Incubate in a shaker at 30°C for 24 hours at a shaking speed of 220 rpm to obtain the first seed culture.
[0102] c) Inoculate the first seed culture into a 5L shake flask containing 2L of YPD liquid culture medium and incubate at 30℃ in a shaker for 16h at a shaker speed of 220rpm to obtain the second seed culture and complete the activation of yeast.
[0103] d) Inoculate 2L of the second seed culture into 20L of liquid culture medium in a 50L fermenter for fermentation. The OD of the fermentation system is then measured. 600 Once the concentration reaches 160, fermentation is terminated, yielding the fermentation broth. The fermentation time is 16 hours, the fermentation temperature is 30°C, the pH of the fermentation system is 6.5, and the dissolved oxygen content of the fermentation system is 30%. During the fermentation, the mixture is stirred at 300 rpm with an aeration rate of 1 vvm. Simultaneously, a 60% acetic acid solution and a 2.8% ammonia solution are continuously added.
[0104] e) Filter the fermentation broth using a plate and frame filter press, and collect the filter cake to obtain a solid material containing yeast cells. The water content in the filter cake is 75% by mass.
[0105] f) After mixing the solid material and deionized water, a mixture with a yeast cell mass percentage of 15% was obtained. The pH of the mixture was then adjusted to 6.5 with dilute sulfuric acid, and the temperature was raised to 55°C. A complex protease was then added for enzymatic hydrolysis for 12 hours, with stirring at 200 rpm during the process, to obtain the first hydrolysate. The pH of the first hydrolysate was then adjusted to 5.5 with dilute sulfuric acid, and the temperature was raised to 70°C. A complex nuclease was then added for enzymatic hydrolysis for 12 hours, with stirring at 200 rpm during the process, to obtain the second hydrolysate. After enzyme inactivation, the enzymatic hydrolysis product was obtained, wherein the mass ratio of the complex protease to the mixture was 0.1%, and the mass ratio of the complex nuclease to the mixture was 0.1%.
[0106] 4) Spray dry the enzymatic hydrolysis product to obtain yeast hydrolysate; wherein, during the spray drying process, the inlet air temperature is 165℃, the outlet air temperature is 105℃, the atomization pressure is 120 bar, the receiving temperature is 35℃, and the feeding rate is 1 mL / min.
[0107] Yeast hydrolysates of Examples 1-5 and Comparative Examples 1-2 were prepared according to the process of Example 1. The differences between each example and the comparative example are shown in Tables 3 and 4. All other conditions were kept the same.
[0108] Table 3
[0109]
[0110] Table 4
[0111]
[0112] 2. The following parameters were tested in the enzymatic hydrolysates of the above examples and comparative examples:
[0113] 1) Percentage of true protein by mass: Refer to GB 5009.124-2016;
[0114] 2) Ratio of aromatic amino acids to sulfur-containing amino acids: Refer to Appendix F of GB / T20886.2-2021 to detect the mass of phenylalanine, tyrosine, cysteine and methionine in yeast hydrolysate, and calculate the mass ratio of aromatic amino acids to sulfur-containing amino acids.
[0115] 3) The ratio of the content of components with a weight average molecular weight of 200~2000 Da to the content of components with a weight average molecular weight greater than 2000 Da, and the ratio of the content of components with a weight average molecular weight of 200~2000 Da to the content of components with a weight average molecular weight less than 200 Da: Refer to GB / T27843-2011 for the detection of molecular weight distribution of yeast hydrolysates and calculate the results;
[0116] 4) The ratio of the sum of the masses of disodium inosinate and disodium guanylate, the sum of the masses of disodium cytidine and disodium uridine, and the mass of disodium nucleotide: Refer to Appendix H of GB / T20886.2-2021 for the detection of the content of free disodium nucleotide in yeast hydrolysate and calculate the results.
[0117] Table 5 Parameters in yeast hydrolysate
[0118]
[0119] Test case
[0120] 1. According to the feed formulation shown in Table 6, the yeast hydrolysates of Examples 1-5 and Comparative Examples 1-2 were added to the feed ingredients to prepare the feed.
[0121] Table 6 Feed Formulation
[0122]
[0123] 2. Prepare the feed of Example 6. The feed formula is the same as that of Example 1, except that the mass percentage of yeast hydrolysate is adjusted to 0.1% and the mass percentage of fishmeal is adjusted to 14.9%; other conditions remain unchanged.
[0124] 3. Prepare the feed of Example 7. The feed formula is the same as that of Example 1, except that the mass percentage of yeast hydrolysate is adjusted to 7.5% and the mass percentage of fishmeal is adjusted to 7.5%; other conditions remain unchanged.
[0125] 4. The feed formulation for the control group is shown in Table 7.
[0126] Table 7
[0127]
[0128] Then, the feeds of Examples 1-7, Comparative Examples 1-2 and the blank group were fed to 10 male piglets aged 3 weeks, and the feeding continued until 60 days of age. The disease incidence and weight gain of the piglets during the feeding period were then recorded. The feeding environment ensured an adequate supply of drinking water. Apart from the feeds mentioned above, the piglets could not obtain other nutrients.
[0129] The test results are as follows.
[0130] Table 8. Feeding results of the feed in Example 1
[0131]
[0132] Calculations showed that the average daily weight gain of piglets fed with the feed in Example 1 was 0.7105 kg, and the disease incidence rate was 30%.
[0133] Table 9 Examples 2-7
[0134]
[0135] Table 10 Comparative Examples 1-2
[0136]
[0137] Table 11 Feeding results in the control group
[0138]
[0139] Calculations showed that the average daily weight gain of piglets fed the control group was 0.5285 kg, and the disease incidence rate was 70%.
[0140] In summary, the application of yeast single-cell protein hydrolysate prepared by the method disclosed in this patent to piglets resulted in a 34% increase in average daily weight gain and a 57% reduction in disease incidence compared to the original feed formula (blank group), demonstrating significant effects.
[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A yeast hydrolysate, characterized in that, The yeast hydrolysate comprises 35% to 65% true protein by weight. The yeast hydrolysate also includes aromatic amino acids and sulfur-containing amino acids, wherein the mass ratio of the aromatic amino acids to the sulfur-containing amino acids is 2 to 5. In the yeast hydrolysate, the mass ratio of the component with a weight average molecular weight of 200-2000 Da to the component with a weight average molecular weight greater than 2000 Da is 4-10, and the mass ratio of the component with a weight average molecular weight of 200-2000 Da to the component with a weight average molecular weight less than 200 Da is 4-9. The yeast hydrolysate further includes disodium nucleotides, which include disodium inosinate, disodium guanylate, disodium cytidine, and disodium uridine, wherein the ratio of the sum of the masses of disodium inosinate and disodium guanylate, the sum of the masses of disodium cytidine and disodium uridine, and the disodium nucleotide is 1:(0.6~1.2):(1.6~2.2).
2. The yeast hydrolysate according to claim 1, characterized in that, The aromatic amino acids include phenylalanine, tryptophan, and tyrosine; And / or, the sulfur-containing amino acids include cysteine and methionine; And / or, the mass ratio of the aromatic amino acid to the sulfur-containing amino acid is 3 to 4.
3. The yeast hydrolysate according to claim 1 or 2, characterized in that, In the yeast hydrolysate, the mass ratio of the component with a weight average molecular weight of 200-2000 Da to the component with a weight average molecular weight greater than 2000 Da is 6-8. And / or, the mass ratio of the component with a weight average molecular weight of 200 to 2000 Da to the component with a weight average molecular weight of less than 200 Da is 5 to 7. And / or, the ratio of the sum of the masses of the inosine disodium and the guanylate disodium to the sum of the masses of the cytidine disodium and the uridine disodium is 1:(0.7~0.8).
4. The yeast hydrolysate according to any one of claims 1-3, characterized in that, The yeast hydrolysate D 50 The range is 0.3µm to 5.0µm; And / or, at 25°C and 101 kPa, the angle of repose of the yeast hydrolysate is 25.5°~37.6°.
5. A method for preparing the yeast hydrolysate according to any one of claims 1-4, characterized in that, include: 1) Inoculate yeast into a liquid culture medium for fermentation, and measure the OD of the fermentation system. 600 Once the temperature reaches 120-160, fermentation is terminated to obtain a fermentation broth; wherein the yeast includes one or more of Yeastia lipolytica, Pichia pastoris, Saccharomyces cerevisiae, and Candida tropicalis, and the fermentation time is 16-24 hours; 2) The fermentation broth is subjected to solid-liquid separation to obtain solid material containing yeast cells; 3) After mixing the solid material with water, the mixture is sequentially hydrolyzed by protease and nuclease, and then the protease and nuclease are inactivated to obtain the hydrolysis product; 4) The enzymatic hydrolysate is spray-dried to obtain the yeast hydrolysate.
6. The method for preparing yeast hydrolysate according to claim 5, characterized in that, The protease includes one or more of papain, alkaline protease, neutral protease, and acidic protease. And / or, the mass ratio of the protease to the mixture is 0.1% to 0.5%; And / or, the pH for enzymatic hydrolysis of the protease is 5.0-7.0, preferably 6.0-6.5; And / or, the temperature for enzymatic hydrolysis of the protease is 45℃~60℃, preferably 50℃~55℃; And / or, the enzymatic hydrolysis time of the protease is 8h~16h, preferably 10h~13h.
7. The method for preparing yeast hydrolysate according to claim 5 or 6, characterized in that, And / or, the nuclease includes one or more of ribonuclease, deoxyribonuclease, and deaminase; And / or, the mass ratio of the nuclease to the mixture is 0.2% to 0.3%; And / or, the pH for enzymatic hydrolysis of the nuclease is 4.5-6.0, preferably 5.0-5.5; And / or, the temperature for enzymatic hydrolysis of the nuclease is 60℃~80℃, preferably 70℃~75℃; And / or, the enzymatic hydrolysis time of the nuclease is 8h~16h, preferably 10h~13h.
8. The method for preparing yeast hydrolysate according to any one of claims 5-7, characterized in that, The dry weight percentage of yeast cells in the mixture is 10% to 25%, preferably 12% to 17%. And / or, during the spray drying process, the inlet air temperature is 165~180℃, the outlet air temperature is 70~105℃, the atomization pressure is 100~120 bar, and the material collection temperature is 25~35℃.
9. A feed, characterized in that, Includes the yeast hydrolysate according to any one of claims 1-4 or the yeast hydrolysate obtained according to the preparation method according to any one of claims 5-8.
10. The feed according to claim 9, characterized in that, The yeast hydrolysate has a mass percentage of 0.1% to 7.5% in the feed, preferably 1.5% to 5.0%.