A method for improving the structure of eel myofibrillar protein
The myofibrillar and bolognese extracts are used to improve the myofibrillar protein structure of eels and prepare the myofibrillar protein conjugate, which solves the problem of difficulty in extracting the eel protein structure, achieves high solubility, antioxidant and flavor enhancement, and is suitable for the processing of low-salt and low-sugar eel products.
Patent Information
- Application Number
- CN202311630312.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-30
- Publication Date
- 2025-08-12
- Estimated Expiration
- 2043-11-30
AI Technical Summary
The structure of eel myofibrillar protein is special, which makes it difficult to extract during processing, poor oxidation resistance, poor flavor, and difficult to digest and absorb.
The extracts of rosemary and bolognese are used to work together to improve the structure of eel myofibrillary protein, and the solubility, antioxidant and digestibility are improved by preparing eel myofibrillary protein conjugates.
It significantly improves the solubility and antioxidant properties of eel myofibrillar protein, improves its flavor characteristics, provides low-salt and low-saccharide eel seasoning products, and enhances the processing and edible value of eel products.
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Abstract
Description
Technical Field
[0001] The invention relates to a method for improving the structure of eel myofibrillar protein and belongs to the field of food processing. Background Art
[0002] Eel, also known as anguilla, primarily grows in tropical and temperate waters. Eel is a nutritious, high-quality protein and essential amino acids, making it a valuable aquatic resource. Eel is also rich in unsaturated fatty acids, which have beneficial health benefits, at levels far higher than those found in similar aquatic and meat products. However, this also makes eel meat susceptible to oxidation during production and processing, making it difficult to preserve and add value.
[0003] Most eel products on the market are processed using methods like grilling and smoking, leveraging the interaction between the protein and lipids in the eel meat to achieve a delicious taste. However, a large amount of seasonings are added during the production process for flavor and preservation, which, while enhancing the taste, inevitably leads to high sugar and salt intake. Eel products prepared using traditional methods like grilling and smoking have a monotonous flavor and low protein digestibility, which can increase the burden on the liver and the risk of cardiovascular disease. With consumers' increasing demand for healthier diets, eel foods, which are high in sugar and salt, face the challenge of monotonous flavors and difficulty reducing sugar and salt content. There is a lack of healthy eel-based seasonings.
[0004] Myofibrillar protein is the largest component of protein in muscle tissue, accounting for over half of the total protein, and is a primary target of research in food protein processing. Conventional meat protein fibers are relatively long, with a high proportion of heavy chains, the largest molecular weight proteins. Methods for their extraction, modification, and utilization are well-established. Fish protein, such as carp, has relatively short fibers, a loosely organized protein structure, and a high water content. Its molecular structure is similar to that of red meat. While the pH environment differs, the methods and mechanisms for improving physicochemical properties such as solubility and hydrophobicity are largely the same. Eel protein, in addition to the common heavy chains, contains a significant number of functional peptides and a significantly higher essential amino acid content than chicken or pork of the same weight. Eel protein differs significantly from other meats in its structure, with a more complex composition and a coarser fiber texture. This makes extraction of eel myofibrillar protein difficult, complicating its utilization and modification. Summary of the Invention
[0005] [Technical Issues]
[0006] The protein structure of eel is different from that of regular meat, and its special characteristics make it difficult to process. Currently, there is no literature on improving the protein structure of eel.
[0007] The myofibrillar protein extracted from eels has poor antioxidant properties, poor flavor, and is difficult to digest and absorb.
[0008] [Technical solution]
[0009] To address these issues, the present invention provides a method for improving the structure of eel myofibrillar protein. This method utilizes the synergistic effects of rosemary and nutmeg extracts to improve the structure of eel myofibrillar protein. This improved eel protein not only possesses excellent flavor binding properties but also has enhanced solubility, antioxidant properties, and digestibility. This method provides a reference for improving and optimizing eel cooking methods.
[0010] The first object of the present invention is to provide a method for improving the structure of eel myofibrillar protein, comprising the following steps:
[0011] (1) Extraction of active ingredients from spices:
[0012] adding ethanol solution to rosemary powder and nutmeg powder respectively, performing ultrasonic extraction, centrifuging, collecting supernatant, rotary evaporating, drying, and crushing to obtain rosemary extract and nutmeg extract; then mixing the rosemary extract and nutmeg extract, and adding them to water to obtain a spice extract solution;
[0013] (2) Extraction of myofibrillar protein:
[0014] Extraction of myofibrillar proteins from eel meat;
[0015] (3) Preparation of protein conjugates:
[0016] The myofibrillar protein is prepared into a myofibrillar protein solution, and then a spice extract solution is added, and the pH is adjusted to 8.0-10.0. The solution is allowed to stand to form a conjugate, and the solution is washed to obtain a paste-like precipitate, i.e., an eel myofibrillar protein conjugate.
[0017] In one embodiment of the present invention, the mass ratio of the rosemary extract to the nutmeg extract in step (1) is 1-3:1-3.
[0018] In one embodiment of the present invention, the ethanol solution in step (1) is an ethanol aqueous solution with a mass fraction of 65-75%.
[0019] In one embodiment of the present invention, the ratio of rosemary powder (or nutmeg powder) to ethanol solution in step (1) is 2-4 g:50 mL.
[0020] In one embodiment of the present invention, the ultrasonic extraction in step (1) is performed at 40-60 kHz and 20-30° C. (normal temperature) for 20-40 min.
[0021] In one embodiment of the present invention, the centrifugation in step (1) is performed at 6000-9000 rpm for 5-20 min.
[0022] In one embodiment of the present invention, the rotary evaporation temperature in step (1) is 30-50°C.
[0023] In one embodiment of the present invention, the drying in step (1) is freeze drying, specifically freeze drying at -80°C for 48 hours.
[0024] In one embodiment of the present invention, the particle size of the powder after pulverization in step (1) is less than 125 microns.
[0025] In one embodiment of the present invention, the method for extracting myofibrillar protein in step (2) is:
[0026] Thaw the eel meat, chop it, add the extract, stir and mash it at 1500-2500 rpm for 0.5-2 min, then heat at 4℃
[0027] Centrifuge at 5000-7000 rpm for 5-15 minutes, discard the supernatant, and add the extract again to the resulting precipitate; repeat the above steps to obtain a centrifuged precipitate;
[0028] Add NaCl solution to the precipitate, stir evenly, filter, adjust the pH to the isoelectric point, centrifuge, and the resulting paste precipitate is myofibrillar protein;
[0029] The extract was 0.01 mol / L phosphate buffer (pH 7.0);
[0030] The dosage ratio (W / V) of eel meat (extraction precipitate) and extract is 1 mg: 4-8 mL;
[0031] The extraction step was repeated 1 to 4 times;
[0032] The ratio of precipitate to NaCl solution (W / V) is 1 mg: 3-5 mL;
[0033] The concentration of NaCl solution is 0.1 mol / L, and the solvent is water;
[0034] Filtration is to filter out foam and impurities through a 100-mesh filter;
[0035] The isoelectric point is 6.25;
[0036] Centrifugation is performed at 4°C and 5000-7000 rpm for 10-20 min.
[0037] In one embodiment of the present invention, the solvent of the myofibrillar protein solution in step (3) is PBS or PIPES buffer with a pH of 6.25 and containing 0.6 mol / L NaCl.
[0038] In one embodiment of the present invention, the concentration of the myofibrillar protein solution in step (3) is 20-40 mg / mL.
[0039] In one embodiment of the present invention, the concentration of the spice extract solution in step (3) is 4 to 800 μg / mL.
[0040] In one embodiment of the present invention, the volume ratio of the myofibrillar protein solution to the spice extract solution in step (3) is 1:0.2-0.6.
[0041] In one embodiment of the present invention, the pH is adjusted in step (3) using 0.1 mol / L sodium hydroxide and 0.1 mol / L hydrochloric acid respectively.
[0042] In one embodiment of the present invention, the standing in step (3) is standing at 0-25° C. for 2-12 hours.
[0043] In one embodiment of the present invention, the washing in step (3) is performed using deionized water.
[0044] The second object of the present invention is the eel myofibrillar protein conjugate prepared by the method of the present invention.
[0045] The third object of the present invention is the application of the eel myofibrillar protein conjugate of the present invention in the food field.
[0046] The fourth object of the present invention is to provide a low-salt and low-sugar seasoned eel product, which uses the eel myofibrillar protein conjugate of the present invention.
[0047] [Technical Effect]
[0048] 1. The present invention uses phenolic substances contained in nutmeg and rosemary to modify the side chains of eel protein, effectively improving the structure of eel protein, regulating the aggregation state of eel protein, and significantly improving the solubility, antioxidant properties and in vitro digestibility of eel myofibrillar protein, providing an optimization direction for low-salt and low-sugar seasoning for existing eel seasoning.
[0049] 2. The present invention couples spice extracts with eel myofibrillar protein to enhance the binding ability of eel myofibrillar protein and flavor substances; the spices provide eel meat with a unique flavor, which is conducive to improving and innovating processes, enhancing the edible quality of eel meat, and producing eel meat products that are more suitable for processing and consumption and are deeply loved by customers.
[0050] 3. The present invention couples nutmeg and rosemary extracts with eel myofibrillar protein, which significantly affects the protein structure, manifested in increased solubility, decreased hydrophobicity, increased antioxidant activity, decreased carbonyl and thiol contents, decreased turbidity, and increased gastrointestinal digestibility. BRIEF DESCRIPTION OF THE DRAWINGS
[0051] Figure 1 This is a comparison of the solubility of eel myofibrillar proteins treated with nutmeg, rosemary, nutmeg-rosemary co-treatment, and gallic acid.
[0052] Figure 2 This is a comparison of the surface hydrophobicity of eel myofibrillar protein treated with nutmeg, rosemary, and nutmeg-rosemary synergistic treatment.
[0053] Figure 3 This is a comparison chart of the DPPH radical scavenging rate of eel myofibrillar protein treated with nutmeg, rosemary, nutmeg-rosemary co-treatment and gallic acid.
[0054] Figure 4 Comparison of carbonyl content in eel myofibrillar protein treated with nutmeg, rosemary, and nutmeg-rosemary combined.
[0055] Figure 5 Comparison of total sulfhydryl content in eel myofibrillar proteins treated with nutmeg, rosemary, nutmeg-rosemary co-treatment, and gallic acid.
[0056] Figure 6 Comparison of the turbidity of eel myofibrillar protein treated with nutmeg, rosemary, and nutmeg-rosemary combined.
[0057] Figure 7 This is a comparison of the in vitro simulated digestibility of eel myofibrillar protein treated with nutmeg, rosemary, nutmeg-rosemary co-treatment, and gallic acid.
[0058] Figure 8 This is a radar chart comparison of the flavor substances in eel myofibrillar protein treated with different spices in Control Example 5.
[0059] Figure 9 This is a comparison chart of the PCA analysis results of flavor substances in eel myofibrillar protein treated with different spices in Control Example 5. DETAILED DESCRIPTION
[0060] The following examples and comparative examples further illustrate the present invention in detail. Unless otherwise specified, all raw materials used in the present invention can be obtained from commercial sources.
[0061] Test method:
[0062] 1. Solubility determination:
[0063] The sample was diluted to 5 mg / mL with 0.1 mol / L phosphate buffer (pH 7.4), stirred at 4°C for 30 min, and centrifuged at 8000 rpm for 15 min. The protein concentration in the supernatant was then determined by the biuret method and converted to protein solubility (%).
[0064] 2. Hydrophobicity determination:
[0065] Dilute the sample to 5 mg / mL in 0.1 mol / L phosphate buffer (pH 7.4). Take 1.5 mL of the dilution and add 200 μL of 0.1 mg / mL bromophenol blue (BPB) solution. Vortex to mix thoroughly and centrifuge at 8000 rpm for 10 minutes. Transfer the supernatant and centrifuge again. Measure the absorbance at 595 nm. Replace the sample solution with buffer as a control. The amount of protein-bound BPB is used as an indicator of hydrophobicity.
[0066] 3. Carbonyl content determination:
[0067] The content of nitropropene was determined by 2,4-dinitrophenylhydrazine (DNPH) method.
[0068] Specifically, the sample was diluted to 4 mg / mL with 0.01 mol / L phosphate buffer (pH 7.0), 1 mL of the dilution was taken, and 3 mL of 10 mmol / L DNPH (dissolved in 2 mol / L HCl) was added. The blank group was 1 mL of the dilution mixed with 3 mL of HCl. The mixture was reacted at room temperature in the dark for 2 h, 4 mL of 20% TCA solution was added, the mixture was allowed to stand for 20 min, centrifuged, and the supernatant was poured off. The resulting precipitate was washed with 5 mL of ethanol / ethyl acetate (1:1, v / v), and the reaction was repeated three times. The resulting precipitate was fully dissolved with 3 mL of 6 mol / L guanidine hydrochloride solution (dissolved in 0.1 mol / L phosphate buffer, pH 7.0), and the absorbance was measured at 370 nm. The molar extinction coefficient was 22000 M -1 cm -1 Perform calculations.
[0069] 4. Determination of total thiol content:
[0070] Dilute the sample to 2 mg / mL in 0.1 mol / L phosphate buffer (pH 7.4); take 0.5 mL of the dilution and add 2.0 mL of urea-SDS solution (0.1 mol / L sodium phosphate buffer, containing 8.0 mol / L urea, 3% SDS, pH 7.4) and 0.5 mL of 10 mmol / L DTNB reagent (dissolved in 0.1 mol / L sodium phosphate buffer, pH 7.4); vortex to mix, incubate in the dark at room temperature for 15 minutes, and measure the absorbance at 412 nm. Buffer solution was used instead of protein solution as a blank. A molar extinction coefficient of 13600 M was used. -1 cm -1 Calculate the total thiol content.
[0071] 5. Turbidity determination:
[0072] The sample was diluted to 2 mg / mL with 0.1 mol / L phosphate buffer (pH 7.4) and the absorbance was measured at 500 nm.
[0073] 6. In vitro simulated digestion assay:
[0074] The sample was diluted to 10 mg / mL with 0.1 mol / L phosphate buffer (pH 7.4). 5 mL of the dilution was added to 37 mL of 10 mM HCI solution and 8 mL of pepsin solution (4% w / w protein, pH 2.0). The mixture was mixed and incubated at 37°C for 1 h to simulate gastric digestion.
[0075] Add an equal volume of 20% trichloroacetic acid solution to ice water for 30 minutes to terminate digestion. Centrifuge at 10,000 rpm for 10 minutes at 4°C. Remove the supernatant and redissolve the resulting precipitate in 1 mL of 1 M NaOH. Determine protein concentration using the biuret method and calculate gastric digestibility according to formula (1):
[0076] Digestibility (%) = (c t -c p ) / c t (1)
[0077] Among them, c t is the initial protein concentration, c p is the protein concentration of TCA precipitation.
[0078] The pH was adjusted to 7.5 to inactivate pepsin, and trypsin (4% w / w protein) was immediately added. The sample was heated and incubated in a 37°C water bath for 1 h. The pancreatic digestibility was determined in the same manner as the gastric digestibility.
[0079] 7. Electronic nose measurement:
[0080] The Heracles II rapid gas phase electronic nose was used to analyze the flavor characteristics of eels treated with different spices, specifically:
[0081] Weigh 1.0 g of sample into an injection vial, cap it, and incubate at 70°C. Samples were injected simultaneously using two chromatographic columns, DB-5 and DB-1701, of varying polarity. Kovat indices and VOCA identification were calculated using a standard solution of alkanes (C6-C16). Compounds were analyzed using the instrument's built-in data processing software, Alphasoft V 12.44, and the Aroma Chem Base database. Experiments were repeated three times.
[0082] 8. DPPH free radical scavenging rate determination:
[0083] Prepare 0.1mmol / L DPPH solution with anhydrous ethanol and store in the dark. Add 50μL of test sample solution and 0.95mL DPPH solution into the same test tube and shake well. Let it stand in the dark at room temperature for 20min and then measure its absorbance A. 样品 , and at the same time, the absorbance A0 of 0.95mL DPPH solution mixed with 50μL solvent was measured; the DPPH radical scavenging rate was calculated according to formula (2):
[0084] DPPH free radical scavenging rate (%) = (A0-A 样品 ) / A0×100(2)
[0085] Where A0 is the absorbance of DPPH at 517 nm without adding sample; A 样品 is the absorbance of DPPH added to the sample at 517 nm.
[0086] The raw materials used in the embodiments and comparative examples are:
[0087] Rosemary powder: Commercially available rosemary was dried at 30°C and ground to obtain a powder with a particle size of less than 125 μm;
[0088] Nutmeg powder: Commercially available rosemary was dried at 30°C and ground to obtain a powder with a particle size of less than 125 μm;
[0089] Eel meat: available in the market;
[0090] Other raw materials were purchased from commercial sources.
[0091] In the examples and comparative examples, % mentioned unless otherwise specified are percentages by mass; solutions mentioned unless otherwise specified are solvents, which are water; reactions performed at room temperature (25° C.) unless otherwise specified.
[0092] Example 1 Nutmeg-Rosemary
[0093] A method for improving the structure of eel myofibrillar protein comprises the following steps:
[0094] (1) Extraction of active ingredients from spices:
[0095] 50 mL of 70% ethanol solution was added to 3 g of rosemary powder, and ultrasonic extraction was performed at room temperature (25° C.) and 50 kHz for 30 minutes. The supernatant was then centrifuged at 8000 rpm for 15 minutes, and the extract was rotary evaporated at 40° C., freeze-dried at -80° C. for 48 hours, and pulverized to obtain a rosemary extract powder with a particle size of less than 125 μm.
[0096] 50 mL of 70% ethanol solution was added to 3 g of nutmeg powder, and ultrasonic extraction was performed at room temperature (25°C) and 50 kHz for 30 minutes. The supernatant was then centrifuged at 8000 rpm for 15 minutes, and the extract was rotary evaporated at 40°C, freeze-dried at -80°C for 48 hours, and pulverized to obtain a nutmeg extract powder with a particle size of less than 125 μm.
[0097] Rosemary extract powder and nutmeg extract powder were mixed in a mass ratio of 1:1 and added to water to obtain a spice extract solution with a total concentration of 400 μg / mL;
[0098] (2) Extraction of myofibrillar protein:
[0099] Thaw the eel and mince it; add 200 mL of 0.01 mol / L sodium phosphate extract (pH 7.0) to 40 g of minced eel meat, stir and mash at 2000 rpm for 1 min, centrifuge at 4°C and 6000 rpm for 10 min, decant the supernatant, add the same volume of 0.01 mol / L sodium phosphate extract (pH 7.0) to the resulting precipitate, and repeat the above steps three times;
[0100] To the precipitate obtained in the above step, add 200 mL of 0.1 mol / L NaCl solution, stir uniformly at 2000 rpm, filter out foam and impurities through a 100-mesh filter, adjust the pH to 6.25, and centrifuge at 4°C and 6000 rpm for 15 min. The resulting paste precipitate is myofibrillar protein. The protein content is determined by the biuret method, using BSA as the standard protein.
[0101] (3) Preparation of protein conjugates:
[0102] The paste myofibrillar protein was diluted to a protein concentration of 20 mg / mL with a phosphate buffer having a pH of 7.0 and containing 0.6 mol / L NaCl to obtain a myofibrillar protein solution; 4 mL of the spice extract solution was added to 10 mL of the myofibrillar protein solution, and the mixture was stirred at 800 rpm for 5 minutes. The pH was adjusted to 9.0 with 0.5 mol / L sodium hydroxide, and the mixture was allowed to stand for 2 hours to form a conjugate. The pH was adjusted to 7.0 with 0.5 mol / L hydrochloric acid, and the mixture was washed three times with deionized water to obtain an eel myofibrillar protein conjugate, which was then subjected to performance testing.
[0103] Control 1: No treatment
[0104] The myofibrillar protein prepared in step (2) of Example 1 was directly used.
[0105] Comparative Example 2 Nutmeg
[0106] The rosemary powder in step (1) of Example 1 was omitted, and 6 g of nutmeg powder was directly used for extraction to prepare the extract; the other steps were the same as in Example 1 to obtain the eel myofibrillar protein conjugate.
[0107] Control Example 3 Rosemary
[0108] The nutmeg powder in step (1) of Example 1 was omitted, and 6 g of rosemary powder was directly used for extraction to prepare the extract; the other steps were the same as in Example 1 to obtain an eel myofibrillar protein conjugate.
[0109] Comparative Example 4 Gallic acid
[0110] Omit step (1) of Example 1;
[0111] In step (3), the added spice extract was adjusted to gallic acid, and the gallic acid concentration was 400 μg / mL. Other steps were the same as those in Example 1 to obtain an eel myofibrillar protein conjugate.
[0112] The eel myofibrillar protein conjugates obtained in Example 1 and Comparative Examples 1 to 4 were subjected to performance tests, and the test results are as follows:
[0113] Figure 1 The figure shows the solubility comparison of eel myofibrillar proteins treated with nutmeg, rosemary, nutmeg-rosemary and gallic acid. Figure 1 It can be seen that the solubility of the samples treated with spices is significantly higher than that of the untreated eel myofibrillar protein. The solubility of the samples coupled with nutmeg is greater than that of the samples with rosemary. The solubility of the samples synergistically treated with nutmeg and rosemary is the highest, which is higher than that of the eel myofibrillar protein treated with gallic acid.
[0114] Figure 2A comparison of the surface hydrophobicity of eel myofibrillar protein treated with nutmeg, rosemary, and nutmeg-rosemary co-treatment is shown. Studies have shown that BPB molecules can bind to hydrophobic binding sites on the surface of protein molecules. Therefore, the amount of BPB bound by a protein can be used as an indicator of protein surface hydrophobicity. Increased surface hydrophobicity is a key indicator of protein structural unfolding. Oxidation causes protein structural unfolding, exposing more hydrophobic amino acid residues and increasing protein surface hydrophobicity. Generally speaking, protein surface hydrophobicity is negatively correlated with solubility, so changes in solubility indirectly reflect changes in myofibrillar protein surface hydrophobicity. This is consistent with the results for eel myofibrillar protein treated with nutmeg, rosemary, and co-treatment. The surface hydrophobicity of eel myofibrillar protein treated with nutmeg-rosemary co-treatment was the lowest compared to the other samples. However, the surface hydrophobicity of untreated eel myofibrillar protein was lower than that of samples treated with the individual spices and higher than that of samples treated with nutmeg-rosemary co-treatment. This may be because the untreated protein does not undergo coupling reactions in solution, resulting in structural unfolding. Hydrophobicity, to a certain extent, indicates the aggregation of proteins in solution. Aggregates exhibit inherent conformational fluctuations or local structural perturbations, which may be caused by external factors and further accelerate protein aggregation through intermolecular interactions. Hydrophobicity indicates that the aggregation of eel myofibrillar proteins in the co-treated group is much lower than that in other groups.
[0115] Figure 3 The figure shows the comparison of DPPH free radical scavenging rate of eel myofibrillar protein treated with nutmeg, rosemary, nutmeg-rosemary co-treatment and gallic acid. Figure 3 It can be seen that the DPPH free radical scavenging rate of the samples treated with spices increased, and the antioxidant activity was significantly improved compared with the untreated eel myofibrillar protein. The antioxidant effect of the samples with rosemary added was better than that of the samples with nutmeg added. The samples treated with nutmeg and rosemary had the best antioxidant capacity, and the DPPH free radical scavenging rate was higher than that of the eel myofibrillar protein treated with gallic acid.
[0116] Many side-chain amino acid functional groups in proteins are easily oxidized to form carbonyl derivatives. Therefore, carbonyl content is generally considered one of the indicators for judging the degree of protein oxidation. It is generally believed that a higher carbonyl content indicates a higher degree of protein oxidation. Figure 4 This is a comparison of the carbonyl content of eel myofibrillar protein treated with nutmeg, rosemary, and nutmeg-rosemary. Figure 4 It can be seen that the carbonyl content of the samples treated with spices decreased significantly. The carbonyl content of the samples treated with rosemary was lower than that of the samples treated with nutmeg, and the carbonyl content of the sample treated with nutmeg and rosemary was the lowest. This can be attributed to the free radical scavenging and metal ion chelating abilities of polyphenols.
[0117] Figure 5Comparison of the total thiol content of eel myofibrillar proteins treated with nutmeg, rosemary, nutmeg-rosemary co-treatment, and gallic acid. Sulfhydryl groups are susceptible to attack by hydroxyl radicals, converting them into intra- and intermolecular disulfide bonds, thereby causing protein cross-linking and aggregation. Therefore, a reduction in thiol content and the formation of disulfide bonds, or the conversion of thiol groups to disulfide bonds, are important early indicators of free radical-induced protein oxidation. With the exception of the sample treated with rosemary, the thiol content of samples treated with spices decreased, demonstrating a superior antioxidant effect compared to that of nutmeg. The thiol content of samples co-treated with nutmeg-rosemary was lower than that of samples treated with nutmeg, but higher than that of samples treated with gallic acid.
[0118] Figure 6 This figure compares the turbidity of eel myofibrillar protein treated with nutmeg, rosemary, and nutmeg-rosemary. Turbidity can analyze the concentration of soluble and insoluble aggregates, indicating protein aggregation. Furthermore, the oxidation process exposes more reactive sulfhydryl and hydrophobic groups, enhancing thermal aggregation of myofibrillar protein and leading to increased turbidity. The turbidity of samples treated with nutmeg-rosemary was significantly lower than that of the other groups, indicating a reduction in the size of protein aggregates and reduced light scattering. This suggests that nutmeg-rosemary synergistic conjugation of eel myofibrillar protein significantly improves protein aggregation in solution.
[0119] Figure 7 The figure shows the comparison of in vitro simulated digestibility of eel myofibrillar protein treated with nutmeg, rosemary, nutmeg-rosemary co-treatment and gallic acid. Figure 7 It can be seen that the samples treated with nutmeg-rosemary have higher gastrointestinal digestibility than those treated with single spices and gallic acid, indicating that the physicochemical properties of the prepared nutmeg-rosemary group samples have undergone more complex changes compared with single polyphenols, and the structure is more suitable for digestion and decomposition by pepsin and trypsin, thereby improving absorption rate, targeted specific delivery and biological activity.
[0120] Comparative Example 5
[0121] The spices (nutmeg and rosemary) in step (1) were adjusted to nutmeg, rosemary, cloves, cinnamon or chili pepper, pepper, and the other ingredients remained the same as in Example 1 to obtain an eel myofibrillar protein conjugate.
[0122] The eel myofibrillar protein conjugates of Example 1 and Control Example 5 were tested for the flavor characteristics of eels under different spices using the Heracles II ultrafast gas phase electronic nose. The sample data collected by the electronic nose were analyzed to obtain radar charts and principal component analysis charts as shown in FIG. Figure 8 、 Figure 9 shown.
[0123] The fast gas chromatography electronic nose uses two chromatographic columns of different polarities, identified by the numbers 1 and 2 (1 represents a DB-5 column, 2 represents a DB-1701 column). The chromatographic information obtained is the peak area corresponding to the retention time. For example, 19.42-1-A and 20.54-2-A represent the peak area at retention time 19.42 on the DB-5 column and 20.54 on the DB-1701 column, respectively. Figure 8 Each point in the graph represents the average peak area of three replicate samples. Figure 8 It can be seen that the rapid gas chromatography electronic nose has a significant response to the eel meat sample, and there are significant differences in the areas of flavor substances at 18 effective response points between the cooked eel meat prepared with different spices and the blank control group.
[0124] Principal component analysis is a multivariate statistical analysis method that obtains linear classification information by reducing the dimension and transforming the original data. In order to more intuitively and accurately compare the flavor differences and similarities of different samples, such as Figure 9 As shown in the figure: the horizontal axis represents the explanation rate of the first principal component (PC1) in the PCA analysis, and the vertical axis represents the explanation rate of the second principal component (PC2). When the data explanation rate of PC in the PCA analysis reaches more than 85%, PC can be used to replace the original data. Figure 9 The first principal component of the PCA explained 77.097% of the variance, and the second principal component explained 20.227% of the variance. The cumulative variance contribution of the first two principal components was 97.317%, indicating that PC1 and PC2 can well explain the vast majority of the sample information. Differences between samples can be seen in the two-dimensional PCA plot. Samples treated with nutmeg and rosemary were located in different quadrants from the other groups and were farther from the origin. The distances between samples were large, and they differed significantly from untreated cooked eel meat. This indicates that there are significant differences in the aroma components of cooked eel meat marinated with different spices. Nutmeg and rosemary have distinct flavor profiles compared to traditional spices such as cinnamon, cloves, and chili peppers, providing a different flavor experience than other condiments such as chili pepper.
[0125] Example 2 Effect of pH on Conjugates
[0126] After adding the spice extract solution in step (3) of Example 1, the pH was adjusted to 8, 9, 10, and 11, and the other conditions remained the same as in Example 1 to obtain an eel myofibrillar protein conjugate.
[0127] The obtained eel myofibrillar protein conjugate was subjected to performance testing, and the test results are as follows:
[0128] Table 1 Test results of protein conjugates prepared at different pH
[0129] pH Solubility In vitro digestibility DPPH free radical scavenging rate 8 67.65％ 94.11％ 27.85％ 9 (Example 1) 97.45％ 95.28％ 31.17％ 10 64.91％ 94.50％ 29.85％ 11 41.69％ 90.78％ 26.47％
[0130] Example 3: Effect of spice extract concentration on conjugates
[0131] The concentrations of the spice extract solution in step (3) of Example 1 were adjusted to 4, 40, 400, and 1000 μg / mL, while the other conditions remained the same as in Example 1 to obtain an eel myofibrillar protein conjugate.
[0132] The obtained eel myofibrillar protein conjugate was subjected to performance testing, and the test results are as follows:
[0133] Table 2 Test results of protein conjugates prepared from spice extracts at different concentrations
[0134] Concentration (μg / mL) Solubility In vitro digestibility DPPH free radical scavenging rate 4 58.81％ 93.16％ 28.36％ 40 69.43％ 90.28％ 27.33％ 400 97.45％ 95.28％ 31.17％ 1000 87.16％ 91.47％ 29.86％
[0135] Comparative Example 6
[0136] The extraction step (1) of Example 1 was omitted;
[0137] In step (1), the spice extract was replaced with rosmarinic acid and myristic acid in a mass ratio of 1:1, and the other ingredients were kept the same as in Example 1 to obtain a protein conjugate.
[0138] Comparative Example 7
[0139] Omit the extraction of rosemary powder and nutmeg powder in step (1) of Example 1;
[0140] In step (1), the spice extract was replaced with rosmarinic acid, and the other ingredients were kept the same as in Example 1 to obtain a protein conjugate.
[0141] Comparative Example 8
[0142] Omit the extraction of rosemary powder in step (1) of Example 1;
[0143] In step (1), the rosemary extract was replaced with rosmarinic acid, and the other steps were the same as those in Example 1 to obtain a protein conjugate.
[0144] Comparative Example 9
[0145] Omit the extraction of nutmeg powder in step (1) of Example 1;
[0146] In step (1), the nutmeg extract was replaced with myristicin, and the rest was kept the same as in Example 1 to obtain a protein conjugate.
[0147] Comparative Example 10
[0148] The nutmeg in step (1) of Example 1 was replaced with cinnamon; the other ingredients remained the same as in Example 1 to obtain a protein conjugate.
[0149] Comparative Example 11
[0150] The rosemary in step (1) of Example 1 was replaced with clove; the other steps remained the same as in Example 1 to obtain a protein conjugate.
[0151] The obtained protein conjugate was subjected to performance testing, and the test results are as follows:
[0152] Table 3 Test results of protein conjugates prepared in Example 1 and Comparative Examples 6 to 11
[0153] example Solubility In vitro digestibility DPPH free radical scavenging rate Example 1 97.45％ 95.28％ 31.17％ Comparative Example 6 91.39％ 94.28％ 30.93％ Comparative Example 7 56.17％ 91.78％ 29.96％ Comparative Example 8 76.04％ 91.81％ 27.10％ Comparative Example 9 68.52％ 92.52％ 29.91％ Comparative Example 10 53.66％ 93.90％ 27.50％ Comparative Example 11 41.69％ 93.04％ 26.55％
[0154] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. A method for improving the structure of eel myofibrillar protein, characterized in that: The following steps are involved: (1) Extraction of active ingredients from spices: adding ethanol solution to rosemary powder and nutmeg powder respectively, performing ultrasonic extraction, centrifuging, collecting supernatant, rotary evaporating, drying, and crushing to obtain rosemary extract and nutmeg extract; then mixing the rosemary extract and nutmeg extract, and adding them to water to obtain a spice extract solution; (2) Extraction of myofibrillar proteins: Extraction of myofibrillar proteins from eel meat; (3) Preparation of protein conjugates: The myofibrillar protein is prepared into a myofibrillar protein solution, and then a spice extract solution is added, and the pH is adjusted to 8.0-10.0, and the solution is allowed to stand to form a conjugate, and the solution is washed to obtain a paste-like precipitate, i.e., an eel myofibrillar protein conjugate; Wherein, in step (1), the ratio of rosemary powder and nutmeg powder to ethanol solution is 2-4 g:50 mL; The concentration of the myofibrillar protein solution in step (3) is 20-40 mg / mL; the concentration of the spice extract solution is 4-800 μg / mL; The method for extracting myofibrillar protein in step (2) is: Thaw the eel meat, chop it, add the extract, stir and mash it at 1500-2500 rpm for 0.5-2 minutes, then centrifuge it at 4°C and 5000-7000 rpm for 5-15 minutes, remove the supernatant, and add the extract again to the resulting precipitate; Repeat the above steps to obtain a precipitate after centrifugation; Add NaCl solution to the precipitate, stir evenly and filter, adjust the pH to the isoelectric point, centrifuge, and the resulting paste precipitate is myofibrillar protein.
2. The method according to claim 1, characterized in that The mass ratio of the rosemary extract to the nutmeg extract in step (1) is 1-3:1-3.
3. The method according to claim 1, characterized in that The ethanol solution in step (1) is an ethanol aqueous solution with a mass fraction of 65-75%.
4. The method according to claim 1, wherein In step (3), the volume ratio of myofibrillar protein to spice extract is 1:0.2-0.
6.
5. The method according to claim 1, wherein The dosage ratio of eel meat and extract is 1 mg: 4-8 mL.
6. The method according to claim 1, characterized in that The ultrasonic extraction in step (1) is performed at 40-60 kHz and 20-30° C. for 20-40 minutes.
7. The method according to claim 1, characterized in that The rotary evaporation temperature in step (1) is 30-50°C.
8. An eel myofibrillar protein conjugate prepared by the method according to any one of claims 1 to 7.
9. Use of the eel myofibrillar protein conjugate according to claim 8 in the food field.
10. A low-salt and low-sugar seasoned eel product, characterized in that: It is prepared using the eel myofibrillar protein conjugate according to claim 8.