Citrus pectin-phenolic acid compound with antioxidant function as well as preparation method and application of citrus pectin-phenolic acid compound

Abstract

The invention discloses a citrus pectin-phenolic acid compound with an antioxidant function as well as a preparation method and application thereof, and belongs to the technical field of food biology. According to the method, citrus pectin and two phenolic acids (gallic acid / caffeic acid) with different structures are used as raw materials, and the citrus pectin and the phenolic acids are promoted to form a citrus pectin-gallic acid / citrus pectin-caffeic acid compound under a weak alkaline condition. The alkaline condition used in the invention is a weak alkaline condition with the pH value of 8-10, the alkaline adjustment is simple and convenient, the implementation is easy, the preparation cost is lower, and the oxidation resistance of the citrus pectin is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of food biotechnology, and specifically relates to a citrus pectin-phenolic acid complex with antioxidant function, its preparation method and application. Background Technology

[0002] In recent years, with the increasing public awareness of food safety and health, concerns have arisen regarding the potential toxicity and adverse effects of synthetic antioxidants (such as tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), and propyl gallate (PG)). Consequently, there has been a strong interest in exploring plant derivatives as natural antioxidants, leading food manufacturers to increasingly consider replacing synthetic antioxidants with natural ingredients containing antioxidant compounds. Natural antioxidants primarily originate from plant phenolic compounds, which are widely found in the structures of fruits, vegetables, tea, and grains. They are plant secondary metabolites, including flavonoids, phenolic acids, and lignans. Furthermore, plant phenolic compounds possess various biological activities, such as antioxidant, anti-inflammatory, anticancer, antibacterial, antidiabetic, antihypertensive, anti-obesity, and anti-atherosclerotic effects. Phenolic acids are the most common type of dietary polyphenols among non-flavonoid phenolic compounds. They have one or more hydroxyl substituents in the benzene ring and can be divided into two categories: hydroxybenzoic acid (such as gallic acid, syringic acid and vanillic acid) and hydroxycinnamic acid (such as ferulic acid, p-coumaric acid and caffeic acid), with hydroxycinnamic acid being the main one.

[0003] However, phenolic compounds are easily oxidized in the environment, undergoing photodegradation and thermal degradation. Pectin is an acidic heteropolysaccharide derived from plants and is also the most common soluble dietary fiber in plants. It possesses strong gelling, thickening, stability, and biocompatibility. Besides its most common use as a thickener, gelling agent, and stabilizer in industrial production, it also exhibits anti-inflammatory, immunomodulatory, gut microbiota-regulating, anticancer, blood sugar and lipid-lowering, cholesterol-lowering, and antioxidant bioactivities, showing promising applications in food, biomedicine, and cosmetics. Currently, the most commonly used commercial pectin is derived from citrus fruits, followed by apples. Phenolic acids can combine with pectin, forming complexes that not only possess the advantages of both but may also produce synergistic effects, such as enhancing physical, chemical, and biological properties and improving the stability of phenolic acids. Non-covalent and covalent interactions are the two main modes of interaction between phenolic compounds and polysaccharides. Among them, polysaccharides and polyphenols can achieve a more stable structure through covalent interactions. Since covalently bound polysaccharide and polyphenol complexes are difficult to separate and structurally characterize, most people use the method of grafting polyphenols onto polysaccharide structures to form complexes.

[0004] Currently, the main processing methods for pectin-phenolic acid complexes include enzymatic methods, chemical coupling methods, and free radical grafting methods, with chemical coupling and enzymatic methods being the most commonly used. Chemical coupling agents, such as carbodiimide, have a certain degree of toxicity. While enzyme-mediated biocatalysis is superior to chemical coupling in terms of synthesis efficiency, reaction condition specificity, and environmental friendliness, polyphenol oxidase oxidizes the effective functional groups of polyphenols during the catalytic process, inevitably affecting and inhibiting the antioxidant properties of polyphenols, and its manufacturing cost is higher. Free radical grafting utilizes the redox reaction of ascorbic acid and hydrogen peroxide to promote the binding of pectin polyphenols, but its binding mechanism is still unclear.

[0005] A search of domestic and international literature revealed that the preparation of pectin-polyphenol complexes is carried out under acidic conditions, and there are currently no literature reports on promoting the binding of pectin and phenolic acids under alkaline conditions. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the first objective of this application is to provide a safe, low-cost, simple and easy-to-implement method for preparing citrus pectin-phenolic acid complex, so as to promote the application of natural antioxidants in food processing.

[0007] To achieve the above objectives, the inventors conducted extensive experimental research and unremitting exploration, and unexpectedly discovered that the binding of citrus pectin and phenolic acids can also be promoted under weakly alkaline conditions, and the resulting complex has a stronger antioxidant capacity.

[0008] Specifically, the objective of this invention is achieved as follows: a method for preparing a citrus pectin-phenolic acid complex with antioxidant function, the method comprising the following steps:

[0009] 1) Citrus pectin pretreatment: Dissolve citrus pectin (CP) in a sodium dihydrogen phosphate-citric acid buffer solution with a pH of 7.4-7.6 to prepare a citrus pectin solution; after stirring to ensure complete dissolution, adjust the pH of the citrus pectin solution to 7.4-7.6 with NaOH solution;

[0010] 2) Phenolic acid pretreatment: Phenolic acid is fully dissolved in anhydrous methanol to prepare a phenolic acid solution. The phenolic acid is gallic acid (GA) or caffeic acid (CA).

[0011] 3) Alkaline treatment: Add the phenolic acid solution prepared in step 2) to the citrus pectin solution prepared in step 1) while stirring, according to the ratio of 1 g pectin / 2-3 mmol phenolic acid. After mixing, adjust the pH of the mixture to 8-10 with NaOH solution, and then place it in a constant temperature water bath shaker at 28-32℃ and react at 150-200 r / min for 4-24 h.

[0012] 4) Dialysis treatment: Dialysis removes unrecombined polyphenols;

[0013] 5) Rotary evaporation concentration and vacuum freeze-drying: The complex solution after dialysis in step 4) is concentrated by rotary evaporation, and then the concentrated solution is vacuum freeze-dried, pulverized and packaged.

[0014] More preferably, in the method for preparing the citrus pectin-phenolic acid complex with antioxidant function as described above, the citrus pectin in step 1) is a high-ester pectin with an esterification degree > 50% and a galacturonic acid content ≥ 74%; the gallic acid in step 2) has a purity ≥ 99%, and the caffeic acid has a purity ≥ 98%.

[0015] More preferably, in the method for preparing the citrus pectin-phenolic acid complex with antioxidant function as described above, the concentration of the citrus pectin solution prepared in step 1) is 0.5% to 2% (w / v); and the concentration of the phenolic acid solution prepared in step 2) is 5% to 8.5% (w / v).

[0016] More preferably, in the method for preparing the citrus pectin-phenolic acid complex with antioxidant function as described above, in step 3), after mixing the two solutions, the concentration of phenolic acid in the mixture is 20-25 mmol / L.

[0017] More preferably, in the method for preparing the citrus pectin-phenolic acid complex with antioxidant function as described above, after mixing the two solutions in step 3), the pH of the mixture is adjusted to 10 with NaOH solution, and then placed in a constant temperature water bath shaker at 28-32°C and reacted at 150-200 r / min for 22-24 h.

[0018] More preferably, in the method for preparing the citrus pectin-phenolic acid complex with antioxidant function as described above, step 4) involves dialysis with distilled water, and the MWCO of the dialysis bag is 12000-14000 Da.

[0019] More preferably, in the method for preparing the citrus pectin-phenolic acid complex with antioxidant function as described above, the dialysis time is 2 days, with the water changed every 3-4 hours during the day, and at least 2 water changes required in the first 8 hours of dialysis. Water does not need to be changed overnight.

[0020] More preferably, in the method for preparing the citrus pectin-phenolic acid complex with antioxidant function as described above, step 5) involves concentrating the dialyzed complex solution by rotary evaporation at a temperature of 40–50 °C, then cooling the concentrate and freezing it in a refrigerator at -18–-22 °C for 8–12 h, and finally drying it using a vacuum freeze dryer.

[0021] In addition, since the citrus pectin-phenolic acid complex prepared by the present invention has a high phenol content and strong antioxidant capacity (DPPH free radical scavenging rate / ABTS free radical scavenging rate / FRAP iron ion reducing capacity), the second objective of this application is to provide a citrus pectin-phenolic acid complex prepared according to the above preparation method, and the application of the citrus pectin-phenolic acid complex in the antioxidant properties of food or pharmaceuticals.

[0022] Compared with the prior art, the present invention provides a method for preparing a citrus pectin-phenolic acid complex with antioxidant properties, which has the following advantages and significant progress:

[0023] (1) The present invention promotes the formation of covalent complexes between citrus pectin and phenolic acids (gallic acid / caffeic acid) under alkaline conditions, and the citrus pectin-phenolic acid complex prepared under the conditions of pH=10 and reaction for 24 h has the highest bound phenol content and antioxidant capacity.

[0024] (2) The alkaline conditions of pH 8-10 used in this invention are weak alkaline conditions. The alkaline adjustment is relatively simple and easy to implement, the preparation cost is lower, and the antioxidant capacity of citrus pectin is greatly improved.

[0025] (3) Pectin is an acidic polysaccharide. In this invention, the alkaline conditions are first achieved by dissolving the pectin in a buffer solution to make the reaction conditions weakly alkaline at 7 to 7.5, and then a small amount of NaOH solution is added to adjust the pH to 8 to 10, which is safer and greener.

[0026] (4) The pectin-phenolic acid complex prepared by the present invention helps to expand the application of pectin polyphenol complex as a food macromolecule antioxidant additive, thereby improving the utilization value of fruit and vegetable waste. Attached Figure Description

[0027] Figure 1 : UV-Vis absorption spectra of citrus pectin-phenolic acid complexes prepared in each example and comparative example;

[0028] Figure 2 Fourier transform infrared spectra of the citrus pectin-phenolic acid complexes prepared in each embodiment and comparative example;

[0029] Figure 3 Scanning electron microscope images of the citrus pectin-phenolic acid complexes prepared in each example and comparative example;

[0030] Figure 4 : DPPH free radical scavenging capacity of citrus pectin-phenolic acid complexes prepared in each example and comparative example;

[0031] Figure 5 : ABTS free radical scavenging capacity of citrus pectin-phenolic acid complexes prepared in each example and comparative example;

[0032] Figure 6 FRAP (Ferric ion reducing power) graphs of citrus pectin-phenolic acid complexes prepared in each example and comparative example. Detailed Implementation

[0033] The following examples further describe the implementation process and beneficial effects of the method of the present invention. These examples are for illustrative purposes only and do not limit the scope of protection of the present invention. Obvious modifications made by those skilled in the art based on the examples are also included within the scope of the present invention. Furthermore, the citrus pectin (P9135) used in the following examples is a high-ester pectin with a degree of esterification greater than 50% and a galacturonic acid content ≥74%, purchased from Sigma-Aldrich Shanghai Trading Co., Ltd.; the gallic acid used has a purity of 99%, and the caffeic acid has a purity of 98%, both purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0034] Example 1: Preparation of Citrus Pectin-Phenolic Acid Complex

[0035] Step 1) Citrus pectin pretreatment: Dissolve 2 g of citrus pectin in 200 mL of sodium dihydrogen phosphate-citric acid buffer solution with pH=7.5 to prepare a 1% (w / v) citrus pectin solution; stir overnight to fully dissolve, and then take an appropriate amount of 1 mol / L NaOH solution to readjust the pH of the pectin solution to 7.5.

[0036] Step 2) Phenolic acid pretreatment: Dissolve 0.85 g (5 mmol) gallic acid and 0.9 g (5 mmol) caffeic acid in 10 mL and 18 mL of anhydrous methanol respectively to prepare 8.5% (w / v) gallic acid solution and 5% (w / v) caffeic acid solution.

[0037] Step 3) Alkaline treatment: Under stirring conditions, the gallic acid / caffeic acid solution prepared in step 2) was added to the citrus pectin solution prepared in step 1). After mixing the two solutions, the concentration of gallic acid (caffeic acid) in the mixture was approximately 24 mmol / L (23 mmol / L). The pH of the mixture was adjusted to 7 with an appropriate amount of 1 mol / L NaOH solution. Then, it was placed in a constant temperature water bath shaker at 30°C and reacted at 180 r / min for 4 h.

[0038] Step 4) Dialysis to remove unreconstituted gallic acid / caffeic acid: Use distilled water for dialysis. The MWCO of the dialysis bag is 12000-14000 Da. The dialysis time is 2 days. Change the water every 3-4 hours during the day. The water needs to be changed at least twice in the first 8 hours of dialysis. The water does not need to be changed overnight.

[0039] Step 5) Rotary evaporation concentration and vacuum freeze drying: The complex solution after dialysis in Step 4) was placed at 45 °C and rotated at 160 rpm to concentrate the complex solution to about 25-50% of its original volume. The concentrated solution was then placed in a freezer at -20 °C overnight and then dried in a vacuum freeze dryer for 48 h. Finally, it was pulverized and packaged (the ACG-7 / ACC-7 in the attached figure corresponds to the citrus pectin-gallic acid / caffeic acid complex result of Example 1).

[0040] Example 2

[0041] The preparation method of the citrus pectin-phenolic acid complex in Example 2 differs from that in Example 1 in that, in step 3), after the citrus pectin is mixed with gallic acid / caffeic acid, the pH of the mixed solution is adjusted to 8 by NaOH solution. All other operations are the same as in Example 1 and will not be described again (the ACG-8 / ACC-8 in the attached figures correspond to the results of the citrus pectin-gallic acid / caffeic acid complex in Example 2).

[0042] Example 3

[0043] The preparation method of the citrus pectin-phenolic acid complex in Example 3 differs from that in Example 1 in that, in step 3), after the citrus pectin is mixed with gallic acid / caffeic acid, the pH of the mixed solution is adjusted to 9 by NaOH solution. All other operations are the same as in Example 1 and will not be described again (the ACG-9 / ACC-9 in the attached figures correspond to the results of the citrus pectin-gallic acid / caffeic acid complex in Example 3).

[0044] Example 4

[0045] The preparation method of the citrus pectin-phenolic acid complex in Example 4 differs from that in Example 1 in that, in step 3), after the citrus pectin is mixed with gallic acid / caffeic acid, the pH of the mixed solution is adjusted to 10 with NaOH solution. All other operations are the same as in Example 1 and will not be described again (the ACG-10 / ACC-10 in the attached figures correspond to the results of the citrus pectin-gallic acid / caffeic acid complex in Example 4).

[0046] Example 5

[0047] The preparation method of the citrus pectin-phenolic acid complex in Example 5 differs from that in Example 1 in that: in step 3), after the citrus pectin and gallic acid / caffeic acid are mixed, the pH of the mixed solution is adjusted to 10 with NaOH solution, and the mixture is placed in a constant temperature water bath shaker at 30°C and reacted at 180 r / min for 24 h. All other operations are the same as in Example 1 and will not be described again (the ACG-10-24 / ACC-10-24 in the attached figures correspond to the results of the citrus pectin-gallic acid / caffeic acid complex in Example 5).

[0048] Comparative Example

[0049] The preparation method of the citrus pectin-phenolic acid complex in the comparative example differs from that in Example 1 in that: in step 3), 0.6 mL of laccase (purchased from Sigma-Aldrich Shanghai Trading Co., EC 1.10.3.2, derived from Aspergillus sp.) and the gallic acid / caffeic acid solution prepared in step 2) are added to the citrus pectin solution prepared in step 1) while stirring and mixing. Then, the mixture is placed in a constant temperature water bath shaker at 30°C and reacted at 180 r / min for 4 h. The remaining operations are the same as in Example 1 and will not be described again (the LCG / LCC ratio in the attached figure corresponds to the results of the citrus pectin-gallic acid / caffeic acid complex in the comparative example).

[0050] Blank control

[0051] The difference between the blank control and Examples 1-5 is that the blank control does not include steps 1-5 of the complex preparation process, and the blank control is made of citrus pectin raw material (CP in the figure corresponds to the citrus pectin result of the blank control example).

[0052] Example 6: Determination of total phenol content in the complex

[0053] The phenolic acid content in the complex was estimated using the Folin-Ciocalteu assay. The sample solution concentration was 2 mg / mL. 0.2 mL of the sample solution was mixed with 0.2 mL of Folin-Ciocalteu reagent, followed by the addition of 2 mL of distilled water. The mixture was then stirred and allowed to stand for 3 min. 1 mL of 15% Na₂CO₃ solution was added to the mixture, vortexed, and allowed to react in the dark for 1 h. After the reaction, the absorbance of the solution was measured at 765 nm using a microplate reader. The absorbance was then used to calculate the phenolic content in the standard curve. The results were expressed as milliequivalents of gallic acid (mg GAE / g) / milliquid acid (mg CAE / g). A standard curve was plotted with the gallic acid / caffeic acid standard solution concentrations on the x-axis and absorbance on the y-axis. The gallic acid standard curve was: y = 0.0037x + 0.0195, (R² + π / π)² = 0.0037x + 0.0195. 2 = 0.9976), Caffeic acid standard curve: y = 0.0038x - 0.0021, (R = 0.9976) 2 = 0.9998).

[0054] The total phenolic content of the complexes in Examples 1-5, the comparative examples, and the blank control example is shown in Table 1 below. Table 1 shows that the phenolic acid content of the citrus pectin-gallic acid / caffeic acid complexes in Examples 1-5 was significantly higher than that in the blank control example, being 5.7-13 times and 3.5-12.6 times higher, respectively. This indicates that the alkaline method is effective in promoting the formation of the pectin-phenolic acid complex, and the phenolic content of the complex increases with increasing solution pH. The results of Examples 4 and 5 show that, under the same pH conditions, the longer the reaction time, the higher the phenolic content. Compared with the comparative example prepared by the traditional laccase method, the total phenolic content in the examples is slightly lower, but the phenolic content of the citrus pectin-caffeic acid complex in Example 5 is quite close to that of the complex prepared by the enzymatic method in the comparative example.

[0055] Table 1 Comparison of total phenol content in different samples

[0056]

[0057] Example 7: UV-Vis scanning spectral analysis of the complex

[0058] The UV-Vis spectra of phenolic acids (gallic acid / caffeic acid (10 μg / mL)), citrus pectin (0.1 mg / mL), and citrus pectin-gallic acid / caffeic acid complex (0.1 mg / mL) were collected in the 200-400 nm range using a UV-Vis spectrophotometer.

[0059] Figure 1 These are the UV-Vis absorption spectra of Examples 1-5, the comparative examples, and the blank control examples. Figure 1 It can be seen that gallic acid has an absorption peak at a wavelength of 262 nm, while caffeic acid has a maximum absorption peak at a wavelength of 316 nm. Figure 1 In (A), the CP in the blank control showed no absorption peaks at 262 nm and 316 nm, while the ACG-7 / ACC-7, ACG-8 / ACC-8, ACG-9 / ACC-9, ACG-10 / ACC-10, ACG-10-24 / ACC-10-24 in Examples 1-5 and the LCG / LCC in the comparative example all showed higher absorbances at 262 nm and 316 nm than the CP in the blank control, and had more obvious absorption peaks than the CP. This indicates that the alkaline method and the laccase method successfully formed a complex between gallic acid / caffeic acid and citrus pectin, thereby increasing the absorbance value of the complex at this wavelength. In addition, the absorbance values ​​of Examples 1-5 and the comparative example at the maximum absorption peak of the corresponding phenolic acids also generally showed an increasing trend with the increase of phenolic acid content.

[0060] Example 8: Fourier transform infrared spectroscopy analysis of the complex

[0061] Fourier transform infrared spectroscopy (FT-IR) analysis was performed using a Thermo Fisher Scientific, Madison, MA, USA. Citrus pectin, phenolic acids, and pectin-phenolic acid complexes were mixed with KBr at a ratio of 1:100, ground in an agate mortar, and pressed into thin tablets using a tablet press. The infrared spectra were then scanned using a Fourier transform infrared spectroscopy system, with a scanning range of 500–4000 cm⁻¹. −1 The number of scans was 64. The obtained spectra were processed using Omnic 8.2 software.

[0062] Figure 2 The Fourier transform infrared spectra are for Examples 1-5, the comparative examples, and the blank control examples. From... Figure 2 It can be seen that the peak values ​​are located at 3431, 2937, 1749, and 1626 cm. −1 These correspond to the OH and CH bonds, C=O in the esterified carboxyl group, and C=O in the free carboxyl group of citrus pectin in the blank control group, respectively, and are located at 1148~1015 cm. −1 The characteristic peaks represent glycosidic bonds and sugar rings in pectin, which are characteristic peaks of citrus pectin. The citrus pectin-gallic acid / caffeic acid complexes in Examples 1-5 have similar characteristic absorption peaks to citrus pectin, indicating that the complex preparation method in this invention does not change the main structure of citrus pectin. Furthermore, gallic acid shows characteristic absorption peaks at 3496 and 3282 cm⁻¹. −1 It exhibits strong absorption peaks at 3435 and 3234 cm⁻¹. −1 It exhibits a strong absorption peak. Compared to citrus pectin, all citrus pectin-gallic acid / caffeic acid complexes show a peak at 3431 cm⁻¹. −1 The relatively strong absorption peak intensity in the vicinity is likely due to the stretching vibration of the -OH group on the benzene ring in phenolic acids. This is in contrast to the absorption peak intensity of all citrus pectin complexes at 1626 cm⁻¹. −1 The absorption peak intensity is higher than 1749 cm⁻¹ −1 At this location, and ACG-7~ACG-10-24 / ACC-7~ACC-10-24 are at 1626 cm. -1 The peak value near 1626 cm⁻¹ increases with increasing pH. One possible reason is that phenolic acids bind to citrus pectin, and the free carboxyl groups in the phenolic acid structure cause it to peak at 1626 cm⁻¹. −1 Tensile vibrations occur nearby. On the other hand, it's possible that the ester groups of citrus pectin are destroyed under alkaline conditions, converting esterified carboxyl groups into free carboxyl groups. Furthermore, the 1626, 1541, 1415, and 1239 cm⁻¹ groups in the citrus pectin-gallic acid complex... −1 Or 1530, 1414, 1240 cm of citrus pectin-caffeic acid complex −1The presence of benzene ring skeletal vibrations and carboxyl stretching vibrations near the absorption peaks indicates that gallic acid / caffeic acid has formed a complex with citrus pectin.

[0063] Example 9: Microstructure analysis of the complex

[0064] The microstructure of pectin, phenolic acid, and pectin-phenolic acid complexes was observed using scanning electron microscopy. The samples were fixed on the sample stage with conductive adhesive and sputtered with gold, followed by electron microscopy observation. Images of pectin, phenolic acid, and pectin-phenolic acid complexes were collected (magnified at 500x and 1000x).

[0065] Depend on Figure 3 It can be seen that the microstructure of citrus pectin in the blank control example is large granular, gallic acid is rod-shaped, and caffeic acid is relatively small granular. In contrast, the microstructure of the citrus pectin-gallic acid / caffeic acid complex in Examples 1-5 and the comparative example is relatively smooth and sheet-like. This indicates that the microstructure of the complexes prepared by the two different methods is significantly different from that of the single substance, and the different preparation methods (enzymatic and alkaline methods) have no significant impact on the micromorphology of the complexes.

[0066] Example 10: Determination of the DPPH free radical scavenging ability of the complex

[0067] Mix 0.5 mL of sample solution and 0.5 mL of DPPH solution (1×10⁻⁶). -4 The DPPH solution (mol / L, dissolved in anhydrous ethanol) was thoroughly mixed and reacted in the dark for 0.5 h. Subsequently, the absorbance of the mixture was measured at 517 nm (A1). Anhydrous ethanol was used as a blank control (A2), and distilled water was used as a control control (A0). The DPPH radical scavenging rate of the sample was calculated using the formula:

[0068]

[0069] Depend on Figure 4It can be seen that the free radical scavenging rates of the citrus pectin-caffeic acid complex and the citrus pectin-gallic acid complex in Examples 1-5 and the comparative examples were significantly higher than those of the citrus pectin in the blank control example. This indicates that the complexes prepared by the alkaline method and the enzymatic method significantly improved the DPPH free radical scavenging ability of citrus pectin. In addition, the DPPH free radical scavenging rates of the citrus pectin-caffeic acid complexes (ACC-8 / ACC-9 / ACC-10 / ACC-10-24 and LCC) in Examples 1-5 and the comparative examples were significantly higher than those of the citrus pectin-gallic acid complexes (ACG-8 / ACG-9 / ACG-10 / ACG-10-24 and LCG) prepared by the corresponding methods. This may be due to the influence of the phenol content in the complex; the higher the phenol content, the stronger the antioxidant capacity. The DPPH free radical scavenging capacity of the citrus pectin-gallic acid / caffeic acid complex prepared by the alkaline method increased with increasing pH, with ACG-10-24 / ACC-10-24 exhibiting the highest scavenging rate, which is consistent with the trend of phenol content in the complex in Table 1.

[0070] Example 11: Determination of ABTS free radical scavenging ability

[0071] The assay was performed using the ABTS free radical scavenging kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.), following the instructions. Pectin and pectin polyphenol complex were dissolved in the extraction buffer to prepare a 2 mg / mL solution, stirred overnight, and then centrifuged at 10000 r / min for 10 min at 4 ℃. The supernatant was collected and diluted with the extraction buffer to 1 mg / mL. 50 μL of the diluted sample supernatant was added to 100 μL of reagent IV working solution and 850 μL of ABTS working solution, and mixed thoroughly. The mixture was incubated at room temperature in the dark for 6 min, and the absorbance was measured at 405 nm (A1). Distilled water was used as a blank control (A0) instead of the sample solution, and the same procedure was performed as for the sample. For the control group, 50 μL of the diluted sample supernatant was added to 950 μL of reagent I, and the mixture was incubated at room temperature in the dark for 6 min, and the absorbance was measured at 405 nm (A2). The calculation formula is as follows:

[0072]

[0073] Depend on Figure 5 It can be seen that the ABTS radical scavenging rates of Examples 1-5 and the comparative example are significantly higher than those of the blank control example CP, indicating that the complexes all have strong ABTS radical scavenging ability, and their variation pattern is consistent with... Figure 4The ABTS radical scavenging capacity of the citrus pectin-gallic acid / caffeic acid complexes prepared by the alkaline method showed the same trend. The ABTS radical scavenging capacity of the citrus pectin-gallic acid / caffeic acid complexes prepared by the alkaline method increased with increasing pH, with ACG-10-24 / ACC-10-24 exhibiting the highest scavenging rate.

[0074] Example 12: FRAP (Ferric Ion Reduction Antioxidant Capacity) Determination

[0075] Total antioxidant capacity (T-AOC) was measured using a FRAP method (purchased from Nanjing Jiancheng Bioengineering Institute). The procedure was performed according to the instructions. FeSO4·7H2O was used as the standard, and a standard curve was established with concentrations of 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mmol / L (y = 0.4704x + 0.0047, R...). 2 =0.9993). The sample concentration was 5 mg / mL. The sample solution was mixed with the FRAP working solution, incubated at 37°C for 5 min, and the absorbance was measured at 593 nm. Distilled water was used as a blank instead of the sample solution. Antioxidant capacity was expressed as mmol (Fe). 2+ ) / g represents.

[0076] like Figure 6 As shown, the FRAP (ferric reduction ability) of the citrus pectin-gallic acid / caffeic acid complexes in Examples 1-5 and the comparative example were significantly higher than that of the blank control example CP. Among them, the FRAP ability of ACC-10-24 in the complex was the highest, about 46 times that of CP. This indicates that the complexes prepared by the two different methods significantly improved the FRAP ability of citrus pectin.

[0077] And in Figure 4 and Figure 6 In the enzymatic preparation of LCC in citrus pectin-caffeic acid complexes, the DPPH radical scavenging rate and FRAP ability were lower than those of complexes prepared by alkaline methods under pH conditions of 9-10 (ACC-9, ACC-10, ACC-10-24). Figure 5 In addition, the ABTS radical scavenging rate of LCC was also lower than that of ACC-10-24. Furthermore, in Figure 6 In the study, the FRAP activity of the citrus pectin-gallic acid complex (ACG-10-24) prepared by the alkaline method was higher than that of the LCG prepared by the enzymatic method. The phenol content of the complex prepared by the alkaline method was lower than that prepared by the enzymatic method, while some of the complexes prepared by the alkaline method showed stronger antioxidant capacity. This indicates that the citrus pectin-phenolic acid complex prepared by the alkaline method has a certain advantage over the enzymatic method in improving the antioxidant properties of citrus pectin.

[0078] In summary, this invention utilizes an alkaline method to prepare citrus pectin-gallic acid / caffeic acid complexes under different pH conditions (pH=7~10) and reaction times (4 h / 24 h). Comparison of different reaction conditions shows that the citrus pectin-phenolic acid complex prepared at pH=10 and a reaction time of 24 h exhibits the highest phenol content and the highest antioxidant capacity. Furthermore, comparison with citrus pectin reveals that the citrus pectin-phenolic acid complex provided by this invention possesses strong antioxidant capabilities (DPPH free radical scavenging rate / ABTS free radical scavenging rate / FRAP iron ion reducing capacity). Comparison of the alkaline-prepared complex with the traditional laccase-prepared citrus pectin-gallic acid / caffeic acid complex shows no significant difference in microstructure. Although the phenol content of the alkaline-prepared complex is slightly lower than that of the enzymatically prepared complex, when the difference in phenol content is small, the antioxidant capacity of the alkaline-prepared complex is stronger than that of the enzymatically prepared complex, such as ACC-10-24. This indicates that the alkaline method in this invention has certain advantages over the enzymatic method in preparing citrus pectin-phenolic acid complexes with antioxidant capacity, and is a method for preparing citrus pectin-phenolic acid complexes that can improve antioxidant capacity.

Claims

1. A method for preparing a citrus pectin-phenolic acid complex with antioxidant function, characterized in that, The method includes the following steps: 1) Citrus pectin pretreatment: Dissolve citrus pectin in a sodium dihydrogen phosphate-citric acid buffer solution with a pH of 7.4-7.6 to prepare a citrus pectin solution; after stirring to ensure complete dissolution, adjust the pH of the citrus pectin solution to 7.4-7.6 with NaOH solution; 2) Phenolic acid pretreatment: Phenolic acid is fully dissolved in anhydrous methanol to prepare a phenolic acid solution, wherein the phenolic acid is gallic acid or caffeic acid; 3) Alkaline treatment: Add the phenolic acid solution prepared in step 2) to the citrus pectin solution prepared in step 1) while stirring, according to the ratio of 1 g pectin / 2-3 mmol phenolic acid. After mixing, adjust the pH of the mixture to 8-10 with NaOH solution, and then place it in a constant temperature water bath shaker at 28-32℃ and react at 150-200 r / min for 4-24 h. 4) Dialysis treatment: Dialysis removes unrecombined polyphenols; 5) Rotary evaporation concentration and vacuum freeze-drying: The complex solution after dialysis in step 4) is concentrated by rotary evaporation, and then the concentrated solution is vacuum freeze-dried, pulverized and packaged.

2. The method for preparing the citrus pectin-phenolic acid complex with antioxidant function according to claim 1, characterized in that, The citrus pectin mentioned in step 1) is a high-ester pectin with an esterification degree > 50% and a galacturonic acid content ≥ 74%; the gallic acid mentioned in step 2) has a purity ≥ 99% and the caffeic acid has a purity ≥ 98%.

3. The method for preparing the citrus pectin-phenolic acid complex with antioxidant function according to claim 1, characterized in that, Step 1) The concentration of the prepared citrus pectin solution is 0.5% to 2% (w / v).

4. The method for preparing the citrus pectin-phenolic acid complex with antioxidant function according to claim 1, characterized in that, Step 2) The concentration of the prepared phenolic acid solution is 5% to 8.5% (w / v).

5. The method for preparing the citrus pectin-phenolic acid complex with antioxidant function according to claim 1, characterized in that, After the two solutions are mixed in step 3), the concentration of phenolic acid in the mixture is 20-25 mmol / L.

6. The method for preparing the citrus pectin-phenolic acid complex with antioxidant function according to claim 5, characterized in that, After mixing the two solutions in step 3), the pH of the mixture is adjusted to 10 with NaOH solution, and then placed in a constant temperature water bath shaker at 28-32 ℃ and reacted at 150-200 r / min for 22-24 h.

7. The method for preparing the citrus pectin-phenolic acid complex with antioxidant function according to claim 1, characterized in that, In step 4), distilled water is used for dialysis, and the MWCO of the dialysis bag is 12000-14000 Da. The preferred dialysis time is 2 days, with the water changed every 3-4 hours during the day, and the water should be changed at least twice in the first 8 hours of dialysis.

8. The method for preparing the citrus pectin-phenolic acid complex with antioxidant function according to claim 1, characterized in that, Step 5) The dialyzed complex solution is concentrated by rotary evaporation at a temperature of 40-50 °C. After cooling, the concentrate is frozen in a refrigerator at -18-22 °C for 8-12 hours and finally dried using a vacuum freeze dryer.

9. A citrus pectin-phenolic acid complex prepared by the preparation method according to any one of claims 1-8.

10. The use of the citrus pectin-phenolic acid complex according to claim 9 in the antioxidant activity of food or pharmaceuticals.

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