Method for improving hydrolysis degree of oxidized protein

Abstract

The invention belongs to the technical field of food processing, and relates to a method for improving the hydrolysis degree of oxidized protein. According to the method, chlorogenic acid with different concentrations is used for carrying out digestion intervention on the gold pomfret myofibrillar protein oxidized by malonaldehyde, and when the concentration of the chlorogenic acid is 5-200 [mu] mol / L, the hydrolysis degree and digestibility of the gold pomfret myofibrillar protein are both improved. In addition, the chlorogenic acid can also improve the oxidation resistance of myofibrillar protein digestion products, such as ABTS < + > free radical scavenging capacity, DPPH free radical scavenging capacity and total reducing capacity. The method solves the problems of digestibility reduction and nutritional ingredient loss caused by protein oxidation in the storage and processing process of aquatic products, and provides a new way for improving the nutritional value of the aquatic products.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing and relates to a method for improving the hydrolysis degree of oxidized protein. Background Art

[0002] Golden pomfret, a large, flavorful, and tender edible fish, is an important commercial species for marine aquaculture. Myofibrillar protein is the primary component of golden pomfret muscle, accounting for over 50% of total protein and significantly impacting the quality and nutritional value of golden pomfret meat. However, protein oxidation can affect the quality and nutritional value of aquatic products during storage and processing. This oxidation not only causes irreversible changes in their structure and function, but also reduces digestibility, thereby impairing the body's ability to effectively absorb and utilize nutrients from aquatic products.

[0003] Chlorogenic acid is widely present in the plant kingdom and is a natural phenolic substance with multiple biological activities. Studies have shown that chlorogenic acid can effectively inhibit lipid oxidation and the production of free radicals, and has a significant protective effect on various oxidative damage models. In food systems, chlorogenic acid can exert its antioxidant functions, such as scavenging free radicals, chelating metal ions, and inhibiting oxidase activity, thereby reducing oxidative deterioration of food. Currently, there are relatively few studies on the effects of chlorogenic acid on the digestion of oxidized myofibrillar protein in aquatic products. In-depth exploration of the effects of chlorogenic acid on the digestion of myofibrillar protein under the malondialdehyde-mediated oxidation system and its mechanism of action has important theoretical and practical significance for improving the nutritional value of aquatic products, protecting consumer health, and promoting the development of the aquatic product processing industry. Summary of the Invention

[0004] In response to the above problems and defects, the present invention provides a method for improving the hydrolysis degree of oxidized protein. The present invention uses chlorogenic acid at different concentrations to intervene in malondialdehyde-mediated oxidized myofibrillar protein, thereby improving the digestibility of oxidized protein and providing a basis for the application of chlorogenic acid in the preservation and quality improvement of aquatic products.

[0005] In a first aspect, the present invention provides a method for improving the hydrolysis degree of oxidized protein, comprising mixing chlorogenic acid with oxidized protein to increase the hydrolysis degree of the oxidized protein.

[0006] Furthermore, in the method for improving the hydrolysis degree of oxidized protein provided by the present invention, the concentration of chlorogenic acid is 5-200 μmol / L.

[0007] Furthermore, in the method for improving the hydrolysis degree of oxidized protein provided by the present invention, the preparation of the oxidized protein comprises: mixing a myofibrillar protein suspension with malondialdehyde, incubating in the dark, and removing the malondialdehyde by dialysis.

[0008] Furthermore, in the method for improving the hydrolysis degree of oxidized protein provided by the present invention, the myofibrillar protein is derived from golden pomfret.

[0009] Furthermore, in the method for improving the hydrolysis degree of oxidized protein provided by the present invention, the preparation of the myofibrillar protein suspension comprises: dissolving the myofibrillar protein in a sodium phosphate buffer solution.

[0010] Furthermore, in the method for improving the hydrolysis degree of oxidized protein provided by the present invention, the concentration of the myofibrillar protein suspension is 20 mg / mL; The concentration of the malondialdehyde is 1-10 mM.

[0011] Furthermore, in the method for improving the hydrolysis degree of oxidized protein provided by the present invention, before the chlorogenic acid is mixed with the oxidized protein, the oxidized protein is diluted to 10 mg / mL with a phosphate buffer solution.

[0012] In a second aspect, the present invention provides an application of a method for improving the hydrolysis degree of oxidized protein in improving the nutritional value of aquatic products.

[0013] Furthermore, in the application of the method for improving the hydrolysis degree of oxidized protein provided by the present invention in improving the nutritional value of aquatic products, the aquatic product is golden pomfret.

[0014] Furthermore, the method for improving the hydrolysis degree of oxidized protein provided by the present invention is used in improving the nutritional value of aquatic products, wherein improving the nutritional value of aquatic products includes improving the antioxidant capacity of oxidized protein digestion products.

[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: The present invention intervened with oxidized myofibrillar protein using different concentrations of chlorogenic acid (0, 5, 10, 50, 100, 200 μmol / L) and found that chlorogenic acid can increase the hydrolysis degree of oxidized protein, and with the increase of chlorogenic acid concentration, the hydrolysis degree and digestibility of oxidized myofibrillar protein increased. In addition, chlorogenic acid improves the antioxidant capacity of the digestion products of oxidized myofibrillar protein, among which ABTS + The free radical scavenging ability and total reducing capacity increased with increasing chlorogenic acid concentration, while the DPPH free radical scavenging ability decreased at a chlorogenic acid concentration of 200 μmol / L. However, compared with the sample without chlorogenic acid, the DPPH free radical scavenging ability was still enhanced. This invention can improve the digestibility of oxidized protein and contribute to the nutritional value of aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 The effect of different concentrations of malondialdehyde on the hydrolysis degree of myofibrillar protein in golden pomfret. A is the hydrolysis degree of pepsin; B is the hydrolysis degree of trypsin.

[0018] Figure 2 The effect of different concentrations of chlorogenic acid on the hydrolysis degree of myofibrillar protein oxidized by 1 mM malondialdehyde (A) is the hydrolysis degree by pepsin; B is the hydrolysis degree by trypsin.

[0019] Figure 3 The effect of different concentrations of chlorogenic acid on the hydrolysis degree of myofibrillar protein oxidized by 2 mM malondialdehyde (A) is the hydrolysis degree by pepsin; B is the hydrolysis degree by trypsin.

[0020] Figure 4 The effect of different concentrations of chlorogenic acid on the hydrolysis degree of myofibrillar protein oxidized by 5 mM malondialdehyde (A) is the hydrolysis degree by pepsin; B is the hydrolysis degree by trypsin.

[0021] Figure 5 The effect of different concentrations of chlorogenic acid on the hydrolysis degree of myofibrillar protein oxidized by 10 mM malondialdehyde (MDA). A represents the hydrolysis degree by pepsin; B represents the hydrolysis degree by trypsin.

[0022] Figure 6 ABTS of different concentrations of chlorogenic acid on the digestion product of golden pomfret myofibrillar protein oxidized by 5 mM malondialdehyde + Free radical scavenging ability.

[0023] Figure 7 The effect of different concentrations of chlorogenic acid on the DPPH free radical scavenging ability of the digestion products of golden pomfret myofibrillar protein oxidized by 5 mM malondialdehyde.

[0024] Figure 8 The effect of different concentrations of chlorogenic acid on the total reducing capacity of the digestion products of golden pomfret myofibrillar protein oxidized by 5 mM malondialdehyde.

[0025] Figure 9The effect of different concentrations of chlorogenic acid on the UV absorption spectra of the digestion products of myofibrillar protein oxidized by 5 mM malondialdehyde (A: 0 μmol / L, B: 5 μmol / L, C: 10 μmol / L, D: 50 μmol / L, E: 100 μmol / L, and F: 200 μmol / L).

[0026] Figure 10 The effect of different concentrations of chlorogenic acid on the fluorescence absorption spectra of the digestion products of myofibrillar protein oxidized by 5 mM malondialdehyde (A: 0 μmol / L, B: 5 μmol / L, C: 10 μmol / L, D: 50 μmol / L, E: 100 μmol / L, and F: 200 μmol / L).

[0027] Figure 11 The effect of different concentrations of chlorogenic acid on the fluorescence spectra of the adducts of myofibrillar protein digestion products oxidized by 5 mM malondialdehyde (A: 0 μmol / L, B: 5 μmol / L, C: 10 μmol / L, D: 50 μmol / L, E: 100 μmol / L, and F: 200 μmol / L). DETAILED DESCRIPTION

[0028] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified. The percentages in the following examples are percentages by mass unless otherwise specified. The ratios in the following examples are ratios by mass unless otherwise specified.

[0029] Example 1 This example provides a method for constructing a system for improving the digestion of oxidized proteins using chlorogenic acid.

[0030] (1) Preparation of oxidized proteins A suspension of golden pomfret MP (myofibrillar protein) (20 mg / mL, dissolved in 20 mM sodium phosphate buffer, pH 6.0) was mixed with various concentrations of MDA (malondialdehyde, 0, 1, 2, 5, and 10 mM). The mixture was immediately transferred to a tightly sealed glass vial and incubated at 25°C in a dark environment for 24 hours. After incubation, a series of MDA-treated MP samples were obtained as the oxidation system. Following the reaction, these protein solutions were dialyzed to remove unbound MDA, and the proteins in the oxidation system were finally digested.

[0031] (2) Chlorogenic acid improves the construction of the oxidized protein digestion system The MDA-oxidized protein was added to phosphate buffer (pH 6.0, 0.1 mol / L NaCl) and homogenized (5000 rpm, 30 s). To simulate gastric digestion, the protein concentration was adjusted to 10 mg / mL, and the pH of the protein solution was then adjusted to 3.0 using 3 mol / L HCl. The protein solution was mixed with equal volumes of gastric digestion fluid, and chlorogenic acid was added simultaneously. The pepsin activity was 200 U / mg, and the chlorogenic acid concentrations were set at 0, 5, 10, 50, 100, and 200 μmol / L. The mixture was digested at 37°C and 160 rpm for 2 h and then rapidly inactivated in a boiling water bath for 10 min. After the gastric phase reaction was complete, the intestinal phase was simulated. The pH of the mixture was adjusted to 7.0 using 1 mol / L NaOH, and then intestinal digestion fluid was added in equal volumes, with trypsin activity at 100 U / mL. The mixture was then digested at 37°C and 160 rpm for 2 hours and rapidly inactivated in a boiling water bath for 10 minutes. The digestion products collected during the digestion process were cooled to room temperature after inactivation and then centrifuged at 8000 × g for 15 minutes. The supernatant and precipitate were separated and stored at -80°C.

[0032] Example 2 This example provides a method for detecting the effects of chlorogenic acid on the digestibility, structure, and function of oxidized myofibrillar protein.

[0033] (1) Determination of hydrolysis degree Weigh 3.81 g of sodium tetraborate decahydrate solid and dissolve 0.1 g of SDS in distilled water. Then, weigh 0.08 g of o-phthalaldehyde (OPA) and dissolve it in 4 mL of anhydrous ethanol. Once completely dissolved, weigh 0.088 g of dithiothreitol (DTT) and add the mixture to 100 mL. The resulting solution is the OPA solution. A standard curve for free amino groups using L-serine was prepared by weighing 10.509 g of L-serine and diluting the volume to 100 mL with distilled water to obtain the L-serine solution. Dispense 0, 0.2, 0.4, 0.6, 0.8, and 1 mL of the L-serine solution, respectively, and dilute to 1 mL with pure water. Then, aspirate 0.6 mL and add 4.5 mL of the OPA solution. Mix and shake for 30 seconds. Measure the absorbance at 340 nm. Adjust the concentration of the digestion product supernatant to 1 mg / mL, take 600 μL and add 4.5 mL of the OPA mixed reagent, shake well, and keep it in the dark for 2 minutes. Measure the absorbance at a wavelength of 340 nm. The absorbance of the sample at the hydrolysis endpoint is A1, and the absorbance of the hydrolyzed sample is A2. The degree of hydrolysis (H) is calculated according to the following formula: H(%)=[(A1-A2)÷A1]×100 (2) ABTS + Determination of free radical scavenging ability Take 88μL 140mmol / L K2S2O8 solution and 5mL 7mmol / L ABTS + After the solution is mixed and shaken, ABTS is obtained. + The stock solution was placed in the dark at room temperature for 12 hours. + Dilution gradient of the stock solution to obtain OD 734 ABTS with values ​​in the range of 0.700±0.002 + Take 1mL of the digestion product supernatant and add it to 4mL of ABTS + Mix well in the working solution, let it stand at room temperature for 6 minutes, and 734 Measured under the conditions of A1; 1 mL of anhydrous ethanol was added to 4 mL of ABTS + The working solution is used as a blank control and its absorbance is measured under the same conditions as the assay group (A1), which is recorded as A2. + Clearance (ABTS + ) formula for calculation: ABTS + (%)=[(A1-A2)÷A1]×100 (3) Determination of DPPH free radical scavenging ability First, take 1 mL of the digestion product supernatant and 1 mL of 25 μg / mL DPPH solution, mix and shake well, and let it stand at room temperature in the dark for 30 min. 517 Then, 1 mL of the digestion product supernatant was mixed with 1 mL of anhydrous ethanol and shaken evenly. After standing for 30 min, the absorbance was measured at OD 517 The absorbance was measured under the conditions of OD 0.05, recorded as Aj; then 1 mL 25 μg / mL DPPH was mixed with 1 mL anhydrous ethanol and allowed to stand for 30 min. 517 The absorbance was measured under the conditions of , recorded as Ao, as a blank control. Finally, the DPPH clearance (DPPH) was calculated according to the following formula: DPPH(%)=[1-[(Ai-Aj)÷Ao]]×100 (4) Determination of total reducing capacity Transfer 0.5 mL of the digestion supernatant to a test tube. Add 1 mL of 1% potassium ferricyanide and 2.5 mL of 0.2 mmol / L phosphate buffer (pH 6.6) to the tube, mix thoroughly, and incubate at 50°C for 20 minutes. Immediately cool the tube quickly, add 2.5 mL of 10% trichloroacetic acid (TCA), and shake well. Then, add 2.5 mL of double-distilled water and 0.45 mL of 0.1% FeCl₃ solution, mix thoroughly, and let stand at room temperature for 10 minutes. Measure the absorbance at 700 nm. The absorbance is proportional to the total reducing capacity.

[0034] (5) Determination of UV spectrum 200 μL of the digestion product supernatant was added to a quartz 96-well plate and the supernatant was spectrally scanned using a spectrophotometer in the UV range of 200–800 nm with 1 nm intervals.

[0035] (6) Determination of endogenous fluorescence intensity 1 mL of the undigested product supernatant was placed in a fluorescent cuvette with four sides transparent to light. The sample solution was then scanned using a fluorescence spectrometer. Parameters were set as follows: excitation wavelength of 290 nm, scan speed of 1500 nm / min, scan range of 310–400 nm, and 1 nm interval.

[0036] (7) Determination of adduct fluorescence intensity 1 mL of the undigested product supernatant was placed in a fluorescent cuvette with four sides transparent to light. The sample solution was then scanned using a fluorescence spectrometer. Parameters were set as follows: excitation wavelength of 390 nm, scan speed of 1500 nm / min, scan range of 400 nm to 600 nm, and 1 nm interval.

[0037] Example 3 This example provides the hydrolysis of proteins oxidized by different MDA concentrations.

[0038] Under the action of pepsin, proteins will be hydrolyzed to produce polypeptides and amino acids. The degree of enzymatic hydrolysis of proteins can be characterized by measuring the free amino group content in the supernatant of the enzymatic hydrolysis product. Figure 1 As shown in Figure A, the free amino group content in the supernatant of the enzymatic hydrolysis products of all oxidation systems showed an increasing trend. As the malondialdehyde concentration increased, the amount of free amino group released decreased significantly. At the 30th minute of the enzymatic hydrolysis process, the amount of free amino groups released that were not oxidized by MDA was 770.85 nmol / mg, while the amount released by 10 mM MDA was 251.38 nmol / mg, indicating that increasing MDA concentration hindered the normal enzymatic hydrolysis of proteins.

[0039] In order to further observe the hydrolysis degree of each oxidation system during the enzymatic hydrolysis process, the hydrolysis degree of different oxidation systems at the same time was compared. The results are as follows: Figure 1 As shown in Figure B. The degree of hydrolysis of proteins not oxidized by MDA exceeds 90%, indicating that golden pomfret is a high-quality protein source that is easily digested and absorbed by the human body. Over the same time period, as the concentration of malondialdehyde increases, the degree of hydrolysis decreases. This indicates that malondialdehyde can hinder the normal progress of enzymatic hydrolysis. At the 60th minute of enzymatic hydrolysis, the free amino group content in the oxidation system with an MDA concentration of 10 mM no longer significantly increases. This indicates that even under acidic conditions, high concentrations of MDA significantly disrupt protein enzymatic hydrolysis and have a highly significant inhibitory effect on the hydrolysis degree of myofibrillar protein. Therefore, the decrease in the hydrolysis degree of golden pomfret myofibrillar protein can be attributed to the destruction of the protein structure.

[0040] Example 4 This example provides the effect of chlorogenic acid on the digestibility of oxidized myofibrillar protein.

[0041] The degree of protein hydrolysis refers to the percentage of peptide bonds cleaved during catalytic hydrolysis of a protein relative to the total number of peptide bonds. It indicates the extent of protein hydrolysis. Each peptide bond hydrolyzed releases a free amino group, which reacts with o-phthalaldehyde (OPA) to form a fluorescent yellow complex whose absorbance can be measured spectrophotometrically. A higher degree of hydrolysis results in more peptide bonds cleaved, leading to a higher content of free amino acids. This, in turn, generates more fluorescent substances that react with OPA, resulting in a higher fluorescence intensity and, therefore, absorbance. Protein digestibility, on the other hand, refers to the extent to which a protein is broken down by digestive enzymes and absorbed and utilized by the body. Generally, a higher degree of protein hydrolysis indicates a higher digestibility. Therefore, the present invention uses degree of hydrolysis instead of digestibility for analysis and research.

[0042] Effects of different concentrations of chlorogenic acid on the hydrolysis degree of oxidized myofibrillar protein Figure 2 (MDA concentration is 1mM), Figure 3 (MDA concentration is 2mM), Figure 4 (MDA concentration was 5 mM) and Figure 5As shown in the MDA-mediated myofibrillar protein digestion system (MDA concentration was 10 mM), the degree of hydrolysis of myofibrillar protein first increased and then decreased with increasing chlorogenic acid concentration. When chlorogenic acid concentrations ranged from 10 to 100 μmol / L, the degree of hydrolysis gradually increased and reached a peak. Later, when chlorogenic acid concentrations ranged from 100 to 200 μmol / L, the degree of hydrolysis decreased somewhat, but the hydrolysis rate was still significantly higher than that of samples without chlorogenic acid, indicating a significant difference. These results indicate that the hydrolysis degree or digestibility of myofibrillar protein is affected by chlorogenic acid. When the chlorogenic acid concentration is 0-100 μmol / L, chlorogenic acid may interact with myofibrillar protein or digestive enzymes to promote the loosening of protein structure, thereby increasing the degree of hydrolysis. When the chlorogenic acid concentration reaches 200 μmol / L, it may inhibit the digestive enzymes or form a more stable complex with the protein, thereby reducing the degree of hydrolysis and digestibility. However, it still promotes the hydrolysis degree and digestibility of oxidized myofibrillar protein.

[0043] Example 5 This example provides the effect of chlorogenic acid on the antioxidant capacity of myofibrillar protein digestion products oxidized by 5 mM malondialdehyde.

[0044] (1) ABTS + Free radical scavenging ability ABTS + It is a commonly used and stable blue free radical. Under certain conditions, antioxidants can convert ABTS into + The color change can be reflected by measuring the change in absorbance, which can be used to evaluate the sample's ABTS + Free radical scavenging ability.

[0045] Effect of different concentrations of chlorogenic acid on the ABTS of myofibrillar protein digestion products oxidized by 5 mM malondialdehyde + Free radical scavenging ability affects Figure 6 As shown in Figure 2, with the increase of chlorogenic acid concentration, ABTS + The free radical scavenging rate showed a gradual upward trend, indicating that chlorogenic acid has a certain ABTS + The free radical scavenging ability of the chlorogenic acid group was significantly different from that of the ABTS group. + There are significant differences in free radical scavenging rates. ABTS samples without chlorogenic acid added + The free radical scavenging rate was the lowest, only 48.54%. Compared with other samples with chlorogenic acid added, the scavenging rate was significantly lower and the difference was significant. There were also differences between the chlorogenic acid concentrations of 50 μmol / L and 200 μmol / L, indicating that the increase in chlorogenic acid concentration had a significant effect on the free radical scavenging rate of ABTS. +The improvement of free radical scavenging ability is dose-dependent.

[0046] (2) DPPH free radical scavenging ability The DPPH free radical is a nitrogen-centered, fat-soluble free radical with a stable chemical structure. The DPPH free radical possesses an unpaired electron, which makes it somewhat oxidizing and capable of reacting with reducing antioxidants. When antioxidants are present in the system, the electrons or hydrogen atoms they donate can react with the DPPH free radical to form a stable DPPH-H molecule, thereby scavenging the DPPH free radical. The DPPH free radical is more stable than the hydroxyl radical and superoxide radical, making it more suitable for evaluating antioxidant activity.

[0047] Effects of different concentrations of chlorogenic acid on the DPPH free radical scavenging ability of 5 mM malondialdehyde oxidized myofibrillar protein digestion products Figure 7 As shown in the results, the DPPH radical scavenging rate showed a slow upward trend with increasing chlorogenic acid concentration, remaining around 80%. At 200 μmol / L, it showed a slight downward trend, but was still higher than the DPPH radical scavenging rate of the sample group without chlorogenic acid. This indicates that chlorogenic acid has a certain DPPH radical scavenging ability, but this ability is not significantly affected by different concentrations. This may be because the structural characteristics of chlorogenic acid cause its DPPH radical scavenging effect to reach saturation within a certain concentration range. Excessively high concentrations of chlorogenic acid (200 μmol / L) may cause oxidation or structural changes under the experimental conditions, which in turn reduces its antioxidant capacity.

[0048] (3) Total reduction capacity The amino acids such as tryptophan contained in myofibrillar protein have reducing ability. These amino acids can react with oxidants as electron donors. The total reducing ability of myofibrillar protein mainly achieves antioxidant effect by providing hydrogen atoms, electrons, etc. 3+ Reduction to Fe 2+ , which proves that the sample contains excess electrons. The stronger the electron supply capacity of the sample, the stronger the total reducing power and the stronger the antioxidant capacity. 3+ Capabilities) ready to use Figure 8 The vertical axis indicates that the greater the absorbance, the greater the reducing ability.

[0049] As the concentration of added chlorogenic acid increased, the absorbance showed an upward trend, indicating a gradual increase in its total reducing capacity. Reducing capacity is closely related to free radical scavenging and antioxidant activity, indicating that higher chlorogenic acid concentrations are associated with greater total reducing capacity and antioxidant capacity. In samples supplemented with chlorogenic acid at concentrations ranging from 5 to 200 μmol / L, significant differences in total reducing capacity were observed in each group. Compared with the group without chlorogenic acid, the group with 5 μmol / L chlorogenic acid showed no significant difference in total reducing capacity, while significant differences were observed in the other groups. In summary, chlorogenic acid can increase the total reducing capacity of myofibrillar protein digestion products, and this is positively correlated with concentration. When chlorogenic acid concentrations range from 0 to 5 μmol / L, there is little effect on the total reducing capacity; however, when chlorogenic acid concentrations range from 5 to 200 μmol / L, there is a significant effect on the total reducing capacity.

[0050] (4) Ultraviolet absorption spectrum Ultraviolet absorption spectroscopy is generally used to study changes in the tertiary structure of proteins because the side chain groups of tyrosine residues and tryptophan absorb ultraviolet light and have a characteristic absorption peak at 275nm. Figure 9 As shown in the figure, the concentrations of chlorogenic acid added were 0, 5, 10, 50, 100, and 200 μmol / L, which were groups A, B, C, D, E, and F, respectively. Figure 9 It can be seen that with the increase in chlorogenic acid concentration, the characteristic absorption peak originally at 275nm showed a significant red shift, accompanied by a significant increase in the ultraviolet absorption intensity. Chlorogenic acid is a phenolic substance containing active groups such as phenolic hydroxyl groups. It may interact with amino acid residues in protein digestion products to form complexes with hydrogen bonds or other intermolecular forces. The formation of this complex changes the electron cloud distribution of protein digestion products, causing the absorption peak at 275nm to red shift. Chlorogenic acid is a compound with a conjugated structure, and protein digestion products contain chromophores such as aromatic amino acids. The conjugation between chlorogenic acid and the benzene ring of the chromophore reduces the energy required for the π-π* transition, thereby causing the absorption peak to red shift; the protein digestion products may have undergone structural rearrangement and unfolding, exposing more chromophores, resulting in enhanced ultraviolet absorption.

[0051] (5) Fluorescence absorption spectrum The change of tryptophan fluorescence intensity is often used to reflect the changes in protein structure. The stronger the polarity of the environment where tryptophan is located, the longer the maximum absorption wavelength of the endogenous fluorescence spectrum. Figure 10As shown in the figure, the concentrations of chlorogenic acid added were 0, 5, 10, 50, 100, and 200 μmol / L, representing Groups A, B, C, D, E, and F, respectively. The main peak position of samples treated with different concentrations of chlorogenic acid exhibited a red shift, while the fluorescence intensity increased with increasing chlorogenic acid concentration. This suggests that chlorogenic acid inhibits amino acid oxidation, reduces the exposure of tryptophan residues within myofibrillar protein, and reduces the binding of malondialdehyde to protein, leading to an increased polarity of the tryptophan microenvironment, resulting in a red shift and increased fluorescence intensity.

[0052] (6) Effect of adduct fluorescence spectrum Schiff bases are organic compounds containing imine or methylimine characteristic groups (-RC=N-). Malondialdehyde reacts with proteins and amino acids to form Schiff bases, and the interaction of their adducts can be shown from the fluorescence spectrum through the strong fluorescence characteristics of Schiff bases. Figure 11 As shown in the figure, the concentrations of chlorogenic acid added were 0, 5, 10, 50, 100, and 200 μmol / L, which were groups A, B, C, D, E, and F, respectively. Figure 11 As can be seen in the fluorescence spectrum, an absorption peak appeared near 460nm. As the concentration of chlorogenic acid increased, the fluorescence absorption intensity underwent a certain blue shift and continued to decrease. This indicates that the reaction between malondialdehyde and MP was inhibited by chlorogenic acid, and the inhibitory effect increased with increasing concentration. The reduction of Schiff bases affected its digestibility.

[0053] The above data show that with the increase of chlorogenic acid concentration, the hydrolysis degree and digestibility of myofibrillar protein increase; UV spectrum, endogenous fluorescence spectrum and adduct fluorescence spectrum all show this result. In addition, chlorogenic acid improves the antioxidant capacity of myofibrillar protein digestion products, ABTS + Both free radical scavenging and total reducing capacity increased with increasing chlorogenic acid concentration. DPPH radical scavenging activity decreased at a chlorogenic acid concentration of 200 μmol / L, but remained enhanced compared to samples without chlorogenic acid. This may be because chlorogenic acid improves the digestibility of oxidized proteins within a certain concentration range, but this effect is inhibited at elevated concentrations. This study may contribute to improving the digestibility of oxidized proteins and enhance the nutritional value of aquatic products.

[0054] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the scope of protection determined by the present invention.

Claims

1. A method for improving the hydrolysis degree of oxidized protein, characterized in that: Chlorogenic acid is mixed with oxidized protein to increase the degree of hydrolysis of the oxidized protein.

2. The method for improving the hydrolysis degree of oxidized protein according to claim 1, wherein: The concentration of the chlorogenic acid is 5-200 μmol / L.

3. The method for improving the hydrolysis degree of oxidized protein according to claim 1, wherein: The preparation of the oxidized protein comprises the following steps: mixing a myofibrillar protein suspension with malondialdehyde, incubating the mixture in the dark, and removing the malondialdehyde through dialysis.

4. The method for improving the hydrolysis degree of oxidized protein according to claim 3, characterized in that: The myofibrillar protein is derived from golden pomfret.

5. The method for improving the hydrolysis degree of oxidized protein according to claim 3, characterized in that: The preparation of the myofibrillar protein suspension comprises the following steps: dissolving the myofibrillar protein in a sodium phosphate buffer solution.

6. The method for improving the hydrolysis degree of oxidized protein according to claim 3, characterized in that: The concentration of the myofibrillar protein suspension is 20 mg / mL; The concentration of the malondialdehyde is 1-10 mM.

7. The method for improving the hydrolysis degree of oxidized protein according to claim 1, wherein: Before the chlorogenic acid is mixed with the oxidized protein, the oxidized protein is diluted to 10 mg / mL with a phosphate buffer solution.

8. Use of the method for improving the hydrolysis degree of oxidized protein according to claim 1 in improving the nutritional value of aquatic products.

9. The use according to claim 8, characterized in that The aquatic product is golden pomfret.

10. The use according to claim 8, characterized in that The method of improving the nutritional value of aquatic products includes improving the antioxidant capacity of oxidized protein digestion products.

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