Moringa oleifera leaf flavone-chitosan composite fruit and vegetable fresh-keeping agent as well as preparation method and application thereof

Abstract

The invention provides a moringa oleifera leaf flavone-chitosan composite fruit and vegetable fresh-keeping agent and a preparation method thereof, relates to the technical field of agricultural product storage and fresh-keeping, and aims to solve the problem that a composite fresh-keeping agent taking natural active ingredients as main raw materials is lacked in the prior art. The method comprises the following steps: step 1, extracting, filtering, centrifuging and freeze-drying flavone in moringa oleifera leaves through ultrasonic assistance to obtain a moringa oleifera leaf flavone crude extract; step 2, preparing the moringa oleifera leaf flavone crude extract into a solution, and then separating and purifying moringa oleifera leaf flavone to obtain moringa oleifera leaf flavone; and step 3, adding the moringa oleifera leaf flavone into the chitosan solution, fully stirring, and performing ultrasonic treatment to obtain the moringa oleifera leaf flavone-chitosan composite preservative. The moringa oleifera leaf flavone-chitosan composite fresh-keeping agent disclosed by the invention is used for fresh-keeping of fruits and vegetables.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural product storage and preservation, and more specifically, to a flavonoid-chitosan composite fruit and vegetable preservative from Moringa leaves and a preparation method thereof. Background Art

[0002] During the storage and transportation of fresh-cut fruits and vegetables, problems such as accelerated physiological metabolism, microbial infection, and oxidative browning caused by tissue damage seriously restrict their shelf life and commercial value. Although traditional chemical preservatives (such as chlorine dioxide and sodium benzoate) can effectively inhibit spoilage, there are safety controversies and environmental pollution risks. Functional composite preservatives based on natural ingredients have become a research hotspot due to their green and safe characteristics. As a cationic polysaccharide, chitosan can block the invasion of oxygen and microorganisms through film-forming, but its single use has defects such as a narrow antibacterial spectrum and limited antioxidant ability.

[0003] Moringa leaf flavonoids, with a polyphenol hydroxyl structure, have both strong antioxidant and antibacterial properties, but the shortcoming of their weak adhesion limits their direct application. The traditional solvent extraction of flavonoids from Moringa takes a long time and has low efficiency, and long-term heating will destroy the structure of flavonoid compounds, significantly reducing their antioxidant and other biological activities. Enzyme-assisted extraction is to dissolve the cell wall through the specificity of enzymes, disrupt the original state of the cytoplasm, and decompose the impurities in the original cytoplasm, thereby increasing the extraction rate of the target substance. Ultrasonic-assisted extraction uses the effects of ultrasonic wave crushing, cavitation, stirring, etc. to promote the dissolution of active ingredients such as flavonoids, and has advantages such as high efficiency and economy.

[0004] As a cationic polysaccharide, chitosan can block the invasion of oxygen and microorganisms through film-forming, but its single use has defects such as a narrow antibacterial spectrum and limited antioxidant ability. Therefore, it is urgent to study the application of composite natural preservatives in extending the shelf life of fruits and vegetables. Summary of the Invention

[0005] The technical problem to be solved by the present invention is:

[0006] There is a lack of composite preservatives mainly composed of natural active ingredients in the prior art.

[0007] The technical solution adopted by the present invention to solve the above technical problem:

[0008] The present invention provides a preparation method of a flavonoid-chitosan composite fruit and vegetable preservative from Moringa leaves, comprising the following steps:

[0009] Step 1: Extract flavonoids from Moringa leaves by ultrasonic assistance, filter, centrifuge, and freeze-dry to obtain a crude extract of Moringa leaf flavonoids;

[0010] Step 2: Prepare the crude extract of Moringa leaf flavonoids into a solution, and then separate and purify the Moringa leaf flavonoids to obtain Moringa leaf flavonoids;

[0011] Step 3: Add the flavonoids from Moringa oleifera leaves into the chitosan solution, stir well and then perform ultrasonic treatment to obtain the flavonoids-chitosan composite fresh-keeping agent.

[0012] Further, the extraction solution in Step 1 is a 50%-55% ethanol solution.

[0013] Further, in Step 1, the flavonoids in Moringa oleifera leaves are extracted at a material-liquid ratio of 1:70.

[0014] Further, the conditions for ultrasonic-assisted extraction of flavonoids from Moringa oleifera leaves in Step 1 are: at (60-62)°C, ultrasonic treatment at 100 W for (40-50) min.

[0015] Further, the separation and purification of the flavonoids from Moringa oleifera leaves in Step 2 is specifically: separating and purifying the flavonoids from Moringa oleifera leaves using macroporous resin.

[0016] Further, for the separation and purification of the flavonoids from Moringa oleifera leaves in Step 2, the sample loading flow rate is (1-2) mL / min, and the elution flow rate is (1-2) mL / min.

[0017] Further, the concentration of the chitosan solution in Step 3 is 1%.

[0018] Further, the concentration of the flavonoids from Moringa oleifera leaves in the flavonoids-chitosan composite fresh-keeping agent is (1-2) mg / mL.

[0019] The present invention provides a flavonoids-chitosan composite fresh-keeping agent, and the fresh-keeping agent is prepared by the preparation method of the flavonoids-chitosan composite fresh-keeping agent for fruits and vegetables according to any one of the above technical solutions.

[0020] The present invention also provides the application of the flavonoids-chitosan composite fresh-keeping agent in the fresh-keeping of fruits and vegetables.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] The present invention extracts flavonoids from Moringa oleifera leaves, optimizes the extraction and purification processes, obtains the optimal process conditions for ultrasonic-assisted extraction of flavonoids from Moringa oleifera leaves, and finally, based on the excellent antibacterial effect of Moringa oleifera leaf flavonoids and the good film-forming effect of chitosan, a composite preservative is prepared by compounding Moringa oleifera leaf flavonoids and chitosan. Due to the electrostatic interaction between the amino group in Moringa oleifera leaf chitosan and the hydroxyl and carboxyl groups in flavonoids, the stability of the composite preservative is enhanced; at the same time, the hydroxyl and amino groups in chitosan form hydrogen bond networks with the hydroxyl and carbonyl groups in flavonoids, enhancing the interaction in the molecule and improving the tensile strength of the composite film; the hydrophobic aromatic ring structure in flavonoids and the hydrophobic region in chitosan are combined through hydrophobic interaction to regulate the slow-release performance of the composite film; the combination of chitosan and flavonoids exhibits a synergistic antibacterial and antioxidant mechanism.

[0023] Experiments have proved that the composite preservative of the present invention can be applied to the preservation of various types of fruits and vegetables, has strong antioxidant and antibacterial abilities, has strong scavenging abilities for ABTS and DPPH free radicals, and the scavenging rates reach 99.12% and 93.64% respectively, and the total reducing power is also strong. At the same time, it can delay the softening and weight loss during the storage of fruits and vegetables, reduce the browning index, and reduce the browning of fruits and vegetables to play a role in protecting the color; delay the increase in malondialdehyde content and maintain the SOD activity.

[0024] The present invention provides a theoretical basis and technical support for the research of safe, effective, and pollution-free natural preservatives, and has important application value for reducing the use of chemical preservatives and extending the shelf life of fruits and vegetables. Brief Description of the Drawings

[0025] Figure 1 It is a graph showing the effect of ethanol concentration on the extraction rate of total flavonoids in the examples of the present invention;

[0026] Figure 2 It is a graph showing the effect of solid-liquid ratio on the extraction rate of total flavonoids in the examples of the present invention;

[0027] Figure 3 It is a graph showing the effect of ultrasonic time on the extraction rate of total flavonoids in the examples of the present invention;

[0028] Figure 4 It is a graph showing the effect of ultrasonic temperature on the extraction rate of total flavonoids in the examples of the present invention;

[0029] Figure 5Response surface plots and contour plots of the interaction between the four factors in the embodiments of the present invention; Figure a, the influence of ethanol concentration and solid-liquid ratio on the total flavonoid extraction rate; Figure b, the influence of ethanol concentration and ultrasonic time on the total flavonoid extraction rate; Figure c, the influence of ethanol concentration and ultrasonic temperature on the total flavonoid extraction rate; Figure d, the influence of solid-liquid ratio and ultrasonic time on the total flavonoid extraction rate; Figure e, the influence of solid-liquid ratio and ultrasonic temperature on the total flavonoid extraction rate; Figure f, the influence of ultrasonic time and ultrasonic temperature on the total flavonoid extraction rate;

[0030] Figure 6 Leakage curve diagrams of different sample loading flow rates in the embodiments of the present invention;

[0031] Figure 7 Elution curve diagrams of different flow rates in the embodiments of the present invention;

[0032] Figure 8 LC-MS negative ion mode ion flow diagram of flavonoids from Moringa oleifera leaves in the embodiments of the present invention;

[0033] Figure 9 LC-MS positive ion mode ion flow diagram of flavonoids from Moringa oleifera leaves in the embodiments of the present invention;

[0034] Figure 10 Infrared spectrum diagram in the embodiments of the present invention;

[0035] Figure 11 Antibacterial activity comparison diagram of Escherichia coli of the composite preservative in the embodiments of the present invention;

[0036] Figure 12 Antibacterial activity comparison diagram of Staphylococcus aureus of the composite preservative in the embodiments of the present invention;

[0037] Figure 13 Influence comparison diagram of the composite preservative on the hardness of fruits and vegetables in the embodiments of the present invention;

[0038] Figure 14 Influence comparison diagram of the composite preservative on the weight loss rate of fruits and vegetables in the embodiments of the present invention;

[0039] Figure 15 Influence comparison diagram of the composite preservative on the chromaticity of fresh-cut lettuce in the embodiments of the present invention;

[0040] Figure 16 Influence comparison diagram of the composite preservative on the chromaticity of fresh-cut lotus root in the embodiments of the present invention;

[0041] Figure 17 Influence comparison diagram of the composite preservative on the chromaticity of blueberries in the embodiments of the present invention;

[0042] Figure 18Comparison chart of the effect of the composite preservative in the embodiment of the present invention on the MDA content of fruits and vegetables;

[0043] Figure 19 Comparison chart of the effect of the composite preservative in the embodiment of the present invention on the SOD activity of fruits and vegetables;

[0044] Figure 20 Comparison chart of the ABTS free radical scavenging ability of the composite preservative in the embodiment of the present invention;

[0045] Figure 21 Comparison chart of the DPPH free radical scavenging ability of the composite preservative in the embodiment of the present invention;

[0046] Figure 22 Comparison chart of the total reducing power of the composite preservative in the embodiment of the present invention. Detailed implementation manners

[0047] In order to enable those skilled in the art to better understand the solution of the present invention, the exemplary embodiments or examples of the present invention will be described below in conjunction with the accompanying drawings. Obviously, the described embodiments or examples are only a part of the embodiments or examples of the present invention, rather than all of them. Based on the embodiments or examples in the present invention, all other embodiments or examples obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0048] To make the above objects, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0049] The beneficial effects of the present invention are described in detail by the following examples.

[0050] Example 1: Single factor experiment on the extraction process of flavonoids from Moringa oleifera leaves

[0051] (1) Effect of ethanol concentration on the extraction rate of total flavonoids from Moringa oleifera leaves

[0052] Weigh 2 g of Moringa oleifera leaf powder and put it into a conical flask. According to the solid-liquid ratio of 1:60 (w / v), add ethanol solutions with mass fractions of 30%, 40%, 50%, 60%, and 70% respectively. Continuously ultrasonic for 35 min at an ultrasonic power of 100 W and an ultrasonic temperature of 50 °C, filter to obtain the filtrate, and centrifuge at 5000 r / min and 4 °C for 10 min. Take the supernatant and make it up to 100 mL with 30% ethanol in a volumetric flask, and take 1 mL of the made-up supernatant for determination.

[0053] In this example, the existing method [1] was used to calculate the yield of total flavonoids. Preparation of the standard curve of rutin standard: Accurately weigh 20 mg of rutin standard, dissolve it in a small amount of 95% ethanol, and under the condition of 30% ethanol, make the volume up to 100 mL in a volumetric flask to obtain a rutin standard solution with a mass concentration of 0.2 mg / mL. Accurately add 0.0, 1.0, 2.0, 3.0, 4.0, 5.0 mL of the rutin standard solution into a 25-mL volumetric flask, add 10 mL of ethanol with a volume fraction of 30% as a diluent, and shake well. Then, add 1 mL of NaNO2 solution with a volume fraction of 5% to each volumetric flask, shake well again and let it stand for 6 min, then add 1 mL of Al(NO3)3 solution with a volume fraction of 10% and continue to let it stand for 6 min. Then add 10 mL of NaOH solution with a volume fraction of 4%, dilute the solution to the scale line with 30% ethanol, and after fully reacting for 15 min, finally measure its absorbance value at a wavelength of 510 nm with an ultraviolet spectrophotometer.

[0054] Absorb 1 mL of the sample solution into a 25-mL volumetric flask, add 1 mL of NaNO2 solution with a volume fraction of 5%, mix well and let it stand for 6 min, add 1 mL of Al(NO3)3 solution with a volume fraction of 10%, mix well and let it stand for 6 min, add 10 mL of NaOH solution with a volume fraction of 4%, finally make the volume up with 30% ethanol, after fully reacting for 15 min, measure its absorbance value at a wavelength of 510 nm, substitute it into the rutin standard curve for calculation, obtain the flavonoid concentration in the sample solution, and calculate the extraction rate of total flavonoids according to the following formula.

[0055]

[0056] In the formula: N, dilution factor; V, the final volume of the extraction solution, mL; C, the total flavonoid concentration in the sample solution obtained by calculation from the standard curve, μg / mL; M, the mass of the moringa leaf powder, mg.

[0057] (2) Effect of the solid-liquid ratio on the extraction rate of total flavonoids from moringa leaves

[0058] Weigh 2 g of moringa leaf powder and put it into a conical flask, add 50% ethanol solution according to the solid-liquid ratios (w / v) of 1:50, 1:60, 1:70, 1:80, 1:90, continuously ultrasonic for 35 min at a power of 100 W and an ultrasonic temperature of 50 °C, filter by suction to obtain the filtrate, centrifuge at 5000 r / min and 4 °C for 10 min, take the supernatant and make the volume up to 100 mL in a volumetric flask with 30% ethanol, take 1 mL of the supernatant after volume making for determination, and calculate the extraction rate of total flavonoids.

[0059] (3) Effect of ultrasonic time on the extraction rate of total flavonoids from moringa leaves

[0060] Weigh 2 g of Moringa oleifera leaf powder and place it in a conical flask. Add 50% ethanol solution according to a solid-liquid ratio of 1:70 (w / v). Under an ultrasonic power of 100 W and an ultrasonic temperature of 50 °C, ultrasonicate for 25 min, 35 min, 45 min, 55 min, and 65 min respectively. After ultrasonication, filter the mixture to obtain the filtrate, and centrifuge at 5000 r / min and 4 °C for 10 min. Take the supernatant and dilute it to 100 mL with 30% ethanol in a volumetric flask. Take 1 mL of the diluted supernatant for determination and calculate the total flavonoid extraction rate.

[0061] (4) Effect of ultrasonic temperature on the total flavonoid extraction rate from Moringa oleifera leaves

[0062] Weigh 2 g of Moringa oleifera leaf powder and place it in a conical flask. Add 50% ethanol solution according to a solid-liquid ratio of 1:70 (w / v). Under an ultrasonic power of 100 W, ultrasonicate continuously for 45 min at temperatures of 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C respectively. After ultrasonication, filter to obtain the filtrate, and centrifuge at 5000 r / min and 4 °C for 10 min. Take the supernatant and dilute it to 100 mL with 30% ethanol in a volumetric flask. Take 1 mL of the diluted supernatant for determination and calculate the total flavonoid extraction rate.

[0063] As can be seen from Figure 1 the figure, the extraction rate shows a trend of first increasing and then decreasing with the increase of ethanol concentration. When the volume fraction of ethanol reaches 50%, the total flavonoid extraction rate can reach the highest point. Subsequently, continuing to increase the ethanol concentration will instead lead to a decrease in the extraction rate. This is because most flavonoid compounds are weakly polar or non-polar. According to the principle of "like dissolves like", when the polarities are similar, the solubility increases. As the volume fraction of ethanol increases, the polarity difference between flavonoids and the solvent increases, resulting in a decrease in the solubility of flavonoids and having a certain impact on the total flavonoid extraction rate.

[0064] According to Figure 2 what is shown in the figure, the total flavonoid level extracted from Moringa oleifera leaves shows a positive correlation trend with the increase of the solid-liquid ratio, and reaches the highest point when the solid-liquid ratio is 1:70. However, when the solid-liquid ratio exceeds 1:70, the extraction rate will start to gradually decrease. This is attributed to the addition of a large amount of organic solvent, which promotes the dissolution of flavonoid substances and increases the degree of penetration of the solvent into the cells. On the other hand, it also leads to the dissolution of other impurities, a decrease in the extraction amount of flavonoids, and a waste of resources.

[0065] According to Figure 3As shown, when the ultrasonic treatment time is extended, the extraction rate of total flavonoids from Moringa oleifera leaves extracted experimentally is significantly increased first. However, as the treatment time continues to extend, this extraction rate shows a gradually decreasing trend and reaches the maximum value when the treatment time reaches 45 min. The reason is that when the ultrasonic time is too short, flavonoid compounds are not fully dissolved; when the extraction time is too long, the structures of some flavonoid substances are damaged, and the dissolution amount of impurities also increases, resulting in a decrease in the flavonoid extraction rate.

[0066] As Figure 4 shown, the extraction rate of total flavonoids from Moringa oleifera leaves increases first and then decreases with the increase of ultrasonic temperature, and reaches the maximum value at 60 °C. This is because as the temperature increases, the dissolution rate of flavonoids in Moringa oleifera leaves accelerates. However, when the temperature in the system is too high, more soluble proteins will denature, increasing the viscosity of the system and affecting the dissolution of flavonoid substances.

[0067] Response surface optimization experiment

[0068] Based on the single-factor experiments, a response surface experiment design was carried out. Four single factors, namely A - ethanol concentration (%), B - solid-to-liquid ratio (w / v), C - ultrasonic time (min), and D - ultrasonic temperature (°C), were selected for the experiment, and the coded levels were set as -1, 0, and 1. The aim was to optimize the flavonoid extraction process by designing a response surface experiment with four factors and three levels. The extraction rate Y was used as the response value in the response surface experiment. The experimental factors and levels of the response surface experiment are shown in Table 1.

[0069] Table 1 Factors and levels of the response surface optimization experiment design

[0070]

[0071] All experiments were repeated three times in parallel for data statistics and analysis. As shown in Table 2, the experimental data of each factor were regressively fitted, and the regression equation was obtained as follows: Y = 5.41 + 0.0251A - 0.0132B + 0.093C + 0.1190D + 0.0437AB + 0.0278AC - 0.0119AD - 0.0556BC - 0.0754BD - 0.0159CD - 0.3779A 2 - 0.2569B 2 - 0.3104C 2 - 0.4315D 2 .

[0072] After analyzing Table 3, it can be seen that the model showed obvious statistical significance in the significance test, with its P value less than 0.01 and R 2 = 0.8760, indicating that the regression model had a good fit. According to the P value, the quadratic terms A 2 , B 2 , C 2 , D2 The response value was extremely significantly affected (P < 0.01); the response value of the first-order term D was significantly affected (P < 0.05). The larger the F value, the greater the influence on the response value. According to the F value (D > A > B > C), it can be seen that the order of the influence degree of the four factors on the total flavonoid extraction rate of Moringa oleifera leaves from deep to shallow is: ultrasonic temperature, ethanol concentration, solid-liquid ratio, and ultrasonic time.

[0073] From Figure 5 The data of the surface plot and contour plot show that there is an interaction between the four variables of ethanol concentration, solid-liquid ratio, ultrasonic time, and ultrasonic temperature. The response surface plot affected by the interaction between these variables has a steep tendency, and at the same time, the contour plot also shows an elliptical trend, indicating a strong interaction between the independent variables. This observation result is consistent with the variance analysis of the regression equation and the influence result on the flavonoid extraction rate of Moringa oleifera leaves.

[0074] To sum up, the optimal process for extracting flavonoids from Moringa oleifera leaves by ultrasonic method is that the ethanol concentration is 50.32%, the solid-liquid ratio is 1:70, the ultrasonic temperature is 61.37 °C, and the ultrasonic time is 45 min. The predicted extraction rate of total flavonoids from Moringa oleifera leaves under this process is 5.42%. Considering the current actual conditions, the ethanol concentration of 50%, the solid-liquid ratio of 1:70, the extraction temperature of 61 °C, and the ultrasonic time of 45 min are selected as the optimal operating conditions. Under these conditions, the extraction rate of total flavonoids extracted from Moringa oleifera leaves is 5.40%.

[0075] Table 2 Results of Box-Behnken test

[0076]

[0077]

[0078] Table 3 Variance analysis

[0079]

[0080]

[0081] Example 2. Process optimization for the separation and purification of the flavonoid extract from Moringa oleifera leaves by macroporous resin

[0082] Pretreatment of AB-8 macroporous resin

[0083] Alcohol washing: Immerse the AB-8 macroporous resin in absolute ethanol for 24 h to ensure that the resin swells sufficiently, and wash it with water until there is no obvious ethanol smell.

[0084] Acid washing: Immerse it in a 4% HCl solution by mass for 4 h, and then wash it with water until it is neutral.

[0085] Alkaline washing: After soaking in 4% NaOH solution by mass for 4 h, rinse with water until neutral, and dry at 30 °C for standby.

[0086] Dynamic adsorption and desorption experiments of AB-8 macroporous resin

[0087] Soak the pretreated AB-8 resin in water for 12 h. Slowly and evenly pour the resin into the chromatography column along the glass rod, and pay attention not to generate bubbles during the process. Open the piston at the bottom of the column to allow the resin to settle naturally. After the resin is balanced, add a flavonoid solution with a concentration of 1.5 mg / mL through a constant flow pump for adsorption. The total sample loading volume is 60 mL. Set the flow rate of the constant flow pump to 1, 1.5, and 2 mL / min, and collect the effluent every 10 min to measure its flavonoid concentration. Draw a dynamic adsorption breakthrough curve with the sample volume - flavonoid concentration. After the dynamic adsorption is completed, first rinse the AB-8 resin with pure water until the eluent is colorless, and then elute with 50% ethanol solution at a rate of 1, 1.5, and 2 mL / min. Collect the effluent every 10 min to measure its flavonoid concentration. When the flavonoid concentration in the eluent is close to 0, it is considered that the resin has been completely desorbed, and draw a dynamic elution curve with the eluent volume - desorption rate.

[0088] As Figure 6 , as the volume of the sample solution increases, the concentration of flavonoid compounds in the breakthrough solution gradually increases. When the sample loading flow rate is 1.5 mL / min and the volume of the sample solution is 50 mL, the concentration of flavonoid compounds in the breakthrough solution reaches 0.132 mg / mL, which is about 10% of the sample solution concentration, reaching the breakthrough point. In addition, too fast a flow rate results in insufficient interaction time between flavonoid compounds and the resin, and too low a flow rate affects the sample loading efficiency. Considering comprehensively, the optimal sample loading flow rate is selected as 1.5 mL / min and the volume of the sample solution is 50 mL.

[0089] As Figure 7 shown, as the flow rate gradually increases, the volume of ethanol solution required for complete desorption of flavonoids from Moringa oleifera leaves continues to increase. When the flow rate is controlled at 1 mL / min and the volume of the effluent consumed reaches 90 mL, the remaining flavonoid content in the solution is lower than 0.01 mg / mL, indicating that the flavonoids adsorbed on the resin are basically completely eluted. To ensure the recovery rate of the product, the volume of the effluent is selected as 90 mL and the elution flow rate is controlled at 1 mL / min.

[0090] Under positive and negative ion modes, a total of 29 monomeric flavonoid compounds were identified from the total flavonoids of Moringa oleifera leaves. The specific data are shown in Table 4. The main flavonoid compounds in Moringa oleifera leaves are isovitexin, isoorientin, quercetin, rutin, pinocembrin, vitexin, liquiritigenin, chrysin, isorhamnetin, luteolin, hesperetin, etc. The LC-MS negative and positive ion mode ion currents of flavonoids from Moringa oleifera leaves are shown in Figure 8 and 9 .

[0091] LC-MS Analysis of Flavonoid Chemical Constituents in Moringa oleifera Lam. Leaves

[0092]

[0093]

[0094] During the dynamic adsorption and desorption process on AB-8 resin, the optimal loading flow rate was selected as 1.5 mL / min and the volume of the loading solution was 50 mL. To ensure the recovery rate of the product, the volume of the eluent was selected as 90 mL and the elution flow rate was controlled at 1 mL / min. The LC-MS identification results showed that the main flavonoid compounds in Moringa oleifera Lam. leaves were isovitexin, isoorientin, quercetin, rutin, pinocembrin, vitexin, liquiritigenin, chrysin, isorhamnetin, luteolin, hesperetin, etc.

[0095] Example 3: Preparation of a Composite Preservative of Flavonoids from Moringa oleifera Lam. Leaves and Chitosan

[0096] Dissolve different masses of Moringa oleifera Lam. leaf flavonoid powder (MLF-1) in 1% chitosan solution and stir at 1800 r / min for 1 h. At room temperature of 25 °C, ultrasonically oscillate for 15 minutes to obtain a composite preservative of Moringa oleifera Lam. leaf flavonoids and chitosan (FCCP). It includes: composite preservatives obtained from 10 mg MLF-1 + 10 mL of 1% chitosan solution (FCCP-1), 15 mg MLF-1 + 10 mL of 1% chitosan solution (FCCP-2), and 20 mg MLF-1 + 10 mL of 1% chitosan solution (FCCP-3); and prepare 1% chitosan (CP) solution.

[0097] Freeze-dry each group of substances and measure the FTIR spectra of each group of substances. The scanning range is 400 cm -1 ~4000 cm -1 . As Figure 10 shown, the sample shows an O-H stretching vibration peak near 3500 cm -1 , indicating the presence of hydroxyl groups or associated hydroxyl groups (such as alcohols, phenols, or adsorbed water). Among them, the O-H peak of CP is located at 3329 cm -1 , while the peak position of the FCCP sample (3334 - 3336 cm -1 ) shifts slightly to a higher wavenumber, reflecting a weakening of hydrogen bond interaction or a change in the chemical environment around the hydroxyl group (such as increased hydrophobicity). The C=O stretching vibration peak near 1600 cm -1 (CP: 1637.72 cm -1 , FCCP sample: 1637.05 - 1637.72 cm -1 ) indicates that the sample contains a carbonyl structure (such as esters, ketones, or carboxylic acids).

[0098] Analysis of the Bacteriostatic Activity of the Composite Preservative of Moringa oleifera Leaf Flavonoids - Chitosan

[0099] On the ultra - clean workbench, Escherichia coli and Staphylococcus aureus were inoculated onto LB medium by the streak plate method, placed in an incubator and cultured at 37 °C for 24 h, and transferred 3 times to fully activate the strains. The activated strains were inoculated onto slant medium and cultured for 24 h to obtain slant cultures. Use a sterilized pipette to aspirate 5 mL of tryptone physiological saline solution (TPS dilution solution), add it into the slant test tube, and repeatedly blow and wash to wash off the bacterial lawn. Transfer the eluted bacterial liquid to another sterile test tube, and oscillate on a shaker for about one minute to make the bacteria evenly suspended. Aspirate 1 mL of the bacterial liquid into another test tube, add 9 mL of dilution solution to obtain a bacterial suspension diluted ten - fold. Shake the bacterial suspension well, aspirate 150 μL of the bacterial suspension, and evenly coat it on the solid medium. Use forceps to stick a sterile filter paper (diameter: 6 mm) on the coated plate, and drop 10 μL of each sample solution in Example 3, with sterile water as the blank control. Conduct three parallel experiments. After incubating upside - down at 37 °C for 24 h, measure the diameter of the inhibition zone (mm). The diameter of the inhibition zone can be divided into four grades: highly sensitive, with a diameter greater than 20 mm; sensitive, with a diameter between 14 and 20 mm; moderately sensitive, with a diameter between 8 and 14 mm; insensitive, with a diameter less than 8 mm. Among them, the minimum concentration at which an inhibition zone appears is the minimum inhibitory concentration (MIC) of the sample.

[0100] As Figure 11 shown, compared with the control group, all experimental groups showed bacteriostatic effects to varying degrees, and the bacteriostatic activity showed significant differences with the changes in the type and concentration of the treatment group. The diameter of the inhibition zone of the chitosan group (CP) was significantly larger than that of the control group, indicating that it has certain antibacterial ability. The bacteriostatic effect of the composite preservative (FCCP) treatment group was significantly better than that of the CP group, and the bacteriostatic activity showed an increasing trend with the increase of flavonoids. Among them, the diameter of the inhibition zone of FCCP - 2 was the largest, significantly larger than that of the CP group, indicating that its bacteriostatic activity was the strongest. This may be because the composite preservative enhances the bacteriostatic effect through multi - target synergistic effects. In summary, the FCCP group all had a significant bacteriostatic effect on Escherichia coli, and the optimization of the composite ratio could significantly improve its antibacterial performance.

[0101] As Figure 12As shown, there are significant differences in the antibacterial effects of each treatment group, and the antibacterial activity of the composite preservative increases with the increase of the flavonoid concentration gradient, indicating its potential synergistic effect. There is no significant antibacterial zone in the Control group, indicating that Staphylococcus aureus can proliferate normally without intervention. The diameter of the antibacterial zone in the CP group is significantly larger than that in the control group, but lower than that in the FCCP group, indicating that although it has antibacterial ability, the effect is limited. This is because the action target of the single component of chitosan is relatively single, and it is difficult to completely inhibit strains with strong drug resistance. The antibacterial effect of the FCCP-2 group is significantly better than that of the CP group, and its antibacterial zone diameter is the largest. The reason is that the composite preservative destroys the bacterial antioxidant system through multi-target synergistic action. The film-forming property of chitosan enables the slow release of flavonoids to extend the antibacterial action time. In summary, FCCP-2 significantly inhibits the growth of Staphylococcus aureus through the synergistic action of multiple components.

[0102] Example 5. Analysis of the effect of the composite preservative of moringa leaf flavonoid-chitosan on the preservation of fruits and vegetables

[0103] Wash the surfaces of lettuce, lotus root and blueberries with deionized water, and then cut them in a super clean bench. Take the middle tissue part and cut it into 1 cm 3 squares. Immerse lettuce, lotus root and washed blueberries in the sample solutions of each group in Example 3 for 10 s, take them out and air dry naturally, put them into disposable fresh-keeping boxes, cover them and store them in a 4°C refrigerator for 7 days. Take samples every day, and use the treatment of soaking in sterile distilled water as a control. Each treatment is done in 3 parallels.

[0104] The hardness is measured using a texture analyzer. Use a cylindrical probe with a diameter of 6 min, convert the test mode to the fruit pulp puncture program, set the range of the force sensor to 250 N, the starting force to 0.2 N, the test rate to 70 mm / min, the puncture distance to 3 min, the return rate to 80 mm / s, and the return distance to 20 mrn, and measure the hardness of fruits and vegetables every day during storage.

[0105] As Figure 13 shown, during the 7-day storage period, there are significant differences in the hardness maintenance effects of different preservative treatments on fresh-cut lettuce, lotus root and blueberries. The hardness of the blank control group decreased significantly in the later stage of storage. Among them, the softening rate of blueberries was the fastest, and the hardness on the 7th day was only 42.3% of the initial value. In the FCCP treatment, the effects of different concentrations on the hardness showed a gradient effect. For lotus root slices, the hardness retention rates of FCCP-1 and FCCP-2 were increased by 24.5% and 31.8% respectively compared with the control group. The FCCP-2 treatment had the best comprehensive preservation effect on the three kinds of fruits and vegetables, and was most capable of delaying the decrease of fruit and vegetable hardness and reducing softening.

[0106] Calculate the weight loss rate by measuring the weight of fruits and vegetables every day with an analytical balance. The weight loss rate is expressed as:

[0107]

[0108] Where: W1 is the weight of fruits and vegetables on day 0; W i It is the weight of fruits and vegetables from day 1 to day 7.

[0109] like Figure 14 As shown in the figure, the weight loss rate of the control group continued to increase with the extension of storage time, among which blueberry had the fastest weight loss rate, reaching 6.8% on the 7th day, which may be related to its thin epidermal wax layer, strong transpiration and vigorous respiratory metabolism. FCCP-2 treatment performed best among the three fruits and vegetables, effectively slowing down the increase in weight loss rate. The reason may be that its moderate coating concentration not only effectively reduced the weight loss caused by transpiration and microorganisms, but also avoided excessive inhibition of gas exchange by high concentration treatment.

[0110] During the storage period, the colorimeter was used to measure the color of fruits and vegetables every day. * 、a * , b * Determination of L * Indicates brightness, a * Indicates red and green, b * Indicates yellow-blue, and the browning index BI is:

[0111]

[0112] Where: BI is the browning index, ΔL * , Δa * and Δb * They are the difference between the measured value on the sampling day and the measured value on day 0.

[0113] like Figure 15 As shown in the figure, different treatments had significant differences in regulating the chromaticity (Lab value) and browning index of fresh-cut lettuce. The L value of the control group dropped from the initial 55.0 to 36.2 on the 7th day, the a value increased from -2.1 to 3.8, and the browning index (BI) increased to 4.5, indicating that the untreated group caused enzymatic browning and tissue browning due to enhanced polyphenol oxidase (PPO) activity and cell damage. Among the FCCP treatments, FCCP-2 had the most significant preservation effect, with its L value maintained at 48.5 (7th day), a value approaching 0.5, and BI of 1.8. This shows that the coating effectively blocks oxygen contact, reduces PPO activity, and inhibits the damage of microbial metabolites to color.

[0114] like Figure 16As shown, the L value of the control group dropped sharply from the initial 85.0 to 62.3 on the 7th day, the a value increased from -1.5 to 3.2, and the BI increased to 5.1, indicating that the non-treated group had phenolic substance oxidation and browning due to enhanced PPO activity and cell damage. Among the FCCP series treatments, FCCP-2 had the best effect, with the L value maintained at 76.5 (on the 7th day), the a value approaching 0.5, and the BI being 2.1, while inhibiting the damage of microbial metabolites to the color. In the later stage of the FCCP-3 treatment, the L value decreased rapidly (68.4 on the 7th day), and the BI increased to 3.8, possibly because the high-concentration coating hindered gas exchange, triggering anaerobic respiration and membrane lipid peroxidation, which exacerbated browning. The results showed that FCCP-2 effectively maintained the color stability of lotus root slices by balancing air permeability.

[0115] As Figure 17 shown, the L value of the control group blueberries dropped from the initial 35.0 to 22.5 on the 7th day, the a value increased from -0.8 to 2.6, and the BI increased to 3.9. The retention rate of the L value of the CP-treated blueberries increased by 18.2% compared with the control group (28.0 on the 7th day), the a value only increased to 1.5, and the BI was 2.7. FCCP-2 had the best effect, with the L value maintained at 30.2 (on the 7th day), the a value approaching 0.3, and the BI being 1.5, effectively delaying the color change of blueberries.

[0116] Fresh-cut lettuce, lotus root, and blueberries on the 0th, 1st, 3rd, 5th, and 7th days were made into homogenates, and the SOD activity and MDA content of the fruits and vegetables were measured according to the steps in the SOD activity kit and MDA content kit instructions, and calculated.

[0117] As Figure 18 shown, the MDA content of the control group continued to increase with the storage time, indicating that the lipid peroxidation of cell membranes was exacerbated under natural storage conditions. Among them, the MDA accumulation rate of blueberries was the fastest, reaching 25.3 nmol / g on the 7th day, which might be related to the imbalance of reactive oxygen species (ROS) metabolism caused by its high respiratory intensity and strong epidermal permeability. The MDA content of the FCCP-2 treatment was the lowest. The preservative utilized the synergistic effect of the film-forming property and antioxidant components to block oxygen, inhibit microbial metabolism, effectively reduce ROS accumulation, thereby inhibiting the lipid peroxidation reaction and delaying the increase of MDA content.

[0118] As Figure 19As shown in the figure, the SOD activity of the control group continued to decline with the extension of storage time. Among them, the decline of blueberries was the largest, and the activity value decreased by 62.1% on the 7th day compared with the initial value (from 150 U / g to 57 U / g), indicating that the accumulation of reactive oxygen species (ROS) under natural storage conditions led to the decline of the enzyme system function. Taking lotus root slices as an example, after treatment with FCCP-2, the SOD activity still maintained 120 U / g on the 7th day (43.6% higher than that of the control group), indicating that the preservative can reduce the generation of ROS and delay the decline of enzyme activity. FCCP-2 treatment effectively maintained the SOD activity of the three kinds of fruits and vegetables by regulating the redox balance and gas exchange requirements, and its mechanism involved ROS scavenging and regulation of enzyme system stability.

[0119] Example 6. Analysis of the antioxidant activity of the composite preservative of moringa leaf flavonoids-chitosan

[0120] Ascorbic acid solutions with concentrations of 0, 1, 1.5, and 2 mg / mL were prepared respectively as the positive control VC group; flavonoid aqueous solutions with concentrations of 0, 1, 1.5, and 2 mg / mL were prepared as the flavonoid group; 1% chitosan solution, 10 mL of 10 mg MLF-1 + 1% chitosan solution, 10 mL of 15 mg MLF-1 + 1% chitosan solution, and 10 mL of 20 mg MLF-1 + 1% chitosan solution were prepared as the chitosan group.

[0121] Determine the antioxidant activity of different groups: Prepare 7 mmol / L ABTS solution. Accurately measure 5 mL of ABTS solution, add 5 mL of 2.45 mmol / L potassium persulfate solution, mix well and react in the dark for 12 h to obtain the ABTS working solution. Before measurement, take 1 mL of the ABTS working solution and gradually dilute it with absolute ethanol until the absorbance value at 734 nm is (0.7 ± 0.02) to obtain the ABTS ethanol solution for standby. Measure 2 mL of each sample solution in this example respectively, add 2 mL of the ABTS ethanol solution, mix well and react in the dark for 10 min, and measure its absorbance value A at 734 nm. Another 2 mL of pure water was taken, added 2 mL of the ABTS ethanol solution, mixed well and reacted in the dark for 10 min, and its absorbance value A0 was measured at 734 nm. The formula for calculating the ABTS free radical scavenging rate is:

[0122]

[0123] In the formula: A0: the absorbance measured by 2 mL of pure water + 2 mL of ABTS ethanol solution; A: the absorbance measured by 2 mL of sample solution + 2 mL of ABTS ethanol solution.

[0124] As Figure 20As shown in the figure, the scavenging rate is relatively high in the range of flavonoid concentration from 1 to 2 mg / mL, indicating that flavonoids have strong antioxidant ability. The phenolic hydroxyl structure of flavonoids can efficiently neutralize free radicals through hydrogen atom transfer (HAT) and single electron transfer (SET) mechanisms, and the increase in concentration can increase the number of active sites. At a mass concentration of 2 mg / mL, the scavenging rates of the flavonoid group, chitosan group, and positive control VC group for ABTS free radicals are 99.12%, 99.11%, and 100% respectively. It can be seen that both Moringa oleifera leaf flavonoids and the preservative have a high scavenging rate for free radicals. After adding Moringa oleifera leaf flavonoids to the chitosan group, the scavenging rate of the chitosan group for ABTS free radicals is significantly increased compared with that of the 1% chitosan solution. It shows that the composite preservative has a good scavenging effect on ABTS free radicals.

[0125] Take 2 mL of the sample solutions of each group in this example, and gradually add 2 mL of DPPH-ethanol solution with a concentration of 0.1 mmol / L to the reaction mixture. Mix well and react for 30 min under dark conditions. Measure the absorbance value of the reaction solution at a wavelength of 517 nm, and record it as A i ; Take another 2 mL of the sample solutions of each group in this example, add 2 mL of absolute ethanol, mix well and react for 30 min under dark conditions. The absorbance value measured at 517 nm is recorded as Ai j ; Take 2 mL of absolute ethanol and 2 mL of DPPH-ethanol solution with a concentration of 0.1 mmol / L, mix well and react for 30 min under dark conditions. The absorbance value measured at 517 nm is A0. The DPPH free radical scavenging rate is calculated by the following formula:

[0126]

[0127] In the formula: A i : The absorbance value after the reaction of 2 mL of the sample solution + 2 mL of DPPH-ethanol solution at a wavelength of 517 nm; Ai: The absorbance value measured after the reaction of 2 mL of the sample solution + 2 mL of absolute ethanol solution at a wavelength of 517 nm; A0: The absorbance value after the reaction of 2 mL of absolute ethanol + 2 mL of DPPH-ethanol solution at a wavelength of 517 nm.

[0128] As Figure 21 shown, at a mass concentration of 2 mg / mL, the scavenging rates of the flavonoid group, chitosan group, and positive control VC group for DPPH free radicals are 95.91%, 95.72%, and 94.53% respectively. It can be seen that both Moringa oleifera leaf flavonoids and the preservative have a relatively high scavenging rate for DPPH free radicals. With the addition of Moringa oleifera leaf flavonoids in the chitosan group, compared with the 1% chitosan solution, the DPPH scavenging rate increases from 18.81% to 94.53%, and the scavenging rate for DPPH increases significantly. The combination of the two is beneficial to expanding the antioxidant effect.

[0129] Take 1.0 mL of the sample solutions of each group in this example in a centrifuge tube, add 2.5 mL of phosphate buffer solution with pH 6.6 and 2.5 mL of 1% potassium ferricyanide solution, mix well, and water bath at 50 °C for 20 min. Take out and add 2.5 mL of 10% trichloroacetic acid solution, and centrifuge at 4000 r / min for 10 min in a centrifuge. Take 1.0 mL of the supernatant in a test tube, add 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride solution. Measure the absorbance value of the mixed solution at 700 nm.

[0130] As Figure 22 shown, the total reducing power of the VC group and the flavonoid group is positively correlated with the content. The higher the content of flavonoids, the higher the total reducing ability. The total reducing ability of the chitosan group is basically equivalent to that of the positive control VC group. After adding flavonoids to the chitosan group, with the increase of the flavonoid mass concentration, the total reducing ability first increases and then decreases, indicating that the addition of flavonoids improves the antioxidant ability of the composite preservative, and the subsequent decrease is due to the relatively high concentration of flavonoids. Due to the long standing time, the effective antioxidant groups formed by the combination of flavonoids and chitosan are unstable, resulting in a decrease in the total antioxidant ability of the composite solution and affecting the ability of the composite preservative to capture free radicals.

[0131] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art of the present invention can make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will all fall within the protection scope of the present invention.

[0132] The literature cited in the present invention includes:

[0133] [1] Wang Y, Duan X, Ren G, et al. Comparative study on the flavonoids extraction rate and antioxidant activity of onions treated by three different drying methods[J]. Drying Technology, 2019, 37(2): 245 - 252.

Claims

1. A method for preparing a Moringa leaf flavonoids-chitosan composite fruit and vegetable preservative, characterized in that: The steps include: Step 1, weighing Moringa leaf powder, extracting flavonoids from Moringa leaves by ultrasound, filtering, centrifuging, and freeze-drying to obtain a crude extract of Moringa leaf flavonoids; Step 2, preparing a solution from the crude extract of Moringa leaf flavonoids, and then separating and purifying the Moringa leaf flavonoids to obtain Moringa leaf flavonoids; Step 3: Add Moringa leaf flavonoids to the chitosan solution, stir thoroughly and then perform ultrasound to obtain a Moringa leaf flavonoids-chitosan composite preservative.

2. The method for preparing the Moringa leaf flavonoids-chitosan composite fruit and vegetable preservative according to claim 1, characterized in that: The extract in step 1 is a 50%-55% ethanol solution.

3. The preparation method of the Moringa leaf flavonoids-chitosan composite fruit and vegetable preservative according to claim 1, characterized in that: In step 1, flavonoids in Moringa leaves are extracted at a solid-liquid ratio of 1:

70.

4. The method for preparing the Moringa leaf flavonoids-chitosan composite fruit and vegetable preservative according to claim 1, characterized in that: The conditions for ultrasonic-assisted extraction of flavonoids from Moringa leaves in step 1 are: at (60-62)°C, 100W ultrasound for (40-50) min.

5. The method for preparing the Moringa leaf flavonoids-chitosan composite fruit and vegetable preservative according to claim 1, 2, 3 or 4, characterized in that: The step 2 is to separate and purify the Moringa leaf flavonoids, specifically: to separate and purify the Moringa leaf flavonoids using a macroporous resin.

6. The method for preparing the Moringa leaf flavonoids-chitosan composite fruit and vegetable preservative according to claim 5, characterized in that: In step 2, the Moringa leaf flavonoids are separated and purified with a loading flow rate of (1-2) mL / min and an elution flow rate of (1-2) mL / min.

7. The method for preparing the Moringa leaf flavonoids-chitosan composite fruit and vegetable preservative according to claim 1, 2, 3 or 4, characterized in that: The concentration of the chitosan solution in step 3 is 1%.

8. The method for preparing the Moringa leaf flavonoids-chitosan composite fruit and vegetable preservative according to claim 7, characterized in that: The concentration of Moringa leaf flavonoids in the Moringa leaf flavonoids-chitosan composite preservative is (1-2) mg / mL.

9. A Moringa leaf flavonoids-chitosan composite preservative, characterized in that: The preservative is prepared by the preparation method of the Moringa leaf flavonoids-chitosan composite fruit and vegetable preservative described in any one of items 1-8 above.

10. Use of the Moringa leaf flavonoids-chitosan composite preservative according to claim 9 in preserving fruits and vegetables.

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