Goat milk small molecule peptide, natural goat fat and preparation method thereof

Abstract

The application discloses a preparation method of goat milk small molecule peptides, which comprises the following steps: constructing a complex enzyme system, and adding glutamine enzyme and other enzymes to enzymatically hydrolyze goat milk, so as to solve the technical problems of low enzymolysis efficiency, heavy odor and insufficient uniformity of goat milk peptide products in the prior art. The goat milk small molecule peptide product prepared by the preparation method has high uniformity, high yield of 500-1000 Da peptide segments, retains biological active substances, and has high odor removal rate. In the process of deep processing of goat milk, short-chain fatty acids and other flavor active substances are enriched in the fat component. If discarded directly, not only resources are wasted, but also environmental burden is introduced. Therefore, the application synchronously recovers and purifies natural goat fat while preparing goat milk small molecule peptides, so as to form a "protein-fat" synergistic high-value utilization system.

Description

Technical Field

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

[0002] Existing research indicates that β-casein accounts for as much as 50-60% of goat milk protein, and its unique amino acid sequence characteristics (rich in Glu-Leu, Leu-Leu, Lys-Val and other enzyme cleavage sites) are significantly different from those of cow milk.

[0003] Currently, the industrial preparation of goat milk suffers from the following technical defects: (1) lack of ability to identify the characteristic amino acid sequence of goat milk protein, resulting in random enzyme cleavage sites and low yield of target small molecule peptides; (2) inability to simultaneously and effectively solve the inherent goaty smell caused by short-chain fatty acids in goat milk and the bitterness generated during enzymatic hydrolysis, resulting in poor product taste; (3) low efficiency of existing enzyme systems in enzymatic hydrolysis of goat milk; (4) wide molecular weight distribution range, low proportion of 2-3 amino acid target peptides, resulting in unstable product quality; (5) harsh conditions in traditional enzymatic hydrolysis processes, leading to severe loss of functional peptide activity; (6) low solubility of goat milk peptides, with small molecule peptides locally aggregating in the system, limiting their biological activity and affecting product uniformity. Therefore, it is necessary to develop a preparation method to achieve precise cleavage of goat milk protein, eliminate the inherent goaty smell of small molecule peptides in goat milk, and improve their solubility. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing small molecule peptides from goat milk, so as to solve the technical problems of low enzymatic hydrolysis efficiency, strong goaty odor, and insufficient uniformity in the preparation of goat milk peptides in the prior art.

[0005] According to a first aspect of the present invention, a method for preparing goat milk small molecule peptides is provided, comprising the following steps:

[0006] (1) After centrifuging, defatting, and sterilizing the goat milk, perform molecular distillation, and then filter and retain the filtrate;

[0007] (2) Adjust the product from step (1) to a temperature of 50℃±1℃ and a pH of 6.8±0.1, add glutaminase, neutral protease and trypsin, react for 2.5h±0.2h and control the degree of hydrolysis to 20%-25%;

[0008] (3) Adjust the pH of the product from step (2) to 7.0±0.1, then add flavor protease, lipase and β-cyclodextrin, react for 2.0h±0.2h and control the degree of hydrolysis to 28%-30%;

[0009] (4) Inactivate the enzyme in the product of step (3) to obtain an enzyme-inactivated solution;

[0010] (5) Pass the enzyme inactivation solution through a 0.22 μm ceramic membrane, and then pass the filtrate through a 50 kDa ultrafiltration membrane and a 300 Da nanofiltration membrane in sequence. Collect the 300 Da-50 kDa nanofiltration retentate to obtain the solution.

[0011] This invention develops an enzymatic hydrolysis scheme targeting characteristic cleavage sites (Glu-Ala, Leu-Leu, Lys-Val, etc.) of goat milk proteins, achieving precise cleavage and significantly improving the targeted yield of peptides in the 500-1000 Da range. Specifically, this invention first uses neutral and alkaline proteases for enzymatic cleavage, and then simultaneously uses glutaminase, flavor protease, and lipase. Glutaminase mainly acts as a synergist in the fine regulation of the flavor and solubility of small molecule peptides in goat milk. On the one hand, by appropriately deamidating the peptides, it improves the hydrophilicity and solubility of small molecule peptides, facilitating further removal of hydrophobic bitter peptides by flavor proteases and reducing bitterness. On the other hand, based on the deamidation of goat odor by lipase targeting short-chain triglycerides, glutaminase helps reduce the aggregation and enrichment of small goat odor molecules in the system, thereby improving the overall flavor quality of goat milk small peptides. Moreover, the deamidation effect of glutaminase exposes hidden cleavage sites in goat milk, reducing protein aggregation and thus improving the catalytic efficiency of neutral and trypsin proteases. Molecular distillation can remove C6-C10 short-chain fatty acids under high vacuum and low temperature conditions, avoiding heat damage to proteins and functional peptides, and is highly selective with high flavor retention.

[0012] In some embodiments, the activity of the neutral protease is 5000-50000 U / g; the activity of the trypsin is 2500-3000 USP U / g.

[0013] In some embodiments, the mass ratio of neutral protease to trypsin is 4.5-5.5:0.9-1.1. Preferably, the mass ratio of neutral protease to trypsin is 5:1.

[0014] In some embodiments, the enzyme activity of the flavor protease is 500-600 LAPU / g; and the enzyme activity of the lipase is 8000-10000 U / g.

[0015] In some implementations, the mass ratio of flavor protease to lipase is 3:1.

[0016] In some embodiments, the enzyme activity of glutaminase is 20,000 U / g.

[0017] In some embodiments, the amount of β-cyclodextrin added is 0.09%-0.11% of the weight of goat milk, preferably, the amount of β-cyclodextrin added is 0.1% of the weight of goat milk.

[0018] In some embodiments, in step (1), the filtration method is to pass the product after molecular distillation through a 0.22 μm ceramic membrane and retain the filtrate.

[0019] In some embodiments, in step (1), the centrifugation method is to centrifuge at 3-5°C and 5000-7000 rpm for 15-20 min.

[0020] In some embodiments, in step (1), the defatting method is to centrifuge the goat milk obtained after centrifugation at 3-5°C and 8000-10000×g for 15-25 min, and then remove the fat phase.

[0021] In some embodiments, in step (1), the molecular distillation method is to distill goat milk at 60-65°C and a vacuum of 80-90 Pa for 15-25 min and collect the fraction.

[0022] In some embodiments, the total mass of glutaminase, neutral protease, trypsin, flavor protease, lipase, and β-cyclodextrin is 1.3%-1.5% of the mass of goat milk. Preferably, the total mass of glutaminase, neutral protease, trypsin, flavor protease, lipase, and β-cyclodextrin is 1.4% ± 0.14% of the mass of goat milk.

[0023] According to a second aspect of the present invention, a goat milk small molecule peptide prepared by the above preparation method is provided, wherein the yield of the 500-1000 Da peptide is 85.3% ± 2.5%.

[0024] In some embodiments, the IC50 of goat milk small molecule peptides 50 The value was 0.48 ± 0.05 mg / mL.

[0025] According to a third aspect of the present invention, natural mutton fat is provided, which is obtained by washing and dehydrating the fat phase obtained after defatting.

[0026] By processing the fat phase obtained after defatting goat milk, the natural sheep fat was recovered.

[0027] The beneficial effects of this invention are as follows:

[0028] (1) The present invention is based on the amino acid sequence characteristics of goat milk protein and designed a composite enzyme system with an enzyme cleavage site fit of more than 95%. By specifically recognizing the characteristic enzyme cleavage sites of goat milk protein, the yield of peptides of 500-1000 Da is increased to 85.3%±2.5%, realizing the molecular-level directional and precise cleavage.

[0029] (2) This invention establishes a quadruple synergistic deodorization mechanism by low-temperature defatting, molecular distillation, targeted hydrolysis of lipase and glutaminase to inhibit the aggregation of odor molecules, and β-cyclodextrin encapsulation, so that the removal rate of odor components in goat milk small molecule peptides is ≥99%, and the specific exocleation effect of flavor protease reduces the bitterness value by 92%±3%, and the product flavor is significantly improved.

[0030] (3) The three-stage membrane separation system of the present invention achieves precise enrichment of target peptides, and the coefficient of variation (CV) of product molecular weight distribution is reduced from 30%-35% in the traditional method to 10%-12%, and the product uniformity is significantly improved.

[0031] (4) The mild process conditions of the present invention, such as low-temperature enzymatic hydrolysis, nitrogen protection, and immediate inactivation, improve the product bioactivity retention rate by 40%±5% and reduce energy consumption by 30%±5%, which has significant industrial application value and economic benefits.

[0032] (5) Achieve simultaneous high-value utilization of goat milk protein and fat components.

[0033] This invention simultaneously recovers and purifies natural sheep fat while preparing highly uniform goat milk small molecule peptides, thereby improving the comprehensive utilization rate of raw materials, reducing production costs, and having significant industrial application value.

[0034] (6) The present invention provides technical support for industrialization promotion by controlling the quality of the entire process from raw materials to finished products. Attached Figure Description

[0035] Figure 1 This is a technical roadmap for the goat milk small molecule peptides of the present invention;

[0036] Figure 2 This is the SEC-HPLC chromatogram of goat milk small molecule peptides in Example 1 of the present invention. 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 goat milk small molecule peptides of the present invention is as follows: Figure 1 As shown, the amino acid sequence of goat milk was first obtained through proteomics analysis. Then, a specialized complex enzyme system was designed based on its unique restriction enzyme sites. A quadruple synergistic deodorization pretreatment was used, followed by stepwise enzymatic hydrolysis and purification via a three-stage membrane separation process. Molecular weight distribution verification and bioactivity evaluation yielded small-molecule goat milk peptides. The 500-1000 Da peptides showed the highest absorption efficiency.

[0039] To address the technical challenges of strong goaty odor and low solubility in goat milk, glutaminase was added. Through deamidation, the solubility of small peptides is altered, reducing the aggregation of odor molecules and exposing cleavage sites. This allows for synergistic enzymatic hydrolysis by lipase and flavor protease to remove the odor. Simultaneously, the deamidation of glutaminase exposes hidden cleavage sites, reducing protein aggregation and improving the catalytic efficiency of neutral proteases and trypsin. Sequence alignment analysis between predicted cleavage sites and measured peptides revealed a match rate exceeding 95%, and MALDI-TOF MS analysis showed that the cleavage site fit was also high.

[0040] I. Analysis of Goat Milk Protein Substrate Characteristics and Identification of Characteristic Enzyme Cleavage Sites

[0041] A systematic analysis of the amino acid sequences of the major protein components of goat milk (β-casein, αs1-casein, αs2-casein, β-lactoglobulin, and α-lactalbumin) identified the following key characteristic enzyme cleavage sites:

[0042] β-casein: Glu 35 -Ala 36 Leu 190 -Leu 191 Lys 107 -Val 108 ;

[0043] αs1-casein: Glu 46 -Ala 47 Leu 143 -Leu 144 ;

[0044] αs2-casein: Lys 150 -Val 151 Leu 165 -Leu 166 ;

[0045] β-lactoglobulin: Lys 75 -Val 76 Leu 95 -Leu 96 ;

[0046] α-lactalbumin: Glu 49 -Ala 50 Leu 115 -Leu 116 ;

[0047] Based on the above-mentioned characteristic enzyme cleavage site analysis, a combination of complex enzyme systems capable of specifically recognizing these sites was designed.

[0048] II. Product Quality Evaluation of Goat Milk Small Molecule Peptides

[0049] Establish a complete quality evaluation system to ensure product quality.

[0050] 1. Verification of molecular directional shearing effect

[0051] (1) MALDI-TOF MS analysis: Analyze the molecular weight distribution of peptides and calculate the coefficient of variation (CV).

[0052] Sample preparation: Take goat 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.

[0053] 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.

[0054] 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.

[0055] Data processing: Mass spectrometry peak signals in the 500-1000 Da range are extracted, the average value and standard deviation of the peak intensity distribution in this range are calculated, and the coefficient of variation (CV) of molecular weight distribution is calculated accordingly to evaluate the uniformity of small molecule peptide distribution.

[0056] (2) RP-HPLC analysis of bitter peptides: detect the content of hydrophobic bitter peptides and evaluate flavor uniformity.

[0057] 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.

[0058] 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.

[0059] Analytical method: Record the chromatographic peaks with retention times of 20-40 min in the chromatogram as hydrophobic peptide peaks, and calculate the ratio of the sum of their peak areas to the total peak area as a relative indicator of bitter peptide content.

[0060] (3) SEC-HPLC

[0061] Used to determine the molecular weight distribution of peptides.

[0062] Size exclusion chromatography (SEC-HPLC) was performed using a gel size exclusion column with phosphate buffer as the mobile phase at a flow rate of 0.5 mL / min, and detection was performed at 220 nm. Molecular weight calibration was performed using standard peptides, and the relative proportions of peptides in each molecular weight range (e.g., <500 Da, 500-3000 Da, >3000 Da) were calculated.

[0063] 2. Evaluation of the effect of removing odor

[0064] (1) GC-MS analysis of fatty acids related to muttony smell

[0065] Sample pretreatment: Take an appropriate amount of sample and place it in a 20 mL headspace vial, add an appropriate amount of saturated sodium chloride solution, and seal.

[0066] Headspace-SPME extraction: DVB / CAR / PDMS composite fiber was used. After equilibration at 50℃ for 10 min, the fiber was inserted for headspace adsorption for 30 min.

[0067] GC-MS analysis conditions: injection port temperature 250℃; column: DB-FFAP capillary column; ion source: EI, 70eV; scan mode: full scan (m / z 35-350).

[0068] Quantitative analysis: A standard curve of C6-C10 fatty acids was established based on the standards, and the content of relevant fatty acids in the samples was quantitatively analyzed to evaluate the deodorization effect.

[0069] (2) Electronic nose sensor array for detecting oxidative odor

[0070] (3) Bitterness value

[0071] Sensory evaluation was conducted using a blind 5-point scale. An evaluation panel of 10-12 trained judges scored the samples for metallic, bloody, bitter, and overall acceptability under randomized coding conditions. A score of 5 indicated no gamey, bitter, or metallic taste, a pure flavor, and excellent overall acceptability; 4 indicated virtually no unpleasant flavor, only a very slight off-flavor that did not affect acceptability; 3 indicated a slightly perceptible gamey or bitter taste, but still acceptable; 2 indicated a noticeable gamey or bitter taste with low acceptability; and 1 indicated a strong unpleasant flavor that was unacceptable.

[0072] 3. Bioactivity identification

[0073] (1) DPPH free radical scavenging rate

[0074] Experimental procedure: Prepare a 0.1 mmol / L DPPH-ethanol solution and store it in the dark. Mix sample solutions of different concentrations (0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL) with DPPH solution at a ratio of 1:1 (v / v) and react in the dark for 30 min.

[0075] Detection conditions: The absorbance was measured at a wavelength of 517 nm using a UV-Vis spectrophotometer.

[0076] Calculation method: Using the DPPH solution without sample as a blank control (A0), and the absorbance value of the sample group as A1, the clearance rate is calculated according to the following formula: Clearance rate (%) = (A0) / (A1) A1) / A0×100.

[0077] (2) Assay for ACE inhibitory activity (HHL method): Determination of IC50 50 Values ​​used to assess blood pressure lowering function

[0078] 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.

[0079] 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.

[0080] 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.

[0081] (3) Cell experiments (RAW264.7 inflammation model: detection of anti-inflammatory activity (such as inhibition of IL-6 expression))

[0082] Cell culture: RAW264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0083] Experimental treatment: Cells were divided into a blank control group, an LPS model group, and LPS+ sample treatment groups with different concentrations. The final LPS concentration was 1 μg / mL. After pretreatment for 2 h, LPS was added, and the cells were cultured for another 24 h.

[0084] Cell supernatant was collected for IL-6 assay, and IL-6 content was detected using an ELISA kit. The results were compared with the LPS model group to calculate the decrease in IL-6 expression.

[0085] III. Construction of a specific, highly adaptable, dedicated complex enzyme system

[0086] 1. Ratio of dedicated compound enzyme system

[0087] An orthogonal experimental design was used, with the yield of the target peptide (500-1000 Da) and bitterness value (5-point sensory evaluation) as the core evaluation indicators.

[0088] 1.1 Experimental Design

[0089] Factors and Levels: Three key factors were selected, with three levels for each factor.

[0090] Factor A: The ratio (w / w) of site-specific endonucleases to flavor-regulating enzymes, with ratios of 6:2, 7:2, and 8:2.

[0091] Factor B: The ratio of neutral protease to trypsin (w / w) is 4:1, 5:1, or 6:1.

[0092] Factor C: The ratio of flavor protease to lipase (w / w), with ratios of 1:1, 2:1, and 3:1.

[0093] Experimental design: L9(3) 4 An orthogonal array was used, and a total of 9 sets of experiments were conducted.

[0094] 1.2 Key Experimental Results and Analysis

[0095] Table 1 shows the results of the nine experiments. As can be seen from Table 1, the goat milk small molecule peptides prepared under the conditions of Group 5 had the lowest bitterness value and the highest yield of the target peptide.

[0096] Table 1 Experimental Results

[0097]

[0098] 1.3 Conclusion of Range Analysis

[0099] The order of influence of each factor on the yield is: A>B>C, indicating that the total amount of endonuclease is the most critical factor determining the yield of the target peptide.

[0100] The optimal combination is A2B2C1, where the mass ratio of endonuclease to flavor-regulating enzyme is 7:2, the mass ratio of neutral protease to trypsin is 5:1, and the mass ratio of flavor protease to lipase is 3:1. This combination yields the highest target peptide yield and the lowest bitterness value.

[0101] Validation experiment: The optimal combination A2B2C1 was tested three times. The yield of the target peptide was 85.3%±1.2%, and the bitterness value was 1.0±0.2. The results were stable and reliable.

[0102] 2. Selection of dedicated complex enzyme systems

[0103] The three functional modules of the dedicated complex enzyme system constructed in this invention are as follows:

[0104] 2.1 Site-specific endonuclease group

[0105] (1) Neutral protease (enzyme activity specification: 45000-50000 U / g): specifically recognizes Glu-Ala and Leu-Leu sites to achieve the initial cleavage of the protein backbone;

[0106] (2) Trypsin (enzyme activity specification: ≥2500 USP U / g): specifically cleaves the Lys-Val site to achieve precise enzymatic hydrolysis;

[0107] (3) Addition ratio: neutral protease: trypsin = 5:1 (w / w).

[0108] 2.2 Flavor-regulating enzyme group

[0109] (1) Flavor protease (enzyme activity specification: 500-600 LAPU / g): hydrolyzes simultaneously from the N-terminus and C-terminus of the peptide chain, effectively eliminating hydrophobic bitter peptides;

[0110] (2) Lipase (enzyme activity specification: 8000-10000 U / g): targets and hydrolyzes residual C6-C 10 Short-chain triglycerides, precursors for eliminating muttony odor;

[0111] (3) Addition ratio: Flavor protease: lipase = 3:1 (w / w).

[0112] 2.3 Synergistic Agent Group

[0113] (1) Glutaminase (enzyme activity specification: 20000 U / g): moderately deamidates peptides, improves the hydrophilicity and solubility of small molecule peptides; by increasing the negative charge of peptides, electrostatic repulsion is enhanced, thereby inhibiting the aggregation of hydrophobic peptides, and the small odor molecules are difficult to form hydrophobic micro-regions, ultimately reducing the aggregation and enrichment of small odor molecules in the system.

[0114] (2) β-cyclodextrin: 0.1% (w / w) is added to mask residual muttony odor molecules through encapsulation.

[0115] 2.4 Total enzyme system ratio

[0116] Site-specific endonuclease group: flavor-regulating enzyme group: synergist group = 7:2:1 (w / w).

[0117] The site-specific endonuclease group first achieves precise cleavage of the goat milk protein backbone; glutaminase is added simultaneously with the site-specific endonuclease group to improve substrate accessibility; subsequently, it is combined with the flavor-regulating enzyme group to further eliminate bitter peptides and muttony precursors. The three work together to achieve molecular-directed cleavage and precise flavor regulation.

[0118] 3. Preparation of a dedicated complex enzyme system

[0119] Prepare a specialized compound enzyme system according to the following proportions:

[0120] Neutral protease (50000 U / g): 50g;

[0121] Trypsin (2500 USP U / g): 10g;

[0122] Flavor protease (550 LAPU / g): 40g;

[0123] Lipase (source: Candida rugosa, manufacturer: Novozymes) (9000 U / g): 20g;

[0124] Glutaminase (20000 U / g): 10g;

[0125] β-Cyclodextrin: 10g;

[0126] The total weight is 140g. When using, the total mass of the special compound enzyme system should be 1.4% of the mass of the substrate (goat milk).

[0127] IV. Preparation of Natural Sheep Fat

[0128] (1) Low-temperature centrifugation degreasing separation

[0129] During the pretreatment of goat milk, it is centrifuged at 4℃ and 9000×g for 20 min to allow the fat to float to the surface and form a distinct fat layer.

[0130] (2) Collection of fat layer

[0131] Using a pipette or scraper, carefully collect the uppermost fat phase to obtain crude mutton fat.

[0132] (3) Wash with warm water to remove impurities

[0133] Transfer the crude lanolin to a clean container, add an equal volume of preheated deionized water (50-55°C), and stir slowly for 5 minutes to transfer any remaining water-soluble peptides, lactose, and inorganic salts to the aqueous phase. After allowing the layers to separate or centrifuging at low speed, discard the lower aqueous phase. This washing step can be repeated 1-2 times.

[0134] (4) Vacuum drying and dehydration

[0135] The washed mutton fat was dried at 45°C and a vacuum of -0.08 MPa for 2 hours to remove residual moisture, resulting in a light yellow natural mutton fat with a mild odor.

[0136] (5) Product characteristics

[0137] The resulting natural lanolin retains its original fatty acid composition and has not undergone high-temperature cracking or chemical modification, making it suitable for use as a food, functional oil, or cosmetic ingredient.

[0138] V. Time-Controlled Stepwise Enzymatic Hydrolysis Process Based on Molecular Directed Shearing

[0139] 1. Distributed enzymatic hydrolysis process

[0140] A time-controlled stepwise enzymatic hydrolysis strategy is adopted, and the specific process parameters are as follows.

[0141] Phase 1: Site-Specific Splicing

[0142] (1) Reaction conditions: temperature 50℃±1℃, pH 6.8±0.1, reaction time 2.5h±0.2h;

[0143] (2) Enzyme addition: site-specific endonucleases (neutral protease and trypsin) and glutaminase were added;

[0144] (3) Environmental control: nitrogen protection, dissolved oxygen concentration (DO) ≤ 0.5 ppm;

[0145] (4) Process monitoring: real-time monitoring of degree of hydrolysis, with the target DH value controlled at 20%-25%.

[0146] Phase Two: Precise Flavor Control

[0147] (1) Reaction conditions: temperature 50℃±1℃, pH 7.0±0.1, reaction time 2.0h±0.2h;

[0148] (2) Enzyme addition: Add flavor-regulating enzyme group (flavor protease and lipase);

[0149] (3) Flavor enhancement: β-cyclodextrin is added simultaneously;

[0150] (4) Process monitoring: The target DH value is controlled at 28%-30%.

[0151] Phase 3: Immediate Termination of the Reaction

[0152] (1) Inactivation conditions: instantaneous inactivation at 85℃±1℃ / 15s±2s;

[0153] (2) Cooling treatment: rapidly cool to 40℃±2℃.

[0154] The percentages of each ingredient added to goat milk by weight are as follows: neutral protease 0.5% ± 0.05%; trypsin 0.1% ± 0.01%; flavor protease 0.4% ± 0.04%; lipase 0.2% ± 0.02%; β-cyclodextrin 0.1% ± 0.01%; and glutaminase 0.1% ± 0.01%.

[0155] VI. Three-stage membrane separation and purification system with precise molecular weight control

[0156] Establish a three-stage membrane separation system to achieve precise separation and enrichment of target peptides:

[0157] 1. Pretreatment filtration system

[0158] (1) Membrane type: 0.22 μm ceramic membrane;

[0159] (2) Function: Remove denatured protein aggregates and microorganisms.

[0160] 2. Molecular weight primary screening system

[0161] (1) Membrane type: 50kDa ultrafiltration membrane;

[0162] (2) Operating parameters: operating pressure 0.6-0.8 MPa, temperature 40℃±2℃;

[0163] (3) Function: Removes unhydrolyzed macromolecular proteins and enzyme preparations.

[0164] 3. Target peptide enrichment system

[0165] (1) Membrane type: 200-400 Da nanofiltration membrane;

[0166] (2) Operating parameters: operating pressure 1.2-1.5 MPa, temperature 35℃±2℃;

[0167] (3) Function: Precisely retains target peptides of 500-1000 Da, while removing small molecule bitter amino acids and inorganic salts.

[0168] Example 1

[0169] This embodiment provides a method for preparing small molecule peptides from goat milk, including the following steps:

[0170] (1) Raw material pretreatment

[0171] 100 kg of fresh goat milk was collected, with a protein content of 3.36% and a fat content of 3.23%. The milk was centrifuged at 4℃ and 6000 rpm to remove impurities and adjust the total solids content to 11%. Then, it was centrifuged at 4℃ and 9000×g for 20 min to defatted the milk, causing the fat to rise to the surface and forming a fat layer. The fat content was reduced to 0.28%. The upper fat phase was separated and collected for the preparation of natural goat fat; the lower defatted milk layer was used for the preparation of goat milk small molecule peptides. The milk was then pasteurized at 85℃ for 15 s, followed by molecular distillation at 62℃ and 85 Pa vacuum for 20 min to remove any goaty odor. Finally, it was cooled to 50℃ and filtered through a 0.22 μm ceramic membrane to obtain pretreated goat milk. (2) Pump the pretreated goat milk into the enzymatic hydrolysis tank, turn on the nitrogen protection (DO≤0.5 ppm), adjust the temperature to 50℃ and pH to 6.8, add glutaminase, neutral protease (50000 U / g) and trypsin (2500 USPU / g) to the enzymatic hydrolysis tank, and react for 2.5 h. At this time, the degree of hydrolysis DH reaches 23.5%.

[0172] (3) Adjust the pH to 7.0, add flavor protease, lipase and β-cyclodextrin to the enzymatic hydrolysis tank, and continue the reaction for 2 hours. At this time, the degree of hydrolysis DH reaches 29.8%. Then, perform instantaneous enzyme inactivation at 85℃ for 15s and quickly cool to 40℃ to obtain the enzyme-inactivated solution.

[0173] (4) The enzyme-inactivating solution was first pretreated with a 0.22 μm ceramic membrane, and then entered a 50 kDa ultrafiltration system (operating pressure 0.7 MPa, temperature 40 °C), and the permeate was collected. The ultrafiltration permeate then entered a 300 Da nanofiltration system (operating pressure 1.3 MPa, temperature 35 °C), and the retentate was collected. The nanofiltration retentate was vacuum concentrated at 50 °C and -0.08 MPa until the solid content was 32%.

[0174] It should be noted that in Example 1, the enzyme and β-cyclodextrin were weighed according to the proportion in "Preparation of Special Complex Enzyme System". When using it, the special complex enzyme system was added at 1.4% of the mass of goat milk, that is, the special complex enzyme system was 140g, of which the mass of neutral protease was 500g.

[0175] In this invention, HCl and NaOH are used for pH adjustment.

[0176] Comparative Example 1

[0177] This comparative example provides a method for preparing small molecule peptides from goat milk, comprising the following steps:

[0178] (1) 100 kg of goat milk was centrifuged at 4℃ and 6000 rpm to remove the milk and adjust the total solids content to 11%. Then, it was centrifuged at 4℃ and 9000×g for 20 min to remove fat, reducing the fat content to 0.28%. Then, it was pasteurized at 85℃ for 15 s, followed by molecular distillation at 62℃ and 85 Pa vacuum for 20 min to remove the goaty smell. Finally, it was cooled to 50℃ and filtered through a 0.22 μm ceramic membrane to obtain pretreated goat milk.

[0179] (2) Add 500g of neutral protease (enzyme activity of 5000U / g) to the pretreated goat milk at 50℃ and pH 7.0, react for 3 h, perform instantaneous enzyme inactivation at 85℃ for 15s, cool rapidly to 40℃, filter through a 10 kDa ultrafiltration membrane, and collect the filtrate to obtain the product.

[0180] Comparative Example 2

[0181] This comparative example provides a method for preparing small molecule peptides from goat milk, comprising the following steps:

[0182] (1) 100 kg of goat milk was centrifuged at 4℃ and 6000 rpm to remove the milk and adjust the total solids content to 11%. Then, it was centrifuged at 4℃ and 9000×g for 20 min to remove fat, reducing the fat content to 0.28%. Then, it was pasteurized at 85℃ for 15 s, followed by molecular distillation at 62℃ and 85 Pa vacuum for 20 min to remove the goaty smell. Finally, it was cooled to 50℃ and filtered through a 0.22 μm ceramic membrane to obtain pretreated goat milk.

[0183] (2) Add 400g of neutral protease (5000 U / g) and 200g of flavor protease to pretreated goat milk at 50℃ and pH 7.2, react for 3 h, perform instantaneous enzyme inactivation at 85℃ for 15s, cool rapidly to 40℃, filter through a 5 kDa ultrafiltration membrane, collect the filtrate, and obtain the product.

[0184] The goat milk small molecule peptides prepared in Example 1 were evaluated for product quality. The yield of goat milk small molecule peptides was 82.5%, with no goaty or bitter taste and a pure flavor. MALDI-TOF MS analysis showed that the proportion of 500-1000 Da (corresponding to characteristic enzyme cleavage sites) in the goat milk small molecule peptides was 85.3% ± 2.5%. RP-HPLC verification showed that the content of bitter peptides (hydrophobic peptides) was reduced by 92% ± 3% compared with unhydrolyzed goat milk.

[0185] Example 1 of this invention: Goat milk and goat milk small molecule peptides were analyzed by GC-MS. The short-chain fatty acids (C6-C50) in the goat milk small molecule peptides were... 10The removal rate relative to goat milk is ≥99%. Electronic nose detection results show that the response value of the oxidative odor sensor drops below the detection limit, indicating that the goat milk small molecule peptides have no oxidative odor.

[0186] In the bioactivity assessment, the DPPH radical scavenging rate was 86.7% ± 3.2% (1 mg / mL). The DPPH radical scavenging experiment results showed that the goat milk small molecule peptide exhibited a significant dose-dependent scavenging effect within the concentration range of 0.2-1.0 mg / mL. When the sample concentrations were 0.2 mg / mL, 0.5 mg / mL, and 1.0 mg / mL, the DPPH radical scavenging rates were 42.3% ± 3.1%, 68.5% ± 2.8%, and 86.7% ± 3.2%, respectively.

[0187] ACE inhibitory activity IC 50 The concentration was 0.48 ± 0.05 mg / mL. Using LPS-induced RAW264.7 macrophages as an inflammation model, the effect of the sample on the expression of the inflammatory factor IL-6 was detected. The results showed that at a concentration of 100 μg / mL of goat milk small molecule peptide in Example 1, the IL-6 secretion level in the goat milk small molecule peptide treatment group was reduced by 48.6% ± 4.3% compared to the LPS model group; at a concentration of 200 μg / mL, the decrease in IL-6 expression further increased to 61.2% ± 5.1%. In contrast, the IL-6 inhibition rate of the goat milk small molecule peptides in Comparative Examples 1 and 2 at the same concentration was only 20%-30%. Cellular experiments indicated that the goat milk small molecule peptide in Example 1 exhibited significant antioxidant and anti-inflammatory activity.

[0188] The molecular weight distribution results are shown in Table 2, and the SEC-HPLC chromatographic results are shown in Table 3 and... Figure 2 As shown.

[0189] Table 2. Peptide molecular weight distribution of small molecule peptides from goat milk

[0190]

[0191] Table 3. SEC-HPLC chromatogram of small molecule peptides from goat milk

[0192]

[0193] Example 2

[0194] This embodiment provides a method for preparing goat milk small molecule peptides. The difference from Example 1 is that after the protein content is detected, each raw material is weighed and added according to the "Preparation of Special Complex Enzyme System".

[0195] The process parameters used in this embodiment are:

[0196] (1) Processing capacity: 1000L / batch;

[0197] (2) Enzymatic hydrolysis tank volume: 5 m 3 It is equipped with an online pH, temperature, and DO monitoring system;

[0198] (3) Membrane system specifications: The ultrafiltration membrane area is 200 m². 2 The nanofiltration membrane area is 150 m². 2 ;

[0199] (4) Drying system: centrifugal spray drying tower with an evaporation rate of 200 kg / h.

[0200] The yield of the prepared goat milk small molecule peptides was 81.5%-83.5%, the proportion of 500-1000 Da peptides was 84.5%-86.5%, and the energy consumption cost was 0.75-0.85 kW·h / kg.

[0201] Example 3

[0202] This embodiment provides a method for preparing goat milk small molecule peptides. The difference from Example 1 is that in step (2), the neutral protease is replaced with an alkaline protease and the pH is adjusted to 7.5.

[0203] The yield of small molecule peptides from goat milk was 79.8%, and the proportion of the target peptide segment (500-1000 Da) was 82.3%.

[0204] Example 4

[0205] This embodiment provides a method for preparing goat milk small molecule peptides. The difference from Example 1 is that in step (4), the 300 Da nanofiltration membrane is replaced with a 300 Da ceramic membrane, the operating pressure is adjusted to 1.0-1.2 MPa, the product yield is 83.1%, the target peptide ratio is 85.0%, and the membrane lifespan is extended by about 30%.

[0206] Compared to 300 Da ceramic membranes, 300 Da nanofiltration membranes offer higher separation accuracy and lower cost, making them suitable for large-scale production.

[0207] Example 5

[0208] This embodiment provides a method for preparing goat milk small molecule peptides. The difference from Example 1 is that in step (3), the lipase is replaced with Thermomyces lanuginosus lipase (enzyme activity 8000 U / g).

[0209] Comparative Example 3

[0210] This comparative example provides a method for preparing goat milk small molecule peptides, which differs from Example 1 in that glutaminase is not added.

[0211] The goat milk small molecule peptides prepared in Example 1 and Comparative Example 3 were tested, including the degree of hydrolysis (DH) and the proportion of target peptides (500-1000 Da), and the content of bitter peptides was analyzed by RP-HPLC.

[0212] The results are shown in Table 4. As can be seen from Table 4, compared to Comparative Example 3, the preparation method of Example 1, after adding glutaminase, significantly increased the degree of hydrolysis of the goat milk small molecule peptides. This indicates that the protein substrate is more easily cleaved by endonucleases, and the addition of glutaminase can improve enzymatic hydrolysis efficiency. This may be because the deamidation of glutaminase exposes hidden cleavage sites, reducing protein aggregation and thus improving the catalytic efficiency of neutral proteases and trypsin. Simultaneously, the target peptide segment (500-1000 Da) of the small molecule peptides in Example 1 has a higher proportion. Glutaminase, by enhancing substrate accessibility, promotes the cleavage of characteristic sites (such as Glu-Ala, Leu-Leu) by other enzymes, thereby increasing the yield of the target small peptide. Furthermore, the goat milk small molecule peptides in Example 1 contain less bitter peptides.

[0213] Table 4 Comparison of the properties of different goat milk small molecule peptides

[0214]

[0215] Comparative Example 4

[0216] This comparative example provides a method for preparing goat milk small molecule peptides. The difference from Example 1 is that the degree of hydrolysis (DH) in step (3) is controlled to be 25%.

[0217] Testing revealed that the yield of the obtained goat milk small molecule peptides was 75%, which was lower than that of Example 1. Furthermore, the bitter peptides in the product were insufficiently hydrolyzed, with RP-HPLC analysis showing a relative content of 25% ± 3%. In addition, the ACE inhibitory activity IC50 of Comparative Example 4 was... 50 An increase in the value to 0.8 mg / mL indicates incomplete release of its functional peptides and loss of biological activity.

[0218] Comparative Example 5

[0219] This comparative example provides a method for preparing goat milk small molecule peptides. The difference from Example 1 is that the degree of hydrolysis (DH) in step (3) is controlled to be 35%.

[0220] Testing revealed that, compared to Example 1, Comparative Example 5 produced excessive free amino acids and small molecule peptides from goat milk, resulting in a rebound in bitterness (exposure of hydrophobic amino acids). Furthermore, bioactive peptides (such as antioxidant peptides) were further hydrolyzed, and the DPPH free radical scavenging rate was 68.0% ± 3.5%, which was lower than 70% for goat milk small molecule peptides in Example 1. The coefficient of variation (CV) of molecular weight distribution increased to over 20%.

[0221] The goat milk small molecule peptides prepared in Comparative Examples 1 and 2 were tested, and the results were compared with those of the goat milk small molecule peptides in Example 1. 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 cost by the product weight (kg). The energy cost of Comparative Example 1 was 0.8 kW·h / kg, and this result was used as the "baseline" (0%).

[0222] The goat milk small molecule peptides prepared in Comparative Examples 1, 2, and 1 were tested, and the bioactivity retention rate was calculated. Bioactivity retention rate refers to the comprehensive bioactivity level retained by the sample after enzymatic hydrolysis and post-treatment relative to the original protein substrate. It was calculated using a multi-index normalized evaluation method, comprehensively considering: antioxidant activity (DPPH free radical scavenging ability), ACE inhibitory activity (IC50...). 50 value).

[0223] (1) Antioxidant activity retention rate

[0224] The antioxidant activity retention rate (%) was determined at the same mass concentration (e.g., 1 mg / mL) as follows: (DPPH scavenging rate of reference sample / DPPH scavenging rate of sample) × 100. The reference sample should be the DPPH scavenging rate of Example 1 at 1 mg / mL, i.e., 86.7%.

[0225] (2) ACE inhibitory activity retention rate: due to IC 50 The smaller the value, the higher the activity; the reverse normalization method is used for calculation. ACE activity retention rate (%) = (sample IC50) / (sample ACE retention rate ... 50 / Reference IC 50 )×100. Wherein, reference IC 50 IC50 for the optimal active sample (or theoretically optimal active peptide system) 50 IC 50 It should be the IC of Example 1 50 The value is 0.48 mg / mL.

[0226] (3) Calculation of bioactivity retention rate

[0227] Bioactivity retention rate (%) = (antioxidant activity retention rate + ACE activity retention rate) / 2. This method avoids the bias of a single indicator and conforms to the evaluation logic commonly used in the field of functional peptides in food.

[0228] The comparison showed that when the sample concentration was 1.0 mg / mL, the DPPH free radical scavenging rate of the goat milk small molecule peptide in Comparative Example 1 was 52%-58%; the DPPH free radical scavenging rate of the goat milk small molecule peptide in Comparative Example 2 was 65%-72%; while the DPPH free radical scavenging rate of the goat milk small molecule peptide in Example 1 was 86.7%±3.2%, which was significantly higher than that of the comparative sample.

[0229] (4) Odor removal rate

[0230] Evaluation method: Sensory evaluation was used to assess the intensity of the muttony odor in the samples. An evaluation team of 10 evaluators with basic sensory training conducted a blind evaluation of the samples under uniform environmental conditions, scoring them on a scale of 1 to 9 based on the intensity of the muttony odor, where 1 point indicates no obvious muttony odor and 9 points indicates an extremely strong muttony odor.

[0231] The odor removal rate is calculated using the following formula:

[0232] Odor removal rate (%) = (S0 - S1) / S0 × 100%.

[0233] Wherein, S0 is the odor score of the untreated raw goat milk sample, and S1 is the odor score of the sample from Comparative Example 1, Comparative Example 2, or Example 1.

[0234] This invention obtains functional peptides with smaller molecular weight and stable structure through a directional shearing process. On the one hand, it reduces the content of muttony and bitter substances, and on the other hand, it promotes the enrichment of bioactive ACE inhibitory peptides.

[0235] The ACE inhibitory activity IC50 of goat milk small molecule peptides in Comparative Example 1 50 The ACE inhibitory activity IC50 of the goat milk small molecule peptide in Comparative Example 2 was 0.95 ± 0.08 mg / mL. 50 The ACE inhibitory activity IC50 of the goat milk small molecule peptide in Example 1 was 0.72 ± 0.06 mg / mL. 50 The concentration was 0.48 ± 0.05 mg / mL. This indicates that the goat milk small molecule peptides prepared in this invention have strong ACE inhibitory activity, suggesting that small molecule peptides obtained through directed cleavage are more conducive to the release of functional peptides.

[0236] The results of various performance tests are shown in Table 5. As can be seen from Table 5, the yield of 500-1000 Da goat milk small molecule peptides prepared in Example 1 is significantly higher than that in Comparative Example 1 and Comparative Example 2, indicating that more 500-1000 Da peptides can be prepared by the preparation method of the present invention. This is not only because membrane separation and enrichment are used, but also because the special enzyme system improves the enzymatic hydrolysis efficiency.

[0237] Table 5. Properties of small molecule peptides from goat milk

[0238]

[0239] A comparison of the molecular weight distribution of goat milk small molecule peptides in Comparative Example 1 and Example 1 shows that the molecular weight distribution of Example 1 is highly concentrated in the 500-1000 Da range, while the distribution of Comparative Example 1 is more dispersed and the CV value is significantly higher, indicating that the present invention achieves stable molecular directional cleavage.

[0240] 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 goat milk, characterized in that, Includes the following steps: (1) After centrifuging, defatting, and sterilizing the goat milk, perform molecular distillation, and then filter and retain the filtrate; (2) Adjust the product from step (1) to a temperature of 50℃±1℃ and a pH of 6.8±0.1, add glutaminase, neutral protease and trypsin, react for 2.5h±0.2h and control the degree of hydrolysis to 20%-25%; (3) Adjust the pH of the product from step (2) to 7.0±0.1, then add flavor protease, lipase and β-cyclodextrin, react for 2.0h±0.2h and control the degree of hydrolysis to 28%-30%; (4) Inactivate the enzyme in the product of step (3) to obtain an enzyme-inactivated solution; (5) Pass the enzyme-inactivating solution through a 0.22 μm ceramic membrane first, and then pass the filtrate through a 50 kDa ultrafiltration membrane and a 300 Da nanofiltration membrane in sequence. Collect the 300 Da-50 kDa nanofiltration retentate to obtain the solution. The mass ratio of neutral protease to trypsin is 5:1; The mass ratio of flavor protease to lipase is 3:1; The total mass of glutaminase, neutral protease, trypsin, flavor protease, lipase, and β-cyclodextrin was 1.3%–1.5% of the mass of goat milk. The ratio of the total mass of neutral protease to trypsin and the total mass of flavor protease to lipase was 7:

2.

2. The preparation method according to claim 1, characterized in that, The amount of β-cyclodextrin added is 0.09%-0.11% of the weight of goat milk.

3. The preparation method according to claim 1, characterized in that, In step (1), the centrifugation method is to centrifuge at 3-5℃ and 5000-7000 rpm for 15-20 min.

4. The preparation method according to claim 1, characterized in that, In step (1), the defatting method is to centrifuge the goat milk obtained after centrifugation at 3-5℃ and 8000-10000×g for 15-25 min to remove the fat phase.

5. The preparation method according to claim 1, characterized in that, The method used in molecular distillation involves distilling goat milk at 60-65℃ and a vacuum of 80-90 Pa for 15-25 minutes and collecting the fraction.

6. Goat milk small molecule peptides, characterized in that, Prepared by the preparation method according to any one of claims 1 to 5; The yield of the 500-1000 Da peptides in the goat milk small molecule peptides was 85.3% ± 2.5%.

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