Pig liver umami peptide, preparation method and application thereof

Abstract

The present application relates to the field of bioactive peptides, and particularly relates to a pig liver umami peptide, a preparation method and application thereof. The pig liver umami peptide is selected from at least one of the following polypeptide sequences: ADAW, WDDG, WGD, GDEF and WDH. A pig liver raw material is hydrolyzed by alkaline protease under appropriate enzymatic hydrolysis conditions to obtain a pig liver hydrolysate, the pig liver hydrolysate is filtered by a ceramic membrane, and then is subjected to ultrafiltration by an ultrafiltration membrane with a molecular weight cut-off of 1 kDa, is subjected to Sephadex G-15 gel chromatography separation, and the umami peptide with the lowest binding energy is selected as the pig liver umami peptide. The pig liver umami peptide has significant umami taste, and has strong ACE enzyme inhibitory activity and DPPH free radical scavenging activity. The pig liver umami peptide is applied to preparation of umami base or seasoning, and is applied to preparation of a blood pressure lowering product or an antioxidant active product. The preparation method of the pig liver umami peptide is simple, can effectively reduce the cost, and is easy to popularize.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptides, and more particularly to a porcine liver umami peptide, its preparation method, and its application. Background Technology

[0002] Hepatic protein hydrolysate is a small-molecule polypeptide active substance extracted and purified from the liver of healthy animals. It contains hepatocyte regeneration stimulating factors, which can significantly stimulate the synthesis of DNA in newly formed hepatocytes, promote the recovery of damaged hepatocyte mitochondria and rough endoplasmic reticulum, promote liver metabolism and repair, and restore liver function. Secondly, it can also enhance the liver's phagocytic function, effectively regulate the function of lymphocyte subsets, and enhance cellular and humoral immune function.

[0003] Bioactive peptides are a class of small molecule peptides composed of 2-30 amino acids that can regulate bodily functions. Their biological activity is often masked by the spatial structure of the parent protein in its natural state, and they generally require enzymatic hydrolysis to exhibit their corresponding physiological regulatory functions. Compared to intact proteins, bioactive peptides have a low molecular weight, making them easier for the body to digest and absorb, thus enabling them to exert specific biological functions.

[0004] Liver protein hydrolysates have high nutritional value, and certain amino acids may play a greater role in health. However, different animal liver sources yield different bioactive peptides, and the types of proteases used also differ, resulting in different cleavage sites and ultimately different amino acid sequences of the peptides obtained through enzymatic hydrolysis. Similarly, differences in specific hydrolysis conditions, such as hydrolysis time, molecular weight cutoff, and dextran coagulation also contribute to variations in the final amino acid sequence. Different gel separation and purification methods can lead to different final peptides. Therefore, clarifying the sequence and function of porcine liver umami peptides is of great significance for the further development of functional products from liver protein hydrolysates. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a porcine liver umami peptide, its preparation method and application.

[0006] This invention is implemented as follows: First, the present invention provides a porcine liver umami peptide selected from at least one of the following polypeptide sequences: ADAW, WDDG, WGD, GDEF and WDH.

[0007] Secondly, the present invention provides a method for preparing the aforementioned porcine liver umami peptide, comprising the following steps: (1) Pig liver raw material is selected by alkaline protease hydrolysis under appropriate enzymatic hydrolysis conditions to obtain pig liver hydrolysate. The hydrolysate is filtered with a ceramic membrane to remove suspended impurities, and then ultrafiltered with an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The ultrafiltrate is freeze-dried to obtain pig liver flavor peptide powder. The enzymatic hydrolysis conditions are: hydrolysis time of 5h, material-to-liquid ratio of 1:40g / ml, and enzyme dosage of 4000U / g. The pig liver raw material is Tibetan pig liver. (2) The pork liver umami peptide powder obtained in step (1) is separated by Sephadex G-15 gel chromatography. The separated components are analyzed by mass spectrometry combined with computer simulation technology to obtain umami peptides with binding energy ≤ -7. The umami peptide with the lowest binding energy is selected as the pork liver umami peptide.

[0008] Further, the conditions for gel chromatography in step (2) are as follows: injection concentration: 1 mg / mL; mobile phase: ultrapure water; flow rate: 1 mL / min; loading volume: 1 mL; UV detection wavelength: 220 nm.

[0009] Finally, the present invention provides the application of pork liver umami peptide powder containing the aforementioned pork liver umami peptide in the preparation of umami base materials or seasonings.

[0010] Furthermore, the umami base material comprises the following raw materials in parts by weight: 20-40 parts pork liver umami peptide powder, 1-2 parts cysteine ​​powder, 1-2 parts glucose powder, 2-5 parts D-ribose powder and 50-70 parts white sugar.

[0011] The present invention also provides the application of the aforementioned pork liver umami peptide powder in the preparation of products that assist in lowering blood pressure or products with antioxidant activity.

[0012] Furthermore, the product is a health food or skin care product.

[0013] The present invention has the following advantages: The pork liver umami peptides of the present invention have a high umami value, and when applied to umami bases or seasonings, they have a significant umami-enhancing effect. In addition to possessing umami flavor and enhancing umami, the pork liver umami peptides of the present invention also have certain blood pressure-lowering functions and antioxidant activity, effectively reducing harmful or undesirable substances produced by oxidation during high-temperature cooking of food. The preparation method of the pork liver umami peptides of the present invention is simple, effectively reducing costs and facilitating widespread application. Attached Figure Description

[0014] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0015] Figure 1 The elution curve of Sephadex G-15 for fraction F1 is shown.

[0016] Figure 2 The activity diagram of DPPH scavenging by umami peptides in pig liver.

[0017] Figure 3 The graph shows the inhibition of ACE enzyme activity by pork liver flavor peptide powder. Detailed Implementation

[0018] The technical solution of the present invention will now be clearly and completely described in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified in the embodiments, conditions are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0019] 1. Study on the enzymatic hydrolysis conditions of umami peptides in pig liver 1.1 Raw Materials The Tibetan pig liver used in the experiment all came from Tibet Mangkang Lehman Industry Co., Ltd. The fresh pig liver was washed, freeze-dried in a vacuum freeze dryer, and then stored at -20℃ for later use.

[0020] Tibetan pig liver has a crude protein content as high as 65.76% and a crude fat content of 8.61%, which reflects its characteristics of high protein and low fat. This provides an ideal material basis for peptide extraction and facilitates the subsequent targeted development of peptide products with specific properties through enzymatic hydrolysis.

[0021] Tibetan pork liver is rich in free amino acids, with glutamic acid (Glu) at 8.809 mg / g significantly higher than other amino acids. Glutamic acid is the main flavor compound, providing a natural advantage for the development of umami seasoning bases. Secondly, the content of lysine (Lys) is as high as 6.572 mg / g, ranking second among all amino acids. As one of the essential amino acids for the human body, its high content highlights the nutritional value of pork liver as a high-quality protein source. In addition, it is also rich in sweet amino acids such as glycine (Gly) and alanine (Ala), providing new research ideas for the development of natural sweeteners or sweetness enhancers.

[0022] 1.2 Screening for the optimal protease The catalytic specificity of a protease for its substrate is determined by the structure of its active site, which directly leads to significant differences in peptide bond selectivity and catalytic activity among different proteases. The hydrolysis efficiency, sensory evaluation, and percentage of different MW peptides in the hydrolysate of porcine liver protein by six proteases—flavor protease, papain, neutral protease, alkaline protease, pepsin, and trypsin—were analyzed under their respective optimal conditions (Table 1).

[0023] Table 1. Content of different MW peptides in hydrolysates of six proteases (sorted by degree of hydrolysis from smallest to largest)

[0024] The results showed significant differences in the degree of hydrolysis among different proteases. The two enzymes with the highest degree of hydrolysis were flavor protease (39.74%) and alkaline protease (32.55%), while the lowest was pepsin (8.67%). This is because flavor protease can widely hydrolyze peptide bonds adjacent to hydrophobic amino acid residues, while pepsin mainly acts on the amino-terminal peptide bonds of phenylalanine, tyrosine, and leucine, thus producing enzymatic hydrolysates with a degree of hydrolysis significantly different from the product composition. A higher degree of hydrolysis indicates a higher content of small molecule peptides (MW < 1k). The highest content was found in flavor protease and alkaline protease, respectively. However, the umami intensity of the alkaline protease hydrolysate was significantly higher than that of the flavor protease. Therefore, alkaline protease was selected as the enzyme for hydrolyzing the umami peptides of pork liver.

[0025] 1.3 Determination of optimal enzymatic hydrolysis conditions Based on the results of single-factor experiments, enzymatic hydrolysis time, material-to-liquid ratio, and enzyme dosage were selected as the main independent variables, with the degree of hydrolysis as the response value. The optimal parameters for enzymatic hydrolysis of pig liver were determined by using the central composite design principle of Box-Behnken in Design Expert 13 software: enzymatic hydrolysis time 5h, material-to-liquid ratio 1:40g / ml, and enzyme dosage 4000U / g.

[0026] 2. Preparation of umami peptides from pork liver 2.1 Preparation of Pork Liver Umami Peptide Powder Pig liver raw material was hydrolyzed using alkaline protease under optimal enzymatic hydrolysis conditions to obtain pig liver hydrolysate. The hydrolysate was filtered through a ceramic membrane to remove suspended impurities. The hydrolysate filtered through the ceramic membrane was then passed sequentially through ultrafiltration membranes with molecular weight cutoffs of 3 kDa and 1 kDa, yielding three components with different molecular weight ranges: F1 (<1 kDa), F2 (1-3 kDa), and F3 (>3 kDa). The umami value of F1 was significantly higher than that of F2 and F3. F1 was collected for subsequent processing.

[0027] Therefore, the preparation process of pork liver umami peptide powder is as follows: pork liver raw material is selected and hydrolyzed by alkaline protease under the best enzymatic hydrolysis conditions to obtain pork liver hydrolysate. The hydrolysate is filtered by ceramic membrane to remove suspended impurities, and then ultrafiltered by ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The ultrafiltrate is freeze-dried to obtain pork liver umami peptide powder.

[0028] 2.2 Gel Chromatography Separation Weigh 1 mg of the ultrafiltered lyophilized powder, dissolve it in 1 mL of ultrapure water, filter it through a 0.22 μm aqueous filter membrane, and then purify it by gel filtration chromatography. Collect the purified fraction and freeze-dry it for later use. Chromatographic conditions: Column: Sephadex G-15 (Φ16 mm × 60 cm); Mobile phase: ultrapure water; Flow rate: 1 mL / min; Sample volume: 1 mL; UV detection wavelength: 220 nm.

[0029] The elution curve of fraction F1 separated by Sephadex G-15 is as follows: Figure 1 As shown, three peaks gradually appeared as the number of collection tubes increased, and were named G1, G2 and G3 in sequence. The three components were collected for mass spectrometry identification.

[0030] 2.3 Identification and Screening Analysis of Umami Peptides Tandem mass spectra were analyzed using PEAKS Studio version 13 (Bioinformatics Solutions Inc., Waterloo, Canada). The database used was uniprot-Sus scrofa (version 2025, 22830 entries). With None digestion set, 2957, 2255, and 1597 matching peptides were identified from fractions G1, G2, and G3, respectively. These 2957, 2255, and 1597 sequences were then screened using four machine learning models: Umami_YYDS, Umami-MRNN, UMPred-FRL, and Umami_TPDM, respectively, to predict and select umami peptides, retaining 208, 478, and 434 sequences, respectively. Further screening using tools such as PeptideRanker, ToxinPred, and Innovagen yielded 53 sequences for G1, 55 for G2, and 38 for G3.

[0031] Mass spectrometry analysis combined with computer simulation technology yielded 37 (G1), 39 (G2), and 22 (G3) umami peptides with binding energies ≤ -7. Among them, the five peptides with the lowest binding energies were ADAW (G2) (-9.91), WDDG (G2, G3) (-9.7), WGD (G2, G3) (-9.63), GDEF (G2) (-9.31), and WDH (G3) (-9.31).

[0032] 3. Application of pork liver flavor peptide powder in seasonings A barbecue seasoning powder for beef and mutton is formulated based on the characteristics of enhancing freshness, removing fishy smell, increasing aroma and caramelizing aroma (Table 2).

[0033] Enhanced flavor: Mainly umami peptide powder, supplemented with salt and sugar for saltiness and sweetness; To remove fishy smell: use garlic powder, ginger powder, and onion powder as the main ingredients, supplemented with cardamom powder and galangal powder; Flavor enhancer: Mainly cumin and black and white pepper powder, supplemented with fennel powder, star anise powder, cinnamon powder, Sichuan pepper powder, coriander seed powder, and dried tangerine peel powder; Caramelized characteristics: The small peptides, cysteine, glucose, ribose and white sugar in the umami peptide powder seasoning base combine with the protein of the raw meat to undergo Maillard reaction and caramelization reaction during high-temperature grilling, supplemented by the smoky and sweet flavor of smoked red pepper powder.

[0034] Table 2. Beef and Lamb Barbecue Seasoning Powder Recipe

[0035] The umami peptide seasoning base is made by fully mixing 30 parts umami peptide powder, 1.5 parts cysteine ​​powder, 1.5 parts glucose powder, 3.0 parts ribose powder and 64 parts white sugar powder.

[0036] After the Maillard reaction, the bitterness of the meat-flavored seasoning base is reduced, the umami value is stable and slightly enhanced, the total free amino acids are reduced, and the ROAV value of the volatile flavor compound 1-octen-3-one is the highest. This adds a rich and prominent complex "meat-flavored" flavor characteristics of cooked meat, roasting, nuts, fruits and butter to the original single aroma of the enzymatic hydrolysate, which is dominated by strong mushroom and grassy fat aroma.

[0037] 4. Activity analysis of umami peptides in pork liver 4.1 Antioxidant Activity of Umami Peptide Powder The ability of umami peptide powder to scavenge DPPH free radicals reflects its antioxidant activity. A 0.1 mg / ml DPPH solution in anhydrous ethanol was prepared and allowed to stand in the dark. Umami peptide solutions with concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg / ml were also prepared. The reaction mixture consisted of 2.0 ml sample solution + 2.0 ml DPPH in the sample group, 2.0 ml sample solution + 2.0 ml anhydrous ethanol in the control group, and 2.0 ml DPPH + 2.0 ml anhydrous ethanol in the blank group. After reacting at room temperature in the dark for 30 min, the absorbance was measured at 517 nm. The inhibition rate of umami peptide against DPPH was calculated using the following formula. An inhibition rate-concentration curve was fitted, and the sample concentration at which the inhibition rate was 50% was calculated, i.e., the IC50.

[0038] In the formula: P – Clearance rate, % A s —Absorbance values ​​of the sample group; A c —Absorbance value of the control group; A b —Absorbance value of the blank group.

[0039] according to Figure 2 The regression equation showed that the IC50 value of the umami peptide powder for scavenging DPPH free radicals was 2.46 mg / ml. This indicates that the pork liver umami peptide of the present invention has high antioxidant activity.

[0040] 4.2 Blood Pressure Lowering Activity of Umami Peptide Powder The inhibition of ACE by umami peptide powder reflects the antihypertensive activity of umami peptides. The angiotensin-converting enzyme (ACE) inhibitory activity of abalone visceral protein peptides was determined spectrophotometrically at 340 nm using a multifunctional microplate reader. The total reaction system consisted of 100 μL of ACE (0.125 U / mL) solution (ACE enzyme dissolved in 0.08 mol / L HEPES buffer containing 0.3 mol / L sodium chloride) mixed with sample solutions of different concentrations in microplates. FAPPGG solution was then added to the mixture to initiate the reaction, which was carried out at 37℃ for 30 min (protected from light). The absorbance before and after the reaction was recorded using the microplate reader. HEPES buffer was used as a blank control instead of the sample solution. The inhibition rate of ACE activity by umami peptides was calculated using the following formula. An inhibition rate-concentration curve was fitted, and the sample concentration at which the inhibition rate was 50% was calculated, i.e., IC50.

[0041]

[0042] I-ACE inhibition rate, % A1—Initial absorbance at 340 nm for blank control group A2—Absorbance at 340 nm after reaction in blank control group A3—Initial absorbance at 340 nm for sample group A4—Absorbance at 340 nm after reaction for sample group according to Figure 3 The regression equation showed that the IC50 value of the umami peptide powder inhibiting ACE enzyme was 6.06 mg / ml. This indicates that the porcine liver umami peptide of the present invention has a certain blood pressure lowering function.

[0043] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A porcine liver umami peptide, characterized in that: Selected from at least one of the following polypeptide sequences: ADAW, WDDG, WGD, GDEF, and WDH.

2. A method for preparing the porcine liver umami peptide as described in claim 1, characterized in that: Includes the following steps: (1) Pig liver raw material is selected by alkaline protease hydrolysis under appropriate enzymatic hydrolysis conditions to obtain pig liver hydrolysate. The hydrolysate is filtered with a ceramic membrane to remove suspended impurities, and then ultrafiltered with an ultrafiltration membrane with a molecular weight cutoff of 1 kDa. The ultrafiltrate is freeze-dried to obtain pig liver flavor peptide powder. The enzymatic hydrolysis conditions are: hydrolysis time of 5 h, material-to-liquid ratio of 1:40 g / ml, and enzyme dosage of 4000 U / g. The pig liver raw material is Tibetan pig liver. (2) The pork liver umami peptide powder obtained in step (1) is separated by Sephadex G-15 gel chromatography. The separated components are analyzed by mass spectrometry combined with computer simulation technology to obtain umami peptides with binding energy ≤ -7. The umami peptide with the lowest binding energy is selected as the pork liver umami peptide.

3. The preparation method according to claim 2, characterized in that: The conditions for gel chromatography in step (2) are as follows: injection concentration: 1 mg / mL; mobile phase: ultrapure water; flow rate: 1 mL / min; loading volume: 1 mL; UV detection wavelength: 220 nm.

4. The application of a pork liver umami peptide powder containing the pork liver umami peptide of claim 1 in the preparation of umami base materials or seasonings.

5. The application according to claim 4, characterized in that: The umami base material comprises the following raw materials in parts by weight: 20-40 parts pork liver umami peptide powder, 1-2 parts cysteine ​​powder, 1-2 parts glucose powder, 2-5 parts D-ribose powder and 50-70 parts white sugar.

6. The use of a pork liver umami peptide powder comprising the pork liver umami peptide of claim 1 in the preparation of products for assisting in lowering blood pressure or products with antioxidant activity.

7. The application according to claim 6, characterized in that: The product in question is a health food or skincare product.

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