Preparation method of an antioxidant and non-fishy heme polypeptide complex

Abstract

The present invention discloses a preparation method of an antioxidant and non-fishy and stinky heme polypeptide complex, which is characterized by including the following steps: 1) dissolving pig blood powder in water to obtain a pig blood powder solution with a mass fraction of 10%, and adjusting the pH value of the pig blood powder solution to 5-9 under stirring conditions; 2) mixing the above-mentioned pig blood powder solution with at least one of subtilisin and neutral aspergillus oryzae protease, and placing it in a constant temperature water bath for enzymatic hydrolysis; 3) centrifuging the enzymatic hydrolysate to obtain the supernatant, separating it with a 3 kDa ultrafiltration tube, collecting the permeate, then separating it with a 1 kDa ultrafiltration tube, taking the retentate on the membrane, and freeze-drying to obtain a polypeptide powder; 4) dissolving the freeze-dried powder in deionized water to form a solution and mixing it with hemin to form a hemin polypeptide complex; the advantages are higher binding rate, stronger antioxidant property, reduced fishy and stinky smell and increased overall sensory acceptance.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of functional polypeptide complexes, and particularly to a preparation method of an antioxidant and non-fishy heme polypeptide complex. Background Art

[0002] Blood meal is made from fresh pig blood and formed into fine blood powder through defibrination filtration and atomization drying. Blood meal contains rich bioactive substances and nutritional value, and is rich in various proteins and amino acids. In China, the resources of blood meal are abundant, but at present, the utilization of blood meal is mainly as a raw material for high-protein feed, with a low utilization rate, resulting in waste of resources and environmental pollution. Deep development of blood meal can achieve a win-win situation of economic and ecological benefits. However, due to problems such as strong fishy smell and poor palatability, the deep development of blood meal is restricted. By adding proteolytic enzymes, the macromolecular proteins in blood meal can be effectively hydrolyzed into peptides and amino acids. Most of the small peptides can interact with small molecules, showing good physiological activities, greatly improving the utilization rate, and effectively improving the sensory characteristics. Subtilisin and neutral aspergillus oryzae protease have the advantages of rich sources and strong activities, but they have not been developed and applied in blood meal processing yet.

[0003] As an important and indispensable nutrient in the human body, insufficient intake of iron can cause anemia, which in turn affects the immune system. At present, anemia has become a global disease, especially for children, adolescents, pregnant and lactating women. To prevent iron deficiency anemia, many drugs have been widely used for taking iron. Among them, ferrous sulfate, ferrous chloride, ferrous gluconate, etc. are widely used. They have rich iron element content and can provide certain nutrition, but due to their side effects, they may cause iron poisoning.

[0004] Heme is a natural porphyrin iron compound and the main active center of myoglobin and hemoglobin. Hemin chloride is a heme product extracted from blood and is an in vitro purified form of natural heme. It can be used as an iron nutritional fortifier. Compared with traditional iron supplements, it has higher bioavailability, will not cause iron accumulation poisoning in the body, and is relatively mild to the gastrointestinal tract. However, hemin chloride has poor water solubility and poor stability in the body. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a preparation method of an antioxidant and non-fishy heme polypeptide complex with higher binding rate, stronger antioxidant property, reduced fishy smell and better stability and affinity.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows: A preparation method of an antioxidant and non-fishy heme polypeptide complex, comprising the following steps:

[0007] (1) Dissolve the blood meal in water to obtain a blood meal solution with a mass fraction of 8%, and adjust the pH value of the blood meal solution to 5 - 9 under stirring conditions.

[0008] (2) Mix the above blood meal solution with at least one of subtilisin and neutral aspergillus oryzae protease, and place it in a constant temperature water bath for enzymatic hydrolysis.

[0009] (3) Centrifuge the enzymatic hydrolysate at a centrifugation rate of 4000 - 9000 rpm for 5 - 30 min. After centrifugation, take the supernatant, separate it with a 3 kDa ultrafiltration tube, collect the permeate, then separate it with a 1 kDa ultrafiltration tube, take the retentate on the membrane, and freeze-dry to obtain polypeptide powder.

[0010] (4) Take the polypeptide powder and dissolve it in deionized water to prepare a polypeptide solution with a concentration of 0.6 mg / mL. Mix the polypeptide solution with a hemin solution with a concentration of 0.3 - 0.6 mg / mL at a volume ratio of 2:1 - 6:1 to form a hemin-polypeptide complex.

[0011] Furthermore, in step (1), a food-grade sodium bicarbonate solution with a concentration of 0.05 - 0.2 mol / L is added to adjust the pH value.

[0012] Furthermore, the pH value in step (1) is 7 - 7.5.

[0013] Furthermore, the dosage of the protease in step (2) is 3000 - 10000 U / g.

[0014] Furthermore, the temperature of the constant temperature water bath in step (2) is in the range of 25 - 60 °C, the enzymatic hydrolysis time is between 2 - 6 h, and the enzymatic hydrolysis process includes one-step enzymatic hydrolysis and stepwise enzymatic hydrolysis.

[0015] Furthermore, the specific process of the stepwise enzymatic hydrolysis is as follows: Add 5500 U / g of subtilisin to the blood meal solution in step (1) and mix well. Place the mixture in a constant temperature water bath at 45 °C for enzymatic hydrolysis for 3 h, inactivate the enzyme at 90 °C for 10 min, cool to room temperature, centrifuge at 4000 r / min for 10 min, and collect the supernatant; Dropwise add a 0.1 moL / L food-grade sodium bicarbonate solution to the supernatant, control the pH to 7.5, then add 2500 U / g of neutral aspergillus oryzae protease to the supernatant, mix well and place it in a constant temperature water bath at 55 °C for enzymatic hydrolysis for 2 h.

[0016] Furthermore, in step (3), the centrifugation rate is 8000 rpm and the centrifugation time is 10 min.

[0017] Further, the peptide segments in the polypeptide in step (4) that can stably bind to hemin include peptide segment 1 AAWGKVGGQAGAHGAEALER, peptide segment 2 FLANVSTVLTSK, and peptide segment 3 AWGKVGGQAGAHGAEALERM.

[0018] Further, in step (4), the polypeptide solution is mixed with a hemin solution with a concentration of 0.5 mg / mL at a volume ratio of 3:1, and the mixing method of the eluent and hemin includes normal temperature and pressure and ultrasonic treatment.

[0019] Compared with the prior art, the advantages of the present invention are as follows: The present invention first discloses a preparation method of an antioxidant and non-fishy-smelling hemin polypeptide complex. Protease is used to hydrolyze pig blood powder to generate polypeptides, and the polypeptides are complexed with hemin to form a stable structure, which can not only enhance solubility but also improve the bioavailability and stability of hemin. The binding rate of hemin and polypeptides is relatively high, and the prepared complex has good functional activity. The present invention uses protease to hydrolyze pig blood powder into polypeptides, removes fishy smell, improves its flavor, complexes the polypeptides with hemin, makes it have antioxidant and iron-supplementing functional activities, has better stability and affinity, and can improve bioavailability, reduce toxicity and immunogenicity. It helps to realize the high-value application of pig blood powder. Description of the Drawings

[0020] Figure 1 Peptide molecular weight distribution of the enzymolysate in different examples;

[0021] Figure 2 Infrared spectrum (a) and secondary structure content (b) of the polypeptide-hemin complex;

[0022] Figure 3 Zeta potential of the polypeptide-hemin complex;

[0023] Figure 4 Binding rate of the polypeptide and hemin;

[0024] Figure 5 Schematic diagram of the DPPH scavenging rate and ABTS scavenging rate (a) and reducing power (b) of the polypeptide-hemin complex;

[0025] Figure 6 Sephadex G-25 chromatography separation chromatogram of the polypeptide solution in Example 5;

[0026] Figure 7 Binding rate of different eluent components and hemin in Example 5;

[0027] Figure 8 Peptide segment abundance map of the A3 eluent component in Example 5;

[0028] Figure 9 It is the molecular docking diagram of the peptide segment of the A3 eluent component and hemin in Example 5. Detailed implementation manners

[0029] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

[0030] I. Experimental methods

[0031] 1. Determination of the binding rate of polypeptide and hemin

[0032] In the mixed solution of porcine blood powder hydrolysate polypeptide and hemin, add 1 mol / L HCl solution to adjust the pH to precipitate the unbound hemin completely. Centrifuge to obtain the precipitate, freeze-dry and weigh it, and calculate the binding rate according to the following formula: Where M and M0 in the formula represent the masses of hemin before and after binding to the peptide segment respectively.

[0033] 2. Determination of antioxidant property

[0034] (1) Determination of DPPH scavenging rate

[0035] Add 8 mL of 1×10 -4 mol / L DPPH solution to 2 mL of the polypeptide-hemin complex solution. After the mixture is allowed to stand in the dark for 30 min, centrifuge at a rate of 4000 r / min for 6 min, and measure the absorbance A1 at a wavelength of 517 nm. Under the same conditions, measure the absorbance of the mixture of 1 mL of the sample hydrolysate and 4 mL of 95% ethanol, denoted as A2, and the absorbance of the mixture of 4 mL of DPPH solution and 1 mL of 95% ethanol, denoted as A3. The DPPH scavenging rate is calculated according to the following formula:

[0036] (2) Determination of ABTS radical scavenging ability

[0037] Mix 0.5 mL of the polypeptide-hemin complex solution with 2 mL of ABTS solution and allow to stand in the dark for 15 min. Measure the absorbance value A at a wavelength of 734 nm. Then mix 0.5 mL of 95% ethanol with 2 mL of ABTS solution evenly and measure the absorbance value A0 under the same conditions. The ABTS scavenging rate is calculated according to the following formula:

[0038]

[0039] (3) Determination of reducing power

[0040] 2.5 mL of 0.2 mol / L PBS (pH 6.6) and 2.5 mL of 1% potassium ferricyanide solution were successively added to 1 mL of the peptide-polypeptide-hemin complex solution, and shaken well. After the mixture was placed in a constant temperature water bath at 50 °C for 20 min, it was immediately taken out, 2.5 mL of 10% TCA solution was added, and after mixing, it was centrifuged at a rate of 4000 rpm for 10 min. 1 mL of the supernatant was mixed with 1 mL of 0.1% ferric chloride solution, and then 5 mL of distilled water was added and shaken well. Finally, the absorbance was measured at 700 nm, and the higher the absorbance.

[0041] 3. Determination of flavor compounds

[0042] The gas chromatography-mass spectrometry (GC-MS) was used to perform 40 min of microextraction on volatile compounds at 50 °C, and then the extraction head was placed into the injection port of the GC-MS instrument and desorbed for 5 min. The peak area and mass spectrum were extracted from the chromatogram, and the NIST database was used to identify the compound composition by analyzing the mass spectrum. Three replicate experiments were performed for each treatment group, and 5 ppm of 4-methyl-1-pentanol was used as the internal standard to quantitatively determine the volatile compounds, and the odor activity value analysis was performed on the detected volatile compounds to determine the flavor substances that made important contributions to the odor composition.

[0043] 4. Sensory evaluation

[0044] The sensory evaluation panel consisted of 10 sensory assessors aged between 20 and 25 years old who had received professional training, including 5 males and 5 females. The assessors respectively performed sensory evaluations on each hemoglobin hydrolysate. The scoring was on a 10-point scale, and the evaluation indexes included: fishy smell (0 - 4 points indicated no obvious fishy smell, 4 - 6 points indicated having a fishy smell, 7 - 10 points indicated an obvious fishy smell), bitter almond smell (0 - 4 points indicated no obvious bitter almond smell, 4 - 6 points indicated having a bitter almond smell, 7 - 10 points indicated an obvious bitter almond smell), caramel smell (0 - 4 points indicated no obvious caramel smell, 4 - 6 points indicated having a caramel smell, 7 - 10 points indicated an obvious caramel smell), rancid smell (0 - 4 points indicated no obvious rancid smell, 4 - 6 points indicated having a rancid smell, 7 - 10 points indicated an obvious rancid smell), and overall acceptance (0 - 4 points indicated non-acceptance or slight acceptance, 4 - 6 points indicated acceptance, 7 - 10 points indicated very acceptance). II. Specific examples

[0046] Example 1

[0047] A preparation method of an antioxidant and non-fishy hemin polypeptide complex, comprising the following steps:

[0048] (1) Dissolve the pig blood powder (Xi'an Wusehua Biotechnology Co., Ltd., food grade, 99%, product number: WSH3-624) in water to prepare a pig blood powder solution with a mass fraction of 8%. Under stirring conditions, gradually add 0.1 moL / L food grade sodium bicarbonate solution dropwise to the pig blood powder solution, and control the pH to 7;

[0049] (2) Add 8000 U / g of Bacillus subtilis protease to the pig blood powder solution in step (1) and mix well. Place the mixture in a constant temperature water bath at 45 °C and enzymatically hydrolyze for 5 h;

[0050] (3) Centrifuge the enzymatic hydrolysate at a centrifugation rate of 8000 rpm for 10 min. After centrifugation, take the supernatant and separate it with a 3 kDa ultrafiltration tube. Collect the permeate, then separate it with a 1 kDa ultrafiltration tube, take the retentate on the membrane, and freeze-dry to obtain polypeptide powder;

[0051] (4) Dissolve the polypeptide powder in deionized water to prepare a polypeptide solution with a concentration of 0.6 mg / mL. Mix the polypeptide solution and 0.5 mg / mL hemin solution at a volume ratio of 3:1 and stir at room temperature for 20 min to form a hemin-polypeptide complex.

[0052] Example 2

[0053] A preparation method of an antioxidant and odorless hemin-polypeptide complex, comprising the following steps:

[0054] (1) Dissolve the pig blood powder in water to prepare a pig blood powder solution with a mass fraction of 8%. Under stirring conditions, gradually add 0.1 moL / L food grade sodium bicarbonate solution dropwise to the pig blood powder solution, and control the pH to 7.5;

[0055] (2) Add 8000 U / g of neutral Aspergillus oryzae protease to the pig blood powder solution in step (1) and mix well. Place the mixture in a constant temperature water bath at 55 °C and enzymatically hydrolyze for 5 h;

[0056] (3) Centrifuge the enzymatic hydrolysate at a centrifugation rate of 8000 rpm for 10 min. After centrifugation, take the supernatant and separate it with a 3 kDa ultrafiltration tube. Collect the permeate, then separate it with a 1 kDa ultrafiltration tube, take the retentate on the membrane, and freeze-dry to obtain polypeptide powder;

[0057] (4) Dissolve the polypeptide powder in deionized water to prepare a polypeptide solution with a concentration of 0.6 mg / mL. Mix the polypeptide solution and 0.5 mg / mL hemin solution at a volume ratio of 3:1 and stir at room temperature for 20 min to form a hemin-polypeptide complex.

[0058] Example 3

[0059] A preparation method of an antioxidant, odorless and tasteless heme polypeptide complex, comprising the following steps:

[0060] (1) Dissolve blood meal in water to prepare a blood meal solution with a mass fraction of 8%, and dropwise add a 0.1 moL / L food-grade sodium bicarbonate solution to the blood meal solution under stirring conditions, controlling the pH to 7.5;

[0061] (2) Add 2500 U / g of neutral Aspergillus oryzae protease and 5500 U / g of Bacillus subtilis protease to the blood meal solution in step (1), mix well, and place the mixture in a constant temperature water bath at 50 °C for enzymatic hydrolysis for 5 h;

[0062] (3) Centrifuge the enzymatic hydrolysate at a centrifugation rate of 8000 rpm for 10 min. After centrifugation, take the supernatant, separate it with a 3 kDa ultrafiltration tube, collect the permeate, then separate it with a 1 kDa ultrafiltration tube, take the retentate on the membrane, and freeze-dry to obtain polypeptide powder;

[0063] (4) Dissolve the polypeptide powder in deionized water to prepare a polypeptide solution with a concentration of 0.6 mg / mL. Mix the polypeptide solution and a 0.5 mg / mL hemin chloride solution at a volume ratio of 3:1, and stir at room temperature for 20 min to form a hemin chloride polypeptide complex.

[0064] Example 4

[0065] A preparation method of an antioxidant, odorless and tasteless heme polypeptide complex, comprising the following steps:

[0066] (1) Dissolve blood meal in water to prepare a blood meal solution with a mass fraction of 8%, and dropwise add a 0.1 moL / L food-grade sodium bicarbonate solution to the blood meal solution under stirring conditions, controlling the pH to 7.5;

[0067] (2) Add 3200 U / g of neutral Aspergillus oryzae protease and 4800 U / g of Bacillus subtilis protease to the blood meal solution in step (1), mix well, and place the mixture in a constant temperature water bath at 50 °C for enzymatic hydrolysis for 5 h;

[0068] (3) Centrifuge the enzymatic hydrolysate at a centrifugation rate of 8000 rpm for 10 min. After centrifugation, take the supernatant, separate it with a 3 kDa ultrafiltration tube, collect the permeate, then separate it with a 1 kDa ultrafiltration tube, take the retentate on the membrane, and freeze-dry to obtain polypeptide powder;

[0069] (4) Dissolve the polypeptide powder in deionized water to prepare a polypeptide solution with a concentration of 0.6 mg / mL. Mix the polypeptide solution and a 0.5 mg / mL hemin chloride solution at a volume ratio of 3:1, and ultrasonically mix for 20 min to form a hemin chloride polypeptide complex, and the ultrasonic power is 400 W.

[0070] Example 5

[0071] A preparation method of an antioxidant and odorless hematochrome polypeptide complex, comprising the following steps:

[0072] (1) Dissolve blood meal in water to prepare a blood meal solution with a mass fraction of 8%, and dropwise add a 0.1 moL / L food-grade sodium bicarbonate solution to the blood meal solution under stirring conditions, controlling the pH to 7;

[0073] (2) Add 5500 U / g of Bacillus subtilis protease to the blood meal solution in step (1), mix well, place the mixture in a constant temperature water bath at 45 °C for enzymatic hydrolysis for 3 h, inactivate the enzyme at 90 °C for 10 min, cool to room temperature, centrifuge at 4000 r / min for 10 min, and collect the supernatant. Dropwise add a 0.1 moL / L food-grade sodium bicarbonate solution to the supernatant, control the pH to 7.5, then add 2500 U / g of neutral Aspergillus oryzae protease to the supernatant, mix well and place it in a constant temperature water bath at 55 °C for enzymatic hydrolysis for 2 h;

[0074] (3) Centrifuge the enzymatic hydrolysate at a centrifugation rate of 8000 rpm for 10 min, take the supernatant after centrifugation, separate it with a 3 kDa ultrafiltration tube, collect the permeate, then separate it with a 1 kDa ultrafiltration tube, take the retentate on the membrane, and freeze-dry to obtain polypeptide powder;

[0075] (4) Take the polypeptide powder and dissolve it in deionized water to prepare a polypeptide solution with a concentration of 0.6 mg / mL. Mix the polypeptide solution and a 0.5 mg / mL hemin chloride solution in a volume ratio of 3:1, and ultrasonically mix for 20 min to form a hemin chloride polypeptide complex, with an ultrasonic power of 400 W.

[0076] III. Experimental Results and Analysis

[0077] 1. Molecular weight determination of blood meal and enzymatic hydrolysates in the examples

[0078] From Figure 1 it can be seen that compared with blood meal, the component contents of the enzymatic hydrolysates in Examples 1-5 in the ranges of 0.5-1 kDa and 1-5 kDa are significantly increased (P < 0.05). This result indicates that the proteins in blood meal are highly degraded by proteases into low-molecular-weight peptides or free amino acids. The blood meal hydrolysate is mainly composed of low-molecular-weight polypeptides (0.5-5 kDa).

[0079] 2. Structural change analysis

[0080] Preparation method of porcine blood powder - hemin complex in the control group: Mix a 0.6 mg / mL porcine blood powder solution with 0.5 mg / mL hemin at a volume ratio of 3:1 and stir at room temperature for 20 min.

[0081] Figure 2 (a) shows that the characteristic absorption peak of the infrared spectrum of the polypeptide - hemin conjugate is around 865 cm -1 Nearby, compared with the porcine blood powder - hemin conjugate in the control group, after enzymatic hydrolysis, the characteristic absorption peak of the polypeptide - hemin conjugate undergoes a blue shift, indicating a change in the structure of the conjugate. Figure 2 (b) shows the secondary structure change of the polypeptide - hemin complex. It can be seen from the figure that after enzymatic hydrolysis, the secondary structure of the polypeptide - hemin changes from an α - helix structure and a β - sheet structure to a random coil and β - turn structure.

[0082] 3. Zeta potential analysis

[0083] Figure 3 Shows the Zeta potential of the polypeptide - hemin complex. Compared with the porcine blood powder - hemin complex in the control group, the absolute value of the Zeta potential of the polypeptide - hemin complex in Examples 1 - 5 decreases. This may be because more positively charged groups are exposed during the enzymatic hydrolysis process.

[0084] 4. Analysis of the binding rate of the peptide - hemin complex

[0085] The peptide - hemin binding rates in Examples 1 - 5 and the porcine blood powder control group are as Figure 4 Shown. It can be seen from the figure that compared with the control group, the binding rate of the polypeptide and hemin in Examples 1 - 5 has increased significantly (P < 0.05). Among them, the binding rate in Example 3 reaches 60%, which is higher than that in Examples 1 - 2. This may be because a double - enzyme complex is used in Example 3, which can improve the hydrolysis efficiency and obtain more small peptides. Small peptides contain more binding sites for hemin. After adding an ultrasonic procedure in Example 4, the binding degree increases by 9.6% compared with Example 3, reaching 69.6%, indicating that appropriate ultrasonic treatment can promote the expansion of the molecular structure of the peptide segment. By increasing the expression of hydrophobic groups, the binding between the peptide segment and hemin can be effectively improved. In the case of step - by - step enzymatic hydrolysis plus ultrasound in Example 5, the binding rate reaches the highest at 75%.

[0086] 5. Antioxidant analysis of the peptide - hemin complex

[0087] The antioxidant property of the polypeptide - hemin complex is as Figure 5As shown, it can be seen from the figure that compared with the porcine blood powder - hemin complex in the control group, the DPPH scavenging rate, ABTS scavenging rate, and A700 of the polypeptide - hemin complexes in Examples 1 - 5 were all significantly increased (P < 0.05). The antioxidant property of the complex in Example 5 reached the highest level.

[0088] 6. Determination of flavor compounds in the peptide - hemin complex

[0089] Through gas chromatography - mass spectrometry analysis of the hemin - polypeptide complex, a total of 35 volatile compounds were detected, and the contents of 11 flavor substances changed significantly (P < 0.05). Table 1 shows the odor activity values (OVA) of the main flavor substances. When the odor activity value is greater than 1, the flavor substance contributes to the formation of flavor.

[0090] Table 1 OVA values of main volatile compounds

[0091]

[0092] Note: Different letters (a - d) in the table indicate significant differences in odor activity values among treatment groups (P < 0.05).

[0093] As can be seen from Table 1, compared with the porcine blood powder - hemin complex in the control group, the contents of flavor compounds such as 1 - octen - 3 - ol, trans - 2 - octenal, 3 - methylbutanal, butyl acetate, 3 - methyl - 2 - butanone in Examples 1 - 5 increased significantly (P < 0.05), and the flavor activity values increased. However, in different examples, the contents of these substances were different. As can be seen from Table 2, the flavor activity values of these several flavor compounds in Example 5 were higher than those in other groups, and the contents of flavor substances with off - flavors such as valeraldehyde and trans - 2 - decenal decreased significantly (P < 0.05).

[0094] 7. Sensory evaluation of the peptide - hemin complex

[0095] The sensory attributes of different hemin - polypeptide complexes are shown in Table 2. Compared with the porcine blood powder - hemin complex in the control group, in the sensory evaluation of the hemin - polypeptide complexes in Examples 1 - 5, the scores for bloody smell, rancid smell, and bitter almond smell decreased significantly (P < 0.05), and the scores for caramel smell and overall acceptance increased significantly (P < 0.05). Among them, Example 5 was superior to other treatment groups in terms of overall acceptance and caramel smell.

[0096] Table 2 Sensory evaluation form

[0097] Control group Example 1 Example 2 Example 3 Example 4 Example 5 Smell of blood 8.3 5.3 5.7 5.5 3.9 3.2 Bitter almond smell 7.5 4.8 4.2 4.3 3.7 3.3 Rancid smell 6.2 4.2 4.1 3.9 2.6 2.1 Burnt aroma 4.6 1.9 2.9 5.2 5.9 6.2 Overall acceptance 3.2 6.3 5.9 7.3 8.8 9.1 。

[0098] 8. Separation by Sephadex G-25 Gel Filtration Chromatography

[0099] Gel chromatography is a method for separating samples based on molecular weight. When a sample passes through gel particles with a porous network structure, larger molecular weight samples cannot enter the gel particles and are eluted from the chromatography column first, while smaller molecular weight samples are eluted later. The polypeptide solution in Example 5 was separated by Sephadex G-25 gel filtration chromatography, and the elution position of the peptide was detected at 220 nm. The chromatogram is as shown in Figure 6 . According to the separation order in the chromatography column, each component was denoted as A1, A2, A3, and A4. Each eluate component was mixed with a 0.5 mg / mL hemin solution at a volume ratio of 3:1 to form a hemin-polypeptide complex. The determination results of the binding rate between the polypeptide and hemin are as shown in Figure 7 , indicating that the binding rate of component A3 and hemin is the highest.

[0100] 3. Peptide segment identification and molecular simulation docking method for eluate component A3: Molecular docking analysis. AutoDockVina (1.1.2) was used for molecular docking. The structure file of the small molecule hemin was downloaded from the Pubchem database. The small peptide sequence was modeled using Maestro Elements 3.6, and the peptide segments were docked with the small molecule hemin using Vina. The torsional bonds of the small molecule hemin were determined using Autodock, hydrogen was added to the peptide segments, the charges were calculated, and the atomic types were set. After docking using Vina, the three-dimensional and two-dimensional angle force analysis and visualization were performed using Pymol and Discovery Studio software.

[0101] As shown in Figure 8 , it is the peptide segment abundance map of component 3. LC-ESI / MS was used to identify the sequence of component 3 with the highest hemin binding rate. According to the abundance values, 10 peptide segments were initially screened, and the peptide sequences were GQAGAHGAEALERM, GQAGAHGAEALER, WGKVNVDEVGGEA, LWGKVNVDEVGGEA, LLVVYPWTQR, AWGKVGGQAGAHGAEALERM, AAWGKVGGQAGAHGAEALER, LWGKVNVDEVGGEALGR, AHGAEALERM, and FLANVSTVLTSK. Then, the source proteins and peptide scores were analyzed, and the results are shown in Table 3.

[0102] Table 3 Peptide segment sources and polypeptide coverage scores in the mass spectrum

[0103]

[0104] After analysis, these peptide segments mainly come from the alpha chain of hemoglobin (P01965) and the domain protein in globin (A0A8D1HFL8). Considering peptide segment abundance, score, and protein source, AAWGKVGGQAGAHGAEALER, FLANVSTVLTSK, and AWGKVGGQAGAHGAEALERM were selected for subsequent molecular simulation docking.

[0105] 4. Molecular simulation docking of peptide segments

[0106] Figure 9 In (A), (B), and (C), they respectively represent the molecular docking simulation diagrams of peptide segment 1: AAWGKVGGQAGAHGAEALER, peptide segment 2: FLANVSTVLTSK, and peptide segment 3: AWGKVGGQAGAHGAEALERM with hemin. Among them, the blue solid lines represent hydrogen bonds, the yellow dashed lines represent salt bridge forces, and the gray dashed lines represent hydrophobic forces. In the two-dimensional diagram, the green dashed lines represent hydrogen bonds, the light green dashed lines represent carbon-hydrogen bonds, and the pink, purple, grass green, and brown dashed lines represent hydrophobic forces. The binding energies of peptide segment 1, peptide segment 2, and peptide segment 3 with hemin are -5.6 kcal / mol, -5.5 kcal / mol, and -5.2 kcal / mol respectively. Generally, if the binding energy of a ligand to a target protein is less than -5, it indicates a stable binding between the protein and the small molecule. Therefore, peptide segment 1, peptide segment 2, and peptide segment 3 all have strong affinity activities with hemin and can bind stably. It can be seen from the figure that hemin can bind to the 5th LYS amino acid residue of peptide segment 1 through 3 hydrogen bonds respectively and and 2 hydrophobic forces respectively and ; it binds to the 6th VAL amino acid residue of peptide segment 1 through 1 hydrophobic force. Hemin can bind to the 5th VAL amino acid residue of peptide segment 2 through 2 hydrophobic forces respectively and ; it binds to the 11th SER amino acid residue and the 1st PHE amino acid residue of peptide segment 2 through 2 hydrogen bonds respectively and hydrophobic forces respectively; hemin can bind to the 14th ALA amino acid residue of peptide segment 3 through 2 hydrogen bonds respectively and and 1 hydrophobic force; it binds to the 12th HIS amino acid residue of peptide segment 3 through 2 hydrogen bonds respectively and salt bridge forces; through 3 and hydrogen bonds and one hydrophobic interaction bind to the 20th MET amino acid residue, 16th ALA amino acid residue, 13th GLY amino acid residue, and 17th LEU amino acid residue of peptide segment 3, respectively.

[0107] In summary, the present invention provides a method for generating polypeptides by hydrolyzing pig blood powder with protease, complexing the polypeptides with hemin, and preparing a hemin polypeptide complex. The binding rate of hemin and polypeptides is relatively high, and the prepared complex has good functional activity. By comparing the antioxidant properties and sensory qualities of the hemin polypeptide complex and pig blood powder, it is found that the hemin polypeptide complex in this case has stronger antioxidant properties, reduced fishy odor, and increased overall sensory acceptance. Therefore, this scheme helps to achieve the high-value application of pig blood powder.

[0108] The above description is not a limitation of the present invention, nor is the present invention limited to the above examples. Changes, modifications, additions, or substitutions made by those of ordinary skill in the art within the substantial scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A preparation method of an antioxidant odorless and tasteless heme polypeptide complex, characterized in that It includes the following steps: (1) Dissolve blood meal in water to obtain a blood meal solution with a mass fraction of 8%, and adjust the pH value of the blood meal solution to 5 - 9 under stirring conditions; (2) First, enzymatically hydrolyze the blood meal solution obtained in step (1) with subtilisin in a constant temperature water bath, and then enzymatically hydrolyze it with neutral aspergillus oryzae protease in a constant temperature water bath; (3) Centrifuge the enzymatic hydrolysate at a centrifugation rate of 4000 - 9000 rpm for 5 - 30 min. After centrifugation, take the supernatant, separate it with a 3 kDa ultrafiltration tube, collect the permeate, then separate it with a 1 kDa ultrafiltration tube, take the retentate on the membrane, and freeze-dry to obtain polypeptide powder; (4) Take the polypeptide powder and dissolve it in deionized water to prepare a polypeptide solution with a concentration of 0.6 mg / mL. Mix the polypeptide solution with a hemin solution with a concentration of 0.3 - 0.6 mg / mL at a volume ratio of 2:1 - 6:1 to form a hemin-polypeptide complex. The peptide segments in the polypeptide solution that can stably bind to hemin include peptide segment 1 AAWGKVGGQAGAHGAEALER, peptide segment 2 FLANVSTVLTSK, and peptide segment 3 AWGKVGGQAGAHGAEALERM.

2. The preparation method of an antioxidant and odorless heme polypeptide complex according to claim 1, characterized in that: In step (1), a food-grade sodium bicarbonate solution with a concentration of 0.05 - 0.2 mol / L is added to adjust the pH value.

3. The preparation method of an antioxidant and odorless heme polypeptide complex according to claim 1, characterized in that: The pH value described in step (1) is 7 - 7.

5.

4. The preparation method of an antioxidant and odorless heme polypeptide complex according to claim 1, characterized in that: In step (2), the temperature of the constant temperature water bath is in the range of 25 - 60 °C, and the enzymatic hydrolysis time is between 2 - 6 h.

5. The preparation method of an antioxidant and non-fishy heme polypeptide complex according to claim 4, characterized in that The specific process of stepwise enzymatic hydrolysis is as follows: Add 5500 U / g of subtilisin to the blood meal solution in step (1), mix well, place it in a constant temperature water bath at 45 °C for enzymatic hydrolysis for 3 h, inactivate the enzyme at 90 °C for 10 min, cool to room temperature, centrifuge at 4000 r / min for 10 min, and collect the supernatant; Dropwise add 0.1 moL / L of food-grade sodium bicarbonate solution to the supernatant, control the pH to 7.5, then add 2500 U / g of neutral aspergillus oryzae protease to the supernatant, mix well and place it in a constant temperature water bath at 55 °C for enzymatic hydrolysis for 2 h.

6. The preparation method of an antioxidant and odorless heme polypeptide complex according to claim 5, characterized in that: In step (3), the centrifugation rate is 8000 rpm, and the centrifugation time is 10 min.

7. The preparation method of an antioxidant and odorless heme polypeptide complex according to claim 1, characterized in that: In step (4), the polypeptide solution is mixed with a hemin solution with a concentration of 0.5 mg / mL at a volume ratio of 3:1.

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