Natural composite antioxidant and its application in duck curing

Abstract

The application provides a natural composite antioxidant and application thereof in duck curing. The natural composite antioxidant is composed of rutin, chlorogenic acid and hyperoside, wherein the rutin is prepared by ultrasonic extraction assisted with pectinase from sophora fruit. After the rutin powder extracted from sophora fruit is compounded with the chlorogenic acid and the hyperoside according to a proportion, the compound is prepared into a curing liquid together with salt, sugar and water, and duck is cured under vacuum tumbling. The synergistic antioxidant effect of the rutin, the chlorogenic acid and the hyperoside in free radical scavenging, metal ion chelation and protein conformation protection can effectively inhibit the accumulation of carbonyl, the loss of sulfhydryl and the exposure of hydrophobic group of myofibrillar protein in the duck curing process. The experimental results show that after the duck is treated by the composite antioxidant, the carbonyl content of the duck is as low as 6.015-6.248 nmol / mg, the total sulfhydryl content reaches 15.107-15.742 μmol / g, the surface hydrophobicity is significantly reduced, and the protein oxidation damage is comprehensively inhibited.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to a natural compound antioxidant and its application in the curing of duck meat. Background Technology

[0002] Duck meat is a crucial raw material for traditional poultry processing in my country, with processed duck products such as pressed duck, salted duck, and braised duck holding a significant position in the meat consumption market. Marinating is a key process in duck product processing, imparting unique flavor and good texture. However, marinating often involves high salt concentrations and a period of open or cyclic soaking, an environment highly conducive to inducing the oxidation of myofibril proteins in the meat. Protein oxidation leads to peptide chain breakage, sulfhydryl loss, and carbonyl accumulation, resulting in a series of quality deterioration problems such as decreased water retention, loose texture, and flavor degradation, severely restricting the quality stability and industrial production efficiency of duck products.

[0003] Currently, the main strategy for controlling protein oxidation in meat processing is the addition of exogenous antioxidants. In the development and application of natural antioxidants, plant polyphenols have become a research hotspot due to their wide availability, high safety, and combined antioxidant and antibacterial activities.

[0004] Chinese patent CN102657340A, "A Fresh Seasoned Duck Breast Product and Its Production Process," discloses a technical solution that involves adding a compound of tea polyphenols and nisin to the marinating solution to delay spoilage and browning during processing and sales. However, this process only addresses the problems of microbial spoilage and fat browning during product storage. It does not design a specific antioxidant formula targeting the myofibrillar protein oxidation mechanism during the marinating process, making it difficult to specifically inhibit the oxidative damage to myofibrillar proteins during marinating. Consequently, it fails to improve the core defects of duck meat hardening and reduced water retention after marinating.

[0005] Chinese patent CN118435991A, "A Method for Color Protection and Antioxidant Effect of Soy-braised Duck," describes a method that uses a water extract of bamboo leaves, after solvent extraction, to select the n-butanol phase, which has the highest total phenol content and antioxidant activity, as an additive. While this method achieves preliminary separation of the bamboo leaf extract, it still uses a single antioxidant component. Its antioxidant effect depends on increasing the concentration of the added component. However, bamboo leaf extract itself contains a yellow-green pigment, and excessive addition would interfere with the inherent color and flavor of the duck meat. This limits the concentration of the added component and makes it difficult to achieve long-lasting protein protection without sacrificing the sensory quality of the product.

[0006] In summary, existing natural antioxidant solutions do not address the synergistic effects between polyphenol components, and single-phase extraction systems are limited by the conflict between dosage and sensory quality, making it difficult to effectively inhibit oxidative damage to duck myofibrillar proteins under marinating conditions. Therefore, there is an urgent need to develop a natural antioxidant that can coordinate the synergistic effects of polyphenol components, adapt to the duck marinating environment, and effectively inhibit excessive oxidation of myofibrillar proteins while ensuring product sensory quality. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a natural compound antioxidant and its application in duck meat curing, solving the technical problem that "a single antioxidant is difficult to effectively inhibit myofibrillar protein oxidation while maintaining the inherent flavor and taste of duck meat during the curing process".

[0008] To achieve the above objectives, the present invention is implemented using the following technical solution: In a first aspect, the present invention provides a natural composite antioxidant comprising rutin powder, chlorogenic acid powder and hyperoside powder; the rutin powder is prepared by adding water to Sophora japonica flowers, adjusting the acidity, enzymatically hydrolyzing, filtering, concentrating, drying and pulverizing.

[0009] Secondly, the present invention provides a method for marinating duck meat, characterized by comprising the following steps: (1) Take dried Sophora japonica flowers, crush them and sieve them. Add water to make Sophora japonica flower liquid, adjust the pH to acidic, add pectinase for enzymatic hydrolysis, heat up to inactivate pectinase after enzymatic hydrolysis, filter, collect the filtrate and concentrate, dry and crush it to obtain rutin powder; (2) The rutin powder obtained in step (1) is mixed with chlorogenic acid powder and hyperoside powder in a certain proportion to obtain a composite antioxidant. (3) Add salt, white sugar and compound antioxidant to water and stir until completely dissolved to prepare marinade; remove excess fat from fresh duck meat, wash, drain and cut into pieces, place in marinade, tumble and marinate under vacuum conditions, and let stand to marinate after tumbling.

[0010] In this invention, the amount of pectinase added in step (1) is 0.04~0.08% of the dry weight of Sophora japonica buds; the enzymatic hydrolysis is performed under constant temperature of 40℃ with ultrasound-assisted enzymatic hydrolysis for 1~2 hours.

[0011] In this invention, the mass ratio of rutin powder, chlorogenic acid powder and hyperoside powder in step (2) is 0.5:(0.35~0.6):(0.1~0.27).

[0012] In this invention, the vacuum condition in step (3) is a vacuum degree of 0.05 MPa, the temperature of the tumbling and marinating is 4°C, and the time is 15~25 min; the tumbling adopts a cycle mode of 10 min continuous operation followed by a 3 min interval; the conditions for static marinating are: temperature 4°C, time 3~5 h.

[0013] In this invention, the amount of the composite antioxidant added in step (3) is 0.1 to 0.5% of the weight of duck meat; the amount of water added is 28 to 35% of the weight of duck meat; the amount of salt added is 1 to 2% of the weight of duck meat; and the amount of white sugar added is 0.5 to 1.5% of the weight of duck meat.

[0014] Compared with the prior art, the beneficial effects achieved by the present invention are: (1) This invention uses pectin-enzymatically assisted ultrasonic extraction combined with freeze-drying technology to prepare rutin powder from Sophora japonica buds. This process ensures the rutin extraction efficiency while effectively preserving the natural aromatic components in Sophora japonica buds, giving the obtained rutin powder both antioxidant activity and flavor-modifying function. Compared with commercially available high-purity rutin, the rutin prepared in this invention, when applied to duck marinating, can not only effectively inhibit protein oxidation but also impart additional effects of deodorizing and enhancing the aroma of the product, avoiding the limitation of commercially available rutin, which only has a single antioxidant effect due to the removal of natural aromatic substances by multi-stage recrystallization.

[0015] (2) This invention involves the targeted compounding of three polyphenolic components—rutin, chlorogenic acid, and hyperoside—resulting in a significant synergistic antioxidant effect among the three components. Experimental results show that the carbonyl content in Examples 1-3 was reduced by approximately 40% compared to the control group; the total thiol content was increased by approximately 60% compared to the control group; and the surface hydrophobicity (bromophenol blue binding amount) was reduced by approximately 52% compared to the control group. The compounding effect of the three components was superior to any pairwise combination or a single component, confirming that the three polyphenols comprehensively inhibited the oxidative damage of myofibril proteins through three pathways: carbonyl blocking, thiol protection, and conformation maintenance, by synergistically scavenging free radicals and interacting with the protein surface at multiple sites.

[0016] (3) This invention achieves highly efficient inhibition of protein oxidation by synergistically combining three polyphenols, with the total amount of the compound antioxidant added being only 0.1-0.5% of the duck meat weight. The effective concentration of each individual component is significantly lower than that of existing single-agent solutions, avoiding the impact of high-dose addition on the inherent flavor and taste of duck meat. All components are of natural origin, have high safety, conform to the consumer trend of clean labeling, and have good prospects for industrial application. Attached Figure Description

[0017] Figure 1 This is a comparison chart showing the determination results of myofibrillar protein carbonyl content in the various embodiments and comparative examples of the present invention.

[0018] Figure 2 This is a comparison chart showing the determination results of total sulfhydryl content of myofibrillar protein in the various embodiments and comparative examples of the present invention.

[0019] Figure 3 This is a comparison chart showing the results of measuring the surface hydrophobicity of myofibrillar protein in the various embodiments and comparative examples of the present invention. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] During the curing process of duck meat, myofibrillar proteins are prone to oxidative damage, which manifests as peptide chain breakage, carbonyl accumulation, thiol group loss and hydrophobic group exposure, leading to decreased water retention, loose texture and deteriorated flavor.

[0022] To address this technical problem, this invention proposes a directional compounding technique using three polyphenols: rutin, chlorogenic acid, and hyperoside. The ortho-dihydroxyl group on the B ring of rutin can efficiently scavenge hydroxyl radicals and superoxide anion radicals; chlorogenic acid molecules contain ortho-dihydroxyl groups, which can chelate with transition metal ions to form stable complexes, blocking the Fenton reaction catalyzed by metal ions and reducing the generation of hydroxyl radicals; the glycosidic side chain of hyperoside enhances the water solubility of the molecule, making it easier to accumulate in the protein hydration layer and protecting the thiol groups of cysteine ​​residues. These three polyphenols are complementary in chemical structure and mechanism of action, and their combined use can achieve multi-dimensional antioxidant protection.

[0023] Meanwhile, the rutin in this invention is prepared by self-extraction from Sophora japonica buds. A gentle process combining pectin enzymatic hydrolysis-assisted ultrasonic extraction with freeze-drying is used to ensure the rutin extraction rate while retaining the natural aromatic components such as esters, alcohols, and terpenes in the Sophora japonica buds. It should be noted that the large-molecule astringent substances such as tannins and tannins in the Sophora japonica buds are removed by filtration under the action of pectinase, while the smaller molecular weight rutin and aromatic substances pass through the filter cloth with the water, accumulating in the filtrate and being collected. This achieves simultaneous removal of astringency and preservation of aroma, giving the rutin powder both antioxidant activity and a refreshing Sophora japonica aroma. In contrast, commercially available high-purity rutin, after multi-stage recrystallization purification, completely removes natural aromatic substances while removing impurities, possessing only a single antioxidant effect and unable to provide flavor modification for marinated duck meat. Based on the above concept, this invention proposes the following specific technical solutions: A method for marinating duck meat includes the following steps: (1) Take dried Sophora japonica flowers, crush them and pass them through a 40-mesh standard sieve. Add water at a material-liquid mass ratio of 1:10 to make Sophora japonica flower material solution. Adjust the pH of the material solution to 5~5.5. Add pectinase at a rate of 0.04~0.08% of the dry weight of Sophora japonica flowers. Under constant temperature of 40℃, use ultrasound-assisted enzymatic hydrolysis for 1~2 hours. After the enzymatic hydrolysis is completed, heat the solution to 85~90℃ in a closed device and keep it at 10~15 minutes to inactivate the pectinase. Filter the solution with a 200-mesh filter cloth to remove solid residue. Collect the filtrate and concentrate it to obtain rutin powder. (2) Weigh 0.5g of rutin powder, 0.35~0.6g of chlorogenic acid powder and 0.1~0.27g of hyperoside powder prepared above, mix them evenly to obtain a composite antioxidant; (3) Remove excess fat from fresh duck meat, wash it clean, drain the surface water, and cut it into meat pieces. Based on the weight of the meat pieces, take 28-35% water, 1-2% salt, 0.5-1.5% white sugar and 0.1-0.5% of the compound antioxidant prepared in step (2). Add salt, white sugar and compound antioxidant to water and stir until completely dissolved to prepare a marinating solution. Place the meat pieces in a marinating container, add the marinating solution, and tumble in a vacuum tumbler with a vacuum degree of 0.05MPa and a temperature of 4℃ for 15-25 minutes. The tumbling adopts a cycle mode of 10 minutes of continuous operation followed by a 3-minute interval. After taking it out, let it stand and marinate for 3-5 hours.

[0024] Example 1; (1) Take dried Sophora japonica flowers, crush them and pass them through a 40-mesh standard sieve. Add water at a material-liquid mass ratio of 1:10 to make Sophora japonica flower material solution. Adjust the pH of the material solution to 5. Add pectinase at 0.04% of the dry weight of Sophora japonica flowers. Under constant temperature of 40℃, use ultrasound-assisted enzymatic hydrolysis for 1 hour. After the enzymatic hydrolysis is completed, heat the solution to 85℃ in a closed device and keep it at 10 min to inactivate the pectinase. Filter the solution with a 200-mesh filter cloth to remove solid residue. Collect the filtrate and concentrate it. After freeze drying, crush it to obtain rutin powder. (2) Weigh out 0.5g of rutin powder, 0.35g of chlorogenic acid powder and 0.1g of hyperoside powder prepared above, mix them evenly to obtain a composite antioxidant; (3) Remove excess fat from fresh duck meat, wash it clean, drain the surface water, and cut it into meat pieces. Based on the weight of the meat pieces, take 28% water, 1% salt, 0.5% white sugar and 0.1% of the compound antioxidant prepared in step (2). Add salt, white sugar and compound antioxidant to water and stir until completely dissolved to prepare a marinade. Place the meat pieces in a marinade container, add the marinade, and tumble in a vacuum tumbler with a vacuum degree of 0.05MPa and a temperature of 4℃ for 15 minutes. The tumbling adopts a cycle mode of 10 minutes of continuous operation followed by a 3-minute interval. After taking it out, let it stand and marinate for 3 hours.

[0025] Example 2; (1) Take dried Sophora japonica flowers, crush them and pass them through a 40-mesh standard sieve. Add water at a material-liquid mass ratio of 1:10 to make Sophora japonica flower material solution. Adjust the pH of the material solution to 5.2. Add pectinase at a dry weight of 0.06% of Sophora japonica flowers. Under constant temperature of 40℃, use ultrasound-assisted enzymatic hydrolysis for 1.5h. After the enzymatic hydrolysis is completed, heat to 90℃ in a closed device and keep warm for 12min to inactivate the pectinase. Filter with a 200-mesh filter cloth to remove solid residue. Collect the filtrate and concentrate it. After freeze drying, crush it to obtain rutin powder. (2) Weigh out 0.5g of rutin powder, 0.47g of chlorogenic acid powder and 0.2g of hyperoside powder prepared above, mix them evenly to obtain a composite antioxidant; (3) Remove excess fat from fresh duck meat, wash it clean, drain the surface water, and cut it into meat pieces. Based on the weight of the meat pieces, take 30% water, 1.5% salt, 1% white sugar and 0.3% of the compound antioxidant prepared in step (2). Add salt, white sugar and compound antioxidant to water and stir until completely dissolved to prepare a marinade. Place the meat pieces in a marinating container, add the marinade, and tumble in a vacuum tumbler with a vacuum degree of 0.05MPa and a temperature of 4℃ for 20 minutes. The tumbling adopts a cycle mode of 10 minutes of continuous operation followed by a 3-minute interval. After taking it out, let it stand and marinate for 4 hours.

[0026] Example 3; (1) Take dried Sophora japonica flowers, crush them and pass them through a 40-mesh standard sieve. Add water at a material-liquid mass ratio of 1:10 to make Sophora japonica flower material solution. Adjust the pH of the material solution to 5.5. Add pectinase at 0.08% of the dry weight of Sophora japonica flowers. Under constant temperature of 40℃, use ultrasound-assisted enzymatic hydrolysis for 2 hours. After the enzymatic hydrolysis is completed, heat to 90℃ in a closed device and keep warm for 15 minutes to inactivate the pectinase. Filter with a 200-mesh filter cloth to remove solid residue. Collect the filtrate and concentrate it. After freeze drying, crush it to obtain rutin powder. (2) Weigh 0.5g of rutin powder, 0.6g of chlorogenic acid powder and 0.27g of hyperoside powder prepared above, mix them evenly to obtain a composite antioxidant; (3) Remove excess fat from fresh duck meat, wash it clean, drain the surface water, and cut it into meat pieces. Based on the weight of the meat pieces, take 35% water, 2% salt, 1.5% white sugar and 0.5% of the compound antioxidant prepared in step (2). Add salt, white sugar and compound antioxidant to water and stir until completely dissolved to prepare a marinade. Place the meat pieces in a marinating container, add the marinade, and tumble in a vacuum tumbler with a vacuum degree of 0.05MPa and a temperature of 4℃ for 25 minutes. The tumbling adopts a cycle mode of 10 minutes of continuous operation followed by a 3-minute interval. After taking it out, let it stand and marinate for 5 hours.

[0027] Example 4; (1) Take dried Sophora japonica flowers, crush them and pass them through a 40-mesh standard sieve. Add water at a material-liquid mass ratio of 1:10 to make Sophora japonica flower material solution. Adjust the pH of the material solution to 5.2. Add pectinase at a rate of 0.06% of the dry weight of Sophora japonica flowers. Under constant temperature of 40℃, use ultrasound-assisted enzymatic hydrolysis for 1.5h. After the enzymatic hydrolysis is completed, filter the solution through a 200-mesh filter cloth to remove solid residue. Collect the filtrate, concentrate it, freeze dry it and then crush it to obtain rutin powder. (2) Weigh 0.5g of rutin powder and 0.47g of chlorogenic acid powder prepared above, mix them evenly to obtain a composite antioxidant; (3) Remove excess fat from fresh duck meat, wash it clean, drain the surface water, and cut it into meat pieces. Based on the weight of the meat pieces, take 30% water, 1.5% salt, 1% white sugar and 0.3% of the compound antioxidant prepared in step (2). Add salt, white sugar and compound antioxidant to water and stir until completely dissolved to prepare a marinade. Place the meat pieces in a marinating container, add the marinade, and tumble in a vacuum tumbler with a vacuum degree of 0.05MPa and a temperature of 4℃ for 20 minutes. The tumbling adopts a cycle mode of 10 minutes of continuous operation followed by a 3-minute interval. After taking it out, let it stand and marinate for 4 hours.

[0028] Example 5; (1) Take dried Sophora japonica flowers, crush them and pass them through a 40-mesh standard sieve. Add water at a material-liquid mass ratio of 1:10 to make Sophora japonica flower material solution. Adjust the pH of the material solution to 5.2. Add pectinase at a dry weight of 0.06% of Sophora japonica flowers. Under constant temperature of 40℃, use ultrasound-assisted enzymatic hydrolysis for 1.5h. After the enzymatic hydrolysis is completed, heat to 90℃ in a closed device and keep warm for 12min to inactivate the pectinase. Filter with a 200-mesh filter cloth to remove solid residue. Collect the filtrate and concentrate it. After freeze drying, crush it to obtain rutin powder. (2) Weigh 0.5g of rutin powder and 0.2g of hyperoside powder prepared above, mix them evenly to obtain a composite antioxidant; (3) Remove excess fat from fresh duck meat, wash it clean, drain the surface water, and cut it into meat pieces. Based on the weight of the meat pieces, take 30% water, 1.5% salt, 1% white sugar and 0.3% of the compound antioxidant prepared in step (2). Add salt, white sugar and compound antioxidant to water and stir until completely dissolved to prepare a marinade. Place the meat pieces in a marinating container, add the marinade, and tumble in a vacuum tumbler with a vacuum degree of 0.05MPa and a temperature of 4℃ for 20 minutes. The tumbling adopts a cycle mode of 10 minutes of continuous operation followed by a 3-minute interval. After taking it out, let it stand and marinate for 4 hours.

[0029] Example 6; (1) Weigh 0.47g of chlorogenic acid powder and 0.2g of hyperoside powder respectively, mix them evenly to obtain a composite antioxidant; (2) Remove excess fat from fresh duck meat, wash it clean, drain the surface water, and cut it into meat pieces. Based on the weight of the meat pieces, take 30% water, 1.5% salt, 1% white sugar and 0.3% of the compound antioxidant prepared in step (1). Add salt, white sugar and compound antioxidant to water and stir until completely dissolved to prepare a marinade. Place the meat pieces in a marinade container, add the marinade, and tumble in a vacuum tumbler with a vacuum degree of 0.05MPa and a temperature of 4℃ for 20 minutes. The tumbling adopts a cycle mode of 10 minutes of continuous operation followed by a 3-minute interval. After taking it out, let it stand and marinate for 4 hours.

[0030] Comparative Example 1; (1) Remove excess fat from fresh duck meat, wash it clean, drain the surface water, and cut it into meat pieces. Based on the weight of the meat pieces, take 30% water, 1.5% salt, and 1% white sugar. Add the salt and white sugar to the water and stir until completely dissolved to make a marinade. Place the meat pieces in a marinating container, add the marinade, and tumble them in a vacuum tumbler with a vacuum degree of 0.05 MPa and a temperature of 4°C for 20 minutes. The tumbling is done in a cycle mode of 10 minutes of continuous operation followed by a 3-minute interval. After taking it out, let it stand and marinate for 4 hours.

[0031] Comparative Example 2; (1) Take dried Sophora japonica flowers, crush them and pass them through a 40-mesh standard sieve. Add water at a material-liquid mass ratio of 1:10 to make Sophora japonica flower material solution. Adjust the pH of the material solution to 5.2. Add pectinase at a dry weight of 0.06% of Sophora japonica flowers. Under constant temperature of 40℃, use ultrasound-assisted enzymatic hydrolysis for 1.5h. After the enzymatic hydrolysis is completed, heat to 90℃ in a closed device and keep warm for 12min to inactivate the pectinase. Filter with a 200-mesh filter cloth to remove solid residue. Collect the filtrate and concentrate it. After freeze drying, crush it to obtain rutin powder. (2) Remove excess fat from fresh duck meat, wash it clean, drain the surface water, and cut it into meat pieces. Based on the weight of the meat pieces, take 30% water, 1.5% salt, 1% white sugar and 0.3% rutin powder obtained in step (1). Add salt, white sugar and rutin powder to water and stir until completely dissolved to obtain a marinade. Place the meat pieces in a marinating container, add the marinade, and tumble in a vacuum tumbler with a vacuum degree of 0.05MPa and a temperature of 4℃ for 20 minutes. The tumbling adopts a cycle mode of 10 minutes of continuous operation followed by a 3-minute interval. After taking it out, let it stand and marinate for 4 hours.

[0032] Comparative Example 3; (1) Remove excess fat from fresh duck meat, wash it clean, drain the surface water, and cut it into meat pieces. Based on the weight of the meat pieces, take 30% water, 1.5% salt, 1% white sugar and 0.3% rutin powder. Add salt, white sugar and rutin powder to water and stir until completely dissolved to make a marinating solution. Place the meat pieces in a marinating container, add the marinating solution, and tumble in a vacuum tumbler with a vacuum degree of 0.05MPa and a temperature of 4℃ for 20 minutes. The tumbling adopts a cycle mode of 10 minutes of continuous operation followed by a 3-minute interval. After taking it out, let it stand and marinate for 4 hours.

[0033] Comparative Example 4; (1) Remove excess fat from fresh duck meat, wash it clean, drain the surface water, and cut it into meat pieces. Based on the weight of the meat pieces, take 30% water, 1.5% salt, 1% white sugar and 0.3% chlorogenic acid powder. Add salt, white sugar and chlorogenic acid powder to water and stir until completely dissolved to make a marinating solution. Place the meat pieces in a marinating container, add the marinating solution, and tumble in a vacuum tumbler with a vacuum degree of 0.05MPa and a temperature of 4℃ for 20 minutes. The tumbling adopts a cycle mode of 10 minutes of continuous operation followed by a 3-minute interval. After taking it out, let it stand and marinate for 4 hours.

[0034] Comparative Example 5; (1) Remove excess fat from fresh duck meat, wash it clean, drain the surface water, and cut it into meat pieces. Based on the weight of the meat pieces, take 30% water, 1.5% salt, 1% white sugar and 0.3% hyperoside powder. Add salt, white sugar and hyperoside powder to water and stir until completely dissolved to make a marinating solution. Place the meat pieces in a marinating container, add the marinating solution, and tumble in a vacuum tumbler with a vacuum degree of 0.05MPa and a temperature of 4℃ for 20 minutes. The tumbling adopts a cycle mode of 10 minutes of continuous operation followed by a 3-minute interval. After taking it out, let it stand and marinate for 4 hours.

[0035] Test and Results Analysis Carbonyl content determination Test objective: To assess the degree of oxidative damage to myofibrillar proteins during the processing and storage of pre-processed meat products.

[0036] Test method: Accurately weigh 2.00 g of pre-processed meat product sample (after removing surface leaf debris and mincing), add 20 mL of pre-cooled buffer solution containing 0.6 mol / L NaCl and 0.05 mol / L phosphate (pH 7.4), homogenize in an ice bath for 2 min, centrifuge at 4℃ and 8000 r / min for 15 min, repeat four times, collect the precipitate, and adjust the protein concentration for later use. Take 1.0 mL of protein extract, add 4.0 mL of 10 mmol / L DNPH solution (dissolved in 2 mol / L HCl), vortex to mix, and react at 25℃ in the dark for 1 h, shaking once every 15 min during the reaction. Add 5.0 mL of 20% trichloroacetic acid solution, incubate on ice for 15 min, centrifuge at 4 °C and 10000 r / min for 10 min, wash the precipitate three times with ethanol-ethyl acetate (1:1) until colorless, dry the residual organic solvent, add 2.0 mL of 6 mol / L guanidine hydrochloride solution (containing 20 mmol / L phosphate buffer, pH 6.5), incubate at 50 °C for 30 min to dissolve the protein, centrifuge, and measure the absorbance at 370 nm. Calculate the carbonyl content using the DNPH molar extinction coefficient of 22.0 L / (mmol·cm), and express the result as nmol / mg prot; Carbonyl content / (nmol / mg) = Sample A / (22000 × protein sample concentration (mg / mL)) × 10 6 The results are shown Figure 1 .

[0037] Determination of total sulfhydryl content Test objective: To evaluate the structural integrity and redox state of myofibrillar proteins in pre-processed meat products during processing and storage.

[0038] Test method: Take 0.5 mL of the above protein extract, add 2.5 mL of Tris-glycine solution (containing 0.086 mol / L Tris, 0.09 mol / L glycine, 4 mmol / L EDTA, 8 mol / L urea, pH 8.0), vortex to mix, then add 20 μL of Ellman's reagent (4 mg 5,5'-dithiobis-2-nitrobenzoic acid dissolved in 1 mL Tris-glycine buffer), react at 25 °C for 1 h, centrifuge at 10000 r / min for 15 min, and measure the absorbance of the supernatant at 412 nm. The absorbance is calculated using a molar extinction coefficient of 13600 M. - ¹cm - ¹Calculations were performed, with results expressed in μmol / g. The thiol content (μmol / g) = 73.53 × sample A / protein sample concentration (mg / mL). See the results below. Figure 2 .

[0039] Determination of the hydrophobicity of myofibril protein surface Test objective: To assess the degree of change in the tertiary conformation of myofibrillar proteins during the processing and storage of pre-processed meat products, reflecting the exposure of hydrophobic groups within the protein molecules.

[0040] Test method: 1 mL of 5 mg / mL myofibrillar protein solution was used as the experimental group; 1 mL of 20 mmol / L phosphate buffer (pH 6.0) was used as the blank control group. 200 μL of 1 mg / mL bromophenol blue solution was added to both the experimental and control groups, and the mixture was vortexed for 10 min to ensure the BPB probe fully binds to the hydrophobic regions of the protein surface. The mixture was centrifuged at 4℃ and 8000 rpm for 15 min, and the absorbance was measured at 595 nm. The bromophenol blue binding amount was calculated as follows: Binding amount (μg) = 200 μg × (A control - A experiment) / A control. The test results are expressed as μg BPB binding amount. See [see attached table]. Figure 3 .

[0041] like Figure 1 As shown, Examples 1-3 used the natural composite antioxidant composed of rutin, chlorogenic acid, and hyperoside described in this invention. The carbonyl content was 6.015~6.248 nmol / mg, which was significantly lower than all the two-component and single-component formulations and the comparative examples without added antioxidants. This indicates that the three-component formulation can effectively scavenge reactive oxygen species that induce protein side chain carbonylation in the pickling system, blocking the conversion pathway of amino acid residues to carbonyl derivatives at the molecular level, and greatly inhibiting oxidative damage to myofibrillar proteins. Examples 4-6 show that after introducing a third polyphenolic component on the basis of the synergistic effect of the two components, a more complete synergistic antioxidant network was formed among the three polyphenols, achieving a more comprehensive inhibition of protein oxidative damage.

[0042] Comparative Example 1, which did not contain any antioxidants, had a carbonyl content as high as 10.497 nmol / mg, indicating that the myofibrillar proteins underwent severe oxidative damage during the pickling process, confirming that natural complex antioxidants are indispensable for inhibiting protein oxidation.

[0043] Comparative Examples 2 and 3 used self-made rutin from Sophora japonica and commercially available rutin as single antioxidants, respectively. The carbonyl content was 7.413 and 7.281 nmol / mg, respectively, which was significantly lower than the blank control. This indicates that rutin itself has a strong free radical scavenging ability and can effectively inhibit the formation of carbonyl groups.

[0044] Comparative Examples 4 and 5, which used chlorogenic acid and hyperoside as single antioxidants, also showed some carbonyl inhibition effects, but both were weaker than rutin alone.

[0045] The above results indicate that while single polyphenols possess antioxidant activity, their action pathways are limited, making it difficult to comprehensively block the attack of protein side chains by multiple oxidation pathways, resulting in limited inhibitory effects. This invention, however, achieves synergistic blocking of different oxidation pathways through the synergistic combination of three polyphenols, overcoming the limitations of single-agent action and further significantly reducing carbonyl content.

[0046] like Figure 2 As shown, the total thiol content of Examples 1-3 was 15.107-15.742 μmol / g, which was significantly higher than that of the two-component, single-component, and combinations without added antioxidants. This indicates that the ternary composite antioxidant of the present invention can effectively protect the thiol groups of cysteine ​​residues in myofibrillar proteins from oxidative damage and maintain the integrity of the protein structure. The total thiol content of Examples 4-6 was 12.641-14.243 μmol / g, indicating that a synergistic protective effect can be produced by combining any two components, with the combination of rutin and chlorogenic acid showing the most prominent protective effect on thiol groups.

[0047] Comparative Example 1, without the addition of any antioxidants, had a total thiol content of only 9.762 μmol / g, indicating that a large number of thiol groups were oxidized into disulfide bonds or sulfonic acid derivatives during the pickling process, resulting in severe oxidative cross-linking of the protein structure.

[0048] When rutin was used as a single agent in Comparative Examples 2 and 3, the total thiol content was 11.046 and 11.225 μmol / g, respectively. When chlorogenic acid and hyperoside were used as single agents in Comparative Examples 4 and 5, the total thiol content was 9.338 and 9.953 μmol / g, respectively. Although both were higher than the blank Comparative Example 1, the increase was limited. Single polyphenols can only mitigate the oxidative loss of thiols by capturing free radicals through their phenolic hydroxyl structure. However, due to the lack of synergistic cooperation among multiple antioxidant pathways, it is difficult to provide comprehensive protection for thiols in the protein hydration layer and hydrophobic core region. In contrast, the three polyphenols in this invention, when combined, synergistically protect thiols through different action sites and mechanisms, effectively inhibiting the oxidative cross-linking of cysteine ​​residues and achieving the highest level of total thiol content.

[0049] like Figure 3 As shown, the bromophenol blue binding amounts in Examples 1-3 were significantly lower than those in the two-component, single-component, and unantioxidant combinations, indicating that the three-component natural composite antioxidant described in this invention can effectively maintain the tertiary conformation of myofibril proteins and prevent hydrophobic groups from being exposed on the molecular surface due to oxidative stress. Examples 4-6 show that pairwise combinations can effectively inhibit protein conformation unfolding. The hydrophobicity of each of the three-component combinations is further lower than that of the pairwise combinations, and it remains stable at all three concentrations. This indicates that the three-component combination forms a more comprehensive conformational protection mechanism through synergistic free radical scavenging and multi-site interaction with the protein surface, allowing hydrophobic groups to be maintained in an embedded state to the greatest extent.

[0050] Comparative Example 1, without the addition of any antioxidants, showed a bromophenol blue binding amount as high as 66.976 μg, indicating that the protein underwent severe conformational unfolding during pickling, resulting in extensive exposure of hydrophobic regions.

[0051] When rutin was used as a single agent in Comparative Examples 2 and 3, the bromophenol blue binding amounts were 39.642 and 38.460 μg, respectively; when chlorogenic acid and hyperoside were used as single agents in Comparative Examples 4 and 5, the amounts were 45.170 and 48.167 μg, respectively. Although these were significantly lower than those in the blank Comparative Example 1, they were still higher than those in the embodiments of this invention. This indicates that single polyphenols mainly inhibit protein conformational changes indirectly by capturing free radicals, but they are difficult to form synergistic protection for different oxidation sites. After the three polyphenols of this invention are combined, on the one hand, they reduce oxidative attack by synergistically scavenging free radicals; on the other hand, each component may bind to different regions of the protein surface through hydrogen bonds, hydrophobic interactions, etc., forming a protective microenvironment, thereby maintaining the natural folded state of the protein to the greatest extent and allowing hydrophobic groups to be embedded inside the molecule.

[0052] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No markings in the claims should be construed as limiting the scope of the claims.

Claims

1. A method for marinating duck meat, characterized in that, Includes the following steps: (1) Take dried Sophora japonica flowers, crush them and sieve them. Add water to make Sophora japonica flower liquid, adjust the pH to acidic, add pectinase for enzymatic hydrolysis, heat up to inactivate pectinase after enzymatic hydrolysis, filter, collect the filtrate and concentrate, dry and crush it to obtain rutin powder; (2) The rutin powder obtained in step (1) is mixed with chlorogenic acid powder and hyperoside powder in proportion to obtain a composite antioxidant. (3) Add salt, white sugar and compound antioxidant to water and stir until completely dissolved to prepare marinade; remove excess fat from fresh duck meat, wash, drain and cut into pieces, place in marinade, tumble and marinate under vacuum conditions, and let stand to marinate after tumbling.

2. The method according to claim 1, characterized in that, The amount of pectinase added in step (1) is 0.04~0.08% of the dry weight of Sophora japonica.

3. The method according to claim 1, characterized in that, The mass ratio of rutin powder, chlorogenic acid powder and hyperoside powder in step (2) is 0.5:(0.35~0.6):(0.1~0.27).

4. The method according to claim 1, characterized in that, The vacuum condition in step (3) is a vacuum degree of 0.05 MPa; the temperature of the tumbling and marinating is 4°C and the time is 15~25 min.

5. The method according to claim 1, characterized in that, The amount of the compound antioxidant added in step (3) is 0.1~0.5% of the weight of duck meat.

6. The method according to claim 1, characterized in that, In step (3), the amount of water added is 28-35% of the weight of duck meat, the amount of salt added is 1-2% of the weight of duck meat, and the amount of white sugar added is 0.5-1.5% of the weight of duck meat.

7. The method according to claim 1, characterized in that, The enzymatic hydrolysis described in step (1) is performed under constant temperature of 40℃ with ultrasound-assisted enzymatic hydrolysis for 1-2 hours.

8. The method according to claim 1, characterized in that, The tumbling process in step (3) adopts a cycle mode of 10 minutes of continuous operation followed by a 3-minute interval.

9. The method according to claim 1, characterized in that, The conditions for static marinating in step (3) are: temperature 4℃, time 3~5h.

10. A natural compound antioxidant, characterized in that, The natural compound antioxidant comprises rutin powder, chlorogenic acid powder, and hyperoside powder; the rutin powder is obtained by enzymatic extraction of Sophora japonica buds; the mass ratio of rutin powder, chlorogenic acid powder, and hyperoside powder is 0.5:(0.35~0.6):(0.1~0.27).

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