Preparation method of fibrinogen proteolysate and application thereof in minced meat products

Abstract

The application discloses a preparation method of fibrinogen enzymolysis product and application of the fibrinogen enzymolysis product in minced meat products, and has the characteristics that the method comprises the following steps: 1) immediately after poultry is slaughtered, blood is collected and sodium citrate and NaCl are added; supernatant is obtained through centrifugation and is frozen; after being thawed, the supernatant is centrifuged, the precipitate is dissolved in normal saline, and the supernatant is obtained through centrifugation; mannitol is added, and fibrinogen powder is obtained through freeze-drying; 2) the freeze-dried fibrinogen powder is dissolved in water, baking soda is used to adjust the pH to 7.5-8.5, trypsin is added to carry out an enzymolysis reaction, citric acid is used to adjust the pH to 5.5-7.0, and flavor protease is added to carry out an enzymolysis reaction; the enzymolysis liquid is collected, is subjected to ultrasonic pretreatment, and is subjected to small-molecule peptide segment interception; and fibrinogen enzymolysis product freeze-dried powder is obtained through freeze-drying, and the method has the advantages that the method can effectively improve the gelatinous characteristics of myofibrillar protein, and improve the water retention and texture characteristics of steamed minced meat products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of myofibrillar protein gel enhancer preparation, and particularly relates to a preparation method of a fibrinogen enzymatic hydrolysate for enhancing the performance of myofibrillar protein gel and application of the fibrinogen enzymatic hydrolysate in a steamed mince meat product. BACKGROUND

[0002] Dietary protein is generally considered to be one of the most important nutrients for human growth and development. Meat and meat products generally provide high-value animal protein for human diets. Lean meat generally contains about 20% protein, of which myofibrillar protein accounts for 60%-70%. During the thermal processing of mince meat products, the thermal gel formation properties of myofibrillar protein play a crucial role in the quality and mouthfeel of the final product. The gel phenomenon is attributed to the formation of a continuous protein network, involving conformational changes within the protein, i.e. denaturation and aggregation upon heating. In this way, myofibrillar proteins gradually unfold under the driving force of thermal energy input, and denatured protein molecules are interconnected through intermolecular forces such as disulfide bonds and hydrophobic bonds in a certain degree of order, ultimately forming a three-dimensional gel network. However, the single myofibrillar protein gel network is rough and uneven, and the gel strength is poor, and the meat products are often accompanied by water and oil loss.

[0003] In actual production, in order to improve the texture and water holding properties of gel meat products, polysaccharides or proteins and other exogenous substances are often added, and at present, the main focus is on carrageenan, soy protein isolate, whey protein, etc. However, these non-meat-derived proteins may introduce potential allergens; in addition, soy protein isolate has a beany odor or other undesirable flavors, the stability of carrageenan is easily affected by temperature fluctuations, and the production cost of whey protein is high, which will all affect the market acceptance and safety of gel meat products and further limit their application range. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a preparation method of a fibrinogen enzymatic hydrolysate which is simple in process and natural in composition and can enhance the performance of myofibrillar protein gel and application of the fibrinogen enzymatic hydrolysate in mince meat products, so as to improve the water holding and texture properties of steamed mince meat products.

[0005] The technical solution adopted by the present application to solve the above technical problem is: a preparation method of a fibrinogen enzymatic hydrolysate, comprising the following steps:

[0006] (1) Extraction of fibrinogen: immediately after poultry is slaughtered, blood is collected, 3.5-4.0wt% sodium citrate and 0.04-0.06wt% NaCl anticoagulants are added, centrifugation is carried out at 3000-5000g for 5-15min, the supernatant is frozen at-20℃ for 12-24h, after thawing, centrifugation is carried out at 3000-5000g for 12-18min, the precipitate is dissolved in normal saline, centrifugation is carried out at 3000-5000g for 10-20min, the supernatant is collected, 1-1.5wt% mannitol is added to the supernatant, and the freeze-dried fibrinogen powder is obtained after freeze-drying;

[0007] (2) Preparation of fibrinogen enzymolysis product: the freeze-dried fibrinogen powder obtained in step (1) is dissolved in water, the pH is adjusted to 7.5-8.5 by using baking soda, and trypsin with an activity of 8000-10000U / g is added to carry out enzymolysis reaction, and the enzymolysis is carried out at 35-38℃ for 3-5h; then the pH is adjusted to 5.5-7.0 by using citric acid, and flavor protease with an activity of 1000-1500U / g is added to carry out enzymolysis reaction, and the enzymolysis is carried out at 48-52℃ for 3-5h, the enzymolysis liquid is collected, then ultrasonic pretreatment is carried out at 120-180W for 10-15min, then a tangential ultrafiltration system equipped with a 3kDa membrane is used to intercept small molecular peptides, and the freeze-dried powder of the fibrinogen enzymolysis product for enhancing the gel property of myofibrillar protein is obtained by freeze-drying.

[0008] Preferably, the addition amount of the trypsin and the flavor protease is 2.5-4% of the mass of the fibrinogen powder.

[0009] Preferably, the fibrinogen enzymolysis product comprises the following hydrolyzed peptides, and the amino acid sequences are as follows: DNDNDKFDGNCAEQDGSGWWMNKCHAG, GGSISHGPGSVPGTGSPGGLKPGS, FDGNCAEQDGSGWWMNKC, LGGDAGDAFDGFDFGDDSSDK, DGNCAEQDGSGWWMNKC, STGTWDSGHPDPGSAGTWKPG, SGQHEHGSVGSWKPGSSGSGSLRP, TETIEGVDAEDGHIPGDQQK, FDGNCAEQDGSGWWMNK, PVVSGKECEEIIRNGGETSEM.

[0010] The application further provides a use of the fibrinogen enzymolysis product in preparation of a myofibrillar protein gel enhancer.

[0011] The application further provides a use of the fibrinogen enzymolysis product in a steamed minced meat product.

[0012] Preferably, the steamed minced meat products include pork balls, beef balls and lion heads.

[0013] Preferably, the method comprises the following steps: weighing 8-15 kg of minced meat, adding fibrinogen enzymatic hydrolysate freeze-dried powder 5-10%, starch 5-8%, sodium chloride 2-3%, composite phosphate salt mixed by sodium tripolyphosphate and sodium pyrophosphate at a mass ratio of 1:1 0.5-1%, pure water 8-10%, egg white 3-4%, pepper 3-4%, cooking wine 3-6%, onion powder 0-1.5%, ginger powder 0-1.5%, and fully chopping and mixing at a speed of 30-40 r / min for 10-15 min to prepare the minced meat product with a particle size of 3.5-8 cm, and then cooking at 75-100 DEG C for 35-120 min and taking out.

[0014] Compared with the prior art, the fibrinogen enzymatic hydrolysate preparation method and application have the advantages that the fibrinogen enzymatic hydrolysate is prepared by using the fibrinogen rich in the discarded blood generated in the animal slaughtering process as raw material and by using the biological directional enzymolysis and ultrafiltration membrane separation technology, the biomass resource is effectively utilized, the added value of the raw material is improved, the production process is simple, the raw material cost is low, the freeze-dried powder is easy to store, and large-scale industrial production is easy to realize. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Table 6 is the relative content of amino acid components of the fibrinogen enzymatic hydrolysate;

[0016] Figure 2 Fig. 6 is the molecular docking visualization result of Pep1 and myosin, wherein A is a 3D model and B is a 2D model;

[0017] Figure 3 Fig. 7 is the molecular dynamics simulation result of myosin and the Pep1 peptide segment complex, wherein A is the root mean square deviation RMSD, B is the root mean square fluctuation RMSF, C is the radius of gyration Rg, and D is the number of hydrogen bonds;

[0018] Figure 4 Fig. 8 is the change of the water holding capacity of myofibrillar protein gel with different fibrinogen enzymatic hydrolysate addition amounts;

[0019] Figure 5Changes of cooking loss of myofibrillar protein gel with different addition amounts of fibrinogen hydrolysate

[0020] Figure 6 Changes of hardness of myofibrillar protein gel with different addition amounts of fibrinogen hydrolysate

[0021] Figure 7 Changes of cohesiveness of myofibrillar protein gel with different addition amounts of fibrinogen hydrolysate

[0022] Figure 8 Changes of gumminess of myofibrillar protein gel with different addition amounts of fibrinogen hydrolysate

[0023] Figure 9 Changes of chewiness of myofibrillar protein gel with different addition amounts of fibrinogen hydrolysate

[0024] Figure 10 Changes of microstructure of myofibrillar protein gel, left: scanning electron microscope results; right: atomic force microscope results, right side from top to bottom: A Comparative Example 1, B Example 1, C Example 2, D Example 3, E Example 4

[0025] Figure 11 Changes of intermolecular force of myofibrillar protein gel. DETAILED DESCRIPTION

[0026] The application will be further described in detail below with reference to the accompanying drawings.

[0027] I. Experimental methods

[0028] 1. Determination of active thiol groups and free amino acid components of fibrinogen hydrolysate

[0029] Determination of active thiol groups: 0.5 mL of 1 mg / mL enzyme solution was mixed with 4.5 mL of Tris-glycine buffer (0.086 mol / L Tris, 0.09 mol / L glycine, 4 mmol / L EDTA, pH 8.0), then 0.5 mL of Ellman reagent was added, vortexed and mixed, and then reacted at 30°C for 30 min, and then the absorbance was determined at a wavelength of 412 nm.

[0030] Thiol content / (nmol / mg) = A / (p x 13600) x 10 6 x 11, where A is the absorbance of the sample group at a wavelength of 412 nm; p is the protein mass concentration / (mg / mL); and 11 is the dilution factor.

[0031] Free amino acid composition determination: The free amino acid composition of the sample was determined using an automatic amino acid analyzer. The enzyme hydrolysate was mixed with sulfosalicylic acid at a ratio of 4:1, centrifuged (12000g, 15 min), and the supernatant was again centrifuged (12000g, 15 min), diluted, filtered through a 0.22 μm filter, and then analyzed.

[0032] 2. Texture determination

[0033] The TPA mode was selected for texture parameter determination, and the deformation of the sample was set to 50% of its own height. A cylindrical probe (model TA / 20) was selected, and the pre-test speed, test speed, and post-test speed were set to 2.00 mm x s -1 , 0.80 mm x s -1 , and 0.80 mm x s -1 , respectively. The gel strength was determined using a TA / 0.5 probe, with a compression deformation of 50% and a trigger force of 5 g. The pre-test speed, test speed, and post-test speed were 1.5 mm x s -1 , 1 mm x s -1 , and 1 mm x s -1 , respectively, and the gel strength was expressed as the breaking strength x breaking distance.

[0034] 3. Water holding capacity and syneresis determination

[0035] The mass of the empty beaker was measured before heating and recorded as C, and the total mass of the beaker and protein solution was recorded as A. After heating and cooling, the evaporated water was removed, and the total mass of the beaker and gel was measured and recorded as B. The syneresis loss was calculated as follows: Syneresis loss (%) = (A-B) / (A-C) x 100. The gel was centrifuged at 10000 g for 10 min. The supernatant was then removed. The water holding capacity (%) was expressed as the percentage of the weight after centrifugation relative to the weight before centrifugation.

[0036] 4. Interaction force determination

[0037] The gel was treated with the following reagents: SA (0.05 M NaCl), SB (0.6 M NaCl), SC (0.6 M NaCl, 1.5 M urea), SD (0.6 M NaCl, 8 M urea), and SE (0.6 M NaCl, 8 M urea, 0.5 M β-mercaptoethanol). The gel was homogeneously dissolved in the above solutions and centrifuged at 8000 g for 10 min. The supernatant was centrifuged at 8000 g for 10 min. The non-specific binding, ionic bonds, hydrogen bonds, hydrophobic interactions, and disulfide bond strengths between proteins were reflected by the differences in protein content in the supernatant in the SA solution, SB and SA, SC and SB, SD and SC, and SE and SD, respectively.

[0038] 5. Atomic force microscopy

[0039] Drop 40 μΐ of protein solution on a fresh glass slide, put into a petri dish, heat in a 75 °C water bath for 20 min to gelate. After heating, the gels were equilibrated at ambient conditions for 30 min. AFM imaging of the samples was performed using AFM in tapping mode on a Bicope (BS3-02). Height images of the gel surface (3.0 x 3.0 μm 2 area) were automatically drawn at a scan speed of 1.0 Hz, and the average roughness (Rq) of the gel surface was calculated using the software.

[0040] 6. Gel micro-network assay

[0041] The microstructure of the gels was observed by scanning electron microscopy. The gels were fixed with liquid nitrogen for 24 h, freeze-dried, gold-sprayed, and imaged at an acceleration voltage of 3 kV and a magnification of 500.

[0042] 7. LC-MS / MS mass spectrometry identification

[0043] The enzyme hydrolysate obtained by separating and filtering the fibrinogen after proteolysis was dissolved with NH4HCO3solution, and DTT solution and iodoacetamide solution were added. A self-filled desalting C18 column (75 μm i.d. x 50 cm, NanoViper C18 1.9 μm, 100 A) was used, and after dissolution with 0.1% formic acid, the sample was detected by mass spectrometry. The elution program is shown in the following table.

[0044] Table 1 LC-MS / MS elution program

[0045]

[0046] 8. Molecular docking and molecular dynamics simulation

[0047] The polypeptide obtained by separating and filtering the fibrinogen double enzyme hydrolysate through a 3 kDa ultrafiltration was subjected to amino acid sequence identification. The amino acid sequence DNDNDKFDGNCAEQDGSGWWMNKCHAG (named: Pep1) was selected for molecular docking, and myosin was selected as PDB ID: 5M05. Docking software: ZDOCK 3.0.2, docking results were predicted using prodigy server: https: / / wenmr.science.uu.nl / prodigy / , and mapping software: PyMOL 2.5.5 and Discovery studio 2020.

[0048] Molecular dynamics simulations were performed on Myosin and Myosin-Pep1 complex, respectively. The topological file of the protein was constructed on a supercomputer platform with the help of GROMACS2021 software, the force field was ff14SB, and the water model was TIP3P. The peptide and protein were placed in a dodecahedron geometry container, and water molecules, Na + and Cl- were filled to achieve the solvation and charge neutralization of the system. The system was energy minimized by performing the steepest descent method, and then temperature control simulation was performed under the NVT ensemble framework until the system temperature stabilized at 310 K and 1 bar. Finally, molecular dynamics simulation was carried out, and the simulation time was set to 50 ns. II. DETAILED EMBODIMENTS

[0050] Example 1

[0051] A preparation method of a fibrinogen hydrolysate, comprising the following steps:

[0052] (1) Extraction of fibrinogen: immediately after the chicken is slaughtered, blood is collected, 3.8 wt% sodium citrate and 0.05 wt% NaCl anticoagulant are added, centrifuged at 4000g for 10 min, the supernatant is frozen at -20℃ for 24h, after thawing, the precipitate is dissolved in physiological saline and centrifuged at 4000g for 15 min, the supernatant is collected, 1.5 wt% mannitol is added to the supernatant, and the freeze-dried fibrinogen powder is obtained after freeze-drying;

[0053] (2) Preparation of fibrinogen hydrolysate: the freeze-dried fibrinogen powder obtained in step (1) is dissolved in water, the pH is adjusted to 7.5 with baking soda, and trypsin with an activity of 9000 U / g protein is added for enzymatic reaction, and the enzymatic reaction is carried out at 37℃ for 5h; then the pH is adjusted to 5.5-7.0 with citric acid, and flavor protease with an activity of 1000-1500 U / g protein is added for enzymatic reaction, and the enzymatic reaction is carried out at 50℃ for 3h, the amount of each enzyme added is 2.5% of the mass of the fibrinogen powder, the enzyme solution is collected, then ultrasonic pretreatment is carried out at 150W for 10min, then a tangential ultrafiltration system equipped with a 3kDa membrane is used to intercept small molecular peptides, and the freeze-dried powder of the fibrinogen hydrolysate for enhancing the gel performance of myofibrillar protein is obtained by freeze-drying, and is stored in a freezer at -20℃ for standby.

[0054] Example 2

[0055] A preparation method of a fibrinogen hydrolysate, comprising the following steps:

[0056] (1) Extraction of fibrinogen: immediately after the chicken is slaughtered, blood is collected, 3.8wt% sodium citrate and 0.05wt% NaCl anticoagulant are added, centrifugation is performed at 4000g for 10min, the supernatant is frozen at -20℃ for 24h, after thawing, centrifugation is performed at 4000g for 10min, the precipitate is dissolved with normal saline, centrifugation is performed at 4000g for 15min, the supernatant is collected, 1wt% mannitol is added to the supernatant, and after freeze-drying, the freeze-dried fibrinogen powder is obtained;

[0057] (2) Preparation of fibrinogen enzymatic hydrolysate: the freeze-dried fibrinogen powder obtained in step (1) is dissolved with water, the pH is adjusted to 8.0 with baking soda, and trypsin with an activity of 9000U / g protein is added for enzymatic hydrolysis reaction, and the enzymatic hydrolysis is performed at 37℃ for 5h; then, the pH is adjusted to 5.5-7.0 with citric acid, and flavor protease with an activity of 1000-1500U / g protein is added for enzymatic hydrolysis reaction, and the enzymatic hydrolysis is performed at 50℃ for 5h, the addition amount of each enzyme is 4% of the mass of the fibrinogen powder, the enzymatic hydrolysate is collected, then ultrasonic pretreatment is performed at 150W for 8min, then a tangential ultrafiltration system equipped with a 3kDa membrane is used to intercept small molecular peptides, and after freeze-drying, the freeze-dried powder of the fibrinogen enzymatic hydrolysate for enhancing the gel property of myofibrillar protein is obtained, and is stored in a freezer at -20℃ for standby use.

[0058] Example 3

[0059] A method for preparing a fibrinogen enzymatic hydrolysate, comprising the following steps:

[0060] (1) Extraction of fibrinogen: immediately after the chicken is slaughtered, blood is collected, 3.8wt% sodium citrate and 0.05wt% NaCl anticoagulant are added, centrifugation is performed at 4000g for 10min, the supernatant is frozen at -20℃ for 12h, after thawing, centrifugation is performed at 4000g for 15min, the precipitate is dissolved with normal saline, centrifugation is performed at 4000g for 15min, the supernatant is collected, 1wt% mannitol is added to the supernatant, and after freeze-drying, the freeze-dried fibrinogen powder is obtained;

[0061] (2) Preparation of fibrinogen hydrolysate: the fibrinogen powder obtained in step (1) was dissolved in water, and the pH was adjusted to 7.5 with baking soda. Trypsin with an activity of 9000 U / g protein was added for enzymatic reaction, and the enzymatic reaction was carried out at 37°C for 4 h. Then, the pH was adjusted to 5.5-7.0 with citric acid, and flavourzyme with an activity of 1000-1500 U / g protein was added for enzymatic reaction, and the enzymatic reaction was carried out at 50°C for 4 h. The amount of each enzyme added was 3% of the mass of the fibrinogen powder. The enzymatic solution was collected, and then ultrasonic pretreatment was carried out at 150 W for 10 min. Then, a tangential ultrafiltration system equipped with a 3 kDa membrane was used to intercept small molecular peptides. The fibrinogen hydrolysate freeze-dried powder for enhancing the gel property of myofibrillar protein was obtained by freeze-drying, and was stored in a freezer at -20°C for standby use.

[0062] Example 4

[0063] A method for preparing a fibrinogen hydrolysate, comprising the following steps:

[0064] (1) Extraction of fibrinogen: immediately after the chicken was slaughtered, blood was collected, and 3.8 wt% sodium citrate and 0.05 wt% NaCl anticoagulant were added. Centrifugation was carried out at 4000 g for 10 min, and the supernatant was frozen at -20°C for 12 h. After thawing, centrifugation was carried out at 4000 g for 15 min, and the precipitate was dissolved in physiological saline. Centrifugation was carried out at 4000 g for 15 min again, and the supernatant was collected. 1 wt% mannitol was added to the supernatant, and freeze-drying was carried out to obtain fibrinogen powder.

[0065] (2) Preparation of fibrinogen hydrolysate: the fibrinogen powder obtained in step (1) was dissolved in water, and the pH was adjusted to 8.5 with baking soda. Trypsin with an activity of 9000 U / g protein was added for enzymatic reaction, and the enzymatic reaction was carried out at 37°C for 5 h. Then, the pH was adjusted to 5.5-7.0 with citric acid, and flavourzyme with an activity of 1000-1500 U / g protein was added for enzymatic reaction, and the enzymatic reaction was carried out at 50°C for 5 h. The amount of each enzyme added was 3% of the mass of the fibrinogen powder. The enzymatic solution was collected, and then ultrasonic pretreatment was carried out at 150 W for 5 min. Then, a tangential ultrafiltration system equipped with a 3 kDa membrane was used to intercept small molecular peptides. The fibrinogen hydrolysate freeze-dried powder for enhancing the gel property of myofibrillar protein was obtained by freeze-drying, and was stored in a freezer at -20°C for standby use.

[0066] III. Analysis of experimental results

[0067] 1. Analysis of fibrinogen hydrolysate

[0068] The fibrinogen proteolysate prepared in Example 1 was subjected to active thiol and free amino acid component determination. It was found that the fibrinogen proteolysate was rich in active thiol fragments of about 750 nmol / mg. As shown in Table 1, the hydrophobic amino acids of the proteolysate accounted for 43%, the ratio of hydrophilic / hydrophobic amino acids was 1.33, the glutamine content reached about 23%, and the cysteine content was 1.7%. Figure 1

[0069] The fibrinogen proteolysate was further subjected to mass spectrometry identification and database retrieval by software PEAKS Studio (10.6) to obtain the identification results, and Table 2 shows the 10 peptide fragments with higher scores.

[0070] Table 2 Hydrolyzed peptide amino acid sequence results of fibrinogen complex proteolysis product

[0071]

[0072] As shown in Table 2, the first polypeptide chain DNDNDKFDGNCAEQDGSGWWMNKCHAG was found to be partially consistent with the sequence of the gamma chain of fibrinogen (PDB: A0A5G2QUU1) through comparative search; the second polypeptide chain GGSISHGPGSVPGTGSPGGLKPGS was found to be partially consistent with the sequence of the alpha chain of fibrinogen (PDB: F1RX36) through comparative search; and the third FDGNCAEQDGSGWWMNKC was consistent with the gamma chain. The results show that the mass spectrometry identified amino acid sequence is consistent with the real protein sequence of fibrinogen in pig blood, showing the accuracy of the amino acid sequence result identification. Further, the computer molecular docking technology was used to simulate the interaction between the DNDNDKFDGNCAEQDGSGWWMNKCHAG peptide (Pep1) and myosin (Table 3). Figure 2

[0073] Table 3 Molecular docking results of the peptide Pep1 and myosin

[0074]

[0075]

[0076] The results show that Pep1 and myosin molecules are combined through various interactions such as hydrophobic interaction, hydrogen bond, salt bridge and van der Waals force, involving multiple amino acid residues, with more hydrophobic interaction and hydrogen bond, showing a high degree of complex combination. The binding energy of Pep1 and myosin is -9.4 kcal / mol, indicating that the combination is a spontaneous dynamic process. Through molecular dynamics simulation of the myosin-Pep1 peptide complex, the overall flexibility change in the simulation process was observed, and the intermolecular interaction mechanism was further explored.​​

[0077] Root mean square deviation (RMSD) values of protein backbone are often used to evaluate the structural deviation of protein from the initial structure conformation to the final position, and lower RMSD values usually represent higher protein structural stability, as shown in Figure 3 A, the complex shows a higher root mean square deviation value (RMSD), indicating that the binding of the Pep1 peptide segment to myosin improves the structural stability. Root mean square fluctuation (RMSF) is used to evaluate the flexibility of each residue to reveal the local changes of protein structure, and lower RMSF values indicate a more rigid secondary structure, as shown in Figure 3 B, the RMSF of the complex is within 0.2 nm, indicating that the protein main structure is very rigid, which may be the effect achieved by binding Pep1. The radius of gyration (Rg) value is often used to evaluate the compactness of the molecular complex, and smaller Rg values usually mean that the molecular complex is more compact, as shown in Figure 3 C, during the simulation, the Rg value of the complex system is large, and the overall system is in a loose state, which can provide more hydrogen bond binding sites. Hydrogen bonding is one of the strongest non-covalent interactions, and the number of hydrogen bonds between the short peptide and the protein during the 50 ns molecular dynamics simulation was monitored, as shown in Figure 3 D, the number of hydrogen bonds formed between the short peptide and the protein during the simulation is 3-16, and the number of hydrogen bonds is 5 in the early simulation and about 8 in the late simulation, indicating that Pep1 plays an important role in the stable binding of the complex.

[0078] 2. Analysis of the gel properties of the complex myofibrillar protein

[0079] Extraction of myofibrillar protein: After the pork was ground into a paste, it was dissolved in 10 mM PBS buffer solution (0.1 M NaCl, 2 mM MgCl2, 1 mM EGTA, pH 7.0) at a ratio of 1:4 (w / v), homogenized at 13500 rpm for 10 s, and then centrifuged at 10000 g for 10 min, and the precipitate was collected. The above steps were repeated 3 times, and the obtained precipitate was used as myofibrillar protein and stored at 4°C.

[0080] Preparation of complex myofibrillar protein gel: The obtained myofibrillar protein was dissolved in 0.6 M NaCl solution, and the protein concentration was adjusted to 45 mg / mL. Fibrinogen proteolysate was added (0.5 mg / mL for Example 1 group, 1 mg / mL for Example 2 group, 2 mg / mL for Example 3 group, and 4 mg / mL for Example 4 group), and then mixed at 8000 rpm for 10 s. The mixture was then placed in a water bath at 30°C for 20 min, and the temperature was increased to 75°C in a linear manner, and the mixture was kept at this temperature for 20 min. After heating, the complex gel was immediately immersed in ice water for cooling, and stored at 4°C.

[0081] Comparative Example 1 was prepared using the same method as above, except that no fibrinogen hydrolysate was added. The gel properties of each group were tested, and the results are as follows.

[0082] Figure 4 The effect of different amounts of fibrinogen hydrolysate added on the water retention of myofibrillar protein gel was demonstrated. With the increase of the amount of fibrinogen hydrolysate added, the water retention of myofibrillar protein gel showed a significant increasing trend. Compared with comparative example 1, the water retention capacity of Example 4 with an addition amount of 4 mg / mL increased significantly by 9.5%, effectively improving the water retention performance of myofibrillar protein gel.

[0083] Figure 5 The effects of different amounts of fibrinogen hydrolysate added on the cooking loss of myofibrillar protein gel were demonstrated. As the amount of fibrinogen hydrolysate added increased, the cooking loss of myofibrillar protein gel showed a significant decreasing trend. Compared with Comparative Example 1, the cooking loss of Example 4 with an addition amount of 4 mg / mL decreased significantly by 18.9%, and there was no significant difference between Example 4 and the groups of Example 2 with an addition amount of 1 mg / mL and Example 3 with an addition amount of 2 mg / mL.

[0084] Figure 6 The effect of different amounts of fibrinogen hydrolysate added on the hardness of myofibrillar protein gel was demonstrated. With the increase in the amount of fibrinogen hydrolysate added, the hardness of myofibrillar protein gel showed a significant increasing trend. Compared with Comparative Example 1, the hardness of Example 4 group with an addition amount of 4 mg / mL increased significantly by 50.4%, and there was no obvious difference between Example 4 and Example 3 group with an addition amount of 2 mg / mL.

[0085] Figure 7 The effects of different fibrinogen hydrolysate addition amounts on the cohesion of myofibrillar protein gel were demonstrated. With the increase in the amount of fibrinogen hydrolysate added, the cohesion of myofibrillar protein gel showed a significant increasing trend. Compared with Comparative Example 1, the cohesion of Example 4 group with an addition amount of 4 mg / mL increased significantly by 76.3%. Considering the cost, cooking loss and hardness, the amount of fibrinogen hydrolysate added was not further increased for testing.

[0086] Figure 8 The effects of different amounts of fibrinogen hydrolysate added on the adhesion of myofibrillar protein gel were demonstrated. With the increase in the amount of fibrinogen hydrolysate added, the adhesion of myofibrillar protein gel showed a significant increasing trend. Compared with Comparative Example 1, the adhesion of Example 4 with an addition amount of 4 mg / mL significantly increased by 150%, and there was no obvious difference between Example 4 and Example 3 with an addition amount of 2 mg / mL.

[0087] Figure 9 The effect of different fibrinogen enzymatic hydrolysate addition amount on the chewiness of myofibrillar protein gel was demonstrated. With the increase of fibrinogen enzymatic hydrolysate addition amount, the chewiness of myofibrillar protein gel showed a significant increasing trend. Compared with Comparative Example 1, the chewiness of Example 4 group with 4 mg / mL addition amount increased by 154%, and Example 3 had a larger increase compared with Example 2 group. Example 4 did not show obvious difference compared with 2 mg / mL addition amount Example 3 group. Considering the cost and actual application effect, the addition amount of fibrinogen enzymatic hydrolysate was not further improved.

[0088] Figure 10 The effect of different fibrinogen enzymatic hydrolysate addition amount on the microstructure of myofibrillar protein was demonstrated. The microstructure of myofibrillar protein single gel network of Comparative Example 1 showed irregular and disordered filamentous network. With the increase of fibrinogen enzymatic hydrolysate addition amount, the cross-linking of protein in the gel structure increased, and the cross-linking degree of protein in Example 4 was the largest. The average roughness of the gel surface decreased, forming a more continuous and uniform gel network structure. This three-dimensional network structure enabled the gel to capture more water and prevent the gel from collapsing, increasing the hardness of the gel. This was consistent with the change of water holding capacity and gel texture characteristics of the above composite gel.

[0089] Figure 11 The effect of different fibrinogen enzymatic hydrolysate addition amount on the intermolecular force of myofibrillar protein was demonstrated. The results showed that the composite gel of different examples was mainly hydrophobic bond and disulfide bond as the main force. With the increase of fibrinogen enzymatic hydrolysate addition amount, the intermolecular hydrophobic bond and disulfide bond of myofibrillar protein gel showed a significant increasing trend. Compared with Comparative Example 1, the content of disulfide bond and hydrophobic bond in Example 4 was the highest.

[0090] In summary, considering the cost and actual application effect, the concentration of myofibrillar protein was about 45 mg / mL, and the addition amount of fibrinogen enzymatic hydrolysate was preferably 2-4 mg / mL, which was equivalent to about 4-9% in myofibrillar protein. Therefore, in the actual application example, 5-10% of blood fibrinogen enzymatic hydrolysate freeze-dried powder was added according to the weight of meat paste.

[0091] Four, application examples

[0092] Application Example 1

[0093] The application of the fibrinogen hydrolysate prepared in a specific embodiment in pork balls includes the following steps: 10 kg of minced pork paste (fat and lean ratio 1:6) is weighed, and 5% of fibrinogen hydrolysate freeze-dried powder, 6% of starch, 3% of sodium chloride, 1% of compound phosphate (sodium tripolyphosphate and sodium pyrophosphate 1:1), 9% of pure water, 4% of egg white, 4% of pepper powder, and 4% of cooking wine are added according to the weight of the paste, and then the mixture is fully chopped and mixed at a speed of 35 r / min for 15 min, and then the pork balls with a particle size of 5 cm are made by using a ball making machine, and then the pork balls are cooked at 85℃ for 45 min, taken out, and then cooled and stored at 4℃.

[0094] Application Example 2

[0095] The application of the fibrinogen hydrolysate prepared in a specific embodiment in beef balls includes the following steps: 8 kg of minced beef paste (fat and lean ratio 1:4) is weighed, and 8% of fibrinogen hydrolysate freeze-dried powder, 5% of starch, 3% of sodium chloride, 0.5% of compound phosphate (sodium tripolyphosphate and sodium pyrophosphate 1:1), 8% of pure water, 3% of egg white, 3% of pepper powder, and 3% of cooking wine are added according to the weight of the paste, and then the mixture is fully chopped and mixed at a speed of 30 r / min for 10 min, and then the beef balls with a particle size of 3.5 cm are made by using a ball making machine, and then the beef balls are cooked at 75℃ for 35 min, taken out, and then cooled and stored at 4℃.

[0096] Application Example 3

[0097] The application of the fibrinogen hydrolysate prepared in a specific embodiment in lion head includes the following steps: 15 kg of minced pork paste (fat and lean ratio 1:2) is weighed, and 10% of fibrinogen hydrolysate freeze-dried powder, 8% of starch, 2% of sodium chloride, 0.8% of compound phosphate (sodium tripolyphosphate and sodium pyrophosphate 1:1), 10% of pure water, 4% of egg white, 4% of pepper powder, 6% of cooking wine, 1.5% of minced onion, and 1.5% of minced ginger are added according to the weight of the paste, and then the mixture is fully chopped and mixed at a speed of 40 r / min for 15 min, and then the lion head with a particle size of about 8 cm is made by using a ball making machine, and then the lion head is cooked in boiling water for 5 min to shape, and then the lion head is cooked at 100℃ for 120 min, taken out, and then cooled and stored at 4℃.

[0098] Application Comparative Example 1

[0099] The same as the above-mentioned application example 1, except that no fibrinogen hydrolysate freeze-dried powder is added during the preparation of the pork balls.

[0100] Application Comparative Example 2

[0101] The same as the above-mentioned application example 1, except that 5% of soybean protein isolate is added during the preparation of the pork balls to replace the fibrinogen hydrolysate freeze-dried powder.

[0102] Application Comparative Example 3

[0103] The same as the above application example 2, except that no blood fibrinogen enzymatic hydrolysate freeze-dried powder was added during the process of making beef balls.

[0104] Application Comparative Example 4

[0105] The same as the above application example 2, except that 5% soybean protein isolate was added during the process of making beef balls to replace the blood fibrinogen enzymatic hydrolysate freeze-dried powder.

[0106] Application Comparative Example 5

[0107] The same as the above application example 3, except that no blood fibrinogen enzymatic hydrolysate freeze-dried powder was added during the process of making lion heads.

[0108] Application Comparative Example 6

[0109] The same as the above application example 3, except that 5% soybean protein isolate was added during the process of making lion heads to replace the blood fibrinogen enzymatic hydrolysate freeze-dried powder.

[0110] The changes in quality indicators of different meat paste products after cooking are shown in Table 4.

[0111] Table 4 Changes in water holding and texture properties of different meat paste products after cooking

[0112]

[0113] As can be seen from Table 4, the fibrinogen enzymatic hydrolysate as a myofibrillar protein gel enhancer effectively improves the water holding and texture quality deterioration problems of the meat emulsion products during cooking. Cooking loss and centrifugal water holding capacity are often used to determine the water holding effect of meat products. Compared with the blank control group and the soybean protein isolate control group, the cooking loss of the application example 1 pork ball product is decreased by 33.3% and 12.6% respectively, and the water holding capacity is increased by 13.0% and 4.0% respectively; the cooking loss of the application example 2 beef ball product is decreased by 29.4% and 12.2% respectively, and the water holding capacity is increased by 9.2% and 3.2% respectively; the cooking loss of the application example 3 lion head is decreased by 21.6% and 11.4% respectively, and the water holding capacity is increased by 13.0% and 3.7% respectively. Hardness, elasticity and chewiness are often considered as important indicators for evaluating the texture characteristics of cooked meat products. Compared with the blank control group and the soybean protein isolate control group, the hardness of the application example 1 pork ball product is increased by 54.0% and 8.5% respectively, the elasticity is increased by 40.2% and 13.9% respectively, and the chewiness is increased by 45.7% and 15.8% respectively; the hardness of the application example 2 beef ball product is increased by 40.7% and 6.3% respectively, the elasticity is increased by 35.5% and 10.8% respectively, and the chewiness is increased by 39.7% and 12.0% respectively; the hardness of the application example 3 lion head is increased by 36.8% and 9.2% respectively, the elasticity is increased by 29.0% and 9.6% respectively, and the chewiness is increased by 37.3% and 14.5% respectively.

[0114] In summary, the fibrinogen enzymatic hydrolysate as a myofibrillar protein gel enhancer effectively improves the quality and microstructure of myofibrillar protein gel, and the influencing mechanism is related to the enhancement of disulfide bond and hydrophobic interaction and other protein intermolecular forces; in addition, the addition of the fibrinogen enzymatic hydrolysate further shows the quality improvement effect on the cooked meat emulsion products such as pork balls, beef balls and lion heads, which can significantly enhance the water holding capacity of the meat products and improve their texture characteristics, which is better than the commonly used soybean protein isolate additive.

[0115] The above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by ordinary skilled in the art within the essential scope of the present application shall also fall within the protection scope of the present application.

Claims

1. A method for preparing a fibrinogen hydrolysate, characterized in that The following steps are involved: (1) Extraction of fibrinogen: Blood was collected immediately after slaughtering poultry, and 3.5-4.0 wt% sodium citrate and 0.04-0.06 wt% NaCl anticoagulant were added to the blood. The blood was centrifuged at 3000-5000 g for 5-15 min. The supernatant was frozen at -20°C for 12-24 h. After thawing, the supernatant was centrifuged at 3000-5000 g for 12-18 min. The precipitate was dissolved with physiological saline and centrifuged again at 3000-5000 g for 10-20 min. The supernatant was collected and 1-1.5 wt% mannitol was added to the supernatant. The supernatant was lyophilized to obtain lyophilized fibrinogen powder. (2) Preparation of fibrinogen hydrolysate: The freeze-dried fibrinogen powder obtained in step (1) is dissolved in water, the pH is adjusted to 7.5-8.5 with baking soda, and trypsin with an activity of 8000-10000 U / g is added for enzymatic hydrolysis, and the enzymatic hydrolysis is carried out at 35-38°C for 3-5 hours; then, the pH is adjusted to 5.5-7.0 with citric acid, and flavor protease with an activity of 1000-1500 U / g is added for enzymatic hydrolysis, and the enzymatic hydrolysis is carried out at 48-52°C for 3-5 hours. The enzymatic hydrolysate is collected and then ultrasonically pretreated at 120-180W for 10-15 minutes. Then, a tangential ultrafiltration system equipped with a 3kDa membrane is used to intercept small molecule peptides, and the fibrinogen hydrolysate freeze-dried powder is obtained by freeze-drying.

2. The method for preparing a fibrinogen hydrolysate according to claim 1, wherein: The added amounts of the trypsin and the flavor protease are both 2.5-4% of the mass of the fibrinogen powder.

3. The method for preparing a fibrinogen hydrolysate according to claim 1, wherein: The fibrinogen hydrolysate includes the following hydrolyzed peptide segments, and the amino acid sequence is as follows: DNDNDKFDGNCAEQDGSGWWMNKCHAG, GGSISHGPGSVPGTGSPGGLKPGS, FDGNCAEQDGSG WWMNKC, LGGDAGDAFDGFDFGDDSSDK, DGNCAEQDGSGWWMNKC, STGT WDSGHPDPGSAGTWKPG, SGQHEHGSVGSWKPGSSGSGSLRP, TETIEGVDAED GHIPGDQQK, FDGNCAEQDGSGWWMNK, PVVSGKECEEIIRNGGETSEM.

4. Use of a fibrinogen hydrolysate prepared by the method according to any one of claims 1 to 3 in preparing a myofibrillar protein gel enhancer.

5. Use of a fibrinogen hydrolysate prepared by the method according to any one of claims 1 to 3 in steamed minced meat products.

6. The use of the fibrinogen hydrolysate according to claim 5 in cooking minced meat products, characterized in that: The steamed and minced meat products include pork balls, beef balls and lion's head meatballs.

7. The use of the fibrinogen hydrolysate according to claim 5 in cooking minced meat products, characterized in that The method comprises the following steps: weighing 8-15 kg of minced meat, adding 5-10% of fibrinogen enzymatic hydrolysate freeze-dried powder, 5-8% of starch, 2-3% of sodium chloride, 0.5-1% of a composite phosphate prepared by mixing sodium tripolyphosphate and sodium pyrophosphate in a mass ratio of 1:1, 8-10% of purified water, 3-4% of egg white, 3-4% of pepper powder, 3-6% of cooking wine, 0-1.5% of chopped green onion, and 0-1.5% of chopped ginger according to the weight of the minced meat, fully chopping and stirring at a speed of 30-40 r / min for 10-15 minutes to prepare a minced meat product with a particle size of 3.5-8 cm, boiling the minced meat at 75-100 DEG C for 35-120 minutes, and then removing the minced meat product.

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