Growth-promoting spleen-tonifying spleen peptide as well as preparation method and application thereof

Abstract

The invention provides a spleen peptide for promoting growth and tonifying spleen as well as a preparation method and application thereof, and belongs to the technical field of biochemistry. The invention provides a preparation method of spleen peptide capable of promoting growth and tonifying spleen. The preparation method comprises the following steps: degreasing spleen particles of healthy animals, fermenting after degreasing, performing subcritical water extraction, performing protease enzymolysis, fermenting an enzymolysis product, clarifying fermentation liquor, and then performing filtration, nanofiltration and spray drying treatment. According to the preparation method, the preparation process of the spleen peptide is reasonably set, so that the prepared spleen peptide is high in protein content, low in average molecular weight, easy to absorb and utilize, free of fat detection, capable of tonifying the spleen and promoting growth, capable of being dissolved into water to form transparent liquid, capable of being processed into tablets, powder and oral liquid, also capable of being used in common food, high in yield and suitable for popularization and application. The method is suitable for industrial popularization.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, and in particular relates to a spleen peptide that promotes growth and strengthens the spleen, its preparation method, and its application. Background Technology

[0002] Spleen peptides are polypeptide components extracted from animal spleens, possessing physiological functions such as immune regulation and spleen and stomach conditioning. Existing extraction methods for spleen peptides are largely similar, generally involving first cutting and washing the spleen of healthy animals (such as pigs and cattle) to create a homogenate, then processing the homogenate through dialysis or ultrafiltration to obtain low molecular weight components, followed by freeze-drying to obtain the finished spleen peptide product. However, existing spleen peptide preparation methods suffer from technical problems such as low yield, high molecular weight, and the need for quality improvement, hindering industrial-scale promotion. Summary of the Invention

[0003] The purpose of this invention is to provide a spleen peptide that promotes growth and strengthens the spleen, as well as its preparation method and application. The spleen peptide prepared by the method of this invention has high protein content, low average molecular weight, no detectable fat, and can be dissolved in water to form a transparent liquid with a high yield, making it suitable for industrial promotion.

[0004] This invention provides a method for preparing spleen peptides that promote growth and strengthen the spleen, comprising the following steps:

[0005] 1) The spleen granules of healthy animals were defatted, and the defatted solids were subjected to a first fermentation using Streptomyces flavus and Yersinia lipophila to obtain the precipitate of the first fermentation.

[0006] 2) The precipitate from the first fermentation was subjected to subcritical water extraction to obtain the extract product;

[0007] 3) The extract is hydrolyzed with a protease to obtain a hydrolysate; the protease includes one or more of bromelain, papain, neutral protease and flavor protease.

[0008] 4) The enzymatic hydrolysate is subjected to a second fermentation using a thermophilic Bacillus stearothermophilus agent to obtain a second fermentation broth; the second fermentation broth is subjected to a third fermentation using a Staphylococcus equi agent to obtain a third fermentation broth;

[0009] 5) The third fermentation broth is clarified using a composite adhesive, and the supernatant is collected; the composite adhesive includes astragalus gum, artemisia gum, and erythrina gum;

[0010] 6) The supernatant is sequentially filtered, nanofiltered and spray-dried to obtain spleen peptides.

[0011] Preferably, in step 1), defatting the spleen particles of healthy animals includes: defatting the spleen particles of healthy animals with n-hexane; the ratio of the mass of the spleen particles to the volume of n-hexane is 1g: 2-10mL; the initial pH value of the first fermentation is 5-8; the fermentation time is 1-5 days; and the fermentation temperature is 20-40℃.

[0012] Preferably, in step 1), the total mass of the *Streptomyces flavus* inoculum and the *Yarrowia lipolytica* inoculum accounts for 1% to 8% of the mass of the spleen granules; the mass ratio of the *Streptomyces flavus* inoculum to the *Yarrowia lipolytica* inoculum is 1:(1-5); and the effective viable count of *Streptomyces flavus* in the *Streptomyces flavus* inoculum is 1 to 8 × 10⁻⁶. 8 cfu / g; the effective viable count of *Yersinia lipolyticis* in the *Yersinia lipolyticis* preparation is 2–8 × 10⁻⁶. 8 cfu / g.

[0013] Preferably, in step 2), the initial pH of the subcritical water extraction system is 5-10; the temperature of the subcritical water extraction is 110-220°C; and the time of the subcritical water extraction is 20-60 min.

[0014] Preferably, in step 3), the initial pH value of the enzymatic hydrolysis is 6-8; the protease accounts for 0.1% w / v to 5% w / v of the extract; the temperature of the enzymatic hydrolysis reaction is 50-60°C; and the time of the enzymatic hydrolysis reaction is 2-8 h.

[0015] Preferably, in step 4), the inoculum amount of the *Bacillus thermophilus* agent is 0.5% w / v to 5% w / v of the enzymatic hydrolysis product; the effective viable count of *Bacillus thermophilus* in the *Bacillus thermophilus* agent is 1 to 8 × 10⁻⁶. 9 cfu / mL; the second fermentation time is 1–5 days; the initial pH of the second fermentation is 3–6; the temperature of the second fermentation is 50–60℃; the inoculum size of the *Staphylococcus equi* agent is 1% w / v–10% w / v of the second fermentation broth; the effective viable count of *Staphylococcus equi* in the *Staphylococcus equi* agent is 1–8 × 10⁻⁶. 9 The concentration of cfu / mL; the time for the third fermentation is 1 to 5 days; the initial pH value for the third fermentation is 6 to 8; and the temperature for the third fermentation is 25 to 35°C.

[0016] Preferably, in step 5), the mass ratio of Astragalus gum, Artemisia gum and Erythrina gum is 1:(1-5):(1-6); the composite gum accounts for 0.5% w / v to 5% w / v of the third fermentation liquid.

[0017] The present invention also provides a spleen peptide prepared by the preparation method described above, wherein the protein content of the spleen peptide is ≥80% and the average molecular weight of the spleen peptide is 700-800.

[0018] The present invention also provides the application of the spleen peptide described above in the preparation of products that strengthen the spleen and / or promote children's growth and development.

[0019] Preferably, promoting children's growth and development includes improving growth retardation caused by malnutrition.

[0020] This invention provides a method for preparing spleen peptides that promote growth and strengthen the spleen, comprising the following steps: defatting spleen granules from healthy animals, followed by fermentation, subcritical water extraction, enzymatic hydrolysis, fermentation of the hydrolysate, clarification of the fermentation broth, and subsequent filtration, nanofiltration, and spray drying. The preparation method of this invention rationally sets the spleen peptide preparation process. By defatting the spleen granules from healthy animals and then further removing fat through fermentation with *Streptomyces flavus* and *Yarrowia lipolyticis*, which have strong fat-decomposing capabilities, the fat is fully extracted through subcritical water extraction, thereby increasing the yield of spleen peptides. Enzymatic hydrolysis of the extracted product, followed by fermentation with *Bacillus stearothermophilus* and *Staphylococcus equi*, which have strong protein-decomposing capabilities, further degrades proteins and large peptides, thus increasing the yield of spleen peptides. Clarification of the fermentation broth further removes insoluble matter. Filtration removes impurities and insoluble matter, ensuring the product becomes a transparent liquid after dissolving in water. Nanofiltration removes inorganic salts and water-soluble free amino acids. The spleen peptide protein prepared by the method of the present invention has a high content, low average molecular weight, is easily absorbed and utilized, and no fat was detected. It can promote growth and strengthen the spleen. After being dissolved in water, it can become a transparent liquid and can be processed into tablets, powders and oral liquids. It can also be used in ordinary foods with a high yield and is suitable for industrial promotion.

[0021] Furthermore, children are in a crucial stage of growth and development, and spleen deficiency is a common constitution type among children. The causes of spleen deficiency include not only congenital factors but also acquired factors such as improper feeding, unbalanced diet, and children's relatively poor self-regulation. Traditional Chinese medicine believes that the spleen governs transformation and transportation, and is the "source of qi and blood production." When the spleen is deficient, it can lead to obesity or thinness, poor appetite, gastrointestinal discomfort, and is prone to respiratory infections, indigestion, malnutrition, and other conditions, affecting children's growth and development. The spleen peptide prepared by the method of this invention has excellent spleen-strengthening and growth-promoting effects. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The molecular weight spectrum for Example 1 shows a peak value of 21.250.

[0024] Figure 2 The molecular weight spectrum for Example 2;

[0025] Figure 3 The molecular weight spectrum for Example 3;

[0026] Figure 4 The molecular weight spectrum for Comparative Example 1 is shown, with a peak value of 19.007.

[0027] Figure 5 The molecular weight spectrum is for comparative example 2;

[0028] Figure 6 The molecular weight spectrum for comparative example 3 is shown, with a peak value of 19.335.

[0029] Figure 7 The graph for comparative example 4 shows the molecular weight spectrum, with a peak value of 19.079. Detailed Implementation

[0030] This invention provides a method for preparing spleen peptides that promote growth and strengthen the spleen, characterized by comprising the following steps:

[0031] 1) The spleen granules of healthy animals were defatted, and the defatted solids were subjected to a first fermentation using Streptomyces flavus and Yersinia lipophila to obtain the precipitate of the first fermentation.

[0032] 2) The precipitate from the first fermentation was subjected to subcritical water extraction to obtain the extract product;

[0033] 3) The extract is hydrolyzed with a protease to obtain a hydrolysate; the protease includes one or more of bromelain, papain, neutral protease and flavor protease.

[0034] 4) The enzymatic hydrolysate is subjected to a second fermentation using a thermophilic Bacillus stearothermophilus agent to obtain a second fermentation broth; the second fermentation broth is subjected to a third fermentation using a Staphylococcus equi agent to obtain a third fermentation broth;

[0035] 5) The third fermentation broth is clarified using a composite adhesive, and the supernatant is collected; the composite adhesive includes astragalus gum, artemisia gum, and erythrina gum;

[0036] 6) The supernatant is sequentially filtered, nanofiltered and spray-dried to obtain spleen peptides.

[0037] The present invention first defatted the spleen granules of healthy animals, and then used Streptomyces flavus and Yeast extract to carry out a first fermentation of the defatted solids to obtain a first fermentation precipitate.

[0038] As one embodiment, the method for preparing the spleen granules includes the following steps: sequentially heat-treating and pulverizing the cleaned animal spleen to obtain spleen granules. As one embodiment, the animal spleen undergoes a cleaning process before processing; the cleaning includes: mixing the animal spleen with water and washing; the mass ratio of the animal spleen to water is 1:(2-10); the number of washing cycles is 2-4; the heat treatment procedure is: heating to 75-95℃ and holding for 20-40 minutes, further heating to 90℃ and holding for 20 minutes; the purpose of the heat treatment is sterilization; the pulverization includes grinding; the grinding is performed using a meat grinder; the particle size of the spleen granules is 3-5 mm; the animal spleen includes frozen or fresh healthy animal spleen; the animal spleen includes bovine spleen.

[0039] As one implementation method, defatting spleen granules from healthy animals includes: defatting the spleen granules from healthy animals with n-hexane; in this invention, n-hexane is used for defatting, and fermentation with *Streptomyces flavus* and *Yersinia lipolyticis* is beneficial for further fat removal; the mass ratio of the spleen granules to the volume of n-hexane is 1 g: 2-10 mL; the defatting of the spleen granules from healthy animals with n-hexane includes the following steps: mixing the spleen granules with n-hexane, soaking and centrifuging in sequence, collecting the first precipitate; washing the first precipitate and mixing it with water to obtain a resuspension. As one implementation method, the soaking time is 2-8 h, more preferably 3-6 h; stirring is also performed during the soaking process; water is used for washing; the amount of water used for each washing is 2-4 times (w / v) of the first precipitate; the number of washings is 2-4 times; during the process of washing the first precipitate and mixing it with water, the ratio of the first precipitate to water is 1:(2-10) (w / v), more preferably 1:4 (w / v).

[0040] In one embodiment, the total mass of the *Streptomyces flavus* inoculum and the *Yarrowia lipolytica* inoculum accounts for 1% to 8% of the spleen granule mass, more specifically 3% to 8%; the mass ratio of the *Streptomyces flavus* inoculum to the *Yarrowia lipolytica* inoculum is 1:(1-5), more specifically 1:(2-4), and even more specifically 1:3; the effective viable count of *Streptomyces flavus* in the *Streptomyces flavus* inoculum is 1 to 8 × 10⁻⁶. 8cfu / g, further increasing to 3–8 × 10⁻⁶ 8 cfu / g, further increased to 4×10 8 cfu / g; the effective viable count of *Yersinia lipolyticis* in the *Yersinia lipolyticis* preparation is 2–8 × 10⁻⁶. 8 cfu / g, further increasing to 4–8 × 10⁻⁶ 8 cfu / g, further increased to 6×10 8 cfu / g.

[0041] In this invention, the spleen granules of healthy animals are defatted, and after defatting, the fat is further removed by inoculating with Streptomyces flavus and Yersinia lipolytica, which have strong fat-decomposing abilities, for fermentation.

[0042] In one embodiment, the initial pH value of the first fermentation is 5-8, more preferably 6-7; the fermentation time is 1-5 days, more preferably 2-4 days; and the fermentation temperature is 20-40°C, more preferably 25-30°C.

[0043] In one implementation, the precipitate from the first fermentation is obtained by a second centrifugation of the first fermentation broth.

[0044] After obtaining the precipitate from the first fermentation, the present invention performs subcritical water extraction on the precipitate to obtain the extraction product.

[0045] In one embodiment, the ratio of the mass of the precipitate from the first fermentation to the volume of water used for subcritical water extraction is 1g:3-10mL, more preferably 1g:5-8mL, and even more preferably 1g:6mL; the initial pH of the subcritical water extraction system is 5-10, more preferably 7.5-9, and even more preferably 8; the temperature of the subcritical water extraction is 110-220℃, more preferably 160-200℃; and the time of the subcritical water extraction is 20-60min, more preferably 30-50min, and even more preferably 40min. This invention obtains spleen protein through subcritical extraction.

[0046] In this invention, protein is fully extracted by subcritical water extraction, thereby improving the yield of spleen peptides in the subsequent process.

[0047] After obtaining the extract, the present invention uses a protease to enzymatically hydrolyze the extract to obtain an enzymatic hydrolysate; the protease includes one or more of bromelain, papain, neutral protease and flavor protease.

[0048] In one embodiment, the initial pH of the enzymatic hydrolysis is 6-8, more preferably 6-7; the protease accounts for 0.1% w / v to 5% w / v of the extract, more preferably 0.5% w / v to 2% w / v, and even more preferably 1% w / v; the temperature of the enzymatic hydrolysis reaction is 50-60°C, more preferably 55°C; the time of the enzymatic hydrolysis reaction is 2-8 h, more preferably 3-6 h, and even more preferably 4 h; the protease is further a neutral protease and a flavor protease; the mass ratio of the neutral protease to the flavor protease is 5:(1-5); the enzyme activity of the neutral protease is 50,000 to 400,000 u / g; and the enzyme activity of the flavor protease is 10,000 to 100,000 u / g.

[0049] After obtaining the enzymatic hydrolysis product, the present invention uses a thermophilic Bacillus stearothermophilus inoculum to carry out a second fermentation of the enzymatic hydrolysis product to obtain a second fermentation broth; and uses a Staphylococcus equi inoculum to carry out a third fermentation of the second fermentation broth to obtain a third fermentation broth.

[0050] In one embodiment, the inoculum amount of the *Bacillus thermophilus* agent is 0.5% w / v to 5% w / v, more specifically 1% w / v to 3% w / v, of the enzymatic hydrolysis product; the effective viable count of *Bacillus thermophilus* in the *Bacillus thermophilus* agent is 1 to 8 × 10⁻⁶. 9 cfu / mL, further 4–8 × 10⁻⁶ 9 cfu / mL, and further increased to 5×10 9 cfu / mL; the second fermentation time is 1–5 days, more preferably 3–5 days; the initial pH value of the second fermentation is 3–6, more preferably 3–5; the temperature of the second fermentation is 50–60°C, more preferably 55–60°C; the inoculum amount of the *Staphylococcus equi* agent is 1% w / v to 10% w / v, more preferably 3% w / v to 10% w / v, and even more preferably 5% w / v; the effective viable count of *Staphylococcus equi* in the *Staphylococcus equi* agent is 1–8 × 10⁻⁶. 9 cfu / mL, further increased to 5 × 10 9 cfu / mL; the third fermentation time is 1-5 days, more specifically 2-3 days; the initial pH value of the third fermentation is 6-8, more specifically 7; the temperature of the third fermentation is 25-35℃, more specifically 28-32℃, and even more specifically 30℃.

[0051] In this invention, by enzymatically hydrolyzing the extract with proteases, and then inoculating the hydrolysate with thermophilic Bacillus stearothermophilus and Staphylococcus equi, which have strong protein-degrading capabilities, the protein and large peptides are further degraded, thereby increasing the yield of spleen peptides.

[0052] After obtaining the third fermentation liquid, the present invention uses a composite adhesive to clarify the third fermentation liquid and takes the supernatant; the composite adhesive includes astragalus gum, artemisia gum and erythrina gum.

[0053] In one embodiment, the clarification of the third fermentation broth using a composite adhesive includes mixing the third fermentation broth and the composite adhesive, and allowing it to stand; the mass ratio of Astragalus membranaceus gum, Artemisia argyi gum, and Erythrina variegata gum is 1:(1-5):(1-6), further 1:(2-4):(2-4), and even further 1:2:3, 1:2:2, or 1:4:4; the composite adhesive accounts for 0.5% w / v to 5% w / v of the third fermentation broth, further 1% w / v to 4% w / v, and even further 3% w / v; the standing temperature is room temperature; the standing time is 1-8 hours, further 3-6 hours; and the composite adhesive is used to clarify the third fermentation broth.

[0054] In this invention, by clarifying the fermentation broth, insoluble matter can be further removed.

[0055] After obtaining the supernatant, the supernatant is sequentially filtered, nanofiltered, and spray-dried to obtain spleen peptides.

[0056] In one embodiment, the filtration includes cotton cake filtration. The present invention does not impose any particular limitations on the specific parameters of the cotton cake filtration, nanofiltration, and spray drying; they can be performed according to conventional conditions in the art. In this invention, filtration removes impurities and insoluble substances, ensuring that the product can be dissolved in water to form a transparent liquid, while nanofiltration removes inorganic salts and water-soluble free amino acids.

[0057] The present invention also provides a spleen peptide prepared by the preparation method described above, wherein the protein content of the spleen peptide is ≥80% and the average molecular weight of the spleen peptide is 700-800.

[0058] In one embodiment, the protein content of the spleen peptide is 82% to 86.7%; the average molecular weight of the spleen peptide is 722 to 790.

[0059] The spleen peptide of this invention has a high protein content, small molecular weight, and is easily absorbed. It becomes a transparent liquid after being dissolved in water and can be processed into tablets, powders, and oral liquids. It can also be used in ordinary foods to meet the needs of children to strengthen their spleen and promote growth.

[0060] The present invention also provides the application of the spleen peptide described above in the preparation of products that strengthen the spleen and / or promote children's growth and development.

[0061] In one implementation, promoting children's growth and development includes improving growth retardation caused by malnutrition; the malnutrition includes insufficient protein intake.

[0062] As one implementation method, promoting children's growth includes promoting the growth and development of children with short stature.

[0063] In this invention, the product includes food, health products, or pharmaceuticals.

[0064] In this invention, the product is an oral preparation; the oral dose of the product, calculated for rats, is 250 mg / kg body weight.

[0065] To further illustrate the present invention, a method for preparing spleen peptide and its application provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0066] In the embodiments of the present invention, *Streptomyces flavus* inoculum was purchased from Shandong Qilu Chemical Technology Co., Ltd., *Yersinia lipolyticis* inoculum was purchased from Wuhan Huanna Biotechnology Co., Ltd., proteases were all purchased from Nanning Pangbo Bioengineering Co., Ltd., *Bacillus stearothermophilus* inoculum was purchased from Shanghai Yushao Biotechnology Co., Ltd., *Staphylococcus equi* inoculum was purchased from Shanghai Bangjing Industrial Co., Ltd., *Astragalus membranaceus* gum was purchased from Shandong Gukang Bioengineering Co., Ltd., *Artemisia argyi* gum was purchased from Shandong Shengmao Biotechnology Co., Ltd., and *Erythrina variegata* gum was purchased from Shandong Aobang Biotechnology Co., Ltd.

[0067] Example 1

[0068] Step S1: Sterilize and grind raw materials: After thawing frozen beef spleen, weigh 1 kg, add 3 times (w / v) of water, wash twice, heat to 90℃, keep warm for 20 minutes; grind into 3-5 mm particles using a meat grinder;

[0069] Step S2 Degreasing: Add 4 times (w / v) n-hexane, soak and stir for 6 hours, centrifuge to remove supernatant, add 2 times (w / v) water, wash 4 times; add 4 times (w / v) water, adjust pH to 6.0, inoculate with 5% Streptomyces flavus (4×10⁻⁶) at 30℃. 8 cfu / g) and Yersinia lipolytica (6×10) 8 CFU / g (ratio 1:3), ferment for 4 days, then centrifuge to remove the supernatant;

[0070] Step S3: Subcritical water extraction of protein: Add 6 times (w / v) water, pH 8, temperature 200℃, extraction time 40 min to obtain bovine spleen protein;

[0071] Preparation of enzymatic hydrolysate in step S4: After extraction, the pH of the protein was adjusted to 7, 1% neutral protease and flavor protease were added, and the enzymatic hydrolysis temperature was 55℃ for 4 hours.

[0072] Step S5: Preparation of enzymatic fermentation broth: Adjust pH to 5, temperature to 55℃, and inoculate with 3% *Bacillus stearothermophilus* (5 × 10⁻⁶). 9Fermentation was carried out for 5 days with cfu / mL; then the temperature was lowered to 30℃, pH 7, and the mixture was inoculated with 5% Staphylococcus equi (5×10⁻⁶ cfu / mL). 9 (cfu / mL), ferment for 2 days; heat to 90℃, keep warm for 30 minutes, and sterilize;

[0073] Step S6 Clarification of enzymatic hydrolysis fermentation broth: Add 3% astragalus gum, artemisia gum and erythrina gum (ratio 1:2:3) to the fermentation hydrolysate, let stand at room temperature for 6 hours, and take the supernatant.

[0074] Step S7 Spray drying: The clarified liquid is filtered through cotton cake to remove impurities, and nanofiltration is used to remove inorganic salts and water-soluble free amino acids. After spray drying, bovine spleen peptides are obtained.

[0075] Example 2

[0076] Step S1: Sterilize and grind raw materials: After thawing frozen beef spleen, weigh 1 kg, add 3 times (w / v) of water, wash twice, heat to 90℃, keep warm for 20 minutes; grind into 3-5 mm particles using a meat grinder;

[0077] Step S2 Degreasing: Add 2 times (w / v) n-hexane, soak and stir for 3 hours, centrifuge to remove supernatant, add 2 times (w / v) water, wash 4 times; add 4 times (w / v) water, adjust pH to 6.0, inoculate with 3% Streptomyces flavus (4×10⁻⁶) at 30℃. 8 cfu / g) and Yersinia lipolytica (6×10) 8 CFU / g (ratio 1:2), ferment for 2 days, then centrifuge to remove the supernatant;

[0078] Step S3: Subcritical water extraction of protein: Add 5 times (w / v) water, pH 7.5, temperature 160℃, extraction time 30 min to obtain bovine spleen protein;

[0079] Preparation of enzymatic hydrolysate in step S4: After extraction, the pH of the protein was adjusted to 6, 0.5% neutral protease and flavor protease were added, and the enzymatic hydrolysis temperature was 55℃ for 3 hours.

[0080] Step S5: Preparation of enzymatic fermentation broth: Adjust pH to 3, temperature to 55℃, and inoculate with 1% *Bacillus stearothermophilus* (5 × 10⁻⁶). 9 Fermentation was carried out for 3 days (cfu / mL); then the temperature was lowered to 30℃, pH 7, and the mixture was inoculated with 3% Staphylococcus equi (5×10⁻⁶ CFU / mL). 9 (cfu / mL), ferment for 2 days; heat to 90℃, keep warm for 30 minutes, and sterilize;

[0081] Step S6 Clarification of enzymatic hydrolysis fermentation broth: Add 1% astragalus gum, artemisia gum and erythrina gum (ratio 1:2:2) to the fermentation hydrolysate, let stand at room temperature for 3 hours, and take the supernatant.

[0082] Step S7 Spray drying: The clarified liquid is filtered through cotton cake to remove impurities, and nanofiltration is used to remove inorganic salts and water-soluble free amino acids. After spray drying, bovine spleen peptides are obtained.

[0083] Example 3

[0084] Step S1: Sterilize and grind raw materials: After thawing frozen beef spleen, weigh 1 kg, add 3 times (w / v) of water, wash twice, heat to 90℃, keep warm for 20 minutes; grind into 3-5 mm particles using a meat grinder;

[0085] Step S2 Degreasing: Add 6 times (w / v) of n-hexane, soak and stir for 6 hours, centrifuge to remove the supernatant, add 2 times (w / v) of water, and wash 4 times; add 4 times (w / v) of water to adjust the pH to 6.0, and inoculate with 8% Streptomyces flavus (4×10⁻⁶) at 30℃. 8 cfu / g) and Yersinia lipolytica (6×10) 8 CFU / g (ratio 1:4), ferment for 4 days, then centrifuge to remove the supernatant;

[0086] Step S3: Subcritical water extraction of protein: Add 8 times (w / v) water, pH 9, temperature 200℃, extraction time 50 min to obtain bovine spleen protein;

[0087] Preparation of enzymatic hydrolysate in step S4: After extraction, the pH of the protein was adjusted to 7, 2% neutral protease and flavor protease were added, and the enzymatic hydrolysis temperature was 55℃ for 6 hours.

[0088] Step S5: Preparation of enzymatic fermentation broth: Adjust pH to 5, temperature to 60℃, and inoculate with 3% *Bacillus stearothermophilus* (5 × 10⁻⁶). 9 Fermentation was carried out for 5 days with cfu / mL; then the temperature was lowered to 32℃, pH 8, and the mixture was inoculated with 10% Staphylococcus equi (5×10⁻⁶ cfu / mL). 9 (cfu / mL), ferment for 3 days; heat to 90℃, keep warm for 30 minutes, and sterilize;

[0089] Step S6 Clarification of enzymatic hydrolysis fermentation broth: Add 4% astragalus gum, artemisia gum and erythrina gum (ratio 1:4:4) to the fermentation hydrolysate, let stand at room temperature for 6 hours, and take the supernatant.

[0090] Step S7 Spray drying: The clarified liquid is filtered through cotton cake to remove impurities, and nanofiltration is used to remove inorganic salts and water-soluble free amino acids. After spray drying, bovine spleen peptides are obtained.

[0091] Comparative Example 1 (defatted, non-fermented)

[0092] Step S1: Sterilize and grind raw materials: After thawing frozen beef spleen, weigh 1 kg, add 3 times (w / v) of water, wash twice, heat to 90℃, keep warm for 20 minutes; grind into 3-5 mm particles using a meat grinder;

[0093] Step S2 Degreasing: Add 4 times (w / v) of n-hexane, soak and stir for 6 hours, centrifuge to remove the supernatant, add 2 times (w / v) of water, and wash 4 times;

[0094] Step S3: Subcritical water extraction of protein: Add 6 times (w / v) water, pH 8, temperature 200℃, extraction time 40 min to obtain bovine spleen protein;

[0095] Preparation of enzymatic hydrolysate in step S4: After defatting, adjust the pH to 7, add 1% neutral protease and flavor protease, enzymatic hydrolysis temperature 55℃, enzymatic hydrolysis for 4h;

[0096] Step S5: Preparation of enzymatic fermentation broth: Adjust pH to 5, temperature to 55℃, and inoculate with 3% *Bacillus stearothermophilus* (5 × 10⁻⁶). 9 Fermentation was carried out for 5 days with cfu / mL; then the temperature was lowered to 30℃, pH 7, and the mixture was inoculated with 5% Staphylococcus equi (5×10⁻⁶ CFU / mL). 9 (cfu / mL), ferment for 2 days; heat to 90℃, keep warm for 30 minutes, and sterilize;

[0097] Step S6 Clarification of enzymatic hydrolysis fermentation broth: Add 3% astragalus gum, artemisia gum and erythrina gum (ratio 1:2:3) to the fermentation hydrolysate, let stand at room temperature for 6 hours, and take the supernatant.

[0098] Step S7 Spray drying: The clarified liquid is filtered through cotton cake to remove impurities, and nanofiltration is used to remove inorganic salts and water-soluble free amino acids. After spray drying, bovine spleen peptides are obtained.

[0099] Comparative Example 2 (subcritical water extraction omitted)

[0100] Step S1: Sterilize and grind raw materials: After thawing frozen beef spleen, weigh 1 kg, add 3 times (w / v) of water, wash twice, heat to 90℃, keep warm for 20 minutes; grind into 3-5 mm particles using a meat grinder;

[0101] Step S2 Degreasing: Add 4 times (w / v) of n-hexane, soak and stir for 6 hours, centrifuge to remove the supernatant, add 2 times (w / v) of water, and wash 4 times;

[0102] Preparation of enzymatic hydrolysate in step S3: After defatting, adjust the pH to 7, add 1% neutral protease and flavor protease, enzymatic hydrolysis temperature 55℃, enzymatic hydrolysis for 4h;

[0103] Step S4: Preparation of enzymatic fermentation broth: Adjust pH to 5, temperature to 55℃, and inoculate with 3% *Bacillus stearothermophilus* (5 × 10⁻⁶). 9 Fermentation was carried out for 5 days with cfu / mL; then the temperature was lowered to 30℃, pH 7, and the mixture was inoculated with 5% Staphylococcus equi (5×10⁻⁶ cfu / mL). 9(cfu / mL), ferment for 2 days; heat to 90℃, keep warm for 30 minutes, and sterilize;

[0104] Step S5 Clarification of enzymatic hydrolysis fermentation broth: Add 3% astragalus gum, artemisia gum and erythrina gum (ratio 1:2:3) to the fermentation hydrolysate, let stand at room temperature for 6 hours, and take the supernatant.

[0105] Step S6 Spray drying: The clarified liquid is filtered through cotton cake to remove impurities, and nanofiltration is used to remove inorganic salts and water-soluble free amino acids. After spray drying, bovine spleen peptides are obtained.

[0106] Comparative Example 3 (enzyme hydrolysate does not ferment)

[0107] Step S1: Sterilize and grind raw materials: After thawing frozen beef spleen, weigh 1 kg, add 3 times (w / v) of water, wash twice, heat to 90℃, keep warm for 20 minutes; grind into 3-5 mm particles using a meat grinder;

[0108] Step S2 Degreasing: Add 4 times (w / v) n-hexane, soak and stir for 6 hours, centrifuge to remove supernatant, add 2 times (w / v) water, wash 4 times; add 4 times (w / v) water, adjust pH to 6.0, inoculate with 5% Streptomyces flavus (4×10⁻⁶) at 30℃. 8 cfu / g) and Yersinia lipolytica (6×10) 8 CFU / g (ratio 1:3), ferment for 4 days, then centrifuge to remove the supernatant;

[0109] Step S3: Subcritical water extraction of protein: Add 6 times (w / v) water, pH 8, temperature 200℃, extraction time 40 min to obtain bovine spleen protein;

[0110] Preparation of enzymatic hydrolysate in step S4: After defatting, adjust the pH to 7, add 1% neutral protease and flavor protease, enzymatic hydrolysis temperature 55℃, enzymatic hydrolysis for 4h;

[0111] Clarification of the enzymatic hydrolysate in step S5: Add 3% Astragalus gum, Artemisia gum and Erythrina gum (ratio 1:2:3) to the enzymatic hydrolysate, let it stand at room temperature for 6 hours, and take the supernatant.

[0112] Step S6 Spray drying: The clarified liquid is filtered through cotton cake to remove impurities, and nanofiltration is used to remove inorganic salts and water-soluble free amino acids. After spray drying, bovine spleen peptides are obtained.

[0113] Comparative Example 4 (clarification omitted)

[0114] Step S1: Sterilize and grind raw materials: After thawing frozen beef spleen, weigh 1 kg, add 3 times (w / v) of water, wash twice, heat to 90℃, keep warm for 20 minutes; grind into 3-5 mm particles using a meat grinder;

[0115] Step S2 Degreasing: Add 4 times (w / v) n-hexane, soak and stir for 6 hours, centrifuge to remove supernatant, add 2 times (w / v) water, wash 4 times; add 4 times (w / v) water, adjust pH to 6.0, inoculate with 5% Streptomyces flavus (4×10⁻⁶) at 30℃. 8 cfu / g) and Yersinia lipolytica (6×10) 8 CFU / g (ratio 1:3), ferment for 4 days, then centrifuge to remove the supernatant;

[0116] Step S3: Subcritical water extraction of protein: Add 6 times (w / v) water, pH 8, temperature 200℃, extraction time 40 min to obtain bovine spleen protein;

[0117] Preparation of enzymatic hydrolysate in step S4: After extraction, the pH of the protein was adjusted to 7, 1% neutral protease and flavor protease were added, and the enzymatic hydrolysis temperature was 55℃ for 4 hours.

[0118] Step S5: Preparation of enzymatic fermentation broth: Adjust pH to 5, temperature to 55℃, and inoculate with 3% *Bacillus stearothermophilus* (5 × 10⁻⁶). 9 Fermentation was carried out for 5 days with cfu / mL; then the temperature was lowered to 30℃, pH 7, and the mixture was inoculated with 5% Staphylococcus equi (5×10⁻⁶ cfu / mL). 9 (cfu / mL), ferment for 2 days; heat to 90℃, keep warm for 30 minutes, and sterilize;

[0119] Step S6 Spray drying: The clarified liquid is filtered through cotton cake to remove impurities, and nanofiltration is used to remove inorganic salts and water-soluble free amino acids. After spray drying, bovine spleen peptides are obtained.

[0120] Experimental Example 1

[0121] Spleen peptide samples from Examples 1-3 and Comparative Examples 1-4 were taken, and the transparency of the aqueous solution for each sample was observed. The yield was calculated, and the protein content, average molecular weight, and fat content were determined. Protein content was determined according to GB 5009.5, molecular weight according to the method specified in GB 22729, and fat content according to GB 5009.6. The test results are shown in Table 1. Figures 1 to 7 .

[0122] Table 1 Sample test results

[0123]

[0124] It can be seen that the solutions of Comparative Example 1 and Comparative Example 4 are both opaque and contain fat, which do not meet the product requirements; the product yield of Comparative Example 2 is very low and not suitable for industrial promotion; the yield of Comparative Example 3 is low and the molecular weight is too large, which also does not meet the product requirements.

[0125] Experiment Example 2

[0126] Forty female and thirty male SD rats, aged 8 weeks, were used. They had free access to water and were kept in an environment with a temperature of 22–26°C, humidity of 40%–70%, and a 12-hour light / 12-hour dark cycle for 7 days to allow for acclimatization. The rats were housed together overnight. The first day after conception, sperm was detected under a microscope in a smear of vaginal irrigation fluid from the female rats. After conception, the female rats were randomly assigned to a low-protein diet group or a standard diet group. Both groups were fed the standard diet after delivery. At 14 days of age, the female pups from both groups were removed, and at 21 days of age, the pups were weaned and fed the standard diet.

[0127] Criteria for judging dwarfism: Newborn mice born in the low-protein diet group were 2 standard deviations shorter than the average length of newborn mice born in the standard diet group after weaning.

[0128] Twenty pups were randomly selected from the pups born to pregnant mice in the standard diet group and weighed, and marked as the normal group. These pups were then divided into two groups. One group was immediately sacrificed and their spleen weight was measured. The other group was fed normally for 3 weeks, and after sacrifice, their body length, body weight, and spleen weight were measured.

[0129] Fifty dwarf model mice were randomly divided into 5 groups of 10 each. One group was euthanized and their spleen weight was measured. The remaining mice were divided into 4 groups of 10 each: a control group, Example 1 group, Example 2 group, and Example 3 group. The Example 3 group received spleen peptides obtained from the Example 1 treatment via gavage at a dose of 250 mg / kg body weight. Three weeks later, the mice were euthanized and their body length, body weight, and spleen weight were measured. The results are shown in Tables 2-4.

[0130] Table 2 Body length of rats in each group at different time points

[0131] Group Before the experiment (cm) 3 weeks later (cm) normal group 13.32±0.43* 21.73±0.67* control group 10.24±0.69 16.25±0.79 Example 1 Group 10.61±0.50 20.21±0.55* Example 2 group 11.38±0.74 21.36±0.82* Example 3 Group 11.06±0.63 20.97±0.60*

[0132] *The difference from the control group was significant, P<0.05.

[0133] Table 3 Body weight of rats in each group at different time points

[0134] Group Before the experiment (g) 3 weeks later (g) normal group 53.82±3.19* 229.54±7.03* control group 43.72±4.35 168.57±5.41 Example 1 Group 44.80±5.47 211.25±6.72* Example 2 group 46.27±6.51 223.31±7.46* Example 3 Group 45.36±5.84 205.74±6.25*

[0135] *The difference from the control group was significant, P<0.05.

[0136] Table 4. Spleen weight of rats in each group at different time points

[0137] Group Before the experiment (mg) 3 weeks later (mg) normal group 226.96±30.21* 514.73±48.65* control group 162.84±27.49 356.03±32.34 Example 1 Group 157.39±22.61 480.77±37.62* Example 2 group 181.27±31.64 504.52±35.19* Example 3 Group 176.55±28.15 501.24±43.83*

[0138] *The difference from the control group was significant, P<0.05.

[0139] It can be seen that the spleen peptides obtained by gavage in Examples 1-3 can significantly promote the growth and development of dwarf rats and increase spleen weight. Their body length, body weight, and spleen weight are basically the same as those of normal rats.

[0140] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing spleen peptides that promote growth and strengthen the spleen, characterized in that, Includes the following steps: 1) The spleen granules of healthy animals were defatted, and the defatted solids were subjected to a first fermentation using Streptomyces flavus and Yersinia lipophila to obtain the precipitate of the first fermentation. 2) The precipitate from the first fermentation was subjected to subcritical water extraction to obtain the extract product; 3) The extract is hydrolyzed with a protease to obtain a hydrolysate; the protease includes one or more of bromelain, papain, neutral protease and flavor protease. 4) The enzymatic hydrolysate is subjected to a second fermentation using a thermophilic Bacillus stearothermophilus agent to obtain a second fermentation broth; the second fermentation broth is subjected to a third fermentation using a Staphylococcus equi agent to obtain a third fermentation broth; 5) The third fermentation broth is clarified using a composite adhesive, and the supernatant is collected; the composite adhesive includes astragalus gum, artemisia gum, and erythrina gum; 6) The supernatant is sequentially filtered, nanofiltered and spray-dried to obtain spleen peptides.

2. The preparation method according to claim 1, characterized in that, In step 1), defatting the spleen particles of healthy animals includes: defatting the spleen particles of healthy animals with n-hexane; the ratio of the mass of the spleen particles to the volume of n-hexane is 1g: 2-10mL; the initial pH of the first fermentation is 5-8; the fermentation time is 1-5 days; and the fermentation temperature is 20-40℃.

3. The preparation method according to claim 1, characterized in that, In step 1), the total mass of the *Streptomyces flavus* inoculum and the *Yarrowia lipolytica* inoculum accounts for 1% to 8% of the spleen granule mass; the mass ratio of the *Streptomyces flavus* inoculum to the *Yarrowia lipolytica* inoculum is 1:(1-5); the effective viable count of *Streptomyces flavus* in the *Streptomyces flavus* inoculum is 1 to 8 × 10⁻⁶. 8 cfu / g; the effective viable count of *Yersinia lipolyticis* in the *Yersinia lipolyticis* preparation is 2–8 × 10⁻⁶. 8 cfu / g.

4. The preparation method according to claim 1, characterized in that, In step 2), the initial pH of the subcritical water extraction system is 5-10; the temperature of the subcritical water extraction is 110-220℃; and the time of the subcritical water extraction is 20-60 min.

5. The preparation method according to claim 1, characterized in that, In step 3), the initial pH of the enzymatic hydrolysis is 6-8; the protease accounts for 0.1% w / v to 5% w / v of the extract; the temperature of the enzymatic hydrolysis reaction is 50-60°C; and the time of the enzymatic hydrolysis reaction is 2-8 h.

6. The preparation method according to claim 1, characterized in that, In step 4), the inoculum amount of the *Bacillus stearothermophilus* agent is 0.5% w / v to 5% w / v of the enzymatic hydrolysis product; the effective viable count of *Bacillus stearothermophilus* in the *Bacillus stearothermophilus* agent is 1 to 8 × 10⁻⁶. 9 cfu / mL; the second fermentation time is 1–5 days; the initial pH of the second fermentation is 3–6; the temperature of the second fermentation is 50–60℃; the inoculum size of the *Staphylococcus equi* agent is 1% w / v–10% w / v of the second fermentation broth; the effective viable count of *Staphylococcus equi* in the *Staphylococcus equi* agent is 1–8 × 10⁻⁶. 9 The concentration of cfu / mL; the time for the third fermentation is 1 to 5 days; the initial pH value for the third fermentation is 6 to 8; and the temperature for the third fermentation is 25 to 35°C.

7. The preparation method according to claim 1, characterized in that, In step 5), the mass ratio of Astragalus gum, Artemisia gum and Erythrina gum is 1:(1-5):(1-6); the composite gum accounts for 0.5% w / v to 5% w / v of the third fermentation liquid.

8. The spleen peptide prepared by the preparation method according to any one of claims 1 to 7, characterized in that, The protein content of the spleen peptide is ≥80%; the average molecular weight of the spleen peptide is 700-800.

9. The use of the spleen peptide according to claim 8 in the preparation of products that strengthen the spleen and / or promote children's growth and development.

10. The application according to claim 9, characterized in that, Promoting children's growth and development includes improving growth retardation caused by malnutrition.

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