Yak milk small molecule peptide and preparation method thereof

Abstract

According to the yak milk small-molecule peptide and the preparation method thereof, the preparation method of the yak milk small-molecule peptide realizes accurate shearing of a proline-enriched region by specifically recognizing the special proline-enriched region of yak milk protein, and solves the technical problem of difficulty in enzyme digestion of the proline region. Moreover, by establishing a triple control mechanism of mild degreasing, antioxidant protection and phospholipase synergistic effect, oxidation peculiar smell caused by high fat content of the yak milk is avoided. The invention also discloses the yak milk small molecule peptide prepared by the preparation method, the yield of the 2-3 amino acid small molecule peptide is 87.5% + / -2.5%, and the retention rate of conjugated linoleic acid is 95.0%-97.0%.

Description

Technical Field

[0001] This invention belongs to the field of dairy product deep processing technology, specifically relating to yak milk small molecule peptides and their preparation methods. Background Technology

[0002] Yak milk, as a unique dairy resource, has a protein composition that differs significantly from that of ordinary cow's milk, posing serious technical challenges in its processing. If the general enzymatic hydrolysis process for ordinary cow's milk is used, yak milk exhibits low casein hydrolysis rate and insufficient yield of small peptides, resulting in increased hydrolysis degree, but the proportion of target 2-3 peptides and proline-specific peptides remains consistently low.

[0003] Chinese patent application CN103382497A discloses a stepwise preparation method for yak milk glycomacropeptides and small peptides. While this method yields glycomacropeptides and small peptides from yak milk through stepwise preparation, the enzyme selection and cleavage site design remain conventional. It lacks a sequence-level "precise cleavage" design to address the imbalance in the β-casein / κ-casein ratio and the proline-rich fragments in yak milk, and it lacks targeted regulatory strategies for the structural differences among different casein subtypes and whey protein components. Therefore, existing technologies have limited research on yak milk proteins and have not proposed specific solutions.

[0004] In addition, due to the difficulty in controlling lipid oxidation, the high fat content easily leads to lipid oxidation off-flavors during enzymatic hydrolysis, and existing defatting techniques severely damage the unique fat globule membrane structure of yak milk. Furthermore, existing technologies do not adequately protect the unique functional components of yak milk, such as conjugated linoleic acid (CLA) and immunoglobulins, resulting in low retention of bioactivity and poor product uniformity. These are also technical problems that need to be solved in the preparation of small molecule peptides from yak milk. Summary of the Invention

[0005] The purpose of this invention is to identify the difficulty in enzymatic digestion of proline-enriched regions as a structural technical bottleneck in the deep processing of yak milk, and to provide a method for preparing yak milk small molecule peptides based on enzymatic digestion of proline-enriched sequence regions of yak milk, so as to improve the yield of 2-3 amino acid small molecule peptides in the preparation of yak milk small molecule peptides.

[0006] A first aspect of the present invention provides a method for preparing small molecule peptides from yak milk, comprising the following steps: (1) Defatting and sterilizing yak milk, then adding antioxidants to yak milk, filtering and retaining the filtrate to obtain pretreated yak milk; (2) The first complex enzyme was added to the pretreated yak milk at 43-48℃ and pH 7.2±0.1, and the reaction was carried out for 3.0h±0.2h while controlling the degree of hydrolysis to 18%-22%; (3) Adjust the temperature of the product obtained in step (2) to 48℃±1℃, adjust the pH to 7.0±0.1, add the second complex enzyme, the third complex enzyme and the synergist, react for 2.5h±0.2h and control the degree of hydrolysis to 30%-32%, and then inactivate the enzyme. (4) Separate the product obtained in step (3) through a membrane and collect the nanofiltration retentate with a molecular weight of 500-5000 Da to obtain the product. The first complex enzyme is mainly composed of prolyl endopeptidase, neutral protease and trypsin; the second complex enzyme is composed of flavor protease and lipase; the third complex enzyme is composed of glutaminase and phospholipase or glutaminase and glycosidase; the synergist is composed of antioxidant and β-cyclodextrin.

[0007] In the first stage, this invention incorporates prolyl endopeptidase, neutral protease, and trypsin as a first complex enzyme into yak milk, thereby achieving complementarity and synergy of substrate sites. Prolyl endopeptidase preferentially cleaves Pro-Leu and Pro-X sites, breaking down the previously continuous proline-rich fragments in yak milk proteins and removing structural "blockades" that traditional enzymes struggle to penetrate. Neutral protease then cleaves Glu-Leu sites, further breaking down the pre-cleaved, medium-length peptide chains. Trypsin specifically recognizes Lys-Pro sites; after the first two enzymes "loosen" the structure, the previously difficult-to-expose Lys-Pro sites are fully exposed and efficiently cleaved. The sequential action of these three enzymes creates a continuous "relay-style cleavage" process at key sites such as Pro-Leu, Glu-Leu, and Lys-Pro in yak milk proteins. The second stage involves the addition of flavor proteases, lipases, glutaminases, and phospholipases for further gentle enzymatic hydrolysis of yak milk. This process further refines flavor profile and molecular weight distribution. Furthermore, the addition of antioxidants and cyclodextrins addresses the challenge of maximizing the retention of unique bioactive substances in yak milk. This two-stage sequential enzymatic hydrolysis approach, which "first targets and opens up structural bottlenecks, then finely controls molecular weight and flavor," specifically identifies the unique proline-rich regions of yak milk proteins. This increases the yield of 2-3 amino acid target small peptides from 55%-60% in traditional methods to 87.5% ± 2.5%, solving the technical challenge of proline region enzymatic cleavage.

[0008] Furthermore, the high immunoglobulin retention rate in yak milk small molecule peptides is due to the fact that the neutral proteases and trypsin in the first stage preferentially cleave characteristic sites of yak milk proteins (such as Glu-Leu and Lys-Pro). These sites are less distributed in immunoglobulins, reducing the risk of hydrolysis. The proteases in the second stage, under the aforementioned conditions, further reduce the attack on immunoglobulins. Moreover, the synergist indirectly protects the conformation of immunoglobulins by reducing oxidative stress. The high CLA retention rate is due to the addition of phospholipase in the third stage, which preferentially hydrolyzes oxidatively sensitive phospholipids (such as phosphatidylcholine), reducing the formation of lipid peroxides. Simultaneously, it preserves the structural integrity of the fat globule membrane by modifying the phospholipids on the membrane surface. The protective effect on the fat globule membrane structure in yak milk is achieved through the selection of preparation method conditions.

[0009] In some embodiments, the lipase is a lipase derived from microorganisms of the genera *Candida*, *Bacillus*, *Trichoderma*, *Aspergillus*, or *Rhizopus*, or a recombinant lipase obtained through genetic engineering. Preferably, the lipase is a lipase prepared by fermentation from microorganisms of the genera *Aspergillus* or *Rhizopus*. It is a food-grade lipase preparation prepared by fermentation from microorganisms of the genera *Aspergillus* or *Rhizopus*. It exhibits stable lipase activity under conditions of pH 6.5-7.0 and 40-50°C.

[0010] In some embodiments, the amino acid sequences of yak milk small molecule peptides are mainly composed of Gly-Pro (GP), Pro-Hyp (PO), Hyp-Gly (OG), Pro-Gly (PG), Gly-Pro-Hyp (GPO), Ala-Gly-Gly (AGG), Val-Pro-Pro (VPP), and Pro-Hyp-Gly (PHG). By designing "precise cleavage" at the sequence level targeting proline-rich fragments, yak milk small molecule peptides with a high content of 2-3 amino acids are obtained.

[0011] In some embodiments, the active site of the prolyl endopeptide is mainly composed of serine, histidine, and aspartic acid.

[0012] In some embodiments, the total mass of the first complex enzyme, the second complex enzyme, the third complex enzyme, and the synergist is 1.2%-1.8% of the mass of yak milk. Preferably, the total mass of the first complex enzyme, the second complex enzyme, the third complex enzyme, and the synergist is 1.5% ± 0.15% of the mass of yak milk.

[0013] Furthermore, in some embodiments, the mass ratio of the first complex enzyme, the second complex enzyme, the third complex enzyme, and the synergist is 5~7:1.5~2.5:0.9~1.1:0.9~1.1. Preferably, the mass ratio of the first complex enzyme, the second complex enzyme, the third complex enzyme, and the synergist is 6:2:1:1.

[0014] In some embodiments, the mass ratio of prolyl endopeptidase, neutral protease, and trypsin in the first complex enzyme is 3.6~4.4:2.7~3.3:0.9~1.1. Preferably, the mass ratio of prolyl endopeptidase, neutral protease, and trypsin is 4:3:1.

[0015] In some embodiments, the prolyl endopeptidase has an enzyme activity of 30,000-35,000 U / g; the neutral protease has an enzyme activity of 45,000-50,000 U / g; and the trypsin has an enzyme activity of 2,500-3,000 USP U / g.

[0016] In some embodiments, the prolyl endopeptidase is a serine protease-type proline endopeptidase, which has higher cleavage efficiency at the Pro-X site.

[0017] In some embodiments, the mass ratio of flavor protease to lipase in the second complex enzyme is 2.7~3.3:0.9~1.1. Preferably, the mass ratio of flavor protease to lipase is 3:1.

[0018] In some embodiments, the enzyme activity of the flavor protease is 500-600 LAPU / g; and the enzyme activity of the lipase is 12000-15000 U / g.

[0019] In some embodiments, the mass ratio of glutaminase to phospholipase in the third complex enzyme is 1.8~2.2:0.9~1.1. Preferably, the mass ratio of glutaminase to phospholipase is 2:1.

[0020] In some embodiments, the activity of glutaminase is 20,000 U / g; the activity of phospholipase is 8,000-10,000 U / g.

[0021] In some embodiments, the antioxidant is selected from at least one of L-cysteine, tea polyphenols, and vitamin E. Preferably, the antioxidant is L-cysteine.

[0022] In some embodiments, the first complex enzyme also includes soybean protease.

[0023] In some embodiments, the antioxidant in the synergist is added at an amount of 0.03%-0.09% of the mass of yak milk; the β-cyclodextrin is added at an amount of 0.03%-0.09% of the mass of protein in yak milk. Preferably, the antioxidant is added at an amount of 0.05% of the mass of yak milk; the β-cyclodextrin is added at an amount of 0.01% of the mass of yak milk.

[0024] In some embodiments, in step (1), the defatting process involves centrifuging the yak milk at 4°C and 6000-7000 rpm to remove excess milk and adjusting the total solids content of the yak milk to 13%. Then, the milk is gently defatted by centrifugation at 4°C and 7000-9000×g for 25 minutes, and the lower liquid layer is retained.

[0025] In some implementations, in step (1), the filtration process is to filter with a 0.45 μm ceramic membrane.

[0026] In some implementations, the temperature in step (1) is 45℃±1℃.

[0027] In some embodiments, in step (3), the enzyme inactivation method is to inactivate the enzyme in the reacted yak milk at 80℃±1℃ for 12s±2s.

[0028] In some embodiments, in step (4), the membrane separation process is to first filter the product obtained in step (3) through a 0.45 μm ceramic membrane, and then pass the filtrate sequentially through a 100 kDa ultrafiltration membrane, a 5 kDa ultrafiltration membrane, and a 500 Da ultrafiltration membrane.

[0029] In a second aspect, the present invention provides a yak milk small molecule peptide prepared by the above preparation method, wherein the yield of the yak milk small molecule peptide with a molecular weight of 500-1000 Da is 87.5%±2.5%, and the retention rate of conjugated linoleic acid (CLA) is 95.0%-97.0%.

[0030] The technical effects of this invention are as follows: (1) This invention increases the yield of 2-3 amino acid target small molecule peptides from 55-60% in traditional methods to 87.5%±2.5% by specifically identifying the special proline-rich region of yak milk protein. It achieves precise cleavage of the proline-rich region, solves the technical problem of difficult enzymatic cleavage of the proline region, and the proline content in the obtained yak milk small molecule peptides is high.

[0031] (2) The present invention establishes a triple control mechanism of mild defatting, antioxidant protection and phospholipase synergistic effect, so that the TBARS value is ≤0.02, which completely prevents the oxidative odor problem caused by the high fat content of yak milk.

[0032] (3) Based on the special amino acid sequence of yak milk protein, the present invention developed a highly specific special complex enzyme system, which improves the enzyme digestion efficiency of the proline-rich region and the enzyme digestion site adaptability reaches more than 96%.

[0033] (4) The low-temperature enzymatic hydrolysis, rapid inactivation and gentle separation process of the present invention ensures that the CLA retention rate is ≥95% and the immunoglobulin activity retention rate is ≥90%, thus maximizing the preservation of the unique functional components of yak milk.

[0034] (5) The present invention establishes a precise four-stage membrane separation system, which realizes the precise enrichment of target peptides. The coefficient of variation (CV) of the molecular weight distribution of the product is reduced from 35%-40% in the traditional method to 8%-10%, and the product uniformity reaches the international advanced level.

[0035] (6) The yak milk small molecule peptides of the present invention have high antioxidant activity and ACE inhibitory activity, and have significant functional characteristics. Attached Figure Description

[0036] Figure 1 This is a flowchart illustrating the preparation of yak milk small molecule peptides according to the present invention; Figure 2 This is the SEC-HPLC chromatogram of small molecule peptides from yak milk. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto. The raw materials and reagents involved in the following embodiments are all commercially available.

[0038] The technical route diagram for preparing yak milk small molecule peptides of the present invention is as follows: Figure 1 As shown in the figure. Through proteomics analysis, characteristic enzyme cleavage sites were identified in the proline-rich regions of yak milk, and a specific, highly adaptable, dedicated complex enzyme system was constructed. A mild and precise defatting pretreatment was designed. Based on the high fat content of yak milk, phospholipase, a mild defatting process, and an antioxidant (L-cysteine) synergistically form a triple control mechanism. Stepwise enzymatic hydrolysis and four-stage membrane separation purification conditions were designed to achieve functional component enrichment. Finally, the molecular weight distribution verified the preparation of high-quality yak milk small molecule peptides. The yield of the target peptide in the yak milk small molecule peptides of this invention was 87.5% ± 2.5%.

[0039] It should be noted that the target peptide of this invention has a molecular weight of 500-1000 Da and is a small molecule peptide of 2-3 amino acids.

[0040] I. Analysis of Yak Milk Protein Substrate Characteristics and Identification of Characteristic Enzyme Cleavage Sites Proteomics analysis of yak milk was performed using LC-MS / MS to conduct in-depth analysis of the amino acid sequences of the main protein components (β-casein, κ-casein, αs1-casein, β-lactoglobulin, and α-lactalbumin), identifying the following key characteristic enzyme cleavage sites: β-Casein: Pro 78-Leu 79 Glu 45 -Leu 46 Lys 98 -Pro 99 .

[0041] κ-casein: Pro 103 -Leu 104 Glu 75 -Leu 76 Lys 116 -Pro 117 .

[0042] αs1-casein: Pro 35 -Leu 36 Glu 65 -Leu 66 .

[0043] β-lactoglobulin: Pro 38 -Leu 39 Glu 55 -Leu 56 .

[0044] α-Lactalbumin: Pro 25 -Leu 26 Glu 49 -Leu 50 .

[0045] By comparing proteomics data of regular cow's milk and yak milk, it was found that the proportion of β-casein and κ-casein was significantly increased in yak milk compared to regular cow's milk, and continuous proline (Pro-X) enriched sequence regions were formed at sites such as Pro-Leu, Glu-Leu, and Lys-Pro.

[0046] II. Construction of a specific, highly adaptable, dedicated complex enzyme system 1. Enzyme type screening The cleavage efficiency of various proteases (including prolyl endopeptidase, neutral protease, trypsin, flavor protease, lipase, glutaminase, phospholipase, etc.) at these sites was tested. Preliminary screening employed single-factor experiments to evaluate the specific hydrolytic capacity of each enzyme on yak milk substrates and the formation of byproducts. Core enzyme preparations suitable for yak milk proteins, especially proline-rich regions, were screened to provide a basis for the construction of complex enzyme systems.

[0047] Experimental methods (1) Substrate: Pretreated yak milk (protein content 4.2%, fat content ≤0.25%) (2) Single enzyme conditions Temperature: 45℃; pH: 7.2; Enzyme addition: 0.5% (w / w) of protein mass; Reaction time: 3 h.

[0048] (3) Detection indicators Degree of hydrolysis (DH, OPA method); bitter peptide content (RP-HPLC, hydrophobic peak area); molecular weight distribution (MALDI-TOF).

[0049] The experimental results are shown in Table 1. As can be seen from Table 1, prolyl endopeptidase has a significant advantage in the Pro-rich region of yak milk, with DH increased by about 18% and the lowest bitter peptide production. Therefore, it was selected as the core enzyme for the first stage.

[0050] Table 1. Enzyme type screening results

[0051] This invention discovers that yak milk small molecule peptides form continuous proline-enriched sequence regions at sites such as Pro-Leu, Glu-Leu, and Lys-Pro.

[0052] 2. Optimization of enzyme combination ratio An orthogonal experimental design was employed to optimize the enzyme system ratio, using the yield of the target peptide, the proportion of proline peptides, the reduction rate of bitter peptides, and the retention rate of CLA as indicators. For example, the ratio of prolyl endonuclease:neutral protease:trypsin in the proline-specific endonuclease group was determined through gradient experiments (e.g., 3:2:1, 4:3:1, 5:3:1). It was found that when the mass ratio of prolyl endonuclease, neutral protease, and trypsin in the proline-specific endonuclease group was 4:3:1, the proportion of the target peptide (500-1000 Da) in the prepared yak milk small molecule peptides was 87.5% ± 1.9%. Finally, 4:3:1 (w / w) was selected as the optimal ratio.

[0053] 3. Composition of complex enzyme systems 3.1 Proline-specific endonuclease group (1) Prolyl endopeptidase (enzyme activity specification: 30000-35000 U / g): specifically recognizes Pro-Leu and Pro-X sites, solving the problem of proline region enzymatic cleavage; (2) Neutral protease (enzyme activity specification: 45000-50000 U / g): specifically recognizes Glu-Leu site; (3) Trypsin (enzyme activity specification: ≥2500 USP U / g): specifically cleaves Lys-Pro sites; (4) Addition ratio: prolyl endopeptidase: neutral protease: trypsin = 4:3:1 (w / w).

[0054] 3.2 Flavor-Precisely Regulated Enzyme Mechanism (1) Flavor protease (enzyme activity specification: 500-600 LAPU / g): hydrolyzes from the end of the peptide chain to eliminate hydrophobic bitter peptides; (2) Lipase A (enzyme activity specification: 12000-15000 U / g): Selected food-grade lipase preparations obtained by fermentation of Aspergillus or Rhizopus microorganisms. It has stable lipase activity under the conditions of pH 6.5-7.0 and 40-50℃. Lipase A specifically hydrolyzes short-chain triglycerides to prevent off-flavors. (3) Addition ratio: Flavor protease: Lipase A = 3:1 (w / w).

[0055] 3.3 Functional components protect the enzyme group (1) Glutaminase (enzyme activity specification: 20000 U / g): enhances the accessibility of protein substrates and reduces the enzymatic hydrolysis temperature; (2) Phospholipase (enzyme activity specification: 8000-10000 U / g): preferentially hydrolyzes oxidation-sensitive phospholipids (such as phosphatidylcholine), reduces the generation of lipid peroxides, and modifies the phospholipids on the membrane surface to form a more stable membrane structure, thereby preventing the oxidation of conjugated linoleic acid (CLA). (3) Addition ratio: glutaminase: phospholipase = 2:1 (w / w).

[0056] 3.4 Synergistic Agent Group (1) L-cysteine: The amount added is 0.05% (w / w) of the weight of yak milk, as an antioxidant; (2) β-cyclodextrin: The amount added is 0.1% (w / w) of the weight of yak milk, which encapsulates and masks off-flavor molecules.

[0057] 3.5 Total enzyme system ratio Proline-specific endonuclease group: flavor-precisely regulating enzyme group: functional component protective enzyme group: synergist group = 6:2:1:1 (w / w).

[0058] 4. Preparation of a dedicated complex enzyme system Prepare a special compound enzyme system according to the following proportions.

[0059] Proline-specific endonuclease group: prolyl endonuclease (32000 U / g) 40g; neutral protease (48000 U / g) 30g; trypsin (2500 USP U / g) 10g.

[0060] Flavor-precision regulation enzyme group: Flavor protease (550 LAPU / g) 30g; Lipase A (13000 U / g) 10g.

[0061] Functional components protecting enzyme group: glutaminase (20000 U / g) 10g; phospholipase (9000 U / g) 5g.

[0062] Synergistic effector group: L-cysteine ​​5g; β-cyclodextrin 10g.

[0063] The total weight is 150g. When using it, the amount of special compound enzyme system added should be 1.5% of the substrate (yak milk) mass.

[0064] IV. Time-Controlled Stepwise Enzymatic Hydrolysis Process Based on Molecular Directed Shearing A stepwise enzymatic hydrolysis strategy with low-temperature time-controlled operation was adopted, and the specific process parameters are as follows: Phase 1: Proline-specific cleavage (1) Reaction conditions: temperature 45℃±1℃, pH 7.2±0.1, reaction time 3.0h±0.2h; (2) Enzyme addition: Add proline-specific endonuclease group (prolyl endonuclease, neutral protease and trypsin); (3) Environmental control: nitrogen protection, dissolved oxygen concentration (DO) ≤ 0.3 ppm; (4) Process monitoring: real-time monitoring of degree of hydrolysis, with the target DH value controlled at 18%-22%.

[0065] Phase Two: Precise Control of Flavor and Functionality (1) Reaction conditions: temperature 48℃±1℃, pH 7.0±0.1, reaction time 2.5h±0.2h; (2) Enzyme addition: Add flavor-precise regulation enzyme group (flavor protease and lipase A) and functional component protection enzyme group (glutaminase and phospholipase). (3) Addition of synergists: L-cysteine ​​and β-cyclodextrin are added simultaneously; (4) Process monitoring: The target DH value is controlled at 30%-32%.

[0066] Phase 3: Mild Termination of the Reaction (1) Enzyme inactivation conditions: Instantaneous enzyme inactivation at 80℃±1℃ / 12s±2s; (2) Cooling treatment: rapidly cool to 35℃±2℃.

[0067] It should be noted that, by mass percentage, the percentages of each ingredient added to the yak milk are as follows: prolyl endopeptidase 0.4%±0.04%, neutral protease 0.3%±0.03%, trypsin 0.1%±0.01%, flavor protease 0.3%±0.03%, lipase A 0.1%±0.01%, glutaminase 0.1%±0.01%, phospholipase 0.05%±0.005%, L-cysteine ​​0.05%±0.005%, and β-cyclodextrin 0.1%±0.01%.

[0068] V. Effect of degree of hydrolysis on small molecule peptides in yak milk Degree of hydrolysis (DH) is a key parameter in the enzymatic hydrolysis process, directly affecting product yield, peptide distribution, and bioactivity. This scheme optimizes product quality by controlling the DH value in stages (18%-22% in the first stage and 30%-32% in the second stage).

[0069] Excessively high DH values ​​in the second stage (e.g., DH > 32%) lead to excessive hydrolysis of yak milk by enzymes. This not only produces too many short peptides (< 500 Da) and free amino acids, reducing the proportion of target peptides (500-1000 Da), but also increases the content of bitter peptides and deteriorates the flavor due to the exposure of hydrophobic amino acids. Simultaneously, excessively high DH values ​​result in the loss of functional substances in yak milk; heat-sensitive components such as immunoglobulins are inactivated due to excessive hydrolysis, and CLA is lost due to accelerated oxidation.

[0070] Preliminary experiments revealed that when the target DH value in the second stage was greater than 35%, the yield of the target peptide dropped to below 70%, while the content of bitter peptides increased by 50%.

[0071] A low DH value (e.g., DH < 18%) will lead to insufficient hydrolysis, resulting in unhydrolyzed large protein molecules and poor product uniformity (molecular weight distribution CV > 30%). Furthermore, the yield will be low, with the target peptide yield at 58% ± 3%, and ACE IC50 low. 50 The concentration was 0.82 mg / mL; the proline peptide content was <30%. Furthermore, the resulting product had a longer peptide chain, leading to decreased ACE inhibitory and antioxidant activity (IC50). 50 (Value > 0.8 mg / mL), indicating weak biological activity.

[0072] VI. A four-stage membrane separation and purification system with precise molecular weight control A four-stage membrane separation system was established to achieve precise separation of target peptides and enrichment of functional components.

[0073] 1. Pretreatment filtration system (1) Membrane type: 0.45 μm ceramic membrane; (2) Function: Remove denatured protein aggregates while retaining functional components.

[0074] 2. Macromolecule removal system (1) Membrane type: 100 kDa ultrafiltration membrane; (2) Operating parameters: operating pressure 0.4-0.6MPa, temperature 38℃±2℃; (3) Function: Removes unhydrolyzed macromolecular proteins.

[0075] 3. Precise Target Peptide Sorting System (1) Membrane type: 5 kDa ultrafiltration membrane; (2) Operating parameters: operating pressure 0.6-0.8MPa, temperature 40℃±2℃; (3) Function: Remove medium molecular weight peptides and screen for target small molecule peptides.

[0076] 4. Target peptide enrichment system (1) Membrane type: 500 Da nanofiltration membrane; (2) Operating parameters: operating pressure 1.0-1.2MPa, temperature 35℃±2℃; (3) Function: Precisely retains 500-1000 Da target peptides while removing bitter amino acids.

[0077] Example 1 This embodiment provides a method for preparing small molecule peptides from yak milk, including the following steps: (1) Raw material pretreatment 100 kg of fresh yak milk was taken, and its protein content was found to be 4.2% and fat content to be 4.3%. First, it was centrifuged at 4℃ and 6500 rpm to remove milk fat and suspended impurities, adjusting the total solids content of the yak milk to 13%. Then, it was gently defatted by centrifugation at 4℃ and 8000×g for 25 min, reducing the fat content to 0.25%. Next, it was pasteurized at 78℃ for 15 s, and then L-cysteine ​​was added. Finally, it was cooled to 45℃ and filtered through a 0.45 μm ceramic membrane to obtain pretreated yak milk.

[0078] (2) The pretreated yak milk was pumped into the enzymatic hydrolysis tank, and nitrogen protection was turned on (DO≤0.3ppm). At 45℃ and pH=7.2, prolyl endopeptidase (32000 U / g), neutral protease (48000 U / g) and trypsin (2500 USP U / g) were added to the enzymatic hydrolysis tank. The reaction was carried out for 3 hours. At this time, the degree of hydrolysis DH reached 20.5%. (3) Adjust the temperature to 48℃ and pH to 7.0, add trypsin (2500 USP U / g), flavor protease (550 LAPU / g), lipase A (13000 U / g), glutaminase (20000 U / g), phospholipase (9000 U / g), L-cysteine ​​and β-cyclodextrin to the enzymatic hydrolysis tank, and continue the reaction for 2.5 h. At this time, the degree of hydrolysis DH reaches 31.2%. Then, perform instantaneous enzyme inactivation at 80℃ for 12s, cool to 35℃, and obtain the enzyme-inactivated solution. (4) Quadruple membrane separation and purification The enzyme-inactivating solution was first filtered through a 0.45 μm ceramic membrane. The filtrate was then sequentially passed through a 100 kDa ultrafiltration membrane (operating pressure 0.5 MPa, temperature 38℃), a 5 kDa ultrafiltration membrane (operating pressure 0.7 MPa, temperature 40℃), and a 500 Da ultrafiltration membrane (operating pressure 1.1 MPa, temperature 35℃), and the nanofiltration retentate was collected. The nanofiltration retentate was then concentrated under vacuum at 45℃ and -0.08 MPa until the solids content reached 35%, yielding a concentrated solution. (5) Spray drying The concentrate was dried by centrifugal spray drying with an inlet air temperature of 158°C and an outlet air temperature of 65°C to obtain yak milk small molecule peptide powder.

[0079] It should be noted that the amounts of enzyme, L-cysteine, and β-cyclodextrin used in the examples were added according to the "Preparation of a Special Complex Enzyme System". In Example 1, the total mass of the special complex enzyme system was 150g, and the amount of L-cysteine ​​added was 50g. The prepared yak milk small molecule peptide powder can be packaged under nitrogen filling in an environment with a humidity of <25% and a residual oxygen content of <0.3%. The total mass of L-cysteine ​​used in the preparation process was 0.05% of the mass of yak milk, and it was added in two steps to cover the oxidation risk in the pretreatment and enzymatic hydrolysis stages.

[0080] The total solids content of yak milk was adjusted by dilution or concentration. Specifically, after centrifugation of the purified milk, the initial total solids content of the yak milk was first determined using a milk component analyzer. When the initial total solids content was higher than the target percentage, quantitative dilution was performed by adding sterilized deionized water. When the initial total solids content was lower than the target percentage, moderate concentration was performed using a low-temperature vacuum concentration method. The total solids content of the yak milk in step (1) was uniformly adjusted to 13% ± 0.2% to ensure the stability and repeatability of the subsequent enzymatic hydrolysis and membrane separation processes. The pH value in the preparation method was adjusted by adding food-grade NaOH / HCl and controlled in real time using a pH electrode.

[0081] Product quality evaluation The yak milk small molecule peptide powder prepared in Example 1 was evaluated for product quality through the following tests.

[0082] 1. Verification of molecular directional shearing effect (1) Molecular weight distribution MALDI-TOF mass spectrometry (linear positive ion mode) Sample preparation: Take yak milk small molecule peptide samples, dissolve them in ultrapure water to prepare a 1 mg / mL solution, and filter it through a 0.22 μm microporous membrane for later use.

[0083] Matrix preparation: α-cyano-4-hydroxycinnamic acid (CHCA) was selected as the matrix and dissolved in a 50% acetonitrile / 0.1% trifluoroacetic acid solution at a concentration of 10 mg / mL.

[0084] Spotting and Detection: The sample solution and matrix solution were mixed at a 1:1 (v / v) ratio, and 1 μL was spotted onto a MALDI target plate. After air drying, the plate was placed in the mass spectrometer for detection. Positive ion reflectance mode was used, and the mass scan range was set to 200-2000 Da.

[0085] Data processing: Extract mass spectrometry peak signals in the 500-1000 Da range, and calculate the mean (mean) and standard deviation (SD) of the peak intensity in this range. Calculate the coefficient of variation (CV) of molecular weight distribution according to the formula: CV(%) = [Standard Deviation (SD) / Mean (Mean)] × 100. The lower the CV value, the more uniform the distribution of small molecule peptides.

[0086] (2) Bitter peptide content The content of bitter peptides (hydrophobic peptides) was verified using RP-HPLC.

[0087] Sample preparation: The sample was dissolved in ultrapure water to prepare a 5 mg / mL solution, which was then filtered through a 0.22 μm filter membrane.

[0088] Chromatographic conditions: The column was a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm); mobile phase A was 0.1% (v / v) trifluoroacetic acid aqueous solution; mobile phase B was 0.1% (v / v) trifluoroacetic acid acetonitrile solution; elution mode was gradient elution; flow rate was 1.0 mL / min; column temperature was 30℃; detection wavelength was 214 nm.

[0089] Analytical method: Record the hydrophobic peptide peaks with retention times of 20-40 min in the chromatogram, and calculate the ratio of the sum of their peak areas to the total peak area as a relative indicator of bitter peptide content.

[0090] (3) Determination of proline peptide content The obtained peptides were separated, identified, and quantified using LC-MS / MS. Based on the peptide sequence information obtained from mass spectrometry, peptides containing proline residues were screened, and their relative abundance was calculated. The proportion (enrichment) of proline peptides was calculated using the following formula: Proline peptide percentage (%) = (∑proline peptide peak area / ∑ total peptide peak area) × 100.

[0091] 2. Evaluation of the effect of lipid oxidation control (1) TBA colorimetric method to obtain TBARS value.

[0092] Trichloroacetic acid (TCA) was added to the sample, vortexed and incubated on ice, then centrifuged at high speed, and the supernatant was collected for subsequent reactions.

[0093] The treated sample supernatant, MDA standard, and a blank control (water) were mixed with TBA reagent under acidic conditions. The mixtures were then incubated in a water bath at 100°C for 60 minutes.

[0094] After the heating reaction is complete, remove the test tube and cool it to room temperature in running water. After centrifugation, add the supernatant to a 96-well plate. Measure the absorbance (OD value) at 535 nm using a microplate reader (or spectrophotometer). This wavelength is the maximum absorption peak of the pink compound.

[0095] Prepare a standard solution of MDA with a known concentration, and simultaneously perform colorimetric reactions and measurements on the same plate as the sample. Plot a standard curve for the standard solution, and then substitute the absorbance of the sample into the standard curve to obtain the TBARS value.

[0096] (2) Use an electronic nose to detect an array of oxidative odor sensors.

[0097] (3) CLA retention rate CLA content was determined by gas chromatography-flame ionization detection (GC-FID). The fatty acids in the samples were first used to prepare methyl esters using the methanol-methyl sulfate method, followed by GC-FID analysis. CLA retention was calculated using the following formula: CLA retention rate (%) = [Yak milk small molecule peptide CLA content (mg / g) / Yak milk CLA content (mg / g)] × 100.

[0098] 3. Bioactivity identification (1) ELISA detection Immunoglobulin activity in the samples was quantitatively analyzed by enzyme-linked immunosorbent assay (ELISA). Treated samples and untreated control samples were added to the wells of an ELISA plate, respectively. Optical signals were generated through antigen-antibody binding and enzymatic substrate reaction. After measuring the 450 nm OD value, the background signal from the blank wells was subtracted, and the immunoglobulin activity retention rate (%) was calculated. Immunoglobulin activity retention rate (%) = [(control absorbance)] Blank absorbance / (sample absorbance) [Blank absorbance] × 100; The higher the immunoglobulin activity retention rate, the better the treatment process preserves the structure and function of immunoglobulins.

[0099] (2) Detection and calculation of DPPH free radical scavenging rate by spectrophotometry Experimental procedure: Prepare a 0.1 mmol / L DPPH-ethanol solution and store it in the dark. Take sample solutions of different concentrations (0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL) and mix them with the DPPH-ethanol solution at a ratio of 1:1 (v / v), and react in the dark for 30 min.

[0100] Detection conditions: The absorbance was measured at a wavelength of 517 nm using a UV-Vis spectrophotometer.

[0101] Calculation method: The DPPH-ethanol solution without sample was used as a blank control (A0), and the absorbance value of the sample group was recorded as A1. The clearance rate was calculated according to the following formula: clearance rate (%) = (A0-A1) / A0×100.

[0102] (3) HHL detection and calculation of ACE inhibitory activity IC 50 value Reaction system construction: Using Hippuryl-His-Leu (HHL) as substrate, ACE enzyme solution and sample solutions of different concentrations were added, and the reaction was carried out at 37℃ for 60 min.

[0103] Reaction termination and detection: Hydrochloric acid was added to terminate the reaction. The hippuric acid produced by extraction with ethyl acetate was evaporated to dryness and dissolved in ultrapure water. The absorbance was measured at a wavelength of 228 nm.

[0104] Data processing: The ACE inhibition rate was calculated, and a nonlinear regression was performed with sample concentration on the x-axis and inhibition rate on the y-axis to calculate IC. 50 value.

[0105] The product yield (mass percentage of yak milk small molecule peptide powder relative to yak milk) in Example 1 was 84.2%, M wThe concentration was 930-980 Da. Proline (Pro-X) peptides accounted for 46.3% of the total. The hydrophobic peak area of ​​bitter peptides was reduced by approximately 38%-45% compared to yak milk, resulting in a 94.2% reduction in bitter peptide content. Qualitative and quantitative analysis by LC-MS / MS showed that the proportion of proline peptides increased from 34% in yak milk to 46.3% ± 3.5%. This is attributed to the selective hydrolysis of specific peptide bonds in milk proteins by the selected enzyme, and the removal of hydrophobic bitter peptides and large peptides during the process, leading to the enrichment and relative increase of proline peptides in short peptides.

[0106] Thiobarbituric acid reactive value ≤ 0.02; ACE inhibitory activity IC50 50 The value was 0.35 mg / mL. The product was detected using an electronic nose sensor, and the response value of the oxidative odor sensor dropped below the detection limit. These results indicate that the yak milk small molecule peptide has no oxidative odor or bitterness, and a pure taste.

[0107] The target peptides in the 500-1000 Da range accounted for 87.5%. The CLA retention rate of the yak milk small molecule peptides in Example 1 was 96.5%, the immunoglobulin activity retention rate was 92.3%, and the DPPH free radical scavenging rate was 72% ± 3%.

[0108] The obtained small molecule peptides were sequenced by liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis, and the following representative sequences were found to be present: Dipeptides: Gly-Pro (GP), Pro-Hyp (PO), Hyp-Gly (OG), Pro-Gly (PG), etc.; Tripeptides: Gly-Pro-Hyp (GPO), Ala-Gly-Gly (AGG), Val-Pro-Pro (VPP), Pro-Hyp-Gly (POG), etc.

[0109] SEC-HPLC chromatographic results are as follows Figure 2 As shown in Table 2, the molecular weight distribution of peptides is shown in Table 3.

[0110] Table 2 Chromatographic Results

[0111] Table 3. Peptide molecular weight distribution of small molecule peptides from yak milk

[0112] In addition, cleavage sites were predicted using proteomics and compared with peptides detected by LC-MS / MS. The results showed that 96.3% of the predicted sites were detected in small peptides, indicating a high degree of adaptation of the cleavage sites.

[0113] The effects of L-cysteine ​​addition (0.01%, 0.05%, 0.1%, 0.2%) on enzyme activity and oxidation parameters (TBARS value, CLA retention rate) were evaluated, and the results are shown in Table 4.

[0114] As shown in Table 4, when the L-cysteine ​​content is 0.05%, the TBARS value is ≤0.02, and the enzyme activity is not significantly inhibited. The degree of hydrolysis (DH) in step (3) remains at 30-32%, achieving an optimal balance between inhibiting oxidation and maintaining enzyme activity. When the L-cysteine ​​content is higher than 0.1%, standard enzyme activity assays show a 10-15% decrease in the activity of neutral protease and flavor protease.

[0115] Table 4. Effects of L-cysteine ​​addition on small molecule peptides in yak milk

[0116] Example 2 This embodiment provides a method for preparing small molecule peptides from yak milk. The difference from Embodiment 1 is that, after the protein content is detected, the raw materials of the special complex enzyme system are added according to the "special complex enzyme system".

[0117] The process parameters used in this embodiment are: (1) Processing capacity: 1200L / batch; (2) Enzymatic hydrolysis tank volume: 6m³ 3 It is equipped with an online precision monitoring system for pH, temperature, and DO (dissolved oxygen concentration); (3) Membrane system specifications: 100kDa ultrafiltration membrane with an area of ​​250 m² 2 The 5kDa ultrafiltration membrane has a surface area of ​​200 m². 2 The 500Da nanofiltration membrane has an area of ​​180 m². 2 ; (4) Drying system: centrifugal spray drying tower with an evaporation rate of 250 kg / h.

[0118] Each batch of yak milk small molecule peptides was tested, and the product yield was 83.5%-85.5%, the proportion of the target peptide fragment (500-1000 Da) was 86.5%-88.5%, the CLA retention rate was 95.0%-97.0%, and the energy consumption cost was 0.70-0.80 kW·h / kg. This indicates that the preparation method of Example 1 can also achieve the production of yak milk small molecule peptides with a high proportion of the target peptide fragment and a high CLA retention rate in large-scale production processes.

[0119] Example 3 This embodiment provides a method for preparing small molecule peptides from yak milk, including the following steps: (1) Raw material pretreatment 100 kg of fresh yak milk was taken, and its protein content was found to be 4.2% and fat content to be 4.3%. First, the milk was centrifuged at 4℃ and 6500 rpm to remove excess fat and adjust the total solids content to 13%. Then, it was gently centrifuged at 4℃ and 8000×g for 25 min to reduce the fat content to 0.25%. Next, it was pasteurized at 78℃ for 15 s, and then 0.05% L-cysteine ​​was added. Finally, it was cooled to 45℃ and filtered through a 0.45 μm ceramic membrane to obtain pretreated yak milk.

[0120] (2) Pump the pretreated yak milk into the enzymatic hydrolysis tank, turn on the nitrogen protection (DO≤0.3ppm), and add 40g prolyl endopeptidase (32000 U / g), 48g soybean protease, 30g neutral protease (48000 U / g) and 10g trypsin (2500 USP U / g) to the enzymatic hydrolysis tank at 45℃ and pH=7.2, and react for 3h; (3) Adjust the temperature to 48℃ and pH to 7.0, add 10 g trypsin (2500 USP U / g), 30 g flavor protease (550 LAPU / g), 10 g lipase A (13,000 U / g), 10 g glutaminase (20000 U / g), 5 g phospholipase (9000 U / g), 5 g L-cysteine ​​and 10 g β-cyclodextrin to the enzymatic hydrolysis tank, continue the reaction for 2.5 h, then perform instantaneous enzyme inactivation at 80℃ for 12s, cool to 35℃ to obtain enzyme-inactivated solution; (4) Quadruple membrane separation and purification The enzyme-inactivating solution was first filtered through a 0.45 μm ceramic membrane. The filtrate was then sequentially passed through a 100 kDa ultrafiltration membrane (operating pressure 0.5 MPa, temperature 38℃), a 5 kDa ultrafiltration membrane (operating pressure 0.7 MPa, temperature 40℃), and a 500 Da ultrafiltration membrane (operating pressure 1.1 MPa, temperature 35℃), and the nanofiltration retentate was collected. The nanofiltration retentate was then concentrated under vacuum at 45℃ and -0.08 MPa until the solids content reached 35%, yielding a concentrated solution. (5) Spray drying The concentrate was dried by centrifugal spray drying with an inlet air temperature of 158°C and an outlet air temperature of 65°C to obtain yak milk small molecule peptide powder.

[0121] The yak milk small molecule peptides prepared in Example 3 were weighed and tested, and it was found that the product yield was 82.1%, the proportion of the target peptide was 85.2%, and the proportion of the proline peptide was 43.5%.

[0122] Example 4 This embodiment provides a method for preparing small molecule peptides from yak milk. The difference from Example 1 is that the 500 Da nanofiltration membrane is replaced with a 0.45 μm ceramic membrane, and the operating pressure is adjusted to 0.9-1.1 MPa.

[0123] The yak milk small molecule peptides prepared in Example 4 were weighed and tested, and it was found that the product yield was 85.0%, the target peptide ratio was 87.2%, the continuous operation time of 500 Da in Example 1 was 200 h, and the 0.45 μm ceramic membrane was 270 h, and the membrane life was extended by about 35%.

[0124] Comparative Example 1 This comparative example provides a method for preparing small molecule peptides from yak milk, comprising the following steps: 100 g of yak milk was gently defatted by centrifugation at 4℃ and 8000×g for 25 min. The pH was adjusted to 7.5 with NaOH. 50 g of neutral protease was added to the yak milk, and the mixture was enzymatically hydrolyzed at 50℃ for 5 hours. Then, the enzyme was inactivated at 85℃ for 15 min. The yak milk was then passed through a 10 kDa ultrafiltration membrane to obtain the final product.

[0125] The yield of 500-1000 Da small molecule peptides from yak milk prepared in Comparative Example 1 was determined to be approximately 60%.

[0126] Comparative Example 2 This comparative example provides a method for preparing small molecule peptides from yak milk, comprising the following steps: After pasteurizing 100 g of yak milk, add 20 g of trypsin and 20 g of flavor protease to the yak milk, enzymatically hydrolyze at 50℃ for 3 hours, and then separate using a 10 kDa ultrafiltration membrane, retaining the liquid with a molecular weight less than 10 kDa, to obtain the final product.

[0127] Comparative Example 3 This comparative example provides a method for preparing yak milk whey protein peptides, including the following steps: Adjust the pH of 5g yak milk whey protein to 7.0 with NaOH, then add 15g trypsin and 15g flavor protease, and enzymatically hydrolyze at 45℃ for 2.5 hours. Couple the mixture with an ultrafiltration system (i.e., use an enzymatic hydrolysis + online ultrafiltration cycle coupling method, which is the existing technology) to obtain the final product.

[0128] Comparative Example 4 This comparative example provides a method for preparing small molecule peptides from yak milk. The difference from Example 1 is that in step (2), the degree of hydrolysis DH is less than 18%.

[0129] Comparative Example 5 This comparative example provides a method for preparing small molecule peptides from yak milk. The difference from Example 1 is that in step (2), the degree of hydrolysis DH is 30%.

[0130] When DH < 18%, the yield of the target peptide was 58% ± 3%, and the ACE IC 50 =0.82 mg / mL; when DH is approximately 30%, the yield of the target peptide is approximately 87%, IC50 50 It decreased to 0.36 mg / mL.

[0131] The products obtained from Comparative Examples 1-3 were tested, and the results were compared with those of the yak milk small molecule peptides from Example 2. The power consumption (kW·h) of the entire preparation process, including the enzymatic hydrolysis tank (online monitoring system), membrane system (ultrafiltration / nanofiltration), and spray drying tower, was recorded. The energy cost was obtained by dividing the energy consumption by the product weight (kg), with the result of Comparative Example 1 as the baseline (100%). The test results are shown in Table 5.

[0132] Three independent parallel preparations were conducted under the same process conditions, and the performance of each batch of samples was tested separately. The data in Table 5 are expressed as "mean ± standard deviation" or "minimum – maximum". When the results are expressed as "±", the point value is the arithmetic mean of the results from multiple parallel experiments; when the results are expressed as an interval, the upper and lower limits of the interval are the minimum and maximum values ​​measured in multiple experiments, respectively. Table 5 Performance of Small Molecule Peptides

[0133] As shown in Table 5, Comparative Example 1 exhibits low yield of the target peptide (500-1000 kDa) from yak milk small molecule peptides; a TBARS value ≥ 0.08, indicating severe lipid oxidation; and a CV of 35%-40%, resulting in poor product homogeneity. Comparative Example 2 shows a target peptide yield of 65%-70%, a CLA retention rate of 80%-85%, and a TBARS value of 0.04-0.06, indicating insufficient oxidation control compared to the yak milk small molecule peptides of Example 2. This is because its preparation process neglects the high fat and proline enrichment characteristics of yak milk. The yak milk small molecule peptides obtained in Example 2 show significant performance improvements in the yield of 2-3 amino acid small molecule peptides and the proportion of proline peptides compared to the comparative example, with a proline peptide proportion of 46.3% ± 3.5%, indicating high proline peptide enrichment. Furthermore, the energy consumption cost of the preparation method in the examples is significantly lower than that in Comparative Example 1, indicating that the preparation method of the examples can be applied to the production process of yak milk small molecule peptides.

[0134] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for preparing small molecule peptides from yak milk, characterized in that, The method comprises the following steps: (1) Defatting and sterilizing the yak milk, then adding an antioxidant to the yak milk, filtering and retaining the filtrate to obtain pretreated yak milk; (2) Adding a first composite enzyme to the pretreated yak milk under the conditions of 45℃±1℃ and pH 7.2±0.1, reacting for 3.0h±0.2h and controlling the degree of hydrolysis to be 18%-22%; (3) Adjusting the temperature of the product obtained in step (2) to 48℃±1℃ and the pH to 7.0±0.1, adding a second composite enzyme, a third composite enzyme and a synergist, reacting for 2.5h±0.2h and controlling the degree of hydrolysis to be 30%-32%, and then inactivating the enzymes; (4) Subjecting the product obtained in step (3) to membrane separation, and collecting the nanofiltration retentate with a molecular weight of 500-5000 Da, to obtain the yak milk small molecular peptide. The first composite enzyme mainly comprises prolyl endopeptidase, neutral protease and trypsin; the second composite enzyme comprises flavourzyme and lipase; the third composite enzyme comprises glutaminase and phospholipase or comprises glutaminase and glycosidase; and the synergist comprises an antioxidant and β-cyclodextrin.

2. The production method according to claim 1, characterized by, The lipase is a lipase prepared by fermentation of microorganisms of the genus Aspergillus or the genus Rhizopus; and the antioxidant is at least one selected from L-cysteine, tea polyphenol and vitamin E.

3. The preparation method according to claim 1, characterized in that, The first composite enzyme further comprises soybean protease.

4. The method of claim 1, wherein, The total mass of the first composite enzyme, the second composite enzyme, the third composite enzyme and the synergist is 1.2%-1.8% of the mass of the yak milk.

5. The preparation method according to claim 1, characterized in that, The mass ratio of the first composite enzyme, the second composite enzyme, the third composite enzyme and the synergist is 5-7:1.5-2.5:0.9-1.1:0.9-1.

1.

6. The method of claim 1, wherein, In the first composite enzyme, the mass ratio of prolyl endopeptidase, neutral protease and trypsin is 3.6-4.4:2.7-3.3:0.9-1.

1. In the second composite enzyme, the mass ratio of flavourzyme and lipase is 2.7-3.3:0.9-1.

1. In the third composite enzyme, the mass ratio of glutaminase and phospholipase is 1.8-2.2:0.9-1.

1.

7. The preparation method according to claim 1, characterized in that, The addition amount of the antioxidant is 0.03%-0.09% of the mass of the yak milk; and the addition amount of the β-cyclodextrin is 0.03%-0.09% of the mass of the yak milk.

8. The method of claim 1, wherein, In step (1), the defatting treatment method is to centrifuge the yak milk at 4℃ and 6000-7000 rpm to obtain clean milk, adjust the total solid content of the yak milk to 13%, and then gently centrifuge the yak milk at 4℃ and 7000-9000×g for 25 min to retain the lower liquid.

9. The method of claim 1, wherein, In step (4), the membrane separation treatment method is to first filter the product obtained in step (3) through a 0.45 μm ceramic membrane, and then sequentially pass the filtrate through a 100 kDa ultrafiltration membrane, a 5 kDa ultrafiltration membrane and a 500 Da ultrafiltration membrane.

10. A small molecule peptide of yak milk characterized in that, The yak milk small molecular peptide is prepared by the preparation method in any one of claims 1-9, the yield of the peptide with a molecular weight of 500-1000 Da in the yak milk small molecular peptide is 87.5%±2.5%, and the retention rate of conjugated linoleic acid is 95.0%-97.0%.

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