Feather peptide rare earth chelate as well as preparation method and application thereof

Abstract

The invention relates to the technical field of rare earth chelates, and particularly discloses a feather peptide rare earth chelate as well as a preparation method and application thereof. The preparation method comprises the steps of feather pretreatment, peptide solution preparation, rare earth pretreatment, chelation reaction, purification treatment and spray drying. Wherein the feather pretreatment comprises the following steps: carrying out dry puffing treatment on feather pieces to obtain feather powder; the method for preparing the peptide solution comprises the following steps: sequentially carrying out reduction treatment (adding deionized water and a stabilizer comprising a reducing agent and a surfactant), enzymolysis treatment (adding compound protease comprising keratinase, alkaline protease and flavourzyme), separation treatment and ultrafiltration treatment on feather meal to obtain the peptide solution. The feather peptide rare earth chelate prepared by the preparation method has high yield and rare earth chelation rate, and can effectively overcome the problems of low chelation efficiency, poor product stability, low raw material utilization rate and high production cost in the production process of the feather peptide rare earth chelate.

Description

Technical Field

[0001] This application relates to the technical field of rare earth chelates. More specifically, it relates to a feather peptide rare earth chelate, its preparation method and application. Background Art

[0002] Feather peptides are a mixture of short peptides formed by decomposing feather keratin through methods such as high-temperature and high-pressure hydrolysis, enzymatic hydrolysis, or biodegradation. Due to their high protein content and digestibility, they have been widely studied and applied in the field of animal feed in recent years. According to statistics from the Ministry of Agriculture and Rural Affairs, the annual poultry slaughter volume in 2022 was approximately 15.7 billion, of which chickens accounted for about 70%, and ducks, geese, etc. accounted for 30%. And the annual output of feathers is about 25 million tons. However, 30 - 40% of the feathers are discarded due to decentralized slaughter or improper handling, which not only pollutes the environment but also causes waste of resources. Therefore, the resource-based and high-value utilization of feathers is of great significance and value.

[0003] However, the proportion of feathers used as feed is less than 5%. The reason is that their special structure is difficult to be absorbed and utilized by animals. The specific reasons are as follows: First, the surface of feathers is covered with a waxy layer, and feathers are composed of a dense network layer formed by the interlocking of hook-like structures such as barbules, which can hinder the penetration of digestive juices. Second, more than 90% of the keratin in feathers is in the form of β-sheets. Keratin is formed by the cross-linking of amino acids such as cysteine through disulfide bonds to form a three-dimensional network structure, which cannot be effectively cleaved by general proteases and has extremely high stability. At the same time, the proportion of non-polar amino acids (such as alanine, valine) in keratin molecules is as high as 40%, forming a hydrophobic barrier that hinders contact with enzymes. Ordinary enzyme preparations are difficult to precisely cleave and cannot form functional peptide segments with antioxidant and strong immune properties. Therefore, the key point in the transformation from feather meal to feather peptides is essentially how to efficiently destroy the stable structure of keratin and precisely control the decomposition process.

[0004] The application of rare earth elements in animal feed can significantly improve animal growth performance (such as promoting daily weight gain, improving feed conversion rate), enhance immunity and antioxidant capacity, and has the potential to replace antibiotics. However, rare earth ions (such as La 3+ , Ce 3+ ) have a high charge density and are easily combined with hydroxyl groups in water to form insoluble hydroxides and precipitate, resulting in low bioavailability, being difficult to be effectively absorbed by organisms, and may accumulate in the body due to insolubility, causing potential toxicity risks. In addition, due to the easy influence of rare earth chelates by the pH value of the system and competitive coordination, there are also problems of low chelation rate and poor stability in actual production applications. At the same time, the selection of ligands also needs to consider effectiveness, stability, and being cheap and easily available. Existing ligands cannot simultaneously meet the characteristics of effectiveness, stability, and being cheap and easily available, so it has always been difficult to promote large-scale application.

[0005] Although feather peptides have the effects of high protein, growth promotion, and strong immunity, problems such as immature production technology and high production costs, together with the functional limitations of rare earth additives (such as low bioavailability of inorganic rare earths and insufficient chelation stability of organic rare earths), jointly restrict their efficient synergistic application in the feed industry. Summary of the Invention

[0006] The present application provides a feather peptide rare earth chelate, a preparation method thereof, and an application.

[0007] The feather peptide rare earth chelate prepared by the preparation method of the present application has a high yield and rare earth chelation rate, and can effectively overcome the problems of low chelation efficiency, poor product stability, low raw material utilization rate, and high production cost in the production process of the feather peptide rare earth chelate.

[0008] In a first aspect, the present application provides a preparation method of a feather peptide rare earth chelate, adopting the following technical scheme:

[0009] A preparation method of a feather peptide rare earth chelate, the preparation method specifically includes the following steps: feather pretreatment, preparation of peptide solution, rare earth pretreatment, chelation reaction, purification treatment, spray drying;

[0010] Among them, the steps of the feather pretreatment are as follows:

[0011] The feather flakes are subjected to dry expansion treatment to obtain feather meal; the particle size D90 of the feather meal ≤ 100 μm;

[0012] Among them, the steps of the preparation of the peptide solution are as follows:

[0013] The feather meal is successively subjected to reduction treatment, enzymatic hydrolysis treatment, separation treatment, and ultrafiltration treatment to obtain a peptide solution;

[0014] (1) Reduction treatment: Deionized water is added to the feather meal to prepare a suspension with a dry matter weight of 20 - 35%; then a stabilizer is added to the suspension to obtain a solution after reduction treatment;

[0015] The stabilizer includes a reducing agent and a surfactant; the addition amount of the reducing agent is 0.5 - 1.5% of the dry matter weight in the suspension; the addition amount of the surfactant is 0.01 - 0.1% of the dry matter weight in the suspension;

[0016] (2) Enzymatic hydrolysis treatment: A compound protease is added to the solution after reduction treatment, and ultrasonic enzymatic hydrolysis is carried out at 40 - 60 °C for 8 - 15 h to obtain an enzymatic hydrolysate;

[0017] The addition amount of the compound protease is 2 - 4% of the dry matter weight in the solution after reduction treatment;

[0018] The compound protease includes keratinase, alkaline protease, and flavor protease;

[0019] (3) Separation treatment: Treat with a ceramic membrane with a particle size of 80 - 200 nm;

[0020] (4) Ultrafiltration treatment: Treat with an ultrafiltration membrane with a molecular weight cut-off of 4000 Da;

[0021] Among them, the steps of the rare earth pretreatment are as follows:

[0022] Mix the rare earth with a weak acid and react for 30 - 60 min to obtain the pretreated rare earth;

[0023] Among them, the steps of the chelation reaction are as follows:

[0024] Carry out a chelation reaction on the peptide solution and the pretreated rare earth to obtain a crude chelate; the addition amount of the rare earth is 20 - 35% of the dry matter weight in the peptide solution; the conditions of the chelation reaction are: react for 90 - 120 min in an environment with a pH of 3 - 4 and a temperature of 85 - 90 °C.

[0025] The preparation method of this application first adopts dry expansion treatment for feather flakes. Through the combined action of high temperature, high pressure, and shear force, it can not only effectively remove the wax layer on the surface of feathers, destroy the hook-like interlocking structure of barbules, but also break a part of the disulfide bonds, thereby destroying the hydrophobic barrier of feathers and increasing the accessibility of the enzymatic hydrolysis substrate, creating convenience for the subsequent enzymatic hydrolysis process. The high temperature during the expansion process can effectively kill pathogenic microorganisms in feathers, and the expansion is a continuous production process with a short processing time (it can be completed in a few minutes), which is suitable for large-scale production and reduces labor costs. The expanded feather meal has a loose texture, reduced fishy smell, and is more easily accepted by animals.

[0026] By optimizing the expansion temperature (190 - 230 °C) and residence time (30 - 60 s), combined with humidity control (3 - 5% moisture) to enhance the thermal cracking effect. Using a twin-screw extruder, the keratin denaturation rate is increased through the synergistic effect of shear force and thermal effect. Using a hammer mill and an air classification process to ensure the consistency of the expanded material, greatly improving the accessibility of the enzymatic hydrolysis substrate, the uniformity of enzymatic hydrolysis, shortening the enzymatic hydrolysis time, and preventing over-enzymatic hydrolysis. At the same time, due to the improved uniformity of the material after crushing and classification, the enzyme dosage is reduced by 30%, thus greatly reducing the production cost.

[0027] Feathers are rich in various amino acids. Cysteine (Cys), serine (Ser), glycine (Gly), valine (Val), leucine (Leu), threonine (Thr), proline (Pro), phenylalanine (Phe), etc. Its amino acid composition is based on homocysteine and highly hydrophobic amino acids (Ser, Val, Leu, Phe), which is the basis of its structural stability and the main obstacle to resource utilization. Some peptides have antioxidant and strong immune functions. For example, Arg-Arg-Leu can activate macrophage secretion, Gln-Gln-Pro can enhance intestinal mucosal immunity, His-Tyr-Pro can chelate metal ions and remove free radicals, and Cys-Gly-Lys can directly reduce free radicals through sulfhydryl groups. Because enzymes have the characteristics of specificity and high efficiency, a single enzyme cannot achieve the enzymatic hydrolysis effect required by this application.

[0028] This application adopts the enzymatic hydrolysis technology scheme of "reducing agent + surfactant + ultrasonic treatment + multi-enzyme synergistic targeted enzymatic hydrolysis". Adding sodium sulfite and sodium bisulfite as reducing agents has the following advantages: (1) It can prevent certain amino acids, such as cysteine, from being oxidized during the enzymatic hydrolysis process, thereby protecting the protein structure and improving the quality of the peptide; (2) The reducing agent can effectively destroy disulfide bonds and reduce them to sulfhydryl groups (-SH), making the protein easier to be enzymatically hydrolyzed and improving the efficiency of enzymatic hydrolysis; (3) The bleaching effect of the reducing agent can improve the color of the final product. Surfactants have the functions of emulsification and dispersion, solubilization and thickening, wetting and lubrication, adsorption and penetration. Tween-80 is a commonly used and cost-effective surfactant that can reduce the surface tension of the solution, allowing water and enzyme solution to penetrate into the feather fiber, weakening hydrogen bonds and hydrophobic interactions; the cavitation effect of ultrasonic treatment produces microjets and shear forces to physically break the disulfide bond network.

[0029] Multi-enzyme synergistic targeted enzymatic hydrolysis utilizes the synergistic action of keratinase + alkaline protease + flavor protease to perform enzymatic hydrolysis. Among them, keratinase mainly attacks the keratin skeleton after the disulfide bond is broken; alkaline protease cuts serine in a targeted manner, destroys the hydrophobic area and releases functional small peptides; flavor protease removes the bitter end to improve the palatability of the product. The three-dimensional enzymatic cleavage synergy of keratinase-alkaline protease-flavor protease allows keratinase to preferentially cut the Cys-Cys site, alkaline protease targets the hydrophobic area, and flavor protease modifies the end, so that the yield of antioxidant and strong immune functional peptides is increased, while the content of bitter peptides is reduced.

[0030] In addition, the present application also adopts a "nanofiltration + ultrafiltration" separation scheme. The ceramic membrane can separate the un-enzymatically hydrolyzed keratin, globin and other macromolecules in the feathers, thereby obtaining a crude solution containing small peptides. Then, the long peptides with larger molecular weight are filtered out through the ultrafiltration membrane, thereby obtaining a solution with higher purity of short-chain peptides, which is conducive to the chelation reaction between the short-chain peptides and rare earth in the later stage.

[0031] The yield rate of the finished product of the rare earth chelate of feather peptides prepared by the preparation method of the present application is above 85%, and the rare earth chelation rate is above 90%.

[0032] Optionally, the dry extrusion treatment includes extrusion treatment at 190-230°C for 30-60 s in a twin-screw extruder, with a humidity of 3-5%, followed by crushing with a hammer mill and air classification.

[0033] Optionally, the moisture content of the feather flakes is 3-5%.

[0034] Optionally, the reducing agent is a mixture of sodium sulfite and sodium bisulfite, and the weight ratio of the two is (2-3):1.

[0035] Optionally, the surfactant is one or two of the Tween series and the Span series.

[0036] Optionally, in the complex protease, the weight ratio of the three is keratinase: alkaline protease: flavor protease = (40-50):(30-40):(15-25).

[0037] In a specific embodiment, in the complex protease, the weight ratio of the three is keratinase: alkaline protease: flavor protease = 45:35:20.

[0038] Optionally, in the enzymatic hydrolysis treatment, the pH of the solution after the reduction treatment is adjusted to 8-10 before adding the complex protease.

[0039] Optionally, in the rare earth pretreatment, the weight ratio of the rare earth to the weak acid is (5-7):(3-5).

[0040] In a specific embodiment, the weight ratio of the rare earth to the weak acid is 6:4.

[0041] Optionally, the weak acid is any one of acetic acid and lactic acid.

[0042] Optionally, the rare earth is selected from one or more of rare earth carbonates, rare earth nitrates, and rare earth chlorides.

[0043] By adopting the above technical solutions, compared with rare earth nitrates and rare earth chlorides, rare earth carbonates have more stable properties and do not generate dangerous gases such as ammonia or chlorine. Rare earth carbonates react with acetic acid to form rare earth acetates. In rare earth acetates, lanthanum and cerium are more soluble in water, and lanthanum and cerium are more likely to chelate with small peptides, free amino acids, etc. in feather peptides in ionic states.

[0044] Optionally, in the purification treatment, a nanofiltration membrane with a cut-off molecular weight of 200-300 Da is used for treatment.

[0045] During the process of animal feeding, animals have a low utilization rate of free rare earths in feed, which leads to the excretion of free rare earths from the animal body and causes waste of resources. In this application, purification treatment can remove free rare earths, improve the utilization rate of rare earths, and protect the environment at the same time. After the purification removes free rare earths, the concentration of free rare earths in the reaction system decreases. According to Le Chatelier's principle, the equilibrium shifts in the direction of forming chelates. At the same time, the high efficiency of the chelation reaction reduces the purification load, and the thoroughness of the purification provides high-purity raw materials for the next round of chelation, forming a process closed-loop.

[0046] The essence of the synergistic effect between the chelation reaction and the purification treatment is a coupling mechanism of "chemical binding - physical separation - resource recycling" in one, that is: the chelation reaction locks rare earths through a thermodynamically stable coordination structure; the purification technology physically screens out free rare earths and recycles them; the two form a positive feedback loop, promoting the process to spontaneously optimize in the direction of high conversion rate and low loss. This synergistic strategy not only solves the problem of low utilization rate of free rare earths, but also realizes the industrial production closed-loop of "precision chelation - efficient separation - green recycling".

[0047] Optionally, the steps of the spray drying are as follows: spray dry the concentrated solution to obtain the feather peptide rare earth chelate.

[0048] Optionally, the operation of the spray drying is specifically: homogenize the concentrated solution at a pressure of 40 - 80 Mpa for 10 - 20 min, and then place it under the conditions of an inlet air temperature of 200 - 220 °C, an outlet air temperature of 75 - 80 °C, and a pressure of 180 - 200 kg / cm 2 for spray drying.

[0049] By homogenizing the concentrated solution, the chelate aggregates can be broken into smaller particles. The increase in specific surface area can reduce the moisture diffusion path during drying, reduce the drying time, avoid excessive heat exposure, and increase the stability between the rare earth and the structure of the chelating ligand, thereby ensuring the stability of the feather peptide rare earth chelate during spray drying. At the same time, during the two homogenizations, the first homogenization can break the soft agglomeration dominated by van der Waals forces, and the second homogenization can further refine the particles to overcome the hard agglomeration caused by electrostatic adsorption.

[0050] Optionally, after the spray drying, the obtained feather peptide rare earth chelate is made into particles with a particle size of 1 mm by a double-roll dry granulation machine to obtain the feather peptide rare earth chelate product.

[0051] In a second aspect, this application provides a feather peptide rare earth chelate prepared by the above preparation method.

[0052] In a third aspect, this application provides the application of the above feather peptide rare earth chelate in the field of animal feed.

[0053] Optionally, the feather peptide rare earth chelate is used for preparing livestock and poultry feed.

[0054] Optionally, the addition amount of the feather peptide rare earth chelate is 0.05 - 0.2% of the total weight of the feed.

[0055] The feather peptide rare earth chelate prepared in this application can be used as a substitute for antibiotics. After adding the feather peptide rare earth chelate to the feed and feeding aquatic products and healthy broilers, it can improve the growth performance of aquatic products and healthy broilers and reduce the incidence of diseases. Through experiments, it is found that the feather peptide rare earth chelate in this application can increase the daily weight gain of healthy broilers and reduce the content of harmful bacteria in the intestines of healthy broilers, thereby reducing the feed-to-meat ratio and the incidence of diseases of healthy broilers.

[0056] In summary, the technical solution of this application has the following beneficial effects:

[0057] (1) By preparing the feather and rare earth into a feather peptide rare earth chelate, the utilization rate of rare earth by animals can be improved while using feather resources. Moreover, through the preparation method of this application, a feather peptide rare earth chelate with a high content of chelated rare earth, a high chelation rate, and a high yield can be obtained.

[0058] (2) By "dry extrusion + hammer milling + air classification", the accessibility of the enzymatic hydrolysis substrate and the uniformity of enzymatic hydrolysis are improved, the enzymatic hydrolysis time is shortened, and the dosage of enzymes is reduced.

[0059] (3) By adopting the enzymatic hydrolysis technical solution of "reducing agent + surfactant + ultrasonic treatment + multi-enzyme synergistic targeted enzymatic hydrolysis", the enzymatic hydrolysis efficiency and the content of functional peptides are improved.

[0060] (4) By purifying the crude chelate, the chelation rate of the feather peptide rare earth chelate reaches more than 85%.

[0061] (5) The addition amount of the feather peptide rare earth chelate is controlled within 0.05 - 0.2% of the total weight of the animal feed. The synergistic effect of functional peptides and rare earth can be exerted. It can increase the daily weight gain, reduce the feed-to-meat ratio, and improve the immunity and survival rate. Detailed Embodiments

[0062] Before describing the embodiments of this application in detail, it should be understood that the terms used herein are only for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this term belongs.

[0063] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present application, unless otherwise specified, "a plurality of" means two or more.

[0064] In the ranges disclosed in the present application, the endpoints and any values of the ranges are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0065] In the present application, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified in the present application, but does not exclude other aspects of the content.

[0066] The present application provides a method for preparing a feather peptide rare earth chelate. The above preparation method specifically includes the following steps: feather pretreatment, preparation of a peptide solution, rare earth pretreatment, chelation reaction, purification treatment, and spray drying. Specifically as follows:

[0067] (I) Feather pretreatment

[0068] Dry-expanded treatment is performed on feather flakes with a moisture content of 3-5% to obtain feather meal.

[0069] Among them, the particle size D90 of the feather meal < 100 μm.

[0070] Among them, the dry-expanded treatment includes dry expansion treatment at 190-230 °C in a twin-screw extruder for 30-60 s, humidity control at 3-5% moisture, and then pulverization with a hammer mill and air classification.

[0071] (II) Preparation of a peptide solution

[0072] The feather meal is successively subjected to reduction treatment, enzymatic hydrolysis treatment, separation treatment, and ultrafiltration treatment to obtain a peptide solution. Specifically as follows:

[0073] A. Reduction treatment: Deionized water is added to the feather meal to prepare a suspension with a dry matter weight of 20-35%; then a stabilizer is added to the suspension to obtain a solution after reduction treatment.

[0074] Among them, the stabilizer includes a reducing agent and a surfactant. The addition amount of the reducing agent is 0.5-1.5% of the dry matter weight in the suspension, and the addition amount of the surfactant is 0.01-0.1% of the dry matter weight in the suspension.

[0075] Among them, the reducing agent is a mixture of sodium sulfite and sodium bisulfite, and the mixing ratio of the two is (2 - 3):1.

[0076] Among them, the surfactant is one or two of the Tween series and the Span series. For example, Tween - 80, Span - 60.

[0077] B. Enzymatic hydrolysis treatment: Add compound protease to the solution after reduction treatment, and carry out enzymatic hydrolysis at 40 - 60 °C for 8 - 15 h to obtain an enzymatic hydrolysate. Ultrasonic treatment is carried out during enzymatic hydrolysis. The ultrasonic treatment parameters are: frequency 20 - 40 kHz, power density 50 - 100 W / L, pulse mode (on / off = 5 s / 3 s).

[0078] Among them, the addition amount of the compound protease is 2 - 4% of the dry matter weight in the solution after reduction treatment.

[0079] Among them, the addition amount of the compound protease is 2%, 3%, 4%, 2 - 3%, 3 - 4% of the dry matter weight in the solution after reduction treatment.

[0080] Among them, the enzymatic hydrolysis time is 8 h, 10 h, 15 h, 8 - 10 h or 10 - 15 h.

[0081] Among them, the compound protease is composed of three kinds: keratinase, alkaline protease, and flavor protease. The weight ratio of the three is keratinase: alkaline protease: flavor protease = (40 - 50):(30 - 40):(15 - 25)

[0082] Among them, the keratinase is KerA keratinase from Shanghai Yuanye Bio - Technology Co., Ltd., with an enzyme activity of 200,000 u / g; the alkaline protease is alkaline protease from Ningxia Xiasheng Industry Group Co., Ltd., with an enzyme activity of 200,000 u / g, targeting the β - sheet structure of serine; the flavor protease is flavor protease from Nanning Dongheng Huadao Bio - Technology Co., Ltd., with an enzyme activity of 30,000 u / g.

[0083] Optionally, before adding the compound protease to the solution after reduction treatment, adjust the pH of the solution after reduction treatment to 8 - 10.

[0084] C. Separation treatment: Separate the enzymatic hydrolysate with a ceramic membrane to obtain a feather peptide solution. Among them, the particle size of the ceramic membrane is 80 - 200 nm.

[0085] D. Ultrafiltration treatment: Separate the feather peptide solution through an ultrafiltration membrane with a cut - off molecular weight of 4000 Da to obtain a peptide solution.

[0086] (Three) Rare - earth pretreatment

[0087] Mix rare earth with weak acid and react for 30 - 60 min to obtain pretreated rare earth.

[0088] Among them, the weight ratio of rare earth to weak acid is (5 - 7):(3 - 5). The weak acid can be any one of acetic acid and lactic acid.

[0089] (IV) Chelation reaction

[0090] Mix the peptide solution and the pretreated rare earth for chelation reaction to obtain a crude chelate.

[0091] Among them, the addition amount of rare earth is 20 - 35% of the dry matter weight in the peptide solution.

[0092] Among them, the chelation conditions are: react for 90 - 120 min in an environment with a pH of 3 - 4 and a temperature of 85 - 90 °C.

[0093] Among them, the rare earth is any one or more of rare earth carbonate, rare earth nitrate, and rare earth chloride.

[0094] In a specific embodiment, the rare earth used is rare earth carbonate.

[0095] (V) Purification treatment

[0096] Filter the crude chelate with a nanofiltration membrane with a molecular weight cut-off of 200 - 300 Da to obtain a concentrated solution and a filtrate.

[0097] (VI) Spray drying

[0098] Perform spray drying on the concentrated solution to obtain a feather peptide rare earth chelate.

[0099] Among them, the specific operation of spray drying is: place the concentrated solution under the conditions of an inlet air temperature of 200 - 220 °C, an outlet air temperature of 75 - 80 °C, and a pressure of 180 - 200 kg / cm 2 for spray drying.

[0100] To make the purpose, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be described clearly and completely below. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope protected by this application. The embodiments described below are exemplary and are only used to explain this application and should not be construed as a limitation of this application.

[0101] For those not specified in the embodiments, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0102] In the following examples, the feather pieces are duck feather pieces, which are commercially available. The manufacturer of the extruder is Muyang, and the model is SJPS215. The ultrafiltration membrane, nanofiltration membrane, and ceramic membrane are all commercially available. The alkaline protease is commercially available, and the enzyme activity of the alkaline protease is 100000 U / ml.

[0103] In the following examples, the rare earth used is rare earth carbonate. The composition of the rare earth carbonate is a mixture of 50% lanthanum carbonate and 50% cerium carbonate, and the source is Ganxian Hongjin Rare Earth Co., Ltd.

[0104] The present application will be further described in detail below in conjunction with the examples and test results.

[0105] Example

[0106] Example 1

[0107] This example provides a method for preparing a feather peptide rare earth chelate.

[0108] The above preparation method specifically includes the following steps: feather pretreatment, peptide solution preparation, rare earth pretreatment, chelation reaction, purification treatment, and spray drying. Specifically as follows:

[0109] (I) Feather pretreatment

[0110] Place the feather pieces with a moisture content of 5% in a twin-screw extruder and process at 200 °C for 60 s, control the humidity to 5% moisture, and then crush and classify by air flow using a hammer mill to obtain feather powder. It is required that the particle size D90 of the feather powder is 50 μm.

[0111] (II) Peptide solution preparation

[0112] The feather powder is successively subjected to reduction treatment, enzymatic hydrolysis treatment, separation treatment, and ultrafiltration treatment to obtain a peptide solution. Specifically as follows:

[0113] A. Reduction treatment: Add deionized water to the feather powder to prepare a suspension with a dry matter weight of 25%; then add a stabilizer to the suspension to obtain a solution after reduction treatment.

[0114] Among them, the stabilizer includes a reducing agent and a surfactant. The addition amount of the reducing agent (sodium sulfite and sodium bisulfite) is 1% of the dry matter weight in the suspension, and the weight ratio of sodium sulfite to sodium bisulfite is 2:1. The addition amount of the surfactant (Tween-80) is 0.05% of the dry matter weight in the suspension.

[0115] B. Enzymatic hydrolysis treatment: Adjust the pH of the solution after reduction treatment to 8 - 10 (adjust the pH with sodium hydroxide or composite acid), adjust the temperature to 55 °C, add composite protease (the addition amount is 3% of the dry matter weight in the solution after reduction treatment), and continuously stir. During this period, maintain the temperature at 55 °C and the pH at 8 - 10, and perform enzymatic hydrolysis for 10 h to obtain an enzymatic hydrolysate. Ultrasonic treatment is carried out during enzymatic hydrolysis. The ultrasonic treatment parameters are: frequency 20 - 40 kHz, power density 50 - 100 W / L, pulse mode (on / off = 5 s / 3 s).

[0116] C. Separation treatment: Separate the enzymatic hydrolysate with a 100 nm ceramic membrane to obtain a feather peptide solution;

[0117] D. Ultrafiltration treatment: Separate the feather peptide solution with an ultrafiltration membrane with a molecular weight cut-off of 4000 Da to obtain a peptide solution.

[0118] (II) Rare earth pretreatment

[0119] Mix rare earth carbonate and acetic acid in a weight ratio of 6:4, and react for 30 - 60 min to obtain rare earth acetate.

[0120] (III) Chelation reaction

[0121] Mix the peptide solution and rare earth acetate, and carry out a chelation reaction at a pH of 3 - 4 and a temperature of 90 °C for 90 min to obtain a crude chelate. The addition amount of rare earth acetate is 20% of the dry matter weight in the peptide solution.

[0122] (IV) Purification treatment

[0123] Filter the crude chelate with a nanofiltration membrane with a molecular weight cut-off of 200 Da to obtain a concentrated solution and a filtrate.

[0124] (V) Spray drying

[0125] Homogenize the concentrated solution at a pressure of 65 Mpa for 10 min, and then place it under the conditions of an inlet air temperature of 200 °C, an outlet air temperature of 80 °C, and a pressure of 180 - 200 kg / cm 2 for spray drying to obtain a feather peptide rare earth chelate.

[0126] Examples 2 - 3

[0127] Examples 2 - 3 respectively provide a preparation method of a feather peptide rare earth chelate. The differences between the above examples and Example 1 lie in the requirements for D90 of feather meal in feather pretreatment, as shown in Table 1 specifically, and the remaining steps are the same as those in Example 1.

[0128] Comparative example

[0129] Comparative example 1

[0130] Comparative Example 1 provides a method for preparing a feather peptide rare earth chelate. The difference between the above comparative example and Example 2 lies in the requirements for feather meal D90 in the feather pretreatment, as shown in Table 1 specifically, and the remaining steps are the same as those in Example 2.

[0131] Comparative Example 2

[0132] Comparative Example 2 provides a method for preparing a feather peptide rare earth chelate. The difference between the above comparative example and Example 2 lies in that during the preparation of the peptide solution, no stabilizer is added during the reduction treatment, as shown in Table 1 specifically, and the remaining steps are the same as those in Example 2.

[0133] Table 1 Partial parameter settings of examples and comparative examples

[0134]

[0135] Examples 4 - 7

[0136] Examples 4 - 7 respectively provide a method for preparing a feather peptide rare earth chelate. The difference between the above examples and Example 2 lies in the parameter control in the enzymatic hydrolysis treatment step, as shown in Table 1 specifically, and the remaining steps are the same as those in Example 2.

[0137] The specific differences are as follows:

[0138] The differences between Examples 4 - 5 and Example 2 are: the addition amount of compound protease.

[0139] The differences between Examples 6 - 7 and Example 2 are: the enzymatic hydrolysis time.

[0140] Comparative Examples 3 - 7

[0141] Comparative Examples 3 - 7 respectively provide a method for preparing a feather peptide rare earth chelate. The difference between the above comparative examples and Example 2 lies in the parameter control in the enzymatic hydrolysis treatment step, as shown in Table 1 specifically, and the remaining steps are the same as those in Example 2.

[0142] The specific differences are as follows:

[0143] The differences between Comparative Examples 3 - 5 and Example 2 are: the types of proteases.

[0144] The differences between Comparative Examples 6 - 7 and Example 2 are: the addition amount of compound protease.

[0145] Performance detection test one

[0146] The following gives the detection of the color of the feather peptide rare earth chelate prepared in the above examples and comparative examples, as well as the content and bitterness of small peptides in the obtained peptide solution.

[0147] (1) Color of the rare earth chelate of feather peptide

[0148] The colors are in sequence: light yellow → medium yellow → dark yellow → brown yellow.

[0149] Among them, the lighter the color, the lower the degree of oxidation of the product, the more complete the preservation of its antioxidant and immune-enhancing functions, and the easier it is to add the material with a light color to the feed; the darker the color, the higher the degree of oxidation of the product, and the reduction of its antioxidant and immune-enhancing functions.

[0150] (2) Content of small peptides in the peptide solution

[0151] Detection method: bicinchoninic acid method.

[0152] Principle of the method: The reducing groups in the peptide chain react with the BCA reagent to form a purple complex, which has a characteristic absorption peak at 562 nm.

[0153] Detection steps: Take an appropriate amount of the peptide solution and add the BCA working solution. React at 37 °C for 30 min, and measure the absorbance at 562 nm. Calculate the peptide content according to the standard curve.

[0154] (3) Evaluation of the bitterness of the peptide solution

[0155] The specific classification is as follows:

[0156] (1) No bitterness: completely without bitterness;

[0157] (2) Slight bitterness: the bitterness is weak and hardly noticeable;

[0158] (3) Moderate bitterness: the bitterness is obvious but there is no discomfort;

[0159] (4) Stronger bitterness: the bitterness is strong, may cause discomfort, but can still be tolerated;

[0160] (5) Extremely bitter: the bitterness is extremely strong and unbearable.

[0161] (4) Detection results

[0162] The detection results are shown in Table 2.

[0163] Table 2 Detection of the color and small peptide content of the solution after reduction treatment

[0164]

[0165] As can be seen from Table 2, by using the preparation method of the present application, the feathers in the solution after reduction treatment can be fully reduced, and the small peptide content in the peptide solution obtained after enzymatic hydrolysis treatment, separation treatment and ultrafiltration treatment can reach more than 85%.

[0166] Performance detection test two

[0167] The yield and rare earth chelation rate of the feather peptide rare earth chelate prepared in the above-mentioned examples and comparative examples were detected as follows.

[0168] (1) Contents of chelated cerium and chelated lanthanum and chelation degree

[0169] The detection principle is as follows: In an HCl-NaAc buffer solution (pH = 3.0) in the presence of ethanol and cetyltrimethylammonium bromide (CTMA), lanthanum La and cerium Ce form a 1:3 complex with arsenazo (Ⅲ), having a maximum absorption peak at 665 nm, and the molar absorption coefficient is εLa = 1.58×10 5 L / mol / cm, εCe = 1.66×10 5 L / mol / cm. Other interfering elements can be masked with Zn-RDTA, and the absorbance of lanthanum La and cerium Ce can be changed by varying the amount of Zn-EDTA used. Then, according to Beer's law, a system of simultaneous equations for the relationship between the concentrations of lanthanum La and cerium Ce and the absorbance can be established, so that the contents of lanthanum and cerium in the feather peptide rare earth chelate can be obtained.

[0170] The reagents used are as follows: Arsenazo (Ⅲ) solution: Weigh 0.25 g of AR-grade arsenazo (Ⅲ) into a 500 mL volumetric flask, add water and dilute to the mark to obtain a 0.05% solution. CTMA solution: Mix AR-grade CTMA with deionized water to prepare a 0.01 mol / L aqueous solution. HCI-NaAc buffer solution: Take equal volumes of a 1 mol / L HCl solution and a 1 mol / L NaAc solution, mix them evenly, and adjust the pH to 3.0 on a pH meter with 5% HCl and 1:1 ammonia water. Zn-EDTA solution: Weigh pure Zn powder (content ≥ 99.9%), dissolve it with appropriate amount of HCl, and prepare a Zn zinc ion (divalent) solution with a concentration of 0.10 mol / L. Additionally, weigh 7.445 g of EDTA Na2·2H20, dissolve it in 50 - 60 mL of water, add the above 20 mL of zinc ion solution, mix evenly, adjust the pH to 3.5, and then dilute to 100 mL. Mixed masking agent: Weigh 10 g of tartaric acid, 10 g of sodium pyrophosphate, and 1 g of citric acid, mix them, dissolve in an appropriate amount of water, adjust the PH to 3.0, and then dilute to 100 ml.

[0171] The instrument used is a 722-type spectrophotometer with a wavelength range of 200 nm - 1000 nm.

[0172] Preparation of standard solutions: Weigh 0.0266 g of CeC13·7H2O (cerous chloride, AR grade, content ≥ 99.0%), dissolve it with 30 mL of 5 mol / L HCl. After complete dissolution, transfer it to a 1000 mL volumetric flask, dilute it to the mark with water, shake well, and prepare a standard solution with a cerium concentration of 10 μg / mL; Weigh 0.0117 g of La2O3 (lanthanum oxide, content ≥ 99.99%), and similarly, prepare a standard solution with a lanthanum concentration of 10 μg / mL using 30 mL of 5 mol / L HCl; Prepare a mixed standard solution of lanthanum and cerium according to the ratio of cerium:lanthanum = 5:10.

[0173] Drawing the working curve: Pipette 0 mL, 0.5 mL, 1.0 mL, 1.5 mL, and 2.0 mL of the mixed standard solution of lanthanum and cerium into 25 mL colorimetric tubes respectively, add 2 mL (for measuring La) or 4 mL (for measuring Ce) of Zn-EDTA solution and mix well. Then add 5.0 mL of HCl-NaAc buffer solution, 3.0 mL of 0.05% arsenazo (Ⅲ) aqueous solution, 1.0 mL of 0.01 mol / L CTMA solution, and 1.2 mL of absolute ethanol in sequence, and dilute to the mark with water to form 5 kinds of standard solutions of lanthanum or cerium with concentrations of 0 μg / 25 mL, 5 μg / 25 mL, 10 μg / 25 mL, 15 μg / 25 mL, and 20 μg / 25 mL; Shake well, let it stand for 5 min, then use a 1 cm colorimetric cell to detect at 665 nm on a 722 type spectrophotometer, using the corresponding reagent blank as the reference, and measure the absorbance; Take the absorbance as the ordinate and the number of micrograms of lanthanum or cerium contained in the pipetted solution as the abscissa to draw the working curve.

[0174] Detection of the total content of lanthanum (cerium) in the feather peptide rare earth chelate: Accurately weigh 0.3000 g of the sample, dissolve it with 3 mL of concentrated HNO3, transfer it to a 1000 mL volumetric flask, dilute it to the mark with water, shake well, then measure 10 mL and dilute it to 100 mL. The content of lanthanum or cerium in this solution is estimated to be 10 - 20 μg / mL (if the result is not appropriate, adjust according to the situation and re-prepare). Then measure the absorbance under the same conditions as the standard solution, and substitute the measured absorbances of lanthanum and cerium into their respective standard curves respectively. Then the content of lanthanum and cerium in the sample can be obtained.

[0175] Detection of the content of free lanthanum (cerium) in the feather peptide rare earth chelate: Take 0.5000 g of the sample, stir it with 250 mL of water for 10 - 20 min, let it stand for 30 - 60 min, then take the upper clear liquid, use 0.05% arsenazo (Ⅲ) solution as the color-developing agent, measure the absorbance at 665 nm on a 722 type spectrophotometer, and calculate the content of unchelated free lanthanum and cerium in the sample according to the working curve method.

[0176] The content of chelated cerium (chelated lanthanum) in the feather peptide rare earth chelate = the total content of lanthanum (cerium) in the feather peptide rare earth chelate - the content of free lanthanum (cerium) in the feather peptide rare earth chelate.

[0177] The rare earth chelation degree (%) = the content of chelated rare earth (lanthanum, cerium) / the total amount of rare earth (lanthanum, cerium) in the sample × 100%.

[0178] (II) Yield

[0179] The calculation method of the yield is as follows:

[0180] The yield (%) = the weight of the feather peptide rare earth chelate / (the weight of the expanded feather meal + the weight of the rare earth + the weight of acetic acid) × 100%.

[0181] (III) Test results

[0182] The test results are shown in Table 3.

[0183] Table 3 Rare earth content and yield in the feather peptide rare earth chelates of the examples and comparative examples

[0184]

[0185] It can be seen from Table 3 that the rare earth chelation rate of the feather peptide rare earth chelate prepared by the preparation method of the present application can reach more than 90%, and the yield can reach more than 85%.

[0186] Application of the feather peptide rare earth chelate

[0187] The feather peptide rare earth chelate provided in Example 2 of the present application is added to animal feed to verify its application in animal feed.

[0188] Select 600 healthy broilers, randomly divide them into 6 groups, with 100 in each group, and conduct a feeding comparison test using feeds added with different feather peptide rare earth chelates (as shown in Table 4, the addition amount of the feather peptide rare earth chelate is the percentage of the weight of the broiler feed), continuously feed for 20 days, and record the growth of the test broilers. Take the ileum contents of 5 healthy broilers from each group back to the laboratory, and then detect the number of Escherichia coli by the plate counting method. The results are shown in Table 5 below.

[0189] Detection method of Escherichia coli: Take the ileum contents of the test broilers, culture them with eosin methylene blue agar medium (EMB), the culture conditions are 37°C for 48h, and then conduct colony counting.

[0190] Immune organ index: At 30 days of age, 8 broiler chickens were selected from each group for weighing. After weighing, the left thymus, spleen, and bursa of Fabricius were dissected, and then the fat was removed and weighed. The average immune organ index of each group was calculated (fresh weight of immune organ / total weight of broiler chickens, unit: g / kg).

[0191] Table 4 Sources and addition amounts of feather peptide rare earth chelates in application examples

[0192]

[0193] Table 5 Data statistics on the growth of broiler chickens and the number of ileal bacteria

[0194]

[0195] As can be seen from Table 5, compared with Application Example 1, the average daily gain, feed-to-weight ratio, and survival rate of broiler chickens in Application Examples 2 - 3 are higher than those in Application Example 1, and the thymus index, spleen index, and bursa of Fabricius index in Application Examples 2 - 3 are all higher than those in Application Example 1. Compared with Application Examples 1 - 3, the average daily gain, feed-to-weight ratio, thymus index, spleen index, bursa of Fabricius index, and survival rate of broiler chickens in Application Example 4 decrease. At the same time, the number of Escherichia coli in the intestinal tract of broiler chickens also increases significantly. Therefore, when the addition amount of the prepared feather peptide chelated rare earth is 0.1 - 0.2% of the total weight of the feed, the feed-to-weight ratio and average daily gain of broiler chickens are improved, and the immunity of broiler chickens is enhanced, thereby increasing the survival rate of broiler chickens.

[0196] Compared with Application Examples 2 - 3, the average daily gain, immune organ index, and survival rate of broiler chickens in Application Examples 5 - 6 are all lower. Therefore, compared with broiler chickens fed with cerium and lanthanum elements in the feed or without adding feather peptide chelated rare earth, when feather peptide chelated rare earth is added to the feed, the growth performance of broiler chickens can be improved, the immunity of broiler chickens can be enhanced, and thus the survival rate of broiler chickens can be increased.

[0197] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0198] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A preparation method of a feather peptide rare earth chelate, characterized in that, The preparation method specifically includes the following steps: feather pretreatment, peptide solution preparation, rare earth pretreatment, chelation reaction, purification treatment, and spray drying; Among them, the steps of the feather pretreatment are as follows: The feather flakes are subjected to dry expansion treatment to obtain feather powder; the particle size D90 of the feather powder ≤ 100 μm; Among them, the steps of the peptide solution preparation are as follows: The feather powder is successively subjected to reduction treatment, enzymatic hydrolysis treatment, separation treatment, and ultrafiltration treatment to obtain a peptide solution; (1) Reduction treatment: Deionized water is added to the feather powder to prepare a suspension with a dry matter weight of 20 - 35%; then a stabilizer is added to the suspension to obtain a solution after reduction treatment; The stabilizer includes a reducing agent and a surfactant; the addition amount of the reducing agent is 0.5 - 1.5% of the dry matter weight in the suspension; the addition amount of the surfactant is 0.01 - 0.1% of the dry matter weight in the suspension; (2) Enzymatic hydrolysis treatment: A compound protease is added to the solution after reduction treatment, and ultrasonic enzymatic hydrolysis is carried out at 40 - 60 °C for 8 - 15 h to obtain an enzymatic hydrolysate; The addition amount of the compound protease is 2 - 4% of the dry matter weight in the solution after reduction treatment; The compound protease includes keratinase, alkaline protease, and flavor protease; (3) Separation treatment: Treatment is carried out using a ceramic membrane with a particle size of 80 - 200 nm; (4) Ultrafiltration treatment: Treatment is carried out using an ultrafiltration membrane with a cut-off molecular weight of 4000 Da; Among them, the steps of the rare earth pretreatment are as follows: The rare earth is mixed and reacted with a weak acid for 30 - 60 min to obtain the pretreated rare earth; Among them, the steps of the chelation reaction are as follows: The peptide solution and the pretreated rare earth are subjected to a chelation reaction to obtain a crude chelate; the addition amount of the rare earth is 20 - 35% of the dry matter weight in the peptide solution; the conditions of the chelation reaction are: reaction is carried out in an environment with a pH of 3 - 4 and a temperature of 85 - 90 °C for 90 - 120 min.

2. The preparation method according to claim 1, wherein The dry expansion treatment includes expansion treatment in a twin-screw extruder at 190 - 230 °C for 30 - 60 s, with a humidity of 3 - 5%, and then pulverization with a hammer mill and air classification; Optionally, the moisture content of the feather flakes is 3 - 5%.

3. The preparation method according to claim 1, characterized in that, The reducing agent is a mixture of sodium sulfite and sodium bisulfite, and the weight ratio of the two is (2 - 3):

1.

4. The preparation method according to claim 1, characterized in that, The surfactant is one or two of the Tween series and the Span series.

5. The preparation method according to claim 1, characterized in that, In the compound protease, the weight ratio of the three is keratinase: alkaline protease: flavor protease = (40 - 50):(30 - 40):(15 - 25); Optionally, in the enzymatic hydrolysis treatment, the pH of the solution after reduction treatment is adjusted to 8 - 10 before adding the compound protease.

6. The preparation method according to claim 1, characterized in that, In the rare earth pretreatment, the weight ratio of the rare earth to the weak acid is (5 - 7):(3 - 5); Optionally, the weak acid is any one of acetic acid and lactic acid.

7. The preparation method according to claim 1, wherein The steps of the purification treatment are as follows: Treatment is carried out using a nanofiltration membrane with a cut-off molecular weight of 200 - 300 Da; Optionally, the steps of spray drying are as follows: spray drying the concentrated solution to obtain feather peptide rare earth chelate; the specific operation of spray drying is: homogenize the concentrated solution at a pressure of 40-80 Mpa for 10-20 min, and then place it under the conditions of an inlet air temperature of 200-220 °C, an exhaust air temperature of 75-80 °C, and a pressure of 180-200 kg / cm 2 for spray drying.

8. A feather peptide rare earth chelate prepared by the preparation method according to any one of claims 1-7.

9. Use of the feather peptide rare earth chelate according to claim 8 in the field of animal feed.

10. The application according to claim 9, wherein The feather peptide rare earth chelate is used for preparing livestock and poultry feed; Optionally, the addition amount of the feather peptide rare earth chelate is 0.05-0.2% of the total weight of the feed.

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