Macromolecular antioxidant as well as preparation method and application thereof

Abstract

The invention discloses a macromolecular antioxidant as well as a preparation method and application thereof, and belongs to the technical field of plant active ingredient extraction. According to the method, kiwi fruit peel is used as a raw material, a pectin macromolecular antioxidant rich in combined polyphenol is extracted through a dilute alkali heating method, the molecular weight of the obtained extract is 56.51 kDa, 17 phenolic acid compounds such as gallic acid, ferulic acid, caffeic acid and salicylic acid are identified, and the content of the phenolic acid compounds is more than or equal to 1%. 18 flavonoid compounds such as catechin, epigallocatechin and quercitrin, 4 coumarin compounds and 3 other phenolic substances have high DPPH free radical scavenging capacity, ABTS free radical scavenging capacity and Fe < 3 + > reducing capacity, and high-value utilization of waste plant resources in the kiwi fruit processing industry is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant active ingredient extraction. Specifically, it relates to a macromolecular antioxidant extracted from fruit and vegetable processing by-products, its preparation method and application. Background Art

[0002] Due to its excellent protective effects against oxidation deterioration in foods and drugs and against pathological processes caused by oxidative stress in the body, antioxidant research has always been a research hotspot. In food systems, the application of antioxidants can delay lipid peroxidation and prevent the formation of secondary lipid peroxidation products, thus helping to maintain the flavor, nutrition and sensory quality of foods during storage. In addition, antioxidants can also protect the human body from the effects of free radicals and reactive oxygen species. They can delay the progression of many chronic diseases as well as lipid peroxidation. Therefore, antioxidants have become an indispensable class of food additives. In recent years, there has been a great deal of interest in identifying natural and safe sources of food antioxidants and in searching for natural antioxidants, especially those of plant origin. Fruits and vegetables have been proven to be the best sources of natural antioxidants due to the discovery of antioxidant compounds such as polyphenols, flavonoids and carotenoids. In addition, there are also some natural macromolecular active ingredients extracted from plants, including proteins, polysaccharides and macromolecular polymers, which can also be used as antioxidants. Some small molecule antioxidants can also covalently bind to biological macromolecules to form macromolecular antioxidants, thereby enhancing their antioxidant activity.

[0003] Pectin is the main component of plant cell walls. It is a complex acidic polysaccharide with various nutritional functional properties such as thickening, gelling, emulsifying, and antioxidant properties. It has been widely used as a thickening agent, gelling agent, emulsifying agent, stabilizer, and drug carrier in the food industry. In addition, pectin exhibits potential nutritional or therapeutic effects in aspects such as chronic metabolic syndrome, intestinal diseases, regulation of intestinal probiotics, and immune regulation. Phenolic compounds in food are one of the main secondary metabolites in plants. As an antioxidant, they can protect food from oxidative rancidity at low concentrations, and their reduction in the risk of cardiovascular diseases and some cognitive diseases is related. Especially insoluble phenolic substances not only show relatively higher contents and richer varieties compared with soluble phenolic substances in plants, but also exhibit strong biological activities, including antioxidant, anti-inflammatory, anti-obesity, and anti-diabetic effects, as well as beneficial effects on central nervous system diseases, etc. Since insoluble phenolic substances are usually bound to plant cell wall substances such as pectin through covalent or non-covalent interactions, most current studies focus on the composition and biological functions of soluble phenolic substances, while the research on insoluble phenolic substances is relatively less. In addition, existing studies usually separately study pectin and insoluble phenolic substances as antioxidants, often ignoring the potential of the natural complex formed by the covalent binding of insoluble phenolic substances with macromolecular substances such as pectin as a new type of dietary macromolecular antioxidant. Therefore, the present invention hereby provides a macromolecular antioxidant and its preparation method. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the first object of the present invention is to provide a preparation method of a macromolecular antioxidant extracted from kiwifruit peel, thereby obtaining a natural complex in which phenolic substances are covalently bound to macromolecular substances such as pectin.

[0005] In order to achieve the above technical object, on the basis of a large number of previous experiments, the present inventors finally selected kiwifruit peel, a by-product of kiwifruit processing, as the raw material. By continuously optimizing the extraction and separation processes and parameters, the following technical solution was finally obtained:

[0006] A preparation method of a macromolecular antioxidant, characterized in that the method comprises the following steps:

[0007] (1) Pretreatment: Freeze-dry kiwifruit peel at -45 to -55 °C and a vacuum degree of 10 to 20 Pa for 40 to 50 h, pulverize and sieve to obtain kiwifruit peel powder;

[0008] (2) Preparation of macromolecular antioxidant:

[0009] S1. Weigh the kiwifruit peel powder obtained in step (1), mix it with 0.008-0.01 mol / L NaOH solution at a solid-liquid ratio of 1: (20-40), adjust the pH value of the mixture to 10-12 using NaOH solution, and then place it in a water bath shaker at 70-90°C for 50-70 min;

[0010] S2. The extract obtained in step S1 is centrifuged for 15 to 25 minutes at a centrifugal speed of 7000 to 9000 rpm, and then filtered, and the filtrate is fully mixed with anhydrous ethanol in a volume ratio of 1: (1 to 5), and suspended matter appears in the mixed solution after standing;

[0011] S3. The suspended matter in step S2 was collected, washed repeatedly with anhydrous ethanol, reconstituted with pure water, centrifuged at 7000-9000 rpm for 15-25 min, filtered, and the filtrate was placed in a dialysis bag with a molecular weight of 13000-15000 Da for dialysis, and the dialysis time was 24-72 h;

[0012] (3) Drying: The retentate obtained in the dialysis bag of step S3 is subjected to rotary evaporation and freeze-dried at -45 to -55°C and a vacuum degree of 10 to 20 Pa for 40 to 50 hours to obtain a macromolecular antioxidant.

[0013] Further preferably, in the method for preparing the macromolecular antioxidant as described above, the freeze-drying temperature in step (1) is -45 to -50°C, the vacuum degree is 10 to 15 Pa, and the time is 45 to 50 hours.

[0014] Further preferably, in the method for preparing the macromolecular antioxidant as described above, the material-liquid ratio in step S1 is 1:(20-30).

[0015] Further preferably, in the method for preparing the macromolecular antioxidant as described above, the pH value in step S1 is 11-12.

[0016] Further preferably, in the method for preparing the macromolecular antioxidant as described above, the temperature of the water bath in step S1 is 80-85° C., and the extraction time is 60-70 min.

[0017] Further preferably, in the method for preparing the macromolecular antioxidant as described above, the volume ratio of the filtrate to anhydrous ethanol in step S2 is 1:(2-3).

[0018] Further preferably, in the method for preparing the macromolecular antioxidant as described above, in step S3, the obtained filtrate is placed in a dialysis bag with a molecular weight of 14000Da for dialysis, and the dialysis time is 36 to 54 hours, during which the water is changed every 4 hours.

[0019] Further preferably, in the preparation method of the macromolecular antioxidant as described above, in step (3), the temperature of freeze-drying is -45 to -50 °C, the vacuum degree is 10 to 15 Pa, and the time is 45 to 50 h.

[0020] The second object of the present invention is to provide the macromolecular antioxidant prepared by the above-mentioned extraction and preparation method.

[0021] The third object of the present invention is to provide the application of the above-mentioned macromolecular antioxidant in the antioxidant of food or medicine.

[0022] Compared with the prior art, the present invention prepares a macromolecular antioxidant - a natural complex in which phenolic substances are covalently bonded to macromolecular substances such as pectin. The preparation method and application thereof have the following advantages and remarkable progress:

[0023] (1) The present invention extracts the macromolecular antioxidant by the dilute alkali heating method. Under the conditions of a material-liquid ratio of 1:20, a pH value of the mixed solution of 11.5, an extraction temperature of 80 °C, and an extraction time of 60 min, the yield and antioxidant activity of the macromolecular antioxidant are the highest, and the molecular weight is 56.51 kDa.

[0024] (2) Compared with the acid method and the enzyme method extraction, the macromolecular antioxidant extracted by the dilute alkali method of the present invention has the advantages of high extraction rate (19.13%), high polyphenol content (31.06 mg GAE / g DW), and high RG-I structure content (75.1%).

[0025] (3) The macromolecular antioxidant prepared by the present invention has high DPPH free radical scavenging ability, ABTS free radical scavenging ability and Fe 3+ reducing ability. In addition, 17 phenolic acid compounds such as gallic acid, ferulic acid, caffeic acid and salicylic acid, 18 flavonoid compounds such as catechin, epigallocatechin and quercetin glycoside, 4 coumarin compounds and 3 other phenolic substances are identified therein.

[0026] (4) The implementation of the present invention not only effectively realizes the high-value utilization of the plant resources of the waste in the kiwifruit processing industry, but also provides a new idea for the development and utilization of macromolecular antioxidants. Description of the Drawings

[0027] Figure 1 : Scanning electron microscope images of macromolecular antioxidants extracted by the alkali method, acid method and enzyme method;

[0028] Figure 2 : Atomic force microscope images of macromolecular antioxidants extracted by the alkali method, acid method and enzyme method and commercially available pectin;

[0029] Figure 3: Fourier transform infrared spectra of macromolecular antioxidants extracted by alkali method, acid method and enzymatic method, and commercially available pectin

[0030] Figure 4 : Comparison chart of DPPH free radical scavenging activities of macromolecular antioxidants extracted by alkali method, acid method and enzymatic method, and commercially available pectin

[0031] Figure 5 : Comparison chart of ABTS free radical scavenging activities of macromolecular antioxidants extracted by alkali method, acid method and enzymatic method, and commercially available pectin

[0032] Figure 6 : Fe of macromolecular antioxidants extracted by alkali method, acid method and enzymatic method, and commercially available pectin 3+ Reductive ability comparison chart. Detailed implementation method

[0033] The following examples are used to further describe the implementation process and beneficial effects of the method of the present invention. The test examples are only for illustrative purposes and do not limit the protection scope of the present invention. At the same time, the obvious changes made by those of ordinary skill in the art according to the examples are also included in the scope of the present invention.

[0034] In the following examples, the macromolecular antioxidant extracted by the alkali method is abbreviated as AK; the macromolecular antioxidant extracted by the citric acid method is abbreviated as CA; the macromolecular antioxidant extracted by the enzymatic method is abbreviated as EA; and the commercially available pectin is abbreviated as CP. Each experiment was repeated three times.

[0035] Example 1. Extraction of macromolecular antioxidants

[0036] 1. Raw material pretreatment

[0037] Wash the kiwifruit peel clean and perform pre-cooling treatment, and dry it in a vacuum freeze dryer for 48 h. The dried kiwifruit peel is crushed and passed through an 80-mesh sieve, and stored in a dry place for later use.

[0038] 2. Calculation of the yield of macromolecular antioxidants

[0039] In this experiment, the yield of the macromolecular antioxidant is the proportion of the mass of the extracted macromolecular antioxidant to the kiwifruit peel powder. The calculation formula is as follows: Y = (Wt / W0) * 100%

[0040] Where: Y is the yield of the macromolecular antioxidant; Wt is the mass (g) of the extracted macromolecular antioxidant; W0 is the mass (g) of the weighed kiwifruit peel powder.

[0041] 3. Extraction of macromolecular antioxidants by citric acid method, alkali method and enzymatic method

[0042] (1) Extraction by citric acid method

[0043] Accurately weigh 10 g of pretreated kiwifruit peel powder, mix it with citric acid solution with a pH of 1.4 at a solid-liquid mass ratio of 1:30, and then place it in a water bath shaker at 85 °C for continuous heating and stirring for 70 min. After cooling to room temperature, immediately centrifuge (8000 rpm, 20 min), filter by suction, and collect the filtrate. Then mix the filtrate with three volumes of absolute ethanol. After standing at 4 °C for 8 h, collect the precipitate. The precipitate is repeatedly washed three times with absolute ethanol and 95% ethanol and then redissolved. After the redissolved solution is centrifuged again (8000 rpm, 20 min), dialysis is carried out using a dialysis bag with a molecular weight cut-off of 14000 Da for 48 h, and the water is changed every 4 h during this period. The dialyzed solution is filtered by suction again, the filtrate is collected and freeze-dried using a vacuum freeze dryer at a temperature of -46 °C and a vacuum degree of 15 Pa for 48 h to obtain the macromolecular antioxidant extracted by the citric acid method. Use an electronic balance to weigh the macromolecular antioxidant extracted by the citric acid method. It can be calculated that the yield of the macromolecular antioxidant obtained by the citric acid method is 11.37%.

[0044] (2) Extraction by alkali method

[0045] Other steps are the same as (1). For extraction by the alkali method, mix it with 0.01 mol / L NaOH solution at a solid-liquid mass ratio of 1:20, and adjust the pH value of the mixture to 11.5 using 0.1 mol / L NaOH solution, and then place it in a water bath shaker at 80 °C for continuous heating and stirring for 60 min. It can be calculated that the yield of the macromolecular antioxidant obtained by the alkali method is 19.13%.

[0046] (3) Extraction by enzyme method

[0047] Other steps are the same as (1). For extraction by the enzyme method, mix it with distilled water at a solid-liquid mass ratio of 1:20, and add the enzyme preparation Celluclast 1.5L at a dose of 100 μL / g of kiwifruit peel powder, and then place it in a water bath shaker at 50 °C for continuous heating and stirring for 24 h. It can be calculated that the yield of the macromolecular antioxidant obtained by the enzyme method is 12.8%.

[0048] Example 2: Physicochemical property analysis of macromolecular antioxidant

[0049] 1. Physicochemical indexes

[0050] (1) Determination of galacturonic acid content

[0051] The m-hydroxybiphenyl method was used to determine the galacturonic acid content of macromolecular antioxidants. Preparation of galacturonic acid standard solution: Accurately weigh 100 mg of galacturonic acid standard and make up the volume to 100 mL with distilled water. Dilute the galacturonic acid solution to 20, 40, 60, 80, and 100 μg / mL respectively to obtain the galacturonic acid standard solution. Drawing of standard curve: Take 6 test tubes and add 1 mL of 0, 20, 40, 60, 80, and 100 μg / mL galacturonic acid standard solutions respectively. Then add 6 mL of 0.0125 mol / L sulfuric acid-sodium tetraborate solution to each. Immediately place them in an ice-water bath and cool to room temperature. After vortexing, heat them in a boiling water bath for 10 min and then cool them in an ice-water bath again. Finally, add 0.10 mL of 0.15% m-phenylphenol solution (m-phenylphenol dissolved in 0.50% sodium hydroxide solution) to each test tube and shake well. After standing for 15 min, measure the absorbance at 520 nm with a spectrophotometer and draw the standard curve.

[0052] Determination of sample: Take 1 mL of 0.10% macromolecular antioxidant solution (w / w), add 11.50 mL of distilled water to dilute it to an 80 μg / mL macromolecular antioxidant solution. Measure its absorbance using the above standard curve drawing method. The reaction reagent added for the blank is distilled water. Calculate the galacturonic acid content of the macromolecular antioxidant through the standard curve.

[0053] (2) Determination of total sugar content

[0054] The phenol-sulfuric acid method was used to determine the total sugar content in macromolecular antioxidants. Preparation of glucose standard solution: Accurately weigh 20 mg of dried glucose standard, dissolve it in distilled water and make up the volume to 100 mL to obtain a 0.2 mg / mL glucose standard solution. Respectively pipette 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard solution into 6 test tubes and supplement distilled water to 2.0 mL. Then add 50 μL of 80% phenol solution to them, shake well, and then add 2.5 mL of concentrated sulfuric acid, shake and mix well. After standing at room temperature for 25 min, measure the absorbance value at a wavelength of 490 nm, and then draw the glucose standard curve, where the abscissa is the glucose mass concentration in each test tube and the ordinate is the absorbance value.

[0055] Determination of sample: Accurately prepare a 100 μg / mL macromolecular antioxidant solution, pipette 1 mL into a test tube, and determine the total sugar content according to the above standard curve drawing method. Substitute the obtained absorbance value into the glucose standard curve to calculate the total sugar content in the macromolecular antioxidant sample.

[0056] (3) Determination of bound polyphenol content

[0057] Extraction of bound polyphenols in macromolecular antioxidants: The bound polyphenols in AK, CA, EA, and CP were extracted by alkaline hydrolysis. Exactly 200 mg of the macromolecular antioxidant sample was weighed and placed in a dark screw-cap bottle. NaOH solution (8 M, 5 mL) was added, and then N2 was injected into the remaining space of the container to exclude air. The mixture was slowly stirred for 4 h with a rotor on a magnetic stirrer at room temperature and in the absence of light. After hydrolysis was completed, the pH of the mixture was adjusted to 2.0 with 6 M HCl solution, and then centrifuged at 5000 rpm for 10 min. The supernatant was extracted 5 times with ethyl acetate (1:1, v / v). The combined ethyl acetate extracts were concentrated in vacuo at 45 °C and dried with N2. The dried extract was redissolved in 1 mL of 60% methanol for determination of total phenol content, or redissolved in 1 mL of pure methanol for quantitative analysis through a 0.22-μm PTFE membrane filter.

[0058] Determination of total phenol content: The Folin-Ciocalteu assay was used to estimate the total phenol content in the macromolecular antioxidant samples. Briefly, 150 μL of the polyphenol extract was mixed with 3 mL of 7.5% Na2CO3 (w / v) and 250 μL of Folin-Ciocalteu reagent. After incubation for 1 h, the absorbance of the solution was measured at 765 nm using a microplate reader and substituted into the standard curve to calculate the total phenol content. Standard curve preparation: Exactly 20 mg of gallic acid was dissolved in distilled water and made up to a volume of 100 mL in a volumetric flask to prepare a standard stock solution of 200 μg / mL. 0, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the gallic acid standard stock solution were respectively pipetted into 10-mL volumetric flasks and made up to volume to prepare standard solutions of different concentrations. The absorbance values were measured by the above method, and a standard curve was plotted with the absorbance value as the ordinate and the concentration of the gallic acid (GAE) standard solution as the abscissa. The total phenol content was expressed as milligrams of gallic acid equivalents per gram of dry weight of the macromolecular antioxidant (mg GAE / g DW).

[0059] (4) Results

[0060] The specific data of the physical and chemical indexes and their contents of AK, CA, EA, and CP are shown in Table 1. The macromolecular antioxidants extracted by the alkaline method can reach a relatively ideal purity. In addition, compared with commercial pectin, the macromolecular antioxidants extracted from kiwifruit peel have significantly higher bound polyphenol contents, among which the content in the macromolecular antioxidants extracted by the alkaline method is the highest, more than 7 times higher than that of commercial pectin.

[0061] Table 1 Physical and chemical indexes and contents of AK, CA, EA, and CP

[0062]

[0063] 2. Determination of monosaccharide composition

[0064] The monosaccharide compositions of macromolecular antioxidants and commercial pectin macromolecules were determined by high performance liquid chromatography. Sample hydrolysis and derivatization: Accurately weigh 4 mg of macromolecular antioxidant and commercial pectin macromolecule samples into clean hydrolysis tubes. After adding 2 mL of ultrapure water to fully dissolve, add 2 mL of 4 mol / L trifluoroacetic acid solution, and hydrolyze at 110 °C for 6 h. Pass nitrogen and blow dry. Add methanol for cleaning and then blow dry again. Repeat the methanol cleaning 3 - 4 times. Subsequently, add 2 mL of ultrapure water to the sample (monosaccharides after acid hydrolysis) to fully dissolve it, pass through a 0.22 μm ultrafiltration membrane, and collect the filtrate. Take 400 μL of the above solution into a 5 mL centrifuge tube, add 450 μL of 0.3 mol / L NaOH solution and 450 μL of 0.5 mol / L PMP methanol solution, vortex and mix well, then place it in a 70 °C water bath and heat for 30 min. After cooling to room temperature, add 450 μL of 0.30 mol / L HCl solution to adjust the pH to neutral, add 1 mL of chloroform and extract 3 times repeatedly. Take the upper layer solution, pass through a 0.22 μm ultrafiltration membrane and place it in a liquid phase injection vial. Preparation of standard solution: Take 10 mg each of fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man) and galacturonic acid (GalA), dissolve with ultrapure water and make up the volume to 10 mL with a volumetric flask to prepare a 1 mg / mL standard solution. And dilute the above mixed standard stock solution to 20, 40, 80, 160, 240 and 320 μg / mL, and the derivatization steps are the same as the above method. Chromatographic conditions: Mobile phase, phase A: 15% (v / v) acetonitrile + 0.05 mol / L phosphate buffer solution (potassium dihydrogen phosphate + sodium hydroxide, pH 6.9), phase B: 40% (v / v) acetonitrile + 0.05 mol / L phosphate buffer solution (potassium dihydrogen phosphate + sodium hydroxide, pH 6.9); Chromatographic column: ZORBAX Eclipse XDB-C18 separation column (4.6×250 mm, 5 μm); Column temperature: 25 °C; Detector: Diode array detector (PDA), detection wavelength: 250 nm; Injection volume: 10 μL. Calculate the monosaccharide composition in the macromolecular antioxidant sample according to the peak elution time and peak area.

[0065] The monosaccharide compositions of the macromolecular antioxidants extracted from kiwifruit peel by three methods and the commercial pectin macromolecules are shown in Table 2. Compared with the commercial pectin macromolecules, the proportion of GalA in all the macromolecular antioxidants extracted from kiwifruit peel is lower, while the proportions of the neutral sugars Ara and Gal are higher. The neutral sugars Ara and Gal are the main components of the side chains of pectin macromolecules. Therefore, the macromolecular antioxidants rich in the rhamnogalacturonan-I (RG-I) region are produced by different extraction methods. This may be related to the raw material source. Among them, the proportions of Gal and Ara in AK are the highest, indicating that the side chains in the RG-I region are effectively released from the cell wall under alkaline conditions and retained. This structure not only endows AK with high biological activities such as antioxidant activity, but also provides more binding sites for small molecules such as polyphenols.

[0066] Table 2 Monosaccharide compositions of AK, CA, EA and CP

[0067]

[0068] 3. Determination of molecular weight and its distribution

[0069] The molecular weight of the macromolecular antioxidant was determined by high performance gel permeation chromatography equipped with a differential refractive index detector (HPGPC-RID). The differential detector was Optilab T-rEX, and the laser light scattering detector was DAWN HELEOSⅡ. Exactly weigh 20 mg of the macromolecular antioxidant sample powder, dissolve it with 10 mL of 0.1 mol / L sodium nitrate solution, then centrifuge it at 14000 rpm for 10 min, and take the supernatant to pass through a 0.22 μm filter membrane. Add the filtered clear liquid to an Ohpak SB-804HQ chromatographic column and elute it with 0.1 mol / L sodium nitrate solution at a flow rate of 0.4 mL / min; column temperature: 25 °C; injection volume: 100 μL; the refractive index increment (dn / dc) of pectin was set to 0.147 cm 3 / g. Use dextran standards with different molecular weights to obtain a linear regression equation and calculate the molecular weight of the macromolecular antioxidant sample.

[0070] The average molecular weights of AK, CA, EA and CP are shown in Table 3. Compared with the commercial pectin macromolecules, the average molecular weights of all the macromolecular antioxidants are reduced to varying degrees, indicating that different extraction methods can all damage the main chain structure of macromolecular substances, which may lead to the reduction of the average molecular weight; in addition, the macromolecular antioxidant extracted by the alkaline method shows the lowest average molecular weight, indicating that its main chain structure is more damaged and more RG-I regions are retained, which not only endows it with good solubility and stability, but also may show higher biological activities such as antioxidant activity.

[0071] Table 3 Average molecular weights of AK, CA, EA and CP

[0072]

[0073] 4. Microstructure Analysis

[0074] (1) Scanning Electron Microscope Image Analysis

[0075] The surface morphology of the dried macromolecular antioxidant was observed using a scanning electron microscope. The macromolecular antioxidant powder was fixed on the sample stage with conductive glue and sputter-coated with gold before being observed on the instrument. The images were taken at an acceleration voltage of 10 kV with magnification factors of 200 and 1000.

[0076] It can be seen from Figure 1 that there are significant differences in the surface morphology of the macromolecular antioxidants obtained by the acid method, alkali method, and enzymatic method. Compared with the acid method and enzymatic method, the structure of the macromolecular antioxidant extracted by the alkali method is looser and rougher. This loose and rough surface structure may endow it with stronger adsorption ability and higher thickening, gelling, and stability, thus enabling more effective control of the release of nutrients.

[0077] (2) Atomic Force Microscope Image Analysis

[0078] The nanostructure of the macromolecular antioxidant was observed using an atomic force microscope. The macromolecular antioxidants extracted by the three methods and commercial pectin macromolecules were dissolved in distilled water, and the solution was stirred at 80 °C for 48 h to promote dissolution, and then diluted to 10 μg / mL with ultrapure water. Then, 5 μL of the solution was dropped onto the mica substrate, and the substrate was air-dried in a dust-free hood before use. At room temperature, the mica substrate was observed using an atomic force microscope in tapping mode. The probe was a classic silicon cantilever (Si3N4) with a spring constant of 0.2 N / m and a resonance frequency of 10 kHz. The scanning area was set to 1×1 μm, and the scanning resolution was 256×256 points.

[0079] It can be seen from Figure 2 that compared with the macromolecular antioxidants extracted by the acid method and enzymatic method and commercial pectin macromolecules, the macromolecular antioxidant extracted by the alkali method exhibits a highly branched molecular morphology and a complex cross-linked network structure. This not only reflects that the macromolecular antioxidant extracted by the alkali method is rich in the RG-I region with high biological activity but also may have higher stability and gelling ability.

[0080] 5. Fourier Transform Infrared Spectroscopy Analysis

[0081] The main functional groups of macromolecular antioxidants and commercial pectin macromolecules were detected using Fourier transform infrared spectroscopy. The macromolecular antioxidant powder was mixed evenly with potassium bromide at a ratio of 1:100, and then the mixture was ground evenly in a dry agate mortar and pressed into a thin slice using a tablet press. The Fourier transform infrared spectrometer was used to scan the infrared spectrum, and within the scanning range of 500 cm -1 -4000 cm -1 , 32 scans were performed at a resolution of 4 cm -1 . The obtained spectrum was analyzed and processed using Omnic 8.2 software.

[0082] As can be seen from Figure 3 , the macromolecular antioxidant exhibited characteristic absorption peaks similar to those of commercial pectin macromolecules, indicating that the different extraction methods did not change the main structure of the macromolecular antioxidant. The stretching vibrations of the phenolic hydroxyl group of phenolic substances and the carboxyl group in phenolic acid compounds were observed near multiple absorption peaks such as 1520, 1690, 1625, and 2932 cm-1. In addition, the macromolecular antioxidant extracted by the alkali method showed stronger absorption peaks at 1010 cm -1 and 891 cm-1, which further verified that the RG-I region and its side chains were better protected during the alkali extraction process.

[0083] Example 3: Determination of the antioxidant activity of macromolecular antioxidants and qualitative analysis of the bound polyphenols therein

[0084] 1. Determination of antioxidant activity

[0085] (1) DPPH radical scavenging activity

[0086] The DPPH radical scavenging activities of macromolecular antioxidants and commercial pectin macromolecules extracted by different methods were compared. 1 mL of sample solutions with different concentrations (0.2 - 1.0 mg / mL) was mixed with 2 mL of freshly prepared DPPH (0.2 mM) ethanol solution. The mixed solution was shaken vigorously and incubated in the dark at room temperature for 1 h, and then the absorbance of the reaction solution was immediately measured at a wavelength of 517 nm against a blank control. The calculation formula for the DPPH radical scavenging activity is as follows:

[0087]

[0088] where: A0 is the absorbance of methanol and DPPH solution, A1 is the absorbance of pectin solution and DPPH solution, and A2 is the absorbance of methanol and pectin solution.

[0089] As can be seen from Figure 4It can be seen that the DPPH radical scavenging activity of all samples increases with the increase in pectin concentration. The DPPH radical scavenging activity of macromolecular antioxidants is much higher than that of commercial pectin macromolecules; at the same concentration gradient, the DPPH radical scavenging activity of macromolecular antioxidants extracted by the alkali method is the highest, reaching more than 6 times that of commercial pectin macromolecules. This is not only related to the rich RG-I domain of macromolecular antioxidants extracted by the alkali method, but also may be closely related to the types and contents of bound polyphenols therein.

[0090] (2) ABTS radical scavenging activity

[0091] Compare the ABTS radical scavenging activities of macromolecular antioxidants and commercial pectin macromolecules extracted by different extraction methods. Mix 5 mL of 7 mM ABTS diammonium salt with 5 mL of 2.5 mM potassium persulfate, and then store it in the dark at room temperature for 24 h to obtain the ABTS stock solution. Dilute the ABTS stock solution with phosphate buffer (10 mmol / L, pH = 7.4). The diluted solution with an absorbance between 0.7 ± 0.02 at a wavelength of 734 nm can be used for the experiment. Mix 150 μL of the diluted solution with 50 μL of sample solutions at different concentrations (0.2 - 1.0 mg / mL), and react at room temperature for 10 min.

[0092] Measure the concentration of the mixture at a wavelength of 734 nm. The calculation formula for the ABTS radical scavenging effect is as follows:

[0093]

[0094] Where: A0 is the absorbance of distilled water and the ABTS solution, A1 is the absorbance of the pectin solution and the ABTS solution, and A2 is the absorbance of distilled water and the pectin solution.

[0095] It can be seen from Figure 5 that the ABTS radical scavenging activity of all samples increases with the increase in pectin concentration. The ABTS radical scavenging activity of macromolecular antioxidants is much higher than that of commercial pectin macromolecules; at the same concentration gradient, the ABTS radical scavenging activity of macromolecular antioxidants extracted by the alkali method is the highest, reaching more than 3 times that of commercial pectin macromolecules.

[0096] (3) Fe 3+ Reducing power

[0097] Compare the Fe 3+ reducing power of macromolecular antioxidants and commercial pectin macromolecules extracted by different extraction methods. Fe 3 +The working solution for the determination of reducing ability (FRAP) was prepared by mixing 0.3 mol / L acetate buffer (pH 3.6), 0.01 mol / L solution of tripyridyltriazine (prepared with 0.04 mol / L hydrochloric acid), and 0.02 mol / L ferric chloride solution in a ratio of 10:1:1. It was preheated in a 37 °C water bath for 30 min before use. 20 μL of the sample solution (10 mg / mL) was evenly mixed with 180 μL of the FRAP reagent, incubated at 37 °C for 5 min, and then the absorbance was measured at a wavelength of 593 nm. Substitute into the standard curve to calculate the Fe 3+ reducing ability of pectin. The Fe 3+ reducing ability of different samples was calculated as μmol Fe 2+ / g of FeSO4 equivalent in the sample.

[0098] It can be seen from Figure 6 that the Fe 3+ reducing ability of the macromolecular antioxidant is much higher than that of the commercial pectin macromolecule; at a concentration of 10 mg / mL, the Fe 3+ reducing ability of the macromolecular antioxidant extracted by the alkaline method is the highest, which can reach more than 100 times that of the commercial pectin and more than 3 times that of the other two macromolecular antioxidants obtained by extraction. This indicates that the rich RG-I domain, low molecular weight, and high bound polyphenol content of the macromolecular antioxidant extracted by the alkaline method may endow it with more reducing and non-reducing ends, more opportunities to contact with iron ions, and thus exhibit stronger reducing ability.

[0099] 2. Qualitative analysis of bound polyphenols in macromolecular antioxidants

[0100] Non-targeted metabolomics was used to qualitatively analyze the bound polyphenolic substances in macromolecular antioxidants. Characterization was performed by connecting an Agilent 1290 UPLC system to an Agilent 6538 hybrid Q-TOF mass spectrometer equipped with an ESI source. UPLC separation was carried out using an Agilent Eclipse XDB-C18 column (2.1 mm × 100 mm, 1.8 μm). The mobile phases were acidified water (0.1% formic acid) and acidified acetonitrile (0.1% formic acid) (eluent B). The gradient elution program was as follows: 5–5%, 0–1.5 min B; 5–60%, 1.5–15 min B; 60–100%, 15–25 min B; 100–100%, 25–30 min B; 100–5%, 30–35 min B. The injection volume of the sample was 3 μL, the column chamber temperature was controlled at 35 °C, and the elution flow rate was maintained at 0.4 mL / min. Polyphenol data were collected at 245, 280, 320, and 350 nm. AccurateMass QTOF was operated in the negative ion mode (ESI), and mass spectra were obtained by scanning the mass range from 50 to 1500 in the MS / MS mode. Nitrogen (N2) was used as the collision, nebulizer, and drying gas, with a temperature of 250 °C, a flow rate of 8 L / min, and a pressure of 45 psi. The source parameters of the capillary voltage, fragmentor, skimmer, and octopole voltage were set at 3000 V, 140 V, 65 V, and 750 V, respectively. MS 2 The MS data and ion molecular weights were processed using previously known standards, published data, and online databases.

[0101] The qualitative results of the bound polyphenols in macromolecular antioxidants are shown in Table 4. Compared with commercial pectin macromolecules, the types of bound polyphenols in the macromolecular antioxidants extracted from kiwifruit peel are relatively more, especially showing a large difference in phenolic acids and flavonoids. The macromolecular antioxidants extracted by the alkaline method exhibit extremely rich bound polyphenolic substances. Seventeen phenolic acid compounds such as gallic acid, salicylic acid, caffeic acid, and ferulic acid, 18 flavonoid compounds such as catechin, epigallocatechin, myricetin, and quercitrin, four coumarin compounds such as esculetin, and three other phenolic substances such as vanillin were detected from them. The types of phenolic acid substances and flavonoid substances are respectively more than twice those in commercial pectin macromolecules. The presence of these polyphenolic substances contributes greatly to the antioxidant activity of macromolecular antioxidants.

[0102] Table 4 Identification of Bound Polyphenol Components in AK, CA, EA, and CP

[0103]

[0104]

[0105] In summary, the present invention uses a dilute alkali method to extract macromolecular antioxidants. By comparing with the citric acid method and the enzymatic method, it can be seen that under the conditions of a material-liquid ratio of 1:20, a mixed solution pH value of 11.5, an extraction temperature of 80 °C, and an extraction time of 60 min, the yield and antioxidant activity of the macromolecular antioxidants are the highest. The present invention also provides a macromolecular antioxidant prepared by the above extraction method. Through detection and analysis, it can be seen that the obtained macromolecular antioxidant not only is rich in the RG-I domain, but also has a relatively low average molecular weight (56.51 kDa) compared with commercial pectin macromolecules (303.38 kDa). In addition, the macromolecular antioxidant extracted by the alkali method in the present invention is rich in a large amount of bound phenolic substances, including phenolic acid compounds such as gallic acid, salicylic acid, caffeic acid, and ferulic acid, flavonoid compounds such as catechin, epigallocatechin, myricetin, and quercitrin, and coumarin compounds such as esculetin. At the same time, the macromolecular antioxidant extracted by the alkali method has significantly higher antioxidant activity. At the same concentration, its DPPH radical scavenging activity, ABTS radical scavenging activity, and Fe 3+ reduction ability exceed 6 times, 3 times, and 100 times that of commercial pectin, respectively, and it can be used as a macromolecular antioxidant in health foods and functional foods.

Claims

1. A method for preparing a macromolecular antioxidant, characterized in that: The method comprises the following steps: (1) Pretreatment: freeze-dry the kiwifruit peel at -45 to -55°C and a vacuum degree of 10 to 20 Pa for 40 to 50 hours, crush and sieve to obtain kiwifruit peel powder; (2) Preparation of macromolecular antioxidants: S1. Weigh the kiwifruit peel powder obtained in step (1), mix it with 0.008-0.01 mol / L NaOH solution at a solid-liquid ratio of 1: (20-40), adjust the pH value of the mixture to 10-12 using NaOH solution, and then place it in a water bath shaker at 70-90°C for 50-70 min; S2. The extract obtained in step S1 is centrifuged for 15 to 25 minutes at a centrifugal speed of 7000 to 9000 rpm, and then filtered, and the filtrate is fully mixed with anhydrous ethanol in a volume ratio of 1: (1 to 5), and suspended matter appears in the mixed solution after standing; S3. The suspended matter in step S2 was collected, washed repeatedly with anhydrous ethanol, reconstituted with pure water, centrifuged at 7000-9000 rpm for 15-25 min, filtered, and the filtrate was placed in a dialysis bag with a molecular weight of 13000-15000 Da for dialysis, and the dialysis time was 24-72 h; (3) Drying: The retentate obtained in the dialysis bag of step S3 is subjected to rotary evaporation and freeze-dried at -45 to -55°C and a vacuum degree of 10 to 20 Pa for 40 to 50 hours to obtain a macromolecular antioxidant.

2. The method for preparing the macromolecular antioxidant according to claim 1, characterized in that: The freeze-drying temperature in step (1) is -45 to -50°C, the vacuum degree is 10 to 15 Pa, and the time is 45 to 50 hours.

3. The method for preparing the macromolecular antioxidant according to claim 1, characterized in that: The material-liquid ratio in step S1 is 1:(20-30).

4. The method for preparing the macromolecular antioxidant according to claim 1, characterized in that: The pH value described in step S1 is 11-12.

5. The method for preparing the macromolecular antioxidant according to claim 1, characterized in that: The temperature of the water bath in step S1 is 80-85° C., and the extraction time is 60-70 min.

6. The method for preparing the macromolecular antioxidant according to claim 1, characterized in that: The volume ratio of the filtrate to anhydrous ethanol in step S2 is 1:(2-3).

7. The method for preparing the macromolecular antioxidant according to claim 1, characterized in that: In step S3, the obtained filtrate is placed in a dialysis bag with a molecular weight of 14,000 Da for dialysis. The dialysis time is 36 to 54 hours, during which the water is changed every 4 hours.

8. The method for preparing the macromolecular antioxidant according to claim 1, characterized in that: The freeze-drying temperature in step (3) is -45 to -50°C, the vacuum degree is 10 to 15 Pa, and the time is 45 to 50 hours.

9. A macromolecular antioxidant obtained according to the preparation method according to any one of claims 1 to 8.

10. Use of the macromolecular antioxidant according to claim 9 in anti-oxidation of food or medicine.

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