Modified porous cowpea starch adsorbing rutin composite film and application thereof

Abstract

The application belongs to the field of cowpea preservative, and particularly relates to a modified cowpea porous starch adsorbing rutin composite film and application thereof. Cowpea starch is used as raw material to prepare porous starch (PS) through composite enzymolysis, and octenyl succinic anhydride (OSA) is used to modify the hydrophobicity of the porous starch, so as to obtain OSA-PS with amphiphilic structure. Then, the porous structure and hydrophobic sites of OSA-PS are used to realize efficient adsorption and loading of rutin (RUT), so as to obtain OSA-PS-RUT composite particles (OPR), and the OPR is introduced into a gelatin / sodium alginate (GS) film base system to prepare OPR / GS active composite films with different addition amounts. The film can realize stable maintenance of cowpea storage quality through synergistic effect of multiple ways such as bacteriostasis, antioxidant, water loss reduction and construction of suitable gas microenvironment, and provides experimental basis and theoretical support for application of natural degradable active packaging materials in the field of fruit and vegetable preservation.

Description

Technical Field

[0001] This invention belongs to the field of cowpea preservatives, specifically relating to a modified cowpea porous starch-adsorbed rutin composite film and its application. Background Technology

[0002] Cowpeas (Vigna unguiculata), an important legume crop, are rich in nutrients, including high levels of protein, vitamins, minerals, and dietary fiber, and are widely cultivated and consumed in many parts of the world. However, cowpeas are susceptible to microbial contamination, oxidative damage, and moisture loss after harvest, resulting in a short shelf life and significant nutrient loss, causing substantial economic losses and food waste. Traditional preservation methods such as refrigeration, chemical preservatives, or modified atmosphere packaging can extend the shelf life of cowpeas to some extent, but they often have drawbacks such as environmental pollution, residual toxicity, or high costs, failing to meet modern consumers' demands for green, safe, and sustainable food packaging.

[0003] Therefore, developing composite films that can combine the advantages of multiple materials plays an important role in preserving cowpeas. Summary of the Invention

[0004] The present invention aims to develop a gelatin / sodium alginate composite film with rutin adsorbed by OSA-modified porous starch (OSA-PS) as the functional component for the preservation of cowpeas.

[0005] The technical solution of this invention is implemented as follows:

[0006] A modified cowpea porous starch adsorbent rutin composite film comprises the following components in parts by weight: 100 g porous starch, 4-12 g octenyl succinic anhydride (OSA), 50 g rutin (RUT), 2.0 g gelatin (G), 0.5 g sodium alginate (SA), and 0.6 g glycerin.

[0007] Furthermore, the weight ratio of gelatin to sodium alginate is 4:1; the mass ratio of rutin to porous starch is set to 1:2, and the porous starch is cowpea starch.

[0008] Furthermore, the method for preparing the porous starch includes the following steps:

[0009] (1) Soak cowpea seeds in 0.05 mol / L NaOH solution, then discard the NaOH solution, add distilled water and grind, collect the mixed solution, add HCl solution to adjust the pH to 5.5-6.5, let stand and discard the supernatant, then wash the precipitate with ether, and dry the precipitate overnight in hot air to obtain crude extract; mix crude extract with distilled water and ethanol, centrifuge the mixture, wash the precipitate with distilled water 3 times and dry to obtain starch, grind the starch and sieve to obtain cowpea starch;

[0010] (2) Cowpea starch was dissolved in sodium acetate buffer at 50°C and 250 r / min in a water bath constant temperature shaker; then α-amylase and glucoamylase were added, and after stirring, sodium hydroxide solution was added to adjust the pH to 10.0 to terminate the reaction. The mixture was centrifuged, and the precipitate was collected by washing with distilled water and dried to obtain porous starch (PS).

[0011] The method for preparing the above-mentioned composite thin film includes the following steps:

[0012] (1) Dissolve porous starch in distilled water, heat in a water bath while stirring, and add NaOH solution dropwise to adjust the pH to 8.5; gradually add octenyl succinic anhydride to obtain a reaction solution within 1 hour of stirring, and then dilute the reaction solution 5 times with anhydrous ethanol; after stirring continuously for 3 hours, add hydrochloric acid dropwise to adjust the pH of the diluted reaction solution to 6.5 to stop the reaction; centrifuge, wash the precipitate three times with distilled water, then wash it three times with 95% ethanol, and dry it to obtain OSAPS;

[0013] (2) A rutin (RUT) solution with a mass fraction of 0.22% was prepared using anhydrous ethanol as a solvent, and an OSAPS solution with a mass fraction of 5 mg / mL was prepared using distilled water as a solvent. The mass ratio of porous starch in the rutin and OSAPS solutions was set to 1:2. The rutin and OSAPS solutions were then sonicated at 100 W for 10 minutes. The rutin ethanol solution was gradually added to the OSAPS solution at 50°C and stirred continuously to obtain a composite solution. The composite solution was then sonicated, and finally the composite solution was centrifuged to obtain a precipitate. The precipitate was freeze-dried to obtain the OSAPSRUT (OPR) complex.

[0014] (3) Under water bath heating conditions, prepare a 2.5% (w / v) concentration aqueous solution of gelatin (G) and sodium alginate (S) under stirring conditions. Mix the two solutions according to the GS ratio of 4:1 (v / v). Add 0.6 g of glycerol to every 100 mL of the mixed solution and stir continuously for 2 h. After the membrane solution is mixed evenly, remove the air bubbles by ultrasonic treatment to obtain the GS membrane solution. Add OPR to the membrane solution and mix evenly to obtain the OPR / GS membrane solution. Pour it into a dish, spread it evenly and dry it to obtain the OPR / GS film.

[0015] Furthermore, in step (1), the weight of octenyl succinic anhydride accounts for 4%-12% of the dry weight of PS.

[0016] Furthermore, in step (2), the degree of substitution of the complex is 0.0227, 0.0416 and 0.0584, respectively.

[0017] Furthermore, in step (3), the weight of OPR added is 10%, 15%, and 20% of the total dry weight of gelatin and sodium alginate, respectively, and the amount of OPR / GS membrane solution used is 0.3145 g / cm³. 2 .

[0018] The composite films described above may be used in any of the following applications:

[0019] (1) Improve the rutin encapsulation rate and loading rate of the composite film; (2) Improve the uniformity of the composite film; (3) Improve the stability of the composite film; (4) Delay the weight loss rate of the composite film; (5) Reduce the water vapor transmission rate of the composite film; (6) Improve the oxygen transmission rate of the composite film; (7) Improve the carbon dioxide transmission rate of the composite film; (8) Reduce the light transmittance of the composite film; (9) Improve the opacity of the composite film; (10) Reduce the elongation at break of the composite film; (11) Improve the tensile strength of the composite film; (12) Improve the hydrophobicity of the composite film surface and / or improve the water stability of the composite film; (14) Improve the thermal anti-fogging properties of the composite film; (15) Improve the biodegradability of the composite film.

[0020] The composite films described above may be used in any of the following applications:

[0021] (1) Preserving cowpeas; (2) Inhibiting post-harvest decay of cowpeas; (3) Delaying the green-to-yellow color change of cowpeas after harvest; (4) Inhibiting cowpea wilting; (5) Inhibiting post-harvest decay of cowpeas; (6) Improving the nutritional quality of cowpeas; (7) Extending the shelf life of cowpeas; (8) Reducing the water loss rate of cowpeas; (9) Reducing the browning index of cowpeas; (10) Reducing the total number of colonies in cowpeas; Inhibiting browning of cowpeas; (11) Inhibiting rust spots on cowpeas.

[0022] Furthermore, the composite film described above can reduce the electrical conductivity of cowpeas, reduce the malondialdehyde content in cowpeas, increase the ascorbic acid content in cowpeas, increase the chlorophyll content in cowpeas, increase the soluble protein content in cowpeas, increase the soluble solids content in cowpeas, reduce the total phenol content in cowpeas, reduce the total flavonoid content in cowpeas, increase the SOD activity in cowpeas, increase the CAT activity in cowpeas, increase the POD activity in cowpeas, increase the APX activity in cowpeas, and / or reduce the formation of reactive oxygen species in cowpeas during the storage process.

[0023] The above-described composite films are used in antibacterial and / or antioxidant applications.

[0024] Furthermore, the composite film can inhibit Escherichia coli and Staphylococcus aureus.

[0025] A method for preserving cowpeas includes the following steps:

[0026] Cowpeas are sealed and preserved using the aforementioned composite film.

[0027] Beneficial effects:

[0028] This invention constructs a bio-based preservation packaging system that combines barrier properties and bioactivity. Using cowpea starch as raw material, porous starch (PS) is prepared through complex enzymatic hydrolysis, and then hydrophobically modified with octenyl succinic anhydride (OSA) to obtain OSAPS with an amphiphilic structure. Subsequently, utilizing the porous structure and hydrophobic sites of OSAPS, efficient adsorption and loading of rutin (RUT) is achieved, yielding OSAPS-RUT composite particles (OPR). These OPR / GS active composite films with different addition amounts are then introduced into a gelatin / sodium alginate (GS) membrane system to prepare OPR / GS active composite films.

[0029] Characterization results showed that a uniform porous structure was formed on the surface of PS particles, providing space for rutin encapsulation. The porous structure remained intact after OSA modification, while the introduction of hydrophobic long chains enhanced rutin dispersibility and drug loading capacity, with the 8% OSA-modified sample showing the best overall performance. FTIR analysis of the composite membrane confirmed the existence of hydrogen bonding interactions in the GS system, and the addition of OPR further enhanced the membrane's compatibility. XRD showed that the composite membrane maintained an amorphous structure, which is beneficial for the uniform dispersion of functional components. Performance tests showed that the introduction of OPR effectively improved the tensile strength of the film and enhanced its light-blocking properties, while significantly reducing water vapor transmission rate and improving water resistance and surface hydrophobicity. The composite membrane maintained a high CO2 / O2 selective permeability ratio, demonstrating potential for use in balanced atmosphere packaging (EMAP). Furthermore, both GS and OPR / GS films exhibited good anti-fogging properties, and soil burial experiments showed that all films were completely degraded within 15 days, demonstrating good environmental friendliness.

[0030] Bioactivity evaluation results showed that the OPR / GS composite film exhibited significant antioxidant and antibacterial activities, which increased with increasing OPR content. In cowpea storage trials, the OPR / GS composite film significantly delayed water loss and browning, reduced total bacterial count, electrolyte leakage, and malondialdehyde (MDA) accumulation, while more effectively maintaining quality indicators such as ascorbic acid, chlorophyll, soluble protein, and soluble solids; the 20% OPR / GS group showed the best overall preservation effect. Antioxidant enzyme system analysis further indicated that the composite film maintained high activities of POD, SOD, CAT, and APX, synergistically inhibiting reactive oxygen species accumulation and membrane lipid peroxidation. Correlation network analysis revealed a significant positive correlation between nutrient retention indicators and antioxidant enzyme activity, while a negative correlation was observed with total bacterial count, water loss, and oxidative degradation indicators, further validating that the composite film delays cowpea senescence and degradation through a synergistic mechanism of "barrier regulation + activity protection."

[0031] In summary, this invention prepared a gelatin / sodium alginate active composite film based on OSAPS-RUT. This film can achieve stable maintenance of cowpea storage quality through multiple synergistic effects such as antibacterial, antioxidant, slowing down water loss and constructing a suitable gas microenvironment. It provides experimental basis and theoretical support for the application of natural biodegradable active packaging materials in the field of fruit and vegetable preservation. Attached Figure Description

[0032] Figure 1 The microstructures of different starch-based materials and rutin-loaded complexes are shown.

[0033] Figure 2 The ATR-FTIR spectra of the powder-based material and its rutin-supported composite are shown.

[0034] Figure 3 X-ray diffraction patterns of starch-based materials and their rutin-loaded complexes.

[0035] Figure 4 The Fourier transform infrared (FTIR) spectrum and X-ray diffraction (XRD) pattern of the composite thin film are shown.

[0036] Figure 5 Thermogravimetric analysis curves of different starch-based materials and their rutin-loaded complexes: TG curve (A); DTG curve (B).

[0037] Figure 6 The appearance morphology of different composite films.

[0038] Figure 7 The results show the transmittance and opacity of the composite film.

[0039] Figure 8 The results represent the mechanical properties of the composite thin film.

[0040] Figure 9 The results show the water contact angle of the composite film.

[0041] Figure 10 The composite film provides thermal anti-fogging properties.

[0042] Figure 11 Morphological changes of GS and OPR / GS composite films buried in soil for 0–15 days.

[0043] Figure 12 The results are the determination of the material's antioxidant capacity.

[0044] Figure 13 The antibacterial effect of the composite film on Staphylococcus aureus (A) and Escherichia coli (B) was studied.

[0045] Figure 14 The changes in appearance and color of cowpeas in different treatment groups.

[0046] Figure 15 The water loss rate and browning index of cowpeas in different treatment groups.

[0047] Figure 16 Changes in total bacterial count, conductivity, and malondialdehyde (MDA) in cowpeas from different treatment groups.

[0048] Figure 17 Changes in key nutrients in cowpeas from different treatment groups.

[0049] Figure 18 The changes in antioxidant enzyme activity in cowpeas under different treatment groups.

[0050] Figure 19 This is a heatmap showing the correlation between cowpea quality and day 8. Detailed Implementation

[0051] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to better understand the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0052] Example 1: Preparation of OPR / GS Composite Thin Film

[0053] 1.1. Materials

[0054] Cowpea, α-amylase, glucoamylase, octenyl succinic anhydride (OSA), rutin (RUT), gelatin, sodium alginate (SA), glycerol.

[0055] 1.2. Extraction of Cowpea Starch

[0056] 1000 g of cowpea seeds were soaked in 4200 mL of 0.05 mol / L NaOH solution at 4 °C for 17 h. After discarding the NaOH solution, the seeds were ground with distilled water and collected through a 150-mesh sieve. The pH was adjusted to approximately 6 by adding 1 mol / L HCl solution, and the mixture was left to stand for 12 h. The supernatant was discarded, and the precipitate was washed with ether to remove fat. The precipitate was dried overnight in a hot air stream to obtain a crude extract. The crude extract was mixed with distilled water and ethanol, and shaken on a shaker for 1 h. The mixture was then centrifuged at 4000 × g for 10 min. The precipitate was washed three times with distilled water and dried. The starch was ground and sieved through a 300-mesh sieve to obtain pure white, fine cowpea starch.

[0057] 1.3. Preparation of PS, OSAPS and OSAPSRUT

[0058] Cowpea starch (100 g) was dissolved in 400 mL of sodium acetate buffer (pH 5.2) at 50°C and 250 r / min in a water bath to prepare a starch suspension. Then, α-amylase and glucoamylase (weight ratio 1:6) of 2% dry cowpea starch were added, and the mixture was stirred for 18 h. The reaction was terminated by adjusting the pH to 10.0 with 1 mol / L sodium hydroxide solution. The suspension was separated by centrifugation and washed with distilled water, and the precipitate was collected. Finally, the collected precipitate was dried at 50°C for 48 h to obtain porous starch (PS).

[0059] 100 g of PS was dissolved in 233.3 g of distilled water, then heated in a 35°C water bath and stirred with a magnetic stirrer at 250 r / min for 5 min. 0.5 mol / L NaOH solution was added dropwise to adjust the pH of each starch paste to 8.5. Over 1 hour of stirring, 4%, 8%, and 12% OSA (based on the dry weight of PS) were gradually added, followed by a 5-fold dilution with anhydrous ethanol. After continuous stirring for 3 hours, 2% hydrochloric acid was added dropwise to adjust the pH of the reaction solution to 6.5, thus stopping the reaction. The samples were centrifuged at 25°C and 5000 g for 10 min. The precipitate was washed three times with distilled water, then three times with 95% ethanol, and finally dried at 37°C for 24 hours. OSAPS with different degrees of substitution (DS) (4%, 8%, and 12%) were obtained. OSAPS with different DS determined by titration were labeled as OSAPS (DS = 0.0227), OSAPS (DS = 0.0416), and OSAPS (DS = 0.0584), respectively.

[0060] A 0.22% (w / w) RUT solution was prepared using anhydrous ethanol as the solvent, and porous starch (PS) sample solutions and OSAPS sample solutions with different degrees of substitution were prepared using distilled water as the solvent. In both samples, the mass ratio of added RUT to PS was set at 1:2, calculated based on the dry weight of the porous starch. The RUT and starch solutions were then sonicated at 100 W for 10 minutes. To prepare the composite solution, the RUT ethanol solution was gradually added to both sample solutions at 50°C, and the mixture was continuously stirred at 500 r / min to obtain two complex solutions. Stirring and heating removed the ethanol from the solutions. The complex solutions were then sonicated at 500 W for 30 min to improve the starch loading efficiency onto the RUT and minimize particle size. Finally, the composite solution was centrifuged at 10,000 rpm for 30 minutes. The precipitate obtained from the PS sample solution was lyophilized to obtain PSRUT, and the precipitate obtained from the OSAPS sample solution was lyophilized to obtain a complex of OSAPSRUT (DS = 0.0227), OSAPSRUT (DS = 0.0416), and OSAPSRUT (DS = 0.0584) (abbreviated as OPR). The supernatant was retained to calculate the free RUT content.

[0061] 1.4. RUT Encapsulation Ratio and Loading Ratio

[0062] The RUT content was determined using the aluminum nitrate colorimetric method. The procedure is briefly described as follows: Take 1 mL of the supernatant, add 1 mL of 5% (w / v) NaNO2 solution, mix well, and let stand for 6 min. Then add 4 mL of 10% (w / v) Al(NO3)3 solution, mix well, and let stand for 6 min. Finally, add 4 mL of 4% (w / v) NaOH solution, dilute to 10 mL with distilled water, mix well, and let stand in the dark for 15 min. Measure the absorbance at 510 nm. Plot a standard curve (0–200 mg / L) using rutin as a standard, and calculate the RUT content in the supernatant. The concentration of free RUT in the supernatant was determined, and the embedding efficiency (LE) and loading rate (LC) of the RUT were calculated using equations.

[0063] Encapsulation rate (%) = (Total mass of RUT used - Mass of RUT in supernatant) / Total mass of RUT used

[0064] Loading rate (%) = (Total mass of RUT used - Mass of RUT in supernatant) / Total mass of RUT and starch used

[0065] The encapsulation efficiency and loading rate of rutin in cowpea porous starch modified with different OSA additions are shown in Table 1. Unmodified porous starch (PS-RUT) showed limited adsorption capacity for rutin, with an encapsulation efficiency of only 66.57% and a loading rate of 24.97%, indicating that relying solely on the physical adsorption sites provided by the porous structure is insufficient for the efficient immobilization and enrichment of hydrophobic rutin. After OSA modification, the encapsulation performance of porous starch was significantly enhanced (P<0.05). Specifically, the encapsulation efficiency and loading rate of 4% OSAPS-RUT (DS=0.0227) increased to 86.73% and 30.25%, respectively; when the OSA addition increased to 8% (DS=0.0416), the encapsulation efficiency further increased to 90.21%, and the loading rate reached 31.08%. Increasing the OSA dosage to 12% (DS=0.0584) resulted in an encapsulation efficiency of 91.09% and a loading rate of 31.27%, showing an upward trend, but the difference compared to the 8% group was not significant (P>0.05). This result indicates that OSA-modified porous starch significantly improved the encapsulation and loading efficiency of rutin. When the OSA addition reaches a certain level (e.g., 8%), the hydrophobic sites on the carrier surface that can participate in binding tend to saturate, and the effective availability of the pore space also approaches its upper limit. Therefore, further increasing the degree of substitution has limited effect on adsorption performance. Considering both adsorption effect and modification cost, OSAPS prepared with 8% OSA (DS=0.0416) exhibits both high encapsulation efficiency and loading rate, and also demonstrates better economic efficiency.

[0066] Table 1. Encapsulation and loading rates of rutin on different carrier materials

[0067]

[0068] 1.5. Preparation of GS and OPR / GS films

[0069] A 2.5% (w / v) gelatin (G) and sodium alginate (S) solution was prepared at 250 r / min under 70°C water bath heating. The solutions were mixed at a GS:1 (v / v) ratio, and 0.6 g of glycerol was added per 100 mL of the mixture. The mixture was stirred continuously for 2 h. After homogeneous mixing, the solution was sonicated (100 W) for 20 min to remove air bubbles, yielding the GS membrane solution. 20 g of the membrane solution was poured into a 9 cm × 9 cm dish and spread evenly. The solution was dried in a 40°C oven for 20 h to obtain the GS membrane.

[0070] After obtaining the GS membrane solution under water bath heating conditions, 10%, 15%, and 20% concentrations of OPR (based on the dry weight of GS) are added to the membrane solution and mixed thoroughly to obtain the OPR / GS membrane solution. For example, to prepare a 10% OPR / GS membrane solution, take 100 mL of GS membrane solution (containing 2.5 g of gelatin + sodium alginate) and add 0.25 g of OPR (10% of the dry weight of gelatin and sodium alginate). Pour into a dish, spread evenly, and dry to obtain the OPR / GS membrane. The membrane solution volume is 0.3145 g / cm³. 2 .

[0071] 1.6. Scanning Electron Microscopy (SEM)

[0072] The morphology of the starch material was observed. An appropriate amount of the powder sample was taken and evenly adhered to the conductive adhesive on the sample stage. Loose particles were removed by gently blowing with a bulb syringe, and then the sample was adhered to the metal conductive stage with conductive adhesive. Vacuum sputtering with gold was required before scanning electron microscopy. The morphology of the starch material, the surface morphology of the film, and the microstructure of the cross-section were observed under appropriate accelerating voltage and magnification.

[0073] Microstructures of different starch-based materials and rutin-supported complexes are as follows: Figure 1As shown, pure rutin (RUT) exhibits an irregular blocky crystalline morphology with a rough surface and significant agglomeration, resulting in poor solubility and dispersibility, thus limiting its uniform distribution in aqueous systems and polymer membrane substrates. Cowpea starch (St) granules are generally elliptical or near-elliptical in shape, with a smooth, flat surface and dense structure, exhibiting no obvious pores, consistent with the typical morphological characteristics of natural starch granules. After enzymatic hydrolysis by a combination of α-amylase and glucoamylase, porous starch (PS) granules show a large number of uniformly distributed pore structures on their surface, with pore sizes in the micrometer range. Simultaneously, the overall particle outline remains intact, indicating that the enzymatic hydrolysis process has a certain degree of selectivity and gentleness. These results suggest that the complex enzyme primarily acts preferentially on the relatively loose amorphous regions of the starch granules, causing localized hydrolysis and dissolution of the internal structure, ultimately forming interconnected pores and significantly increasing the specific surface area and pore volume, providing more binding sites and physical space for subsequent adsorption and loading of active substances. Further observation of OSA-modified porous starch (OSAPS, DS=0.0227) revealed that its particle morphology was basically consistent with that of PS, and the pore structure remained clearly visible without significant collapse or damage. This indicates that under the current degree of substitution, the OSA modification process did not significantly weaken the stability of the porous structure. Simultaneously, OSA introduces hydrophobic alkyl side chains into the starch molecular chain through esterification, transforming the material from purely hydrophilic to amphiphilic, which enhances its affinity for hydrophobic / weakly polar molecules. When porous starch directly adsorbs rutin (PS-RUT), a large number of needle-like or plate-like crystals are deposited on the particle surface, and some pores show blockage, indicating that rutin undergoes significant crystallization and aggregation on the PS surface. The OSA-modified porous starch-rutin adsorbed complex (OSAPS-RUT) exhibits a more uniform loading morphology, and shows a clear regular change with increasing degree of substitution (DS): the higher the degree of substitution, the finer and more dispersed the rutin crystal deposition becomes, allowing more rutin to enter the pores and uniformly adhere to the pore walls or particle surface. These results demonstrate that the hydrophobic long chains introduced by OSA significantly enhance the hydrophobic interaction and interfacial affinity between the support and rutin, transforming rutin from "surface agglomeration and crystallization" to "intrapore / surface dispersion and adsorption," thereby achieving more stable and efficient loading and dispersion. Overall, the porous structure of PS provides adsorption space, while OSA modification further improves the amphiphilicity of the material and the compatibility with the active material; both synergistically promote the effective encapsulation and uniform distribution of rutin.

[0074] 1.7. Fourier Transform Infrared Analysis

[0075] The structural characteristics of the material were analyzed using Fourier transform infrared spectroscopy. A certain amount of sample was weighed, ground with potassium bromide powder, and then pressed into thin sheets. The sample was then subjected to spectral analysis using Fourier transform infrared spectroscopy, with a detection wavenumber range of 400-4000 cm⁻¹. -1 The resolution is set to 4 cm. -1 .

[0076] ATR-FTIR spectra of powder-based materials and their rutin-supported composites are as follows: Figure 2 As shown. Porous starch (PS) from cowpeas at approximately 3400 cm⁻¹ -1 A broad and strong absorption peak appears at 1200–1000 cm⁻¹, corresponding to the stretching vibration of polyhydroxyl groups; while at 1200–1000 cm⁻¹... -1 Typical starch skeletal vibrational peaks were observed in the region, mainly attributed to C–O–C and C–O stretching vibrations, reflecting the basic characteristics of polysaccharide materials. After OSA modification (OSAPS), its infrared spectrum, based on the characteristic PS peak, reached a peak at 1712 cm⁻¹. -1 A new absorption peak appears at [value missing], which can be attributed to the stretching vibration of the ester carbonyl group (C=O). This is a characteristic peak introduced by OSA, indicating that OSA was successfully grafted onto the porous starch molecular chain via esterification. Furthermore, OSAPS retains the PS peak at 1200–1000 cm⁻¹. -1 The main characteristic absorption of the region indicates that the modification process did not change the main chain structure of starch, which is a typical example of surface grafting and functionalization modification.

[0077] After rutin loading, the PS-RUT spectrum simultaneously exhibits characteristic signals of both starch and rutin. Specifically, the 1600-1500 cm⁻¹ spectrum... -1 The vicinity can be attributed to the vibration of the rutin aromatic ring skeletal structure, 1200-1000 cm. -1 The CO stretching vibration in the region was also enhanced, indicating that rutin was successfully introduced into the composite system. However, no significant shift or new covalent bond characteristic peaks were observed in the relevant peak positions in the PS-RUT, suggesting that the interaction between PS and rutin is mainly physical adsorption. In contrast, the OSAPS-RUT showed enhanced peak positions in the 3200-3600 cm⁻¹ range. -1 The broadening and enhancement of the -OH peak within the range indicates a change in the hydroxyl environment of rutin, suggesting a stronger intermolecular interaction between OSAPS and rutin. This result is consistent with the phenomenon observed in SEM of rutin shifting from agglomerated crystallization to uniform dispersion, further demonstrating that OSA modification can effectively enhance the interaction between porous starch carriers and rutin.

[0078] 1.8. Material XRD Analysis

[0079] The diffraction pattern of the sample was acquired using X-ray diffraction. An X-ray diffractometer (MinFlex600) was used for scanning, with a scanning range of 2.00° to 90.00° (5° / min).

[0080] X-ray diffraction patterns of starch-based materials and their rutin-supported complexes are as follows: Figure 3As shown, pure rutin (RUT) exhibits sharp and high-intensity characteristic diffraction peaks at 2θ = 10.4°, 15.6°, 20.5°, and 26.7°, reflecting its high crystallinity and regular crystal structure. Porous starch (PS) and OSA-modified porous starch (OSAPS) both show obvious diffraction peaks at 2θ = 15.0°, 17.0°, 17.9°, and 23.0°, which are characteristic peaks of the common C-type crystal structure in legume starches. Compared with PS, the diffraction peak positions of OSAPS are basically consistent, with no new crystal form characteristic peaks or significant peak shifts. This indicates that within the degree of substitution (DS) range of this study, OSA mainly introduces hydrophobic side chains through surface esterification grafting without disrupting the original crystal arrangement of starch granules, and the modification process has little impact on the starch crystal structure.

[0081] After rutin loading, the diffraction patterns of PS-RUT and OSAPS-RUT generally retained the broad peak background characteristics of starch-based materials. While some characteristic diffraction peaks of rutin could be observed, their peak intensities were significantly weaker than those of pure rutin. This phenomenon indicates that rutin does not exist entirely in a large crystal form within the support system, but is influenced by the porous structure and intermolecular interactions, resulting in reduced crystallinity, smaller crystal size, or dispersion in a partially amorphous state. Therefore, XRD results show that rutin has been successfully introduced into the PS / OSAPS support system. This conclusion is corroborated by the pore loading and surface crystal distribution observed in SEM, and the enhanced physical adsorption and interaction results revealed by FTIR, proving the successful preparation of the OSAPS-RUT composite.

[0082] 1.9 Analysis of thin film molecular interactions and crystal structure

[0083] The structural characteristics of the material were analyzed using Fourier transform infrared spectroscopy. A certain amount of sample was weighed, ground with potassium bromide powder, and then pressed into thin sheets. The sample was then subjected to spectral analysis using Fourier transform infrared spectroscopy, with a detection wavenumber range of 400-4000 cm⁻¹. -1 The resolution is set to 4 cm. -1 The diffraction pattern of the sample was acquired using X-ray diffraction. An X-ray diffractometer (MinFlex 600) was used for scanning, with a scanning range of 2.00° to 90.00° (5° / min).

[0084] Fourier transform infrared (FTIR) spectra of composite thin films, such as Figure 4 As shown in Figure A. Gelatin (GE) at 3283 cm⁻¹ -1 A typical amide A absorption peak appears at 1632 cm⁻¹ (N–H and O–H stretching vibrations). -1 1535 cm -1 and 1241 cm -1These peaks correspond to amide I (C=O stretching vibration), amide II (C–N stretching and N–H bending vibration), and amide III (C–N–H plane vibration), respectively, exhibiting the characteristic structure of protein materials. Sodium alginate (SA) at 1597 cm⁻¹ -1 and 1406 cm -1 Carboxyl groups (–COO) are present at the respective locations. - Antisymmetric and symmetric stretching vibration peaks. In the GS blend film, characteristic peaks of both GE and SA can be observed, while some peak positions show slight shifts, including the 3283 cm⁻¹ peak. -1 The amide A peak near the membrane broadened significantly, indicating a strong hydrogen bonding between the –NH group in the gelatin molecule and the –OH group in the sodium alginate molecule, thus forming a stable composite network structure. After the introduction of OPR, the FTIR spectrum of the OPR / GS film was observed in the 1600–1500 cm⁻¹ range. -1 The region exhibits more pronounced aromatic ring skeletal vibrational absorption, indicating that the rutin structure has been successfully introduced and stably exists in the membrane matrix. Simultaneously, the amide I peak (1632 cm⁻¹) is observed. -1 The slight shift towards lower wavenumbers indicates that the hydroxyl groups in OPR and the hydrophobic segments of OSA may form additional hydrogen bonds or hydrophobic interactions with the amide and carboxyl groups of gelatin / sodium alginate, thereby further promoting the intermolecular bonding and stability of the membrane system.

[0085] X-ray diffraction (XRD) pattern of composite thin films, as shown below Figure 4 As shown in Figure B, the GS membrane exhibits broadened amorphous diffraction peaks, indicating that no obvious crystalline structure was formed after the gelatin and sodium alginate were blended. After adding OPR, the diffraction curves of the 10%, 15%, and 20% OPR / GS composite membranes still showed predominantly broad amorphous peaks, indicating that OPR did not induce a significant crystallization process in the membrane matrix, and that it mainly existed in an amorphous or highly dispersed state in the thin film system, thus ensuring the uniformity and stability of the membrane structure.

[0086] 2.0 Thermal Properties

[0087] The samples (3-5 mg) were heated from 30°C to 600°C at a rate of 10°C / min under nitrogen flow (40 mL / min), and the weight changes were recorded.

[0088] Thermogravimetric (TG) and thermogravimetric (DTG) curves of different starch-based materials and rutin-supported complexes are shown below. Figure 5As shown, under nitrogen protection, all samples exhibited typical multi-stage thermal decomposition behavior. First, within the temperature range of 30-150℃, all samples showed some initial weight loss, mainly attributed to the evaporation of free and bound water in the materials. Among them, porous starch samples (PS, OSAPS, and their supported complexes) exhibited more significant weight loss in this stage due to their larger specific surface area and pore structure, resulting in stronger water adsorption capacity. Subsequently, the main thermal decomposition stage began. Porous starch samples experienced significant and rapid weight loss in the medium-high temperature range (300–350℃). Their main degradation process corresponded primarily to the glycosidic bond breakage, dehydration cleavage, and thermal decomposition to generate volatile products in the starch molecular chains, exhibiting the typical thermal degradation pattern of starch substances. OSAPS showed a slower weight loss rate in the main decomposition region compared to PS, with a decrease / rightward shift in the DTG main peak intensity, indicating that the interaction between starch chains changed after OSA grafting: on the one hand, the introduction of hydrophobic alkyl chains enhanced the hydrophobic association between molecular chains; on the other hand, the presence of ester bonds improved the thermal stability of the material in the main decomposition region.

[0089] Pure rutin (RUT) exhibits different thermal decomposition characteristics compared to starch, with a slower and more widely distributed weight loss process. When porous starch is loaded with rutin (PSRUT), the residual rate in the mid-to-late stage of its TG curve increases, indicating successful rutin loading. OSAPSRUT exhibits a more moderate weight loss rate at high temperatures, thereby enhancing the thermal stability of the material and increasing the pyrolysis residual rate to some extent.

[0090] 2.1 Appearance and color difference

[0091] 2.1.1 Visual Inspection

[0092] Use a camera to photograph each film and compare the differences in appearance.

[0093] 2.1.2 Color difference parameters

[0094] The color difference of the thin film was measured using a colorimeter, which was calibrated using a white standard plate. The color of the thin film was described by L (white / black), a (red / green), and b (yellow / blue), and each thin film sample was measured three times.

[0095] The appearance morphology of different composite films is as follows Figure 6As shown. Due to the strong crystallinity and hydrophobicity of rutin, the RUT / GS composite film has poor compatibility with the gelatin / sodium alginate (GS) hydrophilic matrix, easily exhibiting local aggregation and uneven distribution. This local aggregation and uneven distribution disrupt the continuity and uniformity of the film's internal network structure, affecting its stability and application effect in actual preservation processes. Therefore, the resulting film has an uneven surface, poor film-forming properties, and fails to meet the film's quality requirements. Pure gelatin-sodium alginate (GS) film is a light beige semi-transparent film with a smooth and uniform surface. As the OSAPS-RUT composite (OPR) addition ratio gradually increases from 10% to 15% and 20%, the overall color of the OPR / GS composite film gradually deepens, changing from light yellow to a deeper yellow, and the film surface exhibits uniform color without obvious agglomerated particles or streaky defects. Color difference parameter (L * a * b * The changes in L further quantified the differences in film appearance (Table 2). The L of the GS film... * The value is 39.32, a * The value is 0.68, b * A value of 2.59 indicates low brightness and a weak yellow tint. The addition of OPR significantly altered the color difference parameters of the composite film: the L value of 10% OPR / GS... * The value rose to its highest level (78.55±1.39), indicating a significant improvement in the film's transparency and brightness. Among these, b... * The value showed a continuous upward trend with the increase of OPR addition, increasing from 33.94±1.06 to 36.35±1.56, with a significant enhancement of the yellow tint. Figure 6 The color change of the medium-thickness film remained consistent. This is mainly attributed to the natural yellow characteristic of rutin itself, and the increased loading of rutin directly improves the yellowness of the film.

[0096] 2.2 Thickness Measurement

[0097] The thickness of the membrane samples was measured using a digital micrometer (accuracy 0.001 mm). Three sampling points were selected for each membrane using a random sampling method, and the average value of the measurements was taken as the membrane thickness.

[0098] The thickness measurements of the composite films are shown in Table 2. Significant differences in thickness were observed among the different films (p<0.05), with the PE film being the thinnest (0.80×10⁻⁶). -2 (mm), GS film thickness is 6.31×10 -2 mm; when OPR is added, the film thickness gradually increases with the amount added, reaching 7.89 × 10⁻⁶ mm at 20% OPR / GS. -2mm. This is mainly attributed to the physical filling effect of OPR particles in the membrane matrix and the increased solids content, which makes the membrane structure more compact and forms a thicker composite network.

[0099] Table 2. Thickness and color difference parameters of different films (L) * a * and b * )

[0100]

[0101] 2.3 Gas Barrier Performance

[0102] 2.3.1 Water vapor transmission rate (WVP)

[0103] Weigh 3 g of anhydrous calcium chloride into a weighing bottle (40 mm × 25 mm), and seal the film to be tested at the mouth of the weighing bottle. Then place the weighing bottle in a desiccator and place it at 25℃ and 75% relative humidity (RH) for 48 hours. Each film sample is tested in parallel three times. The WVP value of the film is calculated by the following formula: WAV = (Δm × L) / (A × t × ΔP); where Δm (g) is the mass change of the weighing bottle; L (m) is the average thickness of the film; A (m²) is the effective area of ​​the film; t (h) is the test time; and ΔP (Pa) is the partial pressure difference across the film.

[0104] 2.3.2 Oxygen Transmission Rate (OP)

[0105] The oxygen permeability (OP) of the membrane was determined using the deoxidizer absorption method. The procedure was as follows: 3 g of deoxidizer was weighed into a weighing bottle (40 mm × 25 mm), and the membrane to be tested was sealed to the mouth of the bottle. The weighing bottle was then placed in a desiccator and stored at 25°C and 75% relative humidity (RH) for 48 h. Each membrane sample was tested in triplicate. The OP value of the membrane was calculated using the following formula: OP = (Δm × L) / (A × t × ΔP); where: Δm (g) is the change in mass of the weighing bottle; L (m) is the average thickness of the membrane; A (m²) is the effective area of ​​the membrane; t (h) is the test time; and ΔP (Pa) is the partial pressure difference across the membrane.

[0106] 2.3.3 Carbon Dioxide Transmission Rate (CDP)

[0107] The carbon dioxide permeability (CDP) of the membrane was determined using the potassium hydroxide absorption method. The procedure was as follows: 3 g of potassium hydroxide was weighed into a weighing bottle (40 mm × 25 mm), and the membrane to be tested was sealed at the bottle opening. The weighing bottle was then placed in a desiccator and kept at 25°C and 75% relative humidity (RH) for 48 hours. Each membrane sample was tested in triplicate. The CDP value of the membrane was calculated using the following formula: CDP = (Δm × L) / (A × t × ΔP); where: Δm (g) is the mass change of the weighing bottle; L (m) is the average thickness of the membrane; A (m²) is the effective area of ​​the membrane; t (h) is the test time; and ΔP (Pa) is the partial pressure difference across the membrane. The water vapor and gas barrier performance parameters of the composite membrane are shown in Table 3.

[0108] Table 3. Water vapor and gas barrier performance parameters of the composite film

[0109]

[0110] Regarding water vapor transmission rate (WVP), the PE membrane exhibited extremely strong water-blocking ability, with the lowest WVP, while the GS membrane had the highest WVP, indicating that the pure GE / SA system has a weak water vapor barrier capability. After introducing OPR, the WVP of the OPR / GS composite membrane decreased significantly (p<0.05), with 15% OPR / GS reaching the lowest value, and although 20% OPR / GS showed a slight recovery, it was still significantly lower than that of the GS membrane. This result indicates that the OSA-modified porous starch carrier possesses a certain degree of hydrophobicity, thereby enhancing the water-blocking performance of the membrane.

[0111] Further comparison of gas permeability reveals that the PE film exhibits extremely low oxygen permeability (OP) and carbon dioxide permeability (CDP), indicating excessive gas barrier properties. While this characteristic helps slow down moisture and gas exchange, excessively low oxygen permeability can lead to rapid depletion of O2 inside the packaging, while CO2 accumulation is difficult to release, thus inducing anaerobic respiration, metabolic disorders, and physiological damage, ultimately accelerating quality deterioration. GS base film and its composite film demonstrate significantly higher gas permeability and stronger gas exchange capacity. The OPR / GS composite film maintains moderate oxygen permeability while also possessing high CO2 / O2 selective permeability. This ensures sufficient O2 supply to prevent anaerobic fermentation while promoting moderate CO2 release or maintaining reasonable accumulation, thereby creating a more stable gaseous environment inside the packaging, reducing respiratory metabolic intensity, and slowing down the aging process, ultimately helping to extend the shelf life of cowpeas.

[0112] 2.4 Optical Performance Analysis

[0113] The film was cut into strips of 40 mm × 10 mm, and the thickness was measured using a micrometer. The strips were then attached to the inside of a cuvette. A full wavelength scan was performed from 200 to 800 nm, with a blank cuvette used as a control to measure the transmittance. Three samples were measured for each film, and the film opacity was calculated using the transmittance at 600 nm. The calculation formula is as follows: Opacity (A / m) = -LogT 600 / X; where Opacity is the opacity, A / m; T 600 Transmittance at -600 nm, %; X - Thickness, mm.

[0114] The results of the transmittance and opacity of the composite film are as follows: Figure 7 As shown in Table 4, the transmittance of the GS film is lower than that of the PE preservation film, indicating that the natural polymer composite film has certain advantages in light shielding. At a wavelength of 600 nm, both the PE and GS films still exhibit high transparency. When OPR is introduced, the optical properties of the composite film change significantly (p<0.05), with transmittance continuously decreasing as the OPR content increases, showing a clear trend of enhanced light blocking across the entire test wavelength range. Corresponding to the decrease in transmittance, the opacity increases in a concentration-dependent manner with the amount of OPR added. This result indicates that the OPR content is a key factor determining the light-blocking ability of the composite film. In fruit and vegetable preservation packaging applications, moderately reducing transmittance helps reduce light-induced oxidation reactions, inhibit the degradation of sensitive components such as vitamins, pigments, and lipids, thereby delaying quality deterioration. Therefore, compared to the high-transmittance PE film, the OPR / GS composite film shows a superior potential preservation advantage in terms of light blocking.

[0115] Table 4 Opacity of different films

[0116]

[0117] 2.5 Mechanical Properties

[0118] Tensile strength (TS) is the ratio of the maximum tensile force exerted on the film before tearing to the cross-sectional area of ​​the film; elongation at break (EAB%) refers to the percentage of displacement of the film at the point of breakage under axial tensile force, relative to its original length. The calculation formulas are as follows: TS = F / (L × X); where, TS - tensile strength, MPa; F - axial tensile force, N; L - sample width, mm; X - sample thickness, mm. EAB (%) = (L1 - L0) / L0; where, EAB - elongation at break, %; L1 - length at break, mm; L0 - initial length, mm.

[0119] The mechanical properties of the composite thin film are as follows: Figure 8As shown, commercial PE food preservation film exhibits the highest elongation at break (162.52%), demonstrating excellent flexibility and extensibility. In contrast, the elongation at break of GS-based film is 81.98%, indicating that although the gelatin / sodium alginate blend system possesses some plasticity, its overall flexibility is still lower than that of PE film. With the introduction of OPR, the elongation at break of OPR / GS composite film shows a significant decreasing trend (p<0.05).

[0120] In terms of tensile strength, PE food preservation film exhibited the highest tensile strength, while pure GS film showed the lowest, indicating that GS film still suffers from insufficient tensile strength. With increasing OPR content, the tensile strength of the OPR / GS composite film gradually improved (p<0.05), further increasing to 25.68 MPa with 20% OPR / GS, approaching the level of PE film. This result demonstrates that the addition of OPR produced a significant filling and reinforcing effect. The composite particles, as an effective mechanical reinforcing phase, can disperse stress and improve the overall load-bearing capacity of the film, thereby significantly improving the tensile strength of the GS base film. In other words, a higher OPR content is beneficial for improving film strength and structural stability, while a lower content is better for maintaining flexibility.

[0121] 2.6 Analysis of the hydrophilic properties of the thin film

[0122] 2.6.1 Contact Angle

[0123] The contact angle was measured using a contact angle meter. The method involved attaching the membrane to a coverslip for fixation, and then using a microsyringe to drop 5 μL of ultrapure water onto the membrane surface. Once equilibrium was reached, the droplet pattern was recorded and the contact angle was calculated by simulating the droplet pattern. Three samples were measured for each membrane, and the average value was recorded.

[0124] 2.6.2 Moisture content (MC)

[0125] The procedure was followed according to Elhadef's method. The film was weighed before and after 24 hours of drying at 110°C, and the MC was calculated as follows: MC% = (W i -W f ) / W i ×100; where W i (g): Initial mass of the film, W f (g): The mass of the film after drying.

[0126] 2.6.3 Swelling Index (SI)

[0127] Take a 2 cm × 2 cm film sample and accurately weigh its initial mass. Completely immerse the sample in deionized water (25℃) for 2 minutes. After immersion, quickly remove the swollen sample, gently wipe away any remaining free water with filter paper, and immediately and accurately weigh its wet weight. The SI calculation formula is as follows: SI% = (W i-W f ) / W i ×100; where W i (g): Initial mass of the film, W f (g): Mass of the film after swelling.

[0128] 2.6.4 Water solubility (WS)

[0129] The membrane sample was immersed in 100 mL of deionized water for 24 h to dissolve. After filtration, it was dried in a 60°C drying oven for 24 h until constant weight was achieved, and then weighed. The water solubility of the membrane was calculated using the following formula: WS% = (W i -W f ) / W i ×100; where W i (g): Initial mass of the film, W f (g): The mass of the film after drying.

[0130] The surface wettability of composite films can be reflected by the water contact angle (WCA), and the results are as follows: Figure 9 As shown, the surface WCA of the pure GS film is 56.3°, exhibiting obvious hydrophilicity. With the increase of OPR addition, the contact angle of the OPR / GS composite film gradually increases. When the OPR addition reaches 20%, the contact angle significantly exceeds 65°, and the film changes from hydrophilic to weakly hydrophobic, indicating that its surface wettability decreases significantly and its water resistance increases significantly.

[0131] Consistent with the trend in contact angle variation, the moisture content (MC), swelling index (SI), and water solubility (WS) of the films all decreased significantly with increasing OPR content (Table 5). The GS film exhibited a high WS of 63.29%, indicating its high water sensitivity; while the WS of the 20% OPR / GS film decreased to 31.83%, a reduction of nearly half. Simultaneously, the SI decreased from 160.33% to 96.87%, indicating a significant suppression of the composite membrane's water absorption and swelling behavior. The MC also decreased from 21.51% to 14.13%, further demonstrating a reduction in the membrane's internal water binding capacity.

[0132] In summary, the introduction of OPR not only improves the hydrophobicity of the film surface, but also significantly improves the water stability of the film.

[0133] Table 5. Hydrophilicity-related parameters of different films: moisture content (MC), swelling index (SI), and water solubility (WS).

[0134]

[0135] 2.7 Anti-fog test

[0136] The thermal anti-fogging performance was evaluated by covering beakers containing 60 mL of deionized water with films (PE, GS, 10% OPR / GS, 15% OPR / GS, and 20% OPR / GS films). The water bath temperature was set to 45°C, and the film-covered beakers were placed in the water bath for 30 min, 60 min, 90 min, and 120 min before photographic images were recorded.

[0137] The thermal anti-fogging properties of composite films are as follows Figure 10 As shown, the PE film exhibited significant atomization and condensation under a 45℃ water bath condition: at 30 min, a large area of ​​misty condensation layer appeared on the PE film surface; at 60 min, the mist layer gradually transformed into visible fine condensation droplets; after 90 min, the droplets further coalesced and grew into larger water droplets; by 120 min, the large droplets formed on the surface slid and dripped, indicating that the PE film could not maintain a stable anti-fogging state under temperature difference and high humidity conditions. In contrast, no obvious water droplet formation was observed in the GS film and OPR / GS composite films with different OPR additions throughout the entire test cycle (30–120 min), demonstrating excellent thermal anti-fogging performance. This indicates that the GS base film can effectively inhibit the droplet formation and aggregation of condensate, making water more likely to form a uniform water film or be quickly adsorbed / conducted by the film surface, thereby avoiding "fogging" caused by scattering.

[0138] 2.8 Biodegradability

[0139] The biodegradability of the films was tested through soil degradation. Circular films (r: 45 mm) were buried 4 cm deep in plastic boxes filled with soil. To maintain constant humidity, 20 ml of water was added every two days. Finally, the films were removed from the soil every 5 days to observe their biodegradation status.

[0140] Figure 11The morphological changes of GS and OPR / GS composite films buried in soil from 0 to 15 days were demonstrated. Overall, all films exhibited good soil biodegradability. In the 0-5 day stage, all films gradually transformed from an initial smooth surface to noticeable wrinkles, swelling, and deformation, indicating that degradation had entered the initial water absorption and swelling stage. In the 5-10 day stage, all samples showed significant fragmentation, reflecting the gradual breakage of the polymer backbone and structural disintegration. The GS film degraded the fastest, transforming into small fragments indistinguishable from soil by day 10. Composite films with added OPR (especially 15% and 20% OPR / GS) still retained relatively large fragments by day 10. In the 10-15 day stage, all films completely disappeared and integrated with the soil after 15 days of burial, indicating that the introduction of OPR did not hinder the final biodegradability of the film system. This film system, composed of natural biopolymers such as gelatin, sodium alginate, and starch, can gradually degrade under the action of soil microorganisms and achieve environmentally friendly reversion. Therefore, OPR / GS composite films maintain good soil biodegradability while improving functionality, and have the potential to be used as sustainable packaging materials.

[0141] 2.9 The material's antioxidant capacity

[0142] 2.9.1 Determination of DPPH free radical scavenging ability

[0143] The antioxidant properties of RUT, PS, PSRUT, OSAPS, OSAPSRUT (OPR), GS, and OPR / GS were measured using the DPPH radical scavenging method. In short, films and material solutions of different concentrations were added to 5 mL of 0.1 mM DPPH methanol solution and placed in the dark at room temperature for 30 min. Finally, the absorbance of the samples was measured at 517 nm. The DPPH% was calculated as follows: DPPH (%) = (A0 - A) / A × 100; where A0 is the absorbance of the control group and A is the absorbance of the sample group. Each sample was measured three times.

[0144] 2.9.2 Determination of ABTS free radical scavenging ability

[0145] A 7 mM ABTS solution and a 2.45 mM potassium persulfate solution were thoroughly mixed at a volume ratio of 1:1 (v / v) and reacted in a dark environment for 12 h. The mixture was then serially diluted with ethanol until its absorbance at 734 nm stabilized at 0.70 ± 0.02, yielding the ABTS radical working solution (prepared immediately). Two mL of starch material and film solutions of different concentrations were thoroughly mixed with 2 mL of the ABTS radical working solution and reacted at room temperature in a dark environment for 30 min. Immediately after the reaction, the absorbance of the mixture at 734 nm was measured. The scavenging activity of the starch material and film against ABTS radicals was calculated using the following formula: ABTS (%) = (A0 - A) / A × 100; where A0 is the absorbance of the control group and A is the absorbance of the sample group. Each sample was tested three times.

[0146] Figure 12 A and Figure 12 B shows the scavenging abilities of different samples against DPPH and ABTS free radicals at different mass concentrations. During the composite film preparation process, the GS membrane itself mainly provides the film-forming framework and barrier properties, without any free radical scavenging ability. PS and OSAPS mainly serve as structural support materials, lacking effective hydrogen-donating antioxidant groups in their composition, and therefore did not exhibit free radical scavenging ability in the antioxidant experiments. Among the other materials / films, OPR particles showed the strongest free radical scavenging ability, significantly higher than all other composite films. Furthermore, the antioxidant capacity of the OPR / GS composite film gradually increased with increasing OPR addition, showing 20% ​​OPR / GS > 15% OPR / GS > 10% OPR / GS, indicating that increasing the loading of the active component can significantly improve the antioxidant strength of the film. This is because the abundant phenolic hydroxyl structures in the rutin molecule are the key source of antioxidant activity, and the OPR support achieves stable loading and dispersion of rutin in the porous structure, enabling it to exert a continuous free radical scavenging effect in the composite film.

[0147] 3.0 Antibacterial activity

[0148] To determine the antibacterial activity of the starch material and composite membrane, *Escherichia coli* and *Staphylococcus aureus* were used as experimental bacteria. Starch and composite membrane materials sterilized by ultraviolet irradiation were immersed in 20 mL of culture medium, inoculated with 0.2 mL of bacterial suspension, and then incubated on a shaker at a constant temperature for 14 hours. At set time points (0 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h), equal volumes of bacterial suspension were taken and the absorbance at 600 nm was measured using a microplate reader to obtain the growth curves of the experimental bacteria.

[0149] The composite film exhibits antibacterial effects against Staphylococcus aureus and Escherichia coli as follows: Figure 13 As shown in A and 13B. The results showed that the bacterial growth curves of the control group (CK) and the GS film group almost overlapped, both rapidly entering the logarithmic growth phase in the early stage of culture, with a rapid increase in OD600 value, indicating that the GS film did not have significant antibacterial activity against either indicator bacterium. After the addition of OPR, the composite film showed a significant inhibitory effect on the growth of both bacteria, exhibiting a significant concentration-dependent enhancement trend: as the amount of OPR added increased, the overall OD600 level continuously decreased during culture, and the bacterial growth rate slowed down, indicating that the antibacterial ability of the film significantly increased with the increase of the active component content. This result shows that the introduction of OPR effectively endowed the GS base membrane with antibacterial function. Its antibacterial mechanism is related to rutin: rutin can interact with bacterial cell membrane proteins or phospholipid components, disrupting the integrity of the membrane structure and leading to leakage of cell contents; it may also inhibit bacterial reproduction by inducing oxidative stress or inhibiting the activity of key metabolic enzymes, interfering with cellular energy metabolism and nucleic acid synthesis. In summary, OPR / GS composite films possess both antioxidant and antibacterial synergistic functions, demonstrating excellent application potential in the active packaging of fruits and vegetables. They can provide effective technical support for delaying post-harvest spoilage and quality decline in cowpeas.

[0150] Example 2: The effect of OPR / GS film on the preservation of cowpeas

[0151] Comparative experiments verified the significant effect of the prepared gelatin / sodium alginate composite film (GS) and the gelatin / sodium alginate composite film loaded with rutin and modified with OSA-modified cowpea porous starch (OPR / GS) in extending the shelf life of cowpea.

[0152] The experiment was set up with the following 6 treatment groups:

[0153] CK group (blank control): Cowpeas were not packaged in any way.

[0154] PE group (product comparison): Wrapped in commercially available PE cling film.

[0155] GS group: Encapsulated with gelatin / algae-based membrane without added starch complex.

[0156] 10% OPR / GS group: Encapsulated with OPR / GS composite film at an addition rate of 10%.

[0157] 15% OPR / GS group: Encapsulated with OPR / GS composite film at an addition rate of 15%.

[0158] 20% OPR / GS group: Encapsulated with OPR / GS composite film at an addition rate of 20%.

[0159] During the experiment, the temperature was 25±0.5℃, the relative humidity was 75±5%, there was no light, and the environment was sealed and airtight. Various indicators were measured at 0, 2, 4, 6, 8, 10, and 12 days.

[0160] 2.1 Changes in the appearance and color of cowpeas

[0161] The cowpeas from each treatment were photographed using a camera to compare appearance differences. Color difference was measured using a colorimeter calibrated with a white standard plate. The color of the cowpeas was described using L (white / black), a (red / green), and b (yellow / blue), with each sample measured three times.

[0162] Appearance and color are the primary sensory indicators for consumers to judge the freshness of cowpeas, and are also important indicators reflecting the post-harvest chlorophyll degradation, tissue dehydration, and browning process. Figure 14 As shown in Figure A, in the early stage of storage (0 days), all groups of cowpeas were bright green, plump, and had intact surfaces. With prolonged storage, the control group (CK) showed the most rapid quality deterioration, exhibiting increased yellowing and wilting, accompanied by rust spots and rot. The PE film group showed significant shrinkage and even rot in the later stages, indicating that although it had a certain water-blocking effect, it was insufficient to effectively inhibit quality decline in the later stages of storage; it had already rotted by day 10, making further experiment impossible. In contrast, the GS film and OPR / GS composite film groups significantly delayed the yellowing and appearance deterioration of cowpeas. The OPR / GS group showed a more stable color retention ability overall, which tended to improve with increasing OPR content.

[0163] Further quantification of the color change patterns is shown in Table 6. During storage, the color change patterns of each group a... * The values ​​all showed a continuous upward trend, indicating that the green color of the cowpeas was gradually fading and developing towards yellowing. * The values ​​reflect the degree of yellowing, and all groups showed an overall upward trend, indicating that the yellowing deepened continuously during storage. * The value (brightness) exhibits differentiated changes across different treatments. The overall color difference ΔE is a comprehensive indicator measuring the deviation of the sample color from its initial state. At the end of the experiment, group CK a... * With b * The largest increase was seen in L. * The decline was significant, with ΔE remaining at the highest level, indicating the fastest loss of greenness and a marked yellowing appearance, consistent with decay. The OPR / GS group, however, maintained even lower a values ​​in the later stages. * With b * Horizontal, L *The decrease was significant, and ΔE was also lower, significantly lower than CK and GS, indicating that the composite film can effectively delay the degradation and yellowing process of chlorophyll. Its ability to maintain green color is better than that of CK and GS. This shows that the composite film significantly inhibits the enhancement of yellowing and can delay the sensory deterioration of cowpeas from green to yellow. It also has advantages in delaying structural collapse and maintaining apparent brightness. It can more effectively stabilize the color of cowpeas and reduce the overall color deterioration. Its effect is significantly better than PE and GS.

[0164] Based on comprehensive observation of appearance and color difference data, the OPR / GS composite membrane exhibits significant advantages in maintaining green color, inhibiting yellowing, and slowing down overall color deterioration (ΔE increase). This effect stems from two main factors: firstly, the membrane's regulatory role in water loss and gas exchange mitigates water loss-induced metabolic disorders and chlorophyll degradation; secondly, the rutin loaded in the OPR imparts sustained antioxidant activity to the membrane, helping to scavenge reactive oxygen species accumulated during storage, reducing membrane lipid peroxidation and cell structure damage, thereby inhibiting color deterioration associated with yellowing and browning.

[0165] Table 6. Effects of different film packaging on color difference parameters (L, a, b*) and total color difference (ΔE) of cowpeas during storage.

[0166]

[0167] 2.2 Water loss rate and browning index of cowpea

[0168] The water loss rate of cowpeas was determined by weighing. The initial fresh weight was recorded as W0, and the real-time fresh weight during storage was recorded as W. t Calculate the water loss rate using the formula: WL (%) = (W0 - W t ) / W0×100; where W0 is the initial fresh weight of cowpeas; W t TN represents the fresh weight of cowpeas after storage time t; TN represents the total number of fruits in the treatment group.

[0169] The cowpea rust index (CI, %) is graded based on the percentage of the total area of ​​pitted lesions or rust spots on the fruit surface to the total fruit surface area (Grade 0: no signs of rust spots, Grade 1: <25% of the fruit area shows rust spots, Grade 2: 25–50% of the fruit area shows rust spots, Grade 3: >50% of the fruit area shows rust spots): CI (%) = Σ(CL×NL) / 3TN×100; where CL (%) is the rust spot grade; NL is the number of fruits at that grade; and TN is the total number of fruits in the treatment group.

[0170] like Figure 15As shown in Figure A, the water loss rate of cowpeas in all treatment groups gradually increased with prolonged storage time, but the rate of increase varied significantly. The control group (CK) experienced the most severe water loss, indicating that the surface moisture of cowpeas decreased extremely rapidly under unpackaged conditions, leading to significant wilting and accelerated quality deterioration. The commercial PE film group exhibited the lowest water loss level throughout the entire storage period, remaining below 5% even after 10 days, demonstrating the strong water vapor barrier properties of the PE film, which can significantly inhibit moisture migration and wilting due to water loss. However, excessively strong barrier properties may also restrict gas exchange within the packaging. Compared to the PE film, the GS film and OPR / GS composite film can inhibit water loss to some extent, but their water-blocking ability is weaker than that of the PE film. The addition of OPR further enhanced the moisturizing effect of the composite film, showing a clear concentration dependence. This indicates that OSA-modified porous starch loaded with rutin improved the water-blocking properties of the GS film and slowed down the water loss process of cowpeas, which is consistent with the aforementioned results on improved film barrier properties.

[0171] like Figure 15 As shown in Figure B, the browning index generally increased with storage time, but the timing and rate of increase varied significantly among different treatments. The CK group showed the earliest and most severe browning, with the browning index appearing at 2 days, rising to 9.72% at 6 days, and then sharply increasing to 43.06% at 12 days. This indicates that the control group experienced significant rust and browning in the later stages of storage, resulting in a rapid loss of commercial value. The PE group showed browning starting at 6 days (2.78%), rising to 13.19% at 10 days, meaning that while PE packaging significantly reduced water loss, it did not effectively prevent later browning. The GS group showed significant browning on day 8, with a significantly lower degree of browning than the CK and PE groups. The OPR / GS composite film showed the most outstanding performance in inhibiting browning: both 15% and 20% OPR / GS remained at 0% at 8 days, indicating that the composite film significantly delayed the onset of browning. This result demonstrates that the introduction of OPR not only improved the film's moisture barrier properties but also reduced cowpea browning through the antioxidant activity of rutin, with a superior effect compared to PE and GS.

[0172] 2.3 Changes in total bacterial count, conductivity, and malondialdehyde in cowpeas

[0173] The total bacterial count in cowpeas was determined using the plate count method. 5 g of cowpea sample was weighed and homogenized under aseptic conditions with 45 mL of 0.85% (w / v) NaCl solution, followed by a tenfold serial dilution. 0.1 mL of each appropriate dilution was evenly spread onto nutrient agar plates and incubated at 37°C for 48 h. Plates with colony counts between 30 and 300 were selected for counting. Results were expressed as log CFU·g. -1 express.

[0174] Electrolyte leakage rate was determined using the conductivity method. 5 g of cowpea sample was weighed and soaked in 20 mL of distilled water. The sample was gently shaken at room temperature for 20 min, and the initial conductivity Ci was measured. Subsequently, the sample was treated in a boiling water bath for 20 min, cooled to room temperature, and the final conductivity Cf was measured. Electrolyte leakage rate was calculated using the formula: EL (%) = (Ci / Cf) × 100; where EL (%) is the conductivity; Ci is the initial conductivity; and Cf is the final conductivity.

[0175] The malondialdehyde (MDA) content was determined using the thiobarbituric acid reactant (TBARS) method. After preparing the reaction system according to the method, the absorbance of the reaction product was measured at 532 nm. The MDA content was calculated based on the absorbance and expressed as nmol·g⁻¹. -1 express.

[0176] Depend on Figure 16 As shown in Figure A, the total TVC (TVC) of all groups continuously increased during the initial storage period (0 days). The PE film group exhibited the fastest microbial growth, reaching 5.53 log CFU / g at 2 days, significantly higher than the CK group (5.29 log CFU / g), and further increasing to 8.08 log CFU / g at 10 days, demonstrating the most severe microbial load. The CK group, under unprotected packaging conditions, saw a steady increase in TVC, reaching 7.58 log CFU / g at 12 days, consistent with the gradual proliferation of microorganisms under natural storage conditions. In contrast, the GS film showed a certain inhibitory effect on microbial proliferation, with a TVC of 6.94 log CFU / g at 12 days, significantly lower than the CK group. Furthermore, with increasing OPR addition, the OPR / GS composite film exhibited a significant concentration-dependent antibacterial effect.

[0177] Depend on Figure 16 As shown in Figure B, the electrolyte level (EL) of each group gradually increased with storage. The PE membrane group reached 29.18% at 4 days and further increased to 76.56% at 10 days, significantly higher than all other groups (P<0.05). This result indicates that the PE membrane exacerbated cell membrane system damage, leading to significant electrolyte extravasation. In contrast, the OPR / GS composite membrane exhibited superior membrane stability in a concentration-dependent manner, with EL significantly lower than the CK and GS groups at 12 days (P<0.05). This suggests that the addition of OPR not only provides antioxidant protection and reduces oxidation-induced membrane lipid peroxidation but also synergistically maintains cell membrane integrity.

[0178] Depend on Figure 16As shown in Figure C, the MDA content in all groups increased significantly with storage time, but the rate of increase varied significantly among different treatments. The CK group accumulated MDA the fastest and remained at the highest level throughout, indicating that the control group experienced significant membrane lipid peroxidation during storage, leading to a continuous increase in tissue oxidative damage. The PE membrane group maintained the lowest MDA content throughout, suggesting that its strong barrier properties reduced the diffusion of external oxygen and decreased the probability of lipid oxidation. At 12 days, the OPR / GS ratios for 10%, 15%, and 20% were 1.60, 1.48, and 1.31 nmol / g, respectively, all significantly lower than those in the CK and GS groups (P<0.05). These results demonstrate that OPR / GS has excellent ROS scavenging ability, can block the chain reaction of membrane lipid peroxidation, and thus alleviate oxidative stress and membrane damage in cowpea tissue.

[0179] 2.4 Changes in key nutrients in cowpeas during storage

[0180] Ascorbic acid (VC) content: determined by 2,6-dichlorophenolindophenol titration. The procedure is briefly described as follows: Weigh 5.0 g of sample, add 10 mL of 1% oxalic acid solution, grind into a homogenate in an ice bath, and bring the volume to 10 mL. Filter and collect the supernatant. Take 2 mL of the supernatant, add 1 mL of 0.025% sodium 2,6-dichlorophenolindophenol solution, react for 1 min, and measure the absorbance at 534 nm. Calculate the content according to the ascorbic acid standard curve (0–0.5 mg / mL). Results are expressed as mg / g fresh weight. Each sample was measured in triplicate.

[0181] Soluble solids (SSC) content: determined using a handheld refractometer. A brief description is as follows: Fresh cowpea samples were chopped and ground into a homogenate. After centrifugation at 8000 g for 10 min, a suitable amount of supernatant was dropped onto the detection prism of the refractometer (PAL-1, Atago, Tokyo, Japan). The cover was closed, and the value was recorded after the reading stabilized. Results are expressed as a percentage (%). Each sample was measured in triplicate.

[0182] Chlorophyll content: The determination was based on the method of Xu et al. (2023) with slight modifications. A brief description is as follows: Weigh 1.0 g of frozen cowpea sample, add 5.0 mL of 80% (v / v) acetone solution, and grind into a homogenate in an ice bath. Wash the grinding equipment and residue repeatedly with acetone solution of the same concentration until colorless. Combine all washings with the homogenate and bring the volume to 20 mL. After standing in the dark for 60 minutes, centrifuge at 11000×g for 15 minutes at 4℃. Measure the absorbance of the supernatant at 652 nm. Calculate the chlorophyll content based on the sample mass and extract volume, and express the results as mg / g fresh weight. Each sample was measured in triplicate.

[0183] Soluble protein content: determined using the Coomassie Brilliant Blue G-250 staining method. A brief description is as follows: Weigh 1.0 g of sample, add 5 mL of 0.05 mol / L phosphate buffer (pH 7.0), and homogenize in an ice bath. Centrifuge the homogenate at 10000×g for 15 min at 4℃, and use the supernatant as the test solution. Take 0.1 mL of the test solution, add 5 mL of Coomassie Brilliant Blue G-250 reagent, mix well, and let stand for 5 min. Measure the absorbance at 595 nm. Plot a standard curve (0–100 μg / mL) using bovine serum albumin (BSA) as the standard, and calculate the soluble protein content in the sample. Results are expressed as mg / g fresh weight, and each sample was measured in triplicate.

[0184] Total phenol content: determined using the Folin-Ciocalteu method. A brief description is as follows: Weigh 1.0 g of sample, add 5 mL of 80% ethanol solution, and grind into a homogenate in an ice bath. Centrifuge the homogenate at 8000×g for 10 min at 4℃, and use the supernatant as the test solution. Take 1 mL of the test solution, add 5 mL of 10% (v / v) Folin-Ciocalteu reagent, shake well, and react for 5 min. Then add 4.0 mL of 7.5% (w / v) Na2CO3 solution, mix well, and let stand at room temperature in the dark for 60 min. Plot a standard curve (0~100 mg / L) using gallic acid as a standard, and measure the absorbance at 765 nm. Calculate the total phenol content in the sample, and the result is expressed as mg (GAE) / g fresh weight. Each sample was measured in triplicate.

[0185] Total flavonoid content: determined by aluminum nitrate colorimetric method. Briefly described as follows: Take 1 mL of the test solution prepared for the "Total Phenolic Content" determination, add 1 mL of 5% (w / v) NaNO2 solution, mix well, and let stand for 6 min. Then add 4 mL of 10% (w / v) Al(NO3)3 solution, mix well, and let stand for 6 min. Finally, add 4 mL of 4% (w / v) NaOH solution, dilute to 10 mL with distilled water, mix well, and let stand in the dark for 15 min. Measure the absorbance at 510 nm. Plot a standard curve (0~200 mg / L) using rutin as a standard, calculate the total flavonoid content in the sample, and express the results as mg (RE) / g fresh weight. Each sample was measured in triplicate.

[0186] Changes in key nutrients during postharvest storage of cowpeas, such as Figure 17Among the various groups, vitamin C (VC) showed an overall decreasing trend, with significant differences in degradation rates (P<0.05). The CK group experienced the fastest VC loss, while the GS membrane had a certain maintenance effect on VC. The OPR / GS composite membrane showed a significant dose-dependent advantage, with the 20% OPR / GS group showing the highest VC content at 12 days. Chlorophyll content also decreased over time. The CK group had 0.08677 mg / g at 12 days, while the PE group decreased to 0.05156 mg / g at 10 days, indicating that excessive sealing may accelerate aging and yellowing. In contrast, the 20% OPR / GS membrane maintained 0.10197 mg / g at 12 days, significantly better than other groups. This was mainly attributed to the light-blocking properties of the composite membrane and the antioxidant effect of rutin, which reduced photo-oxidation and enzymatic degradation of chlorophyll.

[0187] SP and SSC reflect tissue metabolic stability and flavor compound retention levels. During the later stages of storage, the PE group showed the most significant decrease in both SP and SSC; the CK group, due to severe water loss, had an SSC of only 2.814% at 12 days. In contrast, the OPR / GS composite membrane better balanced moisture barrier and gas exchange, with significantly higher SP and SSC levels at 12 days compared to the GS and CK groups.

[0188] Changes in TP and TF need to be explained in conjunction with the water loss rate. The CK group showed a significant increase in TP and TF in the later stages, mainly due to severe water loss. The OPR / GS composite membrane groups maintained higher TP and TF in the later stages, with the 20% group showing the best performance. In summary, the OPR / GS composite membrane effectively delays the decline in nutritional and functional quality through a synergistic mechanism of "moisture regulation + antioxidant protection + rational gas exchange," with the 20% OPR / GS group exhibiting the best overall preservation effect.

[0189] 2.5 Changes in antioxidant enzyme activity

[0190] The activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) were determined using the corresponding kits (Nanjing Jiancheng Biotechnology Institute, Nanjing, China). The specific methods are as follows:

[0191] POD activity was determined using the guaiacol colorimetric method. Enzyme activity was defined as the amount of enzyme required to cause a 0.01 μL change in absorbance at 470 nm per minute in the reaction system, which was defined as one unit of activity (U). Results are expressed in U g. -1 Fresh weight indicates.

[0192] SOD activity was determined using the nitroblue tetrazolium (NBT) photoreduction inhibition method. Enzyme activity was defined as the amount of enzyme that inhibited NBT photoreduction by 50% under the reaction conditions as one activity unit (U). Results are expressed in U g. -1 Fresh weight indicates.

[0193] CAT activity: determined by ultraviolet spectrophotometry. Enzyme activity was calculated by monitoring the rate of decrease in absorbance of H2O2 at 240 nm in the reaction system. Enzyme activity was defined as the amount of enzyme required to decompose 1 μmol of H2O2 per minute as one activity unit (U). Results are expressed in U g. -1 Fresh weight indicates.

[0194] APX activity was determined by ultraviolet spectrophotometry. Enzyme activity was calculated by monitoring the rate of decrease in absorbance of ascorbic acid at 290 nm in the reaction system. Enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of ascorbic acid per minute as one activity unit (U). Results are expressed in U g. -1 Fresh weight indicates.

[0195] All enzyme activity assays were performed in accordance with the kit instructions, and each sample was measured in triplicate.

[0196] During storage, cowpeas continuously produce reactive oxygen species. If the removal system is insufficient, it will induce membrane lipid peroxidation and accelerate aging. Figure 18 The results showed that different film treatments significantly affected the stability of the cowpea antioxidant enzyme system (P<0.05), and the OPR / GS composite film could maintain a higher and more stable enzyme activity level throughout the storage period, indicating that it can enhance the endogenous antioxidant defense capacity of cowpea.

[0197] POD and SOD are the core of the antioxidant defense line, responsible for scavenging H2O2 respectively. Regarding POD, the CK group showed an initial decrease followed by a rebound, while the PE group experienced a significant collapse in the later stages, reflecting damage to the membrane system and enzyme function breakdown under closed-loop stress. In contrast, the OPR / GS composite membrane significantly maintained a higher POD level. SOD showed a "rise followed by fall" trend throughout storage, but the CK and PE groups experienced the most drastic decrease in the later stages, while the OPR / GS composite membrane maintained high activity in the later periods.

[0198] CAT and APX both participate in H2O2 metabolism, but their regulatory characteristics differ. In the CK group, CAT levels abnormally increased in the mid-to-late stages, which is a "compensatory stress response under strong stress" and does not necessarily indicate better preservation. In the PE group, both CAT and APX levels significantly decreased in the later stages, indicating that the anaerobic stress induced by the sealed packaging accelerated the imbalance of the antioxidant system. In contrast, the OPR / GS composite membrane maintained CAT at a moderate and stable level and significantly improved APX activity, thereby maintaining the AsA-GSH cycle and promoting vitamin C regeneration and utilization, which corroborates the optimal vitamin C retention result in this group.

[0199] In summary, the OPR / GS composite membrane enhances the adaptability of cowpea to oxidative stress through a synergistic mechanism of exogenous antioxidant (rutin slow-release to scavenge free radicals) and endogenous enzyme system homeostasis maintenance. Among them, 20% OPR / GS has the most outstanding comprehensive regulatory effect on four key antioxidant enzymes, which is the key physiological basis for delaying aging, reducing oxidative damage and maintaining quality.

[0200] 2.6 Correlation Analysis of Quality Indicators

[0201] A network heatmap of cowpea quality correlation on day 8 was constructed using Origin software. Figure 19 This study visually reveals the synergistic and antagonistic relationships between nutrient retention, antioxidant defense, and the deterioration process. AS, Chl, SP, and SSC showed a significant positive correlation overall, and maintained a strong positive correlation with the activities of antioxidant enzymes such as APX, POD, CAT, and SOD, indicating that the maintenance of nutrient components and the stability of the antioxidant system showed a consistent trend in the later stages of storage. Conversely, TVC generally showed a negative correlation with the above-mentioned nutrient / antioxidant indicators, suggesting that microbial proliferation is often accompanied by increased nutrient consumption and decreased tissue antioxidant capacity, making it an important exogenous driving factor for quality decline.

[0202] In terms of appearance quality, L * With a * b * The correlation between these parameters is significant, reflecting the consistency and synchronicity of cowpea color changes. Simultaneously, color parameters show a predominantly negative correlation with AS, Chl, and antioxidant enzyme activities, indicating that when tissues enter a significant discoloration or browning stage, it is usually accompanied by a decline in nutritional value and disruption of antioxidant homeostasis. Notably, TP and TF show a significant positive correlation, and both are positively correlated with enzyme activities such as CAT / POD, suggesting that phenols and flavonoids may participate in the oxidative stress response in conjunction with the enzymatic antioxidant system, playing a synergistic role in delaying quality decline.

[0203] The Mantel network further reinforced the above pattern from the perspective of "deterioration characterization indicators": MDA, EL, CI, and ΔE showed significant correlations with TVC, WL, and some color parameters, but the trend was opposite to that of AS, Chl, SP / SSC, and the antioxidant enzyme system. This indicates that the decline in cowpea quality on day 8 was mainly driven by a chain process of "increased microbial proliferation / water loss → membrane structure damage and oxidative damage (increased MDA and EL) → discoloration and browning deterioration (increased CI and ΔE)". Therefore, treatments that can maintain antioxidant enzyme activity and keep AS and Chl levels can help reduce MDA and EL and delay CI and ΔE deterioration, mechanistically supporting the superior preservation advantages of OPR / GS composite films in the later stages of storage.

[0204] This invention constructs a good preservation system that combines modified atmosphere storage and bioactivity regulation by introducing rutin loaded onto porous starch of cowpea (OSA) into a gelatin / sodium alginate matrix, effectively solving the problem of cowpea's easy dehydration, shrinkage, yellowing, and rotting after harvest.

[0205] The dense cross-linked network of gelatin / sodium alginate significantly enhances the moisture-barrier properties of the film, effectively inhibiting transpiration water loss in cowpeas and solving the common problem of cowpea wilting. Simultaneously, the film's unique gas selective permeability reduces the respiration intensity of vegetables. More importantly, this composite film, through the active ingredient rutin, exerts a dual antioxidant mechanism of "exogenous scavenging + endogenous activation." Experimental verification shows that this film can enhance the activity of antioxidant enzymes such as SOD, CAT, and POD in cowpeas during the later stages of storage, fundamentally inhibiting membrane lipid peroxidation by scavenging generated reactive oxygen species (ROS) (significantly reducing malondialdehyde accumulation). This mechanism effectively maintains the integrity of the cell membrane structure, significantly reduces relative conductivity, thereby inhibiting the formation of tissue browning and rust spots caused by cell breakdown, and extending the shelf life of cowpeas.

[0206] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A modified cowpea porous starch-adsorbed rutin composite film, characterized in that, The product comprises the following components in parts by weight: 100 g porous starch, 4-12 g octenyl succinic anhydride, 50 g rutin, 2.0 g gelatin, 0.5 g sodium alginate, and 0.6 g glycerin.

2. The composite film as described in claim 1, characterized in that, The weight ratio of gelatin to sodium alginate is 4:1; the mass ratio of rutin to porous starch is set at 1:2, and the porous starch is cowpea starch.

3. The composite film as described in claim 1, characterized in that, The method for preparing the porous starch includes the following steps: (1) Soak cowpea seeds in 0.05 mol / L NaOH solution, then discard the NaOH solution, add distilled water and grind, collect the mixed solution, add HCl solution to adjust the pH to 5.5-6.5, let stand and discard the supernatant, then wash the precipitate with ether, and dry the precipitate overnight in hot air to obtain crude extract; mix crude extract with distilled water and ethanol, centrifuge the mixture, wash the precipitate with distilled water 3 times and dry to obtain starch, grind and sieve the starch to obtain cowpea starch; (2) Cowpea starch was dissolved in sodium acetate buffer at 50°C and 250 r / min in a water bath constant temperature shaker; then α-amylase and glucoamylase were added, and after stirring, sodium hydroxide solution was added to adjust the pH to 10.0 to terminate the reaction. The mixture was centrifuged, and the precipitate was collected by washing with distilled water and dried to obtain porous starch (PS).

4. The method for preparing the composite thin film according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Dissolve porous starch in distilled water, heat in a water bath while stirring, and add NaOH solution dropwise to adjust the pH to 8.5; gradually add octenyl succinic anhydride to obtain a reaction solution within 1 hour of stirring, and then dilute the reaction solution 5 times with anhydrous ethanol. After stirring continuously for 3 hours, hydrochloric acid was added dropwise to adjust the pH of the diluted reaction solution to 6.5 to stop the reaction; the precipitate was centrifuged, washed three times with distilled water, then washed three times with 95% ethanol, and dried; Obtain OSAPS; (2) A rutin (RUT) solution with a mass fraction of 0.22% was prepared using anhydrous ethanol as a solvent, and an OSAPS solution with a mass fraction of 5 mg / mL was prepared using distilled water as a solvent; the mass ratio of porous starch in the rutin and OSAPS solutions was set to 1:2; then the rutin and OSAPS solutions were ultrasonically treated for 10 minutes at 100 W. The rutin ethanol solution was gradually added to the OSAPS solution at 50°C, and the mixture was stirred continuously to obtain a composite solution. The composite solution was then treated with ultrasound, and finally the composite solution was centrifuged to obtain a precipitate. The precipitate was freeze-dried to obtain the OSAPSRUT (OPR) composite. (3) Under water bath heating conditions, prepare a 2.5% (w / v) concentration aqueous solution of gelatin (G) and sodium alginate (S) under stirring conditions. Mix the two solutions according to the GS ratio of 4:1 (v / v). Add 0.6 g of glycerol to every 100 mL of the mixed solution and stir continuously for 2 h. After the membrane solution is mixed evenly, remove the air bubbles by ultrasonic treatment to obtain the GS membrane solution. Add OPR to the membrane solution and mix evenly to obtain the OPR / GS membrane solution. Pour it into a dish, spread it evenly and dry it to obtain the OPR / GS film.

5. The preparation method according to claim 4, characterized in that, In step (1), the weight of octenyl succinic anhydride accounts for 4%-12% of the dry weight of PS; in step (2), the degree of substitution of the complex is 0.0227, 0.0416 and 0.0584, respectively; in step (3), the weight of OPR added is 10%, 15% and 20% of the total dry weight of gelatin and sodium alginate, respectively, and the amount of OPR / GS membrane solution used is 0.3145 g / cm³. 2 .

6. The application of the composite film as described in claim 1 or 2 in any of the following: (1) Improve the rutin encapsulation rate and loading rate of the composite film; (2) Improve the uniformity of the composite film; (3) Improve the stability of the composite film; (4) Delay the weight loss rate of the composite film; (5) Reduce the water vapor transmission rate of the composite film; (6) Improve the oxygen transmission rate of the composite film; (7) Improve the carbon dioxide transmission rate of the composite film; (8) Reduce the light transmittance of the composite film; (9) Improve the opacity of the composite film; (10) Reduce the elongation at break of the composite film; (11) Improve the tensile strength of the composite film; (12) Improve the hydrophobicity of the composite film surface and / or improve the water stability of the composite film; (14) Improve the thermal anti-fogging properties of the composite film; (15) Improve the biodegradability of the composite film.

7. The application of the composite film as described in claim 1 or 2 in any of the following: (1) Preserving cowpeas; (2) Inhibiting post-harvest decay of cowpeas; (3) Delaying the green-to-yellow color change of cowpeas after harvest; (4) Inhibiting cowpea wilting; (5) Inhibiting post-harvest decay of cowpeas; (6) Improving the nutritional quality of cowpeas; (7) Extending the shelf life of cowpeas; (8) Reducing the water loss rate of cowpeas; (9) Reducing the browning index of cowpeas; (10) Reducing the total number of colonies in cowpeas; Inhibiting browning of cowpeas; (11) Inhibiting rust spots on cowpeas.

8. The application as described in claim 7, characterized in that, Composite films can reduce the electrical conductivity of cowpeas, decrease the malondialdehyde content, increase the ascorbic acid content, increase the chlorophyll content, increase the soluble protein content, increase the soluble solids content, decrease the total phenol content, decrease the total flavonoid content, increase the SOD activity, increase the CAT activity, increase the POD activity, increase the APX activity, and / or reduce the formation of reactive oxygen species in cowpeas during storage.

9. The application of the composite film described above in antibacterial and / or antioxidant applications; preferably, the composite film is capable of inhibiting Escherichia coli and Staphylococcus aureus.

10. A method for preserving cowpeas, characterized in that, Includes the following steps: Cowpeas are sealed and preserved using the aforementioned composite film.

Images