Method for improving functional properties of fish gelatin by ultrasonic-assisted phosphorylation modification
By using an ultrasound-assisted phosphorylation modification method, the phosphorylation degree of fish gelatin was increased, thereby improving its gelation and digestibility properties. This solved the problem of low phosphorylation degree in existing technologies and enhanced the functional properties of fish gelatin.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- DALIAN POLYTECHNIC UNIVERSITY
- Filing Date
- 2023-12-20
- Publication Date
- 2026-05-19
AI Technical Summary
In existing technologies, the degree of protein phosphorylation modification is low and cannot be effectively increased by extending the time or increasing the phosphate concentration, and the functional properties of phosphorylated fish gelatin are not fully utilized.
Phosphorylated fish gelatin was prepared by ultrasound-assisted phosphorylation modification, which involved mixing fish gelatin with sodium tripolyphosphate and sodium hydroxide solutions, followed by ultrasonic treatment, desalting by dialysis, and freeze-drying.
It significantly improved the phosphorylation level of fish gelatin, enhanced its gelling and digestibility properties, increased the antioxidant activity of digestion products, and expanded the application range of fish gelatin.
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Figure CN117843988B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of food technology, and specifically relates to a method for improving the functional properties of fish gelatin through ultrasound-assisted phosphorylation modification. Background Technology
[0002] Phosphorylation is an effective method for improving protein functional properties. Phosphorylation mainly occurs at the hydroxyl groups of the side chains of tyrosine, serine, and threonine residues in proteins. By modifying the active side chain groups, the charge state of the protein can be altered, thereby changing its properties. However, protein phosphorylation is often of low degree and cannot be improved by prolonging phosphorylation time or increasing phosphate concentration. Summary of the Invention
[0003] This invention provides a method for improving the functional properties of fish gelatin through ultrasound-assisted phosphorylation modification. This method increases the degree of phosphorylation in fish gelatin, improves its gelling properties, and expands its application range. Simultaneously, the digestibility of phosphorylated fish gelatin is also improved, with digestion products exhibiting better antioxidant activity, thus enhancing the nutritional value of the fish gelatin.
[0004] The purpose of this invention is to develop a highly efficient, low-cost, and pollution-free method for improving the functional properties of fish gelatin.
[0005] To achieve the above objectives, the present invention provides a method for improving the functional properties of fish gelatin through ultrasound-assisted phosphorylation modification, comprising the following steps:
[0006] S1. Dissolve fish gelatin powder in water by stirring to obtain a fish gelatin solution;
[0007] S2. Mix sodium tripolyphosphate with the fish gelatin solution from step S1, and adjust the pH value to 8-9 with sodium hydroxide solution to obtain a fish gelatin and phosphate mixture.
[0008] S3. The fish gelatin and phosphate mixture obtained in S2 is sonicated to obtain an ultrasound-assisted phosphorylation modified fish gelatin solution.
[0009] S4. The ultrasound-assisted phosphorylated fish gelatin solution obtained in S3 was dialyzed to remove salt, and then freeze-dried to obtain phosphorylated fish gelatin.
[0010] Furthermore, the stirring described in step S1 is carried out at 45-50°C for 1-1.5 hours at a stirring speed of 1000-1200 rpm.
[0011] Furthermore, the concentration of the fish gelatin solution in step S1 is 6-8% (w / v, g / mL).
[0012] Furthermore, in step S2, the mass ratio of sodium tripolyphosphate to fish gelatin solution is 1:300 to 500.
[0013] Furthermore, the concentration of the sodium hydroxide solution mentioned in step S2 is 1-2 mol / L.
[0014] Furthermore, the ultrasonic conditions described in step S3 are: ultrasonic-assisted reaction for 20 to 120 minutes at 35–45°C and ultrasonic power of 150–250W.
[0015] Preferably, the ultrasound-assisted response time is 60 minutes.
[0016] Furthermore, the dialysis desalination described in step S4 uses a dialysis bag with a size of 44 mm and a molecular weight cutoff of 3500 Da.
[0017] The present invention provides phosphorylated modified fish gelatin prepared according to the above method.
[0018] The application of phosphorylated modified fish gelatin provided by this invention in the food industry.
[0019] Beneficial effects:
[0020] 1. This invention introduces ultrasound into the phosphorylation modification of fish gelatin, and uses ultrasound-assisted phosphorylation modification to modify fish gelatin, thereby achieving a significant improvement in phosphorylation modification efficiency and functional properties.
[0021] 2. The ultrasonic-assisted phosphorylation modification method used in this invention modifies fish gelatin. The modification method has the advantages of being green, safe, pollution-free, and energy-efficient.
[0022] 3. This invention employs an ultrasound-assisted phosphorylation modification method, which improves the gel properties, digestibility, and antioxidant activity of the digestion products of fish gelatin, thereby enhancing its nutritional value and laying the foundation for expanding the application range of fish gelatin.
[0023] 4. The entire technology of this invention is simple and easy to implement, the product process is simple, it is suitable for mass production, and it has good market prospects. Attached Figure Description
[0024] Figure 1 This is a technical roadmap of the present invention.
[0025] Figure 2 The degree of phosphorylation modification is shown in Examples 1, 2, 1, 2 and 7.
[0026] Figure 3 These are the potential diagrams of Example 1, Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 7.
[0027] Figure 4 The gel strengths are those of Example 3, Comparative Example 3, Comparative Example 4, and Comparative Example 5.
[0028] Figure 5 These are moisture distribution analysis diagrams for Example 3, Comparative Example 3, Comparative Example 4, and Comparative Example 5.
[0029] Figure 6 This is an analysis diagram of the phase angle changes during the cooling process of Example 3, Comparative Example 3, Comparative Example 4, and Comparative Example 5.
[0030] Figure 7 This is an analysis diagram of the phase angle changes during the heating process of Example 3, Comparative Example 3, Comparative Example 4, and Comparative Example 5.
[0031] Figure 8 This is a diagram of the gel micronetwork structure of Example 3.
[0032] Figure 9 This is a diagram of the gel micronetwork structure of Comparative Example 3.
[0033] Figure 10 This is a diagram of the gel micronetwork structure of Comparative Example 4.
[0034] Figure 11 This is a diagram of the gel micronetwork structure of Comparative Example 5.
[0035] Figure 12 The free amino acid content of Examples 3, 4, Comparative Examples 3 and 6 after gastrointestinal digestion.
[0036] Figure 13 These are SDS-PAGE electrophoresis images of Examples 3, 4, Comparative Examples 3 and 6 during the small intestine digestion process in the gastrointestinal tract.
[0037] Figure 14 , 15 This is a peptide distribution diagram of Examples 3, 4, Comparative Examples 3 and 6 during the small intestine digestion process in the gastrointestinal tract.
[0038] Figure 16 The DPPH clearance rate of gastrointestinal digestive products in Examples 3, 4, 3, and 6.
[0039] Figure 17 The OH clearance rate of gastrointestinal digestive products in Examples 3, 4, 3, and 6. Detailed Implementation
[0040] The following embodiments further illustrate the technical solution of the present invention and help to understand the present patent. However, the implementation of the present invention is not limited to the embodiments described, and the scope of protection of the present invention is determined by the claims.
[0041] Test method:
[0042] 1. Test of phosphorylation degree: The phosphorylated fish gelatin sample was carbonized at high temperature and then ashed in a muffle furnace at 550℃. The inorganic residue was transferred to a 100mL volumetric flask with deionized water and diluted to the mark with water. The absorbance of the sample was measured at 660nm using a microplate reader with hydroquinone and sodium sulfite reduction method, and the phosphorus content was calculated from the standard curve.
[0043] The specific steps for plotting the phosphorus content standard curve are as follows:
[0044] ① Accurately weigh 0.4394 g of potassium dihydrogen phosphate (accurate to 0.0001 g) and place it in a beaker. Add an appropriate amount of water to dissolve it and transfer it to a 1000 mL volumetric flask. Add water to make up to the mark and mix well to obtain the phosphorus standard stock solution (100.0 mg / L).
[0045] ② Accurately pipette 10 mL of phosphorus standard stock solution (100.0 mg / L) into a 100 mL volumetric flask, dilute with water to the mark, and mix well to obtain phosphorus standard working solution.
[0046] ③ Accurately pipette 0 mL, 0.500 mL, 1.00 mL, 2.00 mL, 3.00 mL, 4.00 mL, and 5.00 mL of phosphorus standard working solution, equivalent to phosphorus contents of 0 μg, 5.00 μg, 10.0 μg, 20.0 μg, 30.0 μg, 40.0 μg, and 50.0 μg, respectively, into 25 mL stoppered test tubes. Add 2 mL of ammonium molybdate solution (50 g / L) sequentially, shake well, and let stand. Add 1 mL of sodium sulfite solution (200 g / L) and 1 mL of hydroquinone solution (5 g / L), and shake well. Add water to the mark and mix well. After standing for 0.5 h, measure the absorbance at a wavelength of 660 nm. Plot a standard curve using the measured absorbance against the phosphorus content.
[0047] 2. Potential Value Testing: The pH of a 0.05% (w / v) fish gelatin solution was adjusted to 7.0 using 0.1M HCl or 0.1M NaOH. The potential value of the phosphorylated fish gelatin solution was measured at room temperature using a laser particle size analyzer. Three measurements were taken, and the average value was recorded.
[0048] 3. Determination of phosphorylation modification sites:
[0049] Sample pretreatment: Trypsin was added at a ratio of 1:50 (enzyme:protein, mass ratio) to phosphorylated fish gelatin, and the mixture was enzymatically hydrolyzed overnight at 37°C. The sample was then desalted using a C18 desalting column and lyophilized.
[0050] Sample injection: The sample was analyzed by LC-MS / MS with an online nano-spray ion source. The whole system was an Orbitrap Fusion mass spectrometer with EASY-nanoLC 1200 in series. The specific determination method is as follows. The sample volume was 3 μL (analytical column: AcclaimPep Map C18, 75 μm × 25 cm), the sample was separated by a gradient of 60 min, the column flow rate was controlled at 300 nL / min, the column temperature was 40 ℃, the electrospray voltage was 2 kV, the gradient started from 2% B phase, increased to 65% in 55 min with a nonlinear gradient, increased to 100% in 1 min, and maintained for 4 min. Mass spectrometry parameters: (1) MS: scan range (m / z): 350-1800; resolution: 120000; maximum injection time: 50 ms; (2) HCD-MS / MS: resolution: 15000; maximum injection time: 100 ms; dynamic exclusion time: 30 s, protein and peptide card value: -10lgP≥20.
[0051] Data Analysis: Tandem mass spectra were analyzed using PEAKS Studio version 10.6 (BioinformaticsSolutions Inc., Waterloo, Canada). PEAKSD searched the uniprot-Oreochromis niloticus (version 201907, 21656 entries) database and set up trypsin digestion to identify potential phosphorylated peptides.
[0052] 4. Gel strength test: A 6.67% phosphorylated modified fish gelatin solution was prepared and transferred to a 5 mL beaker. The solution was cooled and gelled at 4℃ for 16 h to form a phosphorylated modified fish gel with a diameter of 2 cm and a height of 2.5 cm. The gel strength was measured using a TA.XTplus texture analyzer. The measurement parameters were as follows: probe selection: P / 5, pre-test speed: 1.00 mm / sec, test speed: 0.5 mm / sec, post-test speed: 10 mm / sec, penetration distance: 4 mm.
[0053] 5. Moisture Distribution Test: Phosphorylated fish gelatin gel was placed in a 25 mm diameter cylindrical tube and transferred to a 4 °C nuclear magnetic resonance (NMR) probe to collect Carr-Purcell-Meiboom-Gill (CPMG) attenuation signals. Four scans of the sample yielded 5000 echo data points. The relaxation time T was determined through multi-exponential fitting analysis. 21 T 22 and T 23 The transverse relaxation time of each sample was calculated from the peak position.
[0054] 6. Gel and Melt Temperature Testing: The gel and melt temperatures of phosphorylated fish gelatin were determined using a Discovery HR-2 rheometer. The rheometer was equipped with a 40 mm diameter parallel plate with a 1 mm gap. The prepared phosphorylated fish gelatin gel was slowed down at room temperature and diluted to 4% (w / v). Approximately 1 mL of sample was pipetted onto the sample stage and preheated at 40 °C for 15 min before measurement. Measurement mode: small amplitude temperature variation test; temperature variation mode: temperature ramp; temperature scan range: 40–5 °C, 5–40 °C; temperature change rate: 0.5 °C / min; frequency: 1 Hz; strain: 0.5%. The gel and melt temperatures were obtained when tanδ = 1.
[0055] 7. Gel Microstructure Testing: The microstructure of the phosphorylated fish gelatin gel was observed using cryo-SEM. The phosphorylated fish gelatin gel was slowly melted and mixed at room temperature. 8 μL of the fish gelatin solution was aspirated and dropped onto the sample stage. The sample was placed in liquid nitrogen and completely frozen, then removed. It was then cryogenically fractured using the attached knife. After sublimation at -90°C for 25 min, the sample was sputter-coated with gold to make it conductive. The sample was then transferred to a platform in the SEM at -140°C and observed under an accelerating voltage of 10 kV.
[0056] 8. Gastrointestinal digestion:
[0057] ① Gastric Digestion: Prepare a 3% phosphorylated fish gelatin gel sample. Take one sample before digestion as the initial sample. Mix the artificial gastric juice with the gel sample according to a ratio of gel mass: artificial gastric juice volume = 40g: 1mL. Perform gastric digestion in a water bath at 37℃ and 100r / min, maintaining a pH of 2.0±0.2. The total digestion time is 2h. After sampling, incubate the sample in a water bath at 100℃ for 5min to inactivate the enzymes.
[0058] ② Intestinal digestion: The pH of the sample digested with gastric juice was adjusted to 7.0 using 6M NaOH. Artificial small intestinal fluid was added to the solution digested with simulated gastric juice at a ratio of gel mass to digestion liquid volume of 40g:100μL. The mixture was shaken thoroughly to simulate the small intestinal stage of digestion. The mixture was shaken at 100 rpm for 4 hours at 37℃, maintaining a pH of 7.0±0.2 throughout the digestion process. Samples were taken at 2.5 hours and 4 hours of digestion. After sampling, the samples were incubated in a 100℃ water bath for 5 minutes to inactivate the enzymes.
[0059] 9. Determination of Free Amino Acid Content: Take 2 mL of phosphorylated fish gelatin digestion product and dilute to 50 mL. Take 0.5 mL of the diluted solution in a test tube and add 1.5 mL of deionized water and 1 mL of ninhydrin colorimetric reagent. Mix well, incubate at 100℃ for 15 min, and then cool. Add 5 mL of 40% ethanol solution, mix thoroughly, and let stand for 15 min. Measure the absorbance at 570 nm. Using the same method, use glycine instead of the sample to prepare a standard curve, and calculate the content of free amino acids in the digestion product based on the standard curve.
[0060] 10. SDS-PAGE Test: Take the phosphorylated fish gelatin gel digestion product, dilute it with deionized water, and adjust the protein concentration to 5 mg / mL. Mix the diluted sample solution thoroughly with 5× loading buffer (containing 5% β-mercaptoethanol, 8 mol / L urea, 5% SDS (m / v), 0.25 mol / L pH 7.5 Tris-HCl buffer, and bromophenol blue) at a 1:1 (v / v) ratio. Before electrophoresis, boil the sample in a water bath for 5 min to fully denature the protein, and centrifuge to collect the supernatant for electrophoresis. The specific electrophoresis conditions are as follows: stacking gel concentration: 5%, separating gel concentration: 12%, loading volume: 15 μL. Electrophoresis is performed in constant current mode, with an initial current of 15 mA. When the band reaches the separating gel, the current is adjusted to 30 mA until the end. Place the gel plate in a petri dish and stain with 0.25% Coomassie brilliant blue for 1 h, followed by destaining with destaining solution for 1 h. Finally, the protein bands were decolorized with deionized water until the background was clear, and then imaging was performed using an imaging system.
[0061] 11. Peptide Distribution Determination: The molecular weight distribution of phosphorylated fish gelatin gel digestion products was determined by high performance size exclusion chromatography (HPLC). The test samples were dilute with Wahaha water and filtered through a 0.45 μm aqueous filter membrane. The analytical system was a Shimadzu LC-10A liquid chromatography system. Specific test conditions were as follows: Column: TSK gel G2000SWXL column (7.8 × 300 mm, TOSOH, Tokyo, Japan); Sample concentration: 5 mg / mL; Loading volume: 20 μL; Mobile phase: 45% acetonitrile (containing 0.1% trifluoroacetic acid), isogradient elution; Flow rate: 0.5 mL / min; Column temperature: 30℃; Detection wavelength: 220 nm. The molecular weights of the standards, from smallest to largest, are: glycine (75.07 Da), glutathione (307.32 Da), vitamin B12 (1355.37 Da), aprotinin (6511.44 Da), and cytochrome C (12384 Da). A linear regression was performed on the retention time using the logarithm of the molecular weight (lgMW) to obtain the molecular weight correction curve and its equation.
[0062] 12. DPPH Scavenging Rate Test: Prepare a 6.5 × 10⁻⁵ mol / L DPPH-ethanol solution. Pipette 2 mL of anhydrous ethanol and DPPH-ethanol solution into separate test tubes, vortex, and incubate at room temperature in the dark for 30 min. Measure the absorbance at 517 nm using a microplate reader (A0). Pipette 2 mL of anhydrous ethanol and fish gelatin digestion product into separate test tubes, vortex, and incubate at room temperature in the dark for 30 min. Measure the absorbance at 517 nm (A1). Finally, pipette 2 mL of DPPH-ethanol solution and fish gelatin digestion product into separate test tubes, vortex, and incubate at room temperature in the dark for 30 min. Measure the absorbance at 517 nm (A2). The DPPH scavenging rate is calculated using the following formula:
[0063]
[0064] 13. ·OH scavenging rate test: Take 1 mL of 9 mmol / L salicylic acid-ethanol, 1 mL of 9 mmol / L FeSO4, and 50 μL of fish gelatin digestion product, respectively. Finally, add 1 mL of 8.8 mmol / L H2O2 to start the reaction. Incubate at 37℃ in the dark for 30 min. Using deionized water as a reference, measure the absorbance A1 at 510 nm using a microplate reader. Additionally, use 50 μL of deionized water instead of the fish gelatin digestion product as a blank control and measure the absorbance A0. Use deionized water instead of H2O2 to measure the absorbance A2. The formula for calculating the ·OH scavenging rate is as follows:
[0065]
[0066] Source of raw materials
[0067] Fish gelatin powder is produced by Shanghai Yuanye Biotechnology Co., Ltd.; sodium tripolyphosphate is produced by Tianjin Damao Chemical Reagent Factory.
[0068] Example 1
[0069] S1. Take fish gelatin powder and stir magnetically in deionized water at 45℃ for 1 hour at a speed of 1200 rpm until dissolved to obtain a fish gelatin solution with a concentration of 6% (w / v, g / mL).
[0070] S2. Take solid sodium tripolyphosphate and fish gelatin solution and mix them at a mass ratio of 1:400. Stir the mixture evenly at 800 rpm for 10 minutes at room temperature to ensure complete dissolution. Then, use a pH meter to monitor the pH value of the mixture in real time, and add 1 mol / L sodium hydroxide solution dropwise to the mixture to adjust the pH value to 9, thus obtaining a fish gelatin and phosphate mixture.
[0071] S3. Transfer the fish gelatin and phosphate mixture obtained in S2 to an ultrasonic water bath and perform an ultrasonic-assisted reaction at 40℃ and 200W ultrasonic power for 20 minutes to obtain an ultrasonic-assisted phosphorylated modified fish gelatin solution.
[0072] S4. The ultrasound-assisted phosphorylated fish gelatin solution obtained in S3 was dialyzed with deionized water at 4°C for 36 hours to remove salt, and the dialysate was replaced every 4 hours.
[0073] S5. Freeze-dry the fish gelatin solution obtained in S4 for 36 hours to obtain phosphorylated modified fish gelatin.
[0074] Example 2
[0075] The steps and operations of Example 1 are followed, except that the ultrasound-assisted reaction time in step S3 is adjusted to 80 min.
[0076] Comparative Example 1
[0077] The steps and operations of Example 1 are followed, except that the ultrasonic assistance in step S3 is omitted, and the water bath heating at 40°C is performed directly for 20 minutes.
[0078] Comparative Example 2
[0079] The steps and operations of Example 2 are followed, except that the ultrasonic assistance in step S3 is omitted, and the water bath heating at 40°C is performed directly for 80 minutes.
[0080] Example 3
[0081] The steps and operations of Example 1 are followed, except that the ultrasound-assisted reaction time in step S3 is adjusted to 60 min.
[0082] Example 4
[0083] The steps and operations of Example 1 are followed, except that the ultrasound-assisted reaction time in step S3 is adjusted to 120 min.
[0084] Comparative Example 3
[0085] A method for producing unphosphorylated fish gelatin, comprising the following steps:
[0086] S1. Take fish gelatin powder and magnetically stir it in deionized water at 45℃ for 1-1.5 hours at 1200 rpm until dissolved to obtain a 6% (w / v, g / mL) fish gelatin solution. Then, use a pH meter to monitor the pH value of the mixture in real time, and add 1 mol / L sodium hydroxide solution dropwise to the mixture to adjust the pH value to 9, thus obtaining the fish gelatin solution.
[0087] The fish gelatin solutions obtained from S2 and S1 were freeze-dried to obtain freeze-dried fish gelatin.
[0088] Comparative Example 4
[0089] The preparation was carried out according to the steps in Example 1, except that the ultrasound-assisted reaction in step S3 was changed to 30 min.
[0090] Comparative Example 5
[0091] The preparation was carried out according to the steps in Example 1, except that the ultrasound-assisted reaction in step S3 was changed to 90 min.
[0092] Comparative Example 6
[0093] The preparation was carried out according to the steps in Example 1, except that the ultrasound-assisted reaction in step S3 was changed to 40 min.
[0094] Comparative Example 7
[0095] The preparation is carried out according to the steps in Example 3, wherein the operations of steps S2 and S3 are interchanged and appropriately added, specifically as follows:
[0096] S2. Transfer the fish gelatin solution to an ultrasonic water bath and sonicate it at 40℃ and 200W ultrasonic power for 60 minutes to obtain an ultrasonically treated fish gelatin solution.
[0097] S3. Solid sodium tripolyphosphate and the ultrasonically treated fish gelatin solution obtained in S2 are mixed at a mass ratio of 1:400. The mixture is stirred uniformly at 800 rpm for 10 minutes at room temperature using a magnetic stirrer until fully dissolved. The pH value of the mixture is then monitored in real time using a pH meter. A 1 mol / L sodium hydroxide solution is added dropwise to adjust the pH to 9, resulting in a fish gelatin and phosphate mixture. This mixture is then heated in a 40℃ water bath for 80 minutes to obtain a phosphorylated fish gelatin solution after ultrasonic pretreatment.
[0098] The phosphorylation degree, gel properties (gel strength, moisture distribution, gel and melt temperatures, micronetwork structure), and digestibility characteristics of the phosphorylated fish gelatin obtained in the various embodiments of the present invention and the comparative examples were systematically compared. The results are as follows: Figures 1 to 13 As shown.
[0099] Figure 1 This is a breakdown of the technical roadmap for the present invention. The main step is the preparation of ultrasound-assisted phosphorylation-modified fish gelatin. All process steps are achieved with strict process parameters, ensuring the improvement of the functional properties of fish gelatin.
[0100] Figure 2The degree of phosphorylation modification is shown for Examples 1, 2, 1, 2, and 7. Examples 1 and 2 both showed high phosphorylation degrees, at 1.828 mg / g and 2.051 mg / g, respectively, while Comparative Examples 1 and 2 showed relatively low phosphorylation degrees, at 1.342 mg / g and 1.625 mg / g, respectively. This indicates that ultrasound assistance lowers the activation energy of the reaction, promotes the exposure of phosphorylation sites, and increases the probability of collision between phosphate and phosphorylation sites, thereby increasing the phosphorylation degree of fish gelatin. Furthermore, Comparative Example 7 also showed a low phosphorylation degree of 1.604 mg / g, meaning that pre-treating fish gelatin with ultrasound before phosphorylation modification does not significantly increase the phosphorylation degree of fish gelatin.
[0101] Figure 3 The potential values are for Examples 1, 2, 1, 2, and 7. Phosphate groups carry a negative charge, and the higher the degree of phosphorylation, the lower the potential value. Examples 1 and 2 have relatively low potential values of 1.97 mV and 1.33 mV, respectively, while Comparative Examples 1, 2, and 7 have relatively high potential values of 3.27 mV, 2.57 mV, and 2.63 mV, respectively. This indicates that ultrasound-assisted treatment promotes the electrostatic binding between fish gelatin and phosphate, increasing the degree of phosphorylation of fish gelatin. However, pre-treating fish gelatin with ultrasound before phosphorylation does not significantly increase the degree of phosphorylation.
[0102] Table 1 lists the possible phosphorylated peptides in Example 2 and Comparative Example 2. The results showed that Example 2 and Comparative Example 2 had 8 and 7 phosphorylated peptides, respectively, and 9 and 7 possible phosphorylation sites, respectively. This indicates that compared to phosphorylation modification alone, ultrasound-assisted phosphorylation modification promotes the exposure of phosphorylation sites, which is beneficial for increasing the degree of phosphorylation modification. Furthermore, Example 2 showed a peptide with one double phosphorylation site. 810 GDPGPSGEPGIIGPPGLAGEKGPSGES GPPGSPGAPGTSGPLGLQGFVGLPGSRGDR 871 (T851, S866). Generally, due to steric hindrance, it is difficult for a short peptide to bind to multiple phosphate groups, but the introduction of ultrasound helps gelatin proteins unfold, making it possible for short peptides to bind to multiple phosphate groups.
[0103] The above results indicate that ultrasound-assisted phosphorylation significantly improved the phosphorylation degree of fish gelatin and further reduced its potential value.
[0104] Table 1 shows the phosphorylation modification sites of Example 2 and Comparative Example 2.
[0105]
[0106] Figure 4 The gel strengths of Examples 3, 3, 4, and 5 are shown. Comparative Example 3 has the lowest gel strength at 221.352 g. The gel strengths of Examples 3, 4, and 5 are significantly higher than that of Comparative Example 3, indicating that ultrasound-assisted phosphorylation modification is beneficial to improving the gel strength of fish gelatin. Example 3 has the highest gel strength at 340.604 g, while Comparative Examples 4 and 5 have relatively lower gel strengths at 296.708 g and 272.163 g, respectively. This indicates that fish gelatin obtained by ultrasound-assisted phosphorylation modification for 60 min has the highest gel strength, while the gel strength of fish gelatin obtained by ultrasound-assisted phosphorylation for too short or too long a time is lower than 340.604 g.
[0107] Figure 5 The figures show the water distribution analysis of Examples 3, 3, 4, and 5. Generally, a shorter relaxation time indicates poorer water flow and a tighter binding between the substrate and water; conversely, a shorter relaxation time indicates higher water flow and a looser binding between the substrate and water. It can be observed that Examples 3, 4, and 5 all have shorter relaxation times compared to Comparative Example 3, indicating that ultrasound-assisted phosphorylation modification promotes the binding of fish gelatin molecules with water and reduces the water molecule flow. Example 3 has the shortest relaxation time. This demonstrates that in Example 3, the fish gelatin binds more effectively with water molecules. A tight structure is beneficial for improving gel properties. .
[0108] Figure 6 and Figure 7 The figures show the phase angle changes of Examples 3, 3, 4, and 5 during the cooling and heating processes. These phase angle changes reflect the gel and melt temperatures of the fish gelatin. Analysis revealed that Comparative Example 3 had the lowest gel and melt temperatures, at 16.71℃ and 25.51℃, respectively. After ultrasound-assisted phosphorylation modification, the gel and melt temperatures of Examples 3 (17.96℃, 26.54℃), 4 (17.43℃, 25.94℃), and 5 (16.98℃, 25.57℃) were significantly increased, with Example 3 exhibiting the highest gel and melt temperatures. These results indicate that ultrasound-assisted phosphorylation modification facilitates the transformation of random coils into triple helices during the gelation process of fish gelatin, thereby increasing its gel and melt temperatures.
[0109] Figures 8-11The images show the microstructures of the gels in Examples 3, 3, 4, and 5, respectively. Example 3 exhibits the smallest and densest pores in its gel network, resulting in the highest gel strength. Comparative Example 3 has the worst network structure, with the largest and coarsest pores. While the gel network structures of Comparative Examples 4 and 5 are superior to those of Comparative Example 3, they still exhibit a looser and more porous network structure compared to Example 3. This corresponds to their gel strength, moisture distribution, and gel and melt temperatures. These results indicate that ultrasound-assisted phosphorylation modification is beneficial for the formation of fish gelatin gel networks.
[0110] The above results indicate that ultrasound-assisted phosphorylation promotes the binding of fish gelatin to phosphates and water, which is beneficial for gel network formation and increases the gel strength, gelation temperature, and melting temperature of fish gelatin. However, this promoting effect is not positively correlated with the increase of ultrasound-assisted phosphorylation time; it is only achieved after 60 minutes of ultrasound-assisted phosphorylation modification. Fish gelatin possesses optimal gelling properties, while ultrasound-assisted phosphorylation that is too short or too long will hinder its gelation. Impedes the improvement of gel properties .
[0111] Figure 12 The free amino acid content of Examples 3, 4, Comparative Examples 3, and 6 after gastrointestinal digestion is shown. The free amino acid content reflects the digestion rate during gastrointestinal digestion; a higher free amino acid content indicates easier digestion. After gastrointestinal digestion, Comparative Example 3 had the lowest free amino acid content at 952.53 μg / mL, indicating that fish gelatin without ultrasound-assisted phosphorylation modification had a slower digestion rate in the gastrointestinal tract. Compared to Comparative Example 3, Comparative Example 6 showed a significant increase in free amino acid content to 1019.203 μg / mL. In Examples 3 and 4, the free amino acid content increased to 1123.253 μg / mL and 1252.413 μg / mL, respectively, after gastrointestinal digestion. This result indicates that ultrasound-assisted phosphorylation modification promotes the opening of the gelatin protein structure, facilitating the exposure of pepsin and trypsin digestive sites, thereby increasing the digestion rate in the gastrointestinal tract. Furthermore, the longer the ultrasound-assisted phosphorylation modification time, the faster the digestion rate.
[0112] Figure 13SDS-PAGE electrophoresis images of the small intestine during the gastrointestinal digestion stages of Examples 3, 4, Comparative Examples 3, and 6. Analysis revealed that after gastrointestinal digestion, the protein band changes in Comparative Example 3 were not significant; it still contained a large number of high-molecular-weight protein bands, while low-molecular-weight protein bands remained few and lighter in color, indicating that the digestion rate of fish gelatin without ultrasound-assisted phosphorylation modification was slow during intestinal digestion. In contrast, the digestion rate of fish gelatin in the intestine was significantly increased after phosphorylation modification. Comparative Example 6 showed significant changes in protein bands after gastrointestinal digestion, with high-molecular-weight protein bands gradually becoming lighter in color and low-molecular-weight protein bands gradually increasing. The changes in protein bands in Examples 3 and 4 were even more significant, with high-molecular-weight protein bands disappearing and shifting to low-molecular-weight proteins, resulting in darker bands. This result is consistent with the changes in the content of free amino acids, indicating that the digestibility of fish gelatin is significantly improved after ultrasound-assisted phosphorylation modification, and the longer the ultrasound-assisted phosphorylation modification time, the more conducive it is to the digestion and decomposition of high molecular weight proteins into low molecular weight peptides.
[0113] Figures 14-15 The diagram shows the peptide distribution during the small intestine digestion process in Examples 3, 4, 3, and 6. Figure 14 The peptide distribution of fish gelatin after 2.5 hours of small intestinal digestion was analyzed. In Comparative Example 3, the proportion of peptides with a molecular weight >5 kDa was the highest at 36.41%, followed by Comparative Example 6 (31.18%). Examples 3 and 4 had lower proportions at 27.37% and 19.84%, respectively. In the range of 1 kDa to 3 kDa, Example 4 had the highest proportion at 40.78%, followed by Example 3 (36.45%). Comparative Examples 3 and 6 had relatively lower proportions at 30.14% and 32.85%, respectively. This result indicates that after intestinal digestion, high molecular weight proteins in fish gelatin are digested into low molecular weight proteins, and the molecular weight reduction is more significant in fish gelatin modified by ultrasound-assisted phosphorylation. Figure 15 The peptide distribution of fish gelatin after 4 hours of intestinal digestion was investigated. Results showed that the molecular weight of fish gelatin further decreased after 4 hours of intestinal digestion, but the molecular weight of Comparative Example 3 remained relatively high. Example 4 showed the most significant decrease in molecular weight, followed by Example 3, both of which were significantly smaller than Comparative Examples 3 and 6. In conclusion, ultrasound-assisted phosphorylation modification of fish gelatin improved the intestinal digestion rate of fish gelatin, facilitating the digestion and breakdown of high molecular weight proteins into low molecular weight proteins.
[0114] Figures 16-17 The antioxidant activity of the digestion products of Examples 3, 4, Comparative Examples 3 and 6 after gastrointestinal digestion was analyzed. Figure 16Analysis of the DPPH scavenging rate of the digested products revealed that Comparative Example 3 had the lowest DPPH scavenging rate at 41.525%. After ultrasound-assisted phosphorylation modification, the DPPH scavenging rate improved across the board. Comparative Example 6 had a DPPH scavenging rate of 44.899%, Example 3 had the highest at 50.826%, while Example 4 showed a decrease to 48.725%. This result indicates that ultrasound-assisted phosphorylation modification helps improve the DPPH free radical scavenging rate of fish gelatin gastrointestinal digested products. However, longer ultrasound-assisted phosphorylation modification time does not necessarily lead to a higher DPPH free radical scavenging rate. Conversely, Example 4, due to its longer ultrasound-assisted phosphorylation modification time, had a higher degree of digestion, resulting in a higher content of free amino acids and a lower number of short peptides, thus reducing its ability to capture DPPH free radicals. Example 3, on the other hand, had a moderate degree of digestion, with the product composition mainly consisting of short peptides, which was beneficial for improving the DPPH scavenging rate.
[0115] Figure 17 The ·OH scavenging rate of the digestion products was measured. The results showed that the differences between Examples 3, 4, Comparative Example 3, and Comparative Example 6 were similar to those in DPPH scavenging rate. Comparative Example 3 had the lowest ·OH scavenging rate, at only 24.445%. After ultrasound-assisted phosphorylation modification, the ·OH scavenging rate also significantly increased. Comparative Example 6 had a ·OH scavenging rate of 26.044%, while Example 3 had the highest at 32.154%. Compared to Example 3, Example 4 showed a decrease in ·OH scavenging rate to 30.107%. This indicates that ultrasound-assisted phosphorylation modification increases the degree of digestion of fish gelatin in the gastrointestinal tract, leading to a continuous increase in the number of peptides acting as hydrogen donors, thus increasing the ·OH scavenging rate. However, during digestion, fish gelatin modified by prolonged ultrasound-assisted phosphorylation experienced a decrease in ·OH scavenging rate due to the excessively rapid digestion rate, resulting in the breakdown of a large number of hydrogen donor peptides into free amino acids.
[0116] The above results indicate that ultrasound-assisted phosphorylation modification improves the digestibility of fish gelatin and increases its digestion rate in the gastrointestinal tract. The longer the ultrasound-assisted phosphorylation modification time, the faster the digestion rate of fish gelatin. However, the antioxidant activity of fish gelatin digestion products does not show a continuous upward trend with the increase of ultrasound-assisted phosphorylation modification time. At 60 min of ultrasound-assisted phosphorylation modification, the digestion products of fish gelatin have high antioxidant activity.
[0117] Based on the results of various embodiments and comparative studies, the technology of this invention improves the phosphorylation modification degree of fish gelatin, thereby enhancing its gel properties (gel strength, moisture distribution, gel and melt temperatures) and digestibility (digestion rate, protein bands, peptide distribution, and antioxidant activity of digestion products). This technology can improve the functional properties of fish gelatin, expand its application range, and has promising market prospects.
[0118] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A method for improving the functional properties of fish gelatin through ultrasound-assisted phosphorylation modification, characterized in that, Including the following steps: S1. Dissolve fish gelatin powder in water by stirring to obtain a fish gelatin solution; S2. Mix sodium tripolyphosphate with the fish gelatin solution from step S1, and adjust the pH value to 8-9 with sodium hydroxide solution to obtain a fish gelatin and phosphate mixture. S3. The fish gelatin and phosphate mixture obtained in S2 is sonicated to obtain an ultrasound-assisted phosphorylation modified fish gelatin solution. S4. The ultrasound-assisted phosphorylated fish gelatin solution obtained in S3 was dialyzed to remove salt, and then freeze-dried to obtain phosphorylated fish gelatin.
2. The method according to claim 1, characterized in that, The stirring described in step S1 is to stir at 45-50°C for 1-1.5 hours and at a stirring speed of 1000-1200 rpm.
3. The method according to claim 1, characterized in that, The concentration of the fish gelatin solution in step S1 is 6-8% (w / v, g / mL).
4. The method according to claim 1, characterized in that, In step S2, the mass ratio of sodium tripolyphosphate to fish gelatin solution is 1:300-500.
5. The method according to claim 1, characterized in that, The concentration of the sodium hydroxide solution mentioned in step S2 is 1-2 mol / L.
6. The method according to claim 1, characterized in that, The ultrasonic conditions described in step S3 are: ultrasonic-assisted reaction at 35–45°C and ultrasonic power of 150–250W for 20–120 minutes.
7. The method according to claim 1, characterized in that, The ultrasound-assisted response time was 60 minutes.
8. The method according to claim 1, characterized in that, The dialysis desalination described in step S4 uses a dialysis bag with a size of 44 mm and a molecular weight cutoff of 3500 Da.
9. A phosphorylated modified fish gelatin prepared by the method according to any one of claims 1 to 8.
10. The application of the phosphorylated modified fish gelatin as described in claim 9 in the food industry.