Modified pectin for adsorbing heavy metals and method for preparing the same

Abstract

The application discloses modified pectin for adsorbing heavy metals, which is obtained by the following steps: extracting hawthorn pectin from hawthorn powder by a choline chloride-urea solution, and then modifying the hawthorn pectin by a fermentation liquor of black aspergillus for producing pectinase. On the basis of optimizing the extraction of hawthorn pectin, the hawthorn pectin is modified by the fermentation liquor of black aspergillus for producing pectinase, so that the pectin product with greatly improved heavy metal adsorption rate is obtained. The preparation of the pectin product adopts green production technology, and the prepared pectin product meets the non-toxic and harmless requirements of food additives. The pectin product has extremely significant application significance and value for the purification of food and the repair of non-renewable resources.

Description

Technical Field

[0001] This invention relates to the field of heavy metal adsorbent technology, specifically to a modified pectin for adsorbing heavy metals, and a method for preparing the modified pectin. Background Technology

[0002] Heavy metals accumulate in organisms through environmental media and enter the human body via the food chain, posing a threat to human health. Therefore, the increasingly serious problem of heavy metal contamination in food has severely impacted food safety. Lead contamination is one of the more serious problems in food heavy metal pollution, and excessively high lead levels in the human body can cause certain diseases and even cancer. Current technologies for treating lead contamination typically include chemical precipitation, electrochemistry, membrane filtration, and ion exchange. However, these technologies either have poor removal efficiency or are expensive, both having drawbacks and failing to meet the needs. Biosorption is a new, effective, and environmentally friendly technology for removing heavy metals. It mainly utilizes biological materials to adsorb heavy metal pollutants. Studies have shown that pectin has a certain effect on removing heavy metals, but due to the natural nature of biological materials, the removal efficiency is unstable, resulting in low adsorption rates for some pectins, such as hawthorn pectin, making practical application difficult.

[0003] my country has the world's largest cultivation area and output of hawthorn, possessing resource advantages. However, current development of hawthorn-related products mainly focuses on hawthorn slices, which generate a large amount of waste material. Indiscriminate disposal of this waste inevitably leads to resource waste. Hawthorn slices are rich in pectin, making them a valuable raw material for pectin production. Hawthorn pectin prepared using traditional extraction methods such as microwave extraction, ultrasonic extraction, chelating agent extraction, and enzymatic extraction has limited applications due to its unremarkable performance, failing to effectively and fully utilize hawthorn resources.

[0004] Based on this, solving the application problem of hawthorn pectin, especially developing pectin suitable for heavy metal adsorption, will not only promote the solution of heavy metal pollution and facilitate the development of more economical and environmentally friendly heavy metal removal technologies, but also enable the full utilization of hawthorn resources. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a modified hawthorn pectin for adsorbing heavy metals. Through synergistic optimization of simultaneous extraction and modification of hawthorn pectin, a pectin product with significantly improved heavy metal adsorption rate is obtained. Furthermore, the preparation of this pectin utilizes green production technology, which has extremely significant application value and application significance for food purification and the remediation of non-renewable resources.

[0006] To achieve the above objectives, the modified pectin for adsorbing heavy metals provided by the present invention is a modified hawthorn pectin obtained by extracting hawthorn powder with choline chloride-urea solution, followed by modification treatment with fermentation broth of Aspergillus niger producing pectinase.

[0007] The modified pectin of this invention is modified using the fermentation broth of *Aspergillus niger*, which produces pectinase, rather than purified enzymes, based on optimized hawthorn pectin extraction. This significantly improves the adsorption rate and removal efficiency of pectin molecules for heavy metals. The extraction process utilizes a eutectic solvent, choline chloride-urea solution, instead of traditional solvents, influencing the structure and bioactivity of the pectin. This results in extracted pectin with low esterification, small molecular weight, small particle size, and high extraction rate, thus endowing it with various functional activities. Because pectin is particularly effective against heavy metals, especially Pb... 2+ The adsorption performance of pectin is related to its various molecular structures, such as functional groups, molecular weight, and side chain content. Therefore, based on optimized extraction, the pectin was further modified using Aspergillus niger fermentation broth, which significantly improved the adsorption rate of pectin molecules for heavy metals. This allows the abundant hawthorn raw material to be developed into a heavy metal biosorbent, which not only environmentally and efficiently solves the problem of heavy metal pollution along the food chain and the remediation of non-renewable resources, but also avoids the waste of hawthorn resources and improves their comprehensive utilization value. In addition, eutectic solvents have the advantages of low cost and environmental friendliness compared to traditional solvents, which can promote the development of "green chemistry" and further facilitate the application and promotion of modified pectin in the adsorption of heavy metals.

[0008] As a limitation of the above technical solution, the modification treatment involves dissolving hawthorn pectin in a disodium hydrogen phosphate-citric acid buffer solution, adding Aspergillus niger fermentation broth, and reacting in a constant temperature water bath at 40-50°C.

[0009] As a limitation of the above technical solution, the supernatant of the Aspergillus niger fermentation broth is the supernatant collected by centrifuging a fermentation broth obtained by activating a pectinase-producing Aspergillus niger strain at 25-30℃ and expanding its culture at 25-35℃ at 4-6℃.

[0010] As a limitation of the above technical solution, the choline chloride-urea solution is mainly obtained by mixing choline chloride, urea and water, the molar ratio of choline chloride to urea in the solution is 1:3 to 4, and the water content of the solution is 30 to 40% (water content is expressed as mass concentration wt%).

[0011] By limiting the modification conditions of hawthorn pectin, the acquisition conditions of Aspergillus niger fermentation broth used for modification, and the composition of the choline chloride-urea solution used for extraction, a highly efficient adsorbent suitable for adsorbing heavy metals was obtained.

[0012] As a limitation of the above technical solution, the hawthorn modified pectin is a light yellow powder with a molecular weight of 3.5-4.5 kDa and a degree of esterification of 25%-35%, which meets the requirements for food additives.

[0013] The optimized hawthorn modified pectin meets the high standards for food additives in terms of appearance, properties and safety, and its molecular weight and degree of esterification range are more conducive to improving the adsorption performance of heavy metals.

[0014] Meanwhile, the present invention also provides a method for preparing modified pectin for adsorbing heavy metals as described above, comprising the following steps:

[0015] a. Extraction of hawthorn pectin: Hawthorn powder was heated and extracted using choline chloride-urea solution. After cooling and centrifugation, the supernatant was collected to obtain hawthorn pectin solution. Ethanol was added to the hawthorn pectin solution to precipitate the pectin, which was then washed, purified, and dried to obtain hawthorn pectin.

[0016] b. Modification of hawthorn pectin: The hawthorn pectin prepared in step a was dissolved in disodium hydrogen phosphate-citric acid buffer solution, and the Aspergillus niger fermentation broth was added for constant temperature water bath reaction. Then, it was treated with boiling water bath, cooled to room temperature, and finally purified and dried to obtain modified hawthorn pectin.

[0017] This invention utilizes a choline chloride-urea solution to extract hawthorn pectin, thereby altering the molecular structure and biological activity of the pectin. Based on this, the extracted pectin is modified using fermentation broth from Aspergillus niger, which produces pectinase. By leveraging the multi-enzyme system rich in the fermentation broth to achieve synergistic effects of multiple enzymatic hydrolysis, hawthorn pectin is endowed with excellent functional activity in adsorbing heavy metal ions.

[0018] The purpose of adding ethanol in the above operation is to: (1) precipitate pectin; (2) remove impurities such as pigments and proteins; and (3) remove the eutectic solvent choline chloride-urea solution.

[0019] As a limitation of the above technical solution, the extraction operation of hawthorn pectin includes:

[0020] a1. Use choline chloride-urea solution to heat-extract the sieved hawthorn powder. The ratio of hawthorn powder to choline chloride-urea solution is 1g:30-40mL. The extraction temperature is 70-80℃ and the extraction time is 60-80min. After the extraction is completed, let the extract cool to room temperature, centrifuge and collect the supernatant, which is the hawthorn pectin solution.

[0021] a2. Add 85-95% ethanol to the hawthorn pectin solution obtained in step a1 to precipitate the pectin and wash out the impurities by stirring thoroughly. After centrifugation, discard the supernatant and collect the precipitate. Wash it again with ethanol 1-4 times. After the ethanol evaporates, dissolve the obtained pectin precipitate in distilled water, put it into a dialysis bag, and dialyze it with distilled water as the dialysis solution for purification. Freeze-dry to obtain hawthorn pectin for later use.

[0022] Further refine the operation and conditions for extracting pectin from hawthorn powder, such as the composition and amount of the extractant, the heating and extraction temperature and time, and the ethanol precipitation, washing, and dialysis treatment after extraction, to improve the extraction steps and obtain a hawthorn pectin structure that is more suitable for subsequent modification and conducive to the adsorption of heavy metals.

[0023] As a limitation of the above technical solution, the modification treatment of hawthorn pectin includes:

[0024] b1. After activating and expanding the pectinase-producing Aspergillus niger strain, the fermentation broth is centrifuged at 4-6℃, and the supernatant is collected to obtain Aspergillus niger fermentation broth; the activation conditions are 25-30℃ and 24-48h activation culture; the expansion culture conditions are 2-8% inoculum, 25-35℃ and 36-72h fermentation culture.

[0025] b2. Take the hawthorn pectin prepared in step a2 and dissolve it in a pH 4-5 disodium hydrogen phosphate-citric acid buffer solution to make the concentration of hawthorn pectin 25-30 mg / mL; add Aspergillus niger fermentation broth, react in a constant temperature water bath at 40-50℃ for 2-4 hours, then treat with a boiling water bath, cool to room temperature, dissolve in distilled water, put into a dialysis bag, dialyze and purify with distilled water as the dialysate, freeze dry to obtain modified hawthorn pectin.

[0026] As a limitation of the above technical solution, the amount of Aspergillus niger fermentation broth added in step b2 is 10-200 U / mL; and the mixture is treated in a boiling water bath for 5 min.

[0027] As a limitation of the above technical solution, in steps a2 and b2, the dialysis bag is selected with a capacity of 2000-8000 Da, the dialysis time is 24-48 hours and the dialysis fluid is replaced every 2-6 hours, and the freeze-drying time is 24-48 hours.

[0028] Further refine the modification conditions of hawthorn pectin by Aspergillus niger fermentation broth, such as the fermentation culture conditions of Aspergillus niger strains and the preparation conditions of fermentation broth, and improve the modification treatment operation of hawthorn pectin to obtain hawthorn modified pectin products that can significantly improve heavy metal adsorption performance.

[0029] In summary, the modified pectin of this invention can be used as a heavy metal ion adsorbent. It is inexpensive to produce, easy to prepare in large quantities, has stable physicochemical properties, a large adsorption capacity, and exhibits selectivity for specific heavy metals. Furthermore, it is easily biodegradable and will not cause secondary pollution to the environment, making it extremely significant for the remediation of non-renewable resources. In addition, using the high-quality modified pectin of this invention, which meets the requirements for food additives, for heavy metal adsorption in food can environmentally and efficiently solve the problem of heavy metal pollution along the food chain. Moreover, the preparation of the pectin adopts green production technology, achieving full green utilization of hawthorn resources and effectively avoiding waste and pollution. Attached Figure Description

[0030] Figure 1 The images are scanning electron microscope (SEM) images of the modified hawthorn pectin obtained in the examples at 2000×, 5000×, and 20000× (from left to right).

[0031] Figure 2 The images are scanning electron microscope (SEM) images of hawthorn pectin obtained in Comparative Examples 4-1 to 4-4; the magnifications from left to right are 100×, 500×, and 1000×.

[0032] Figure 3 The images shown are scanning electron microscope (SEM) images of hawthorn pectin obtained in Examples 5-1 and 5-2; where a: Comparative Example 5-1, 200×; b: Comparative Example 5-1, 500×; c: Comparative Example 5-1, 5000×; d: Comparative Example 5-2, 200×; e: Comparative Example 5-2, 500×; f: Comparative Example 5-2, 5000×; g: Example, 200×; h: Example, 500×; i: Example, 5000×.

[0033] Figure 4 The image shows the infrared spectrum of hawthorn pectin obtained in Comparative Examples 4-1 to 4-4.

[0034] Figure 5 The image shows the infrared spectra of hawthorn pectin obtained in Examples 5-1 and 5-2, and Comparative Examples 5-1 and 5-2. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] The chemical raw materials involved in the following examples and comparative examples are all typical products purchased from the market.

[0037] Example

[0038] The hawthorn-modified pectin of this embodiment uses hawthorn as raw material, is extracted with choline chloride-urea solution, and modified with fermentation broth of Aspergillus niger that produces pectinase. The specific preparation method includes the following steps:

[0039] Step a: Extract hawthorn pectin from dried hawthorn berries;

[0040] a1. Take dried hawthorn (which can be obtained by drying the residue left over after producing hawthorn products), crush and sieve it to a particle size of 70 mesh; then use choline chloride-urea solution to heat and extract the sieved hawthorn powder. The choline chloride-urea solution is prepared by mixing choline chloride, urea and water. The molar ratio of choline chloride to urea in the solution is 1:3, the water content is 30wt%, and the material-liquid ratio of hawthorn powder to choline chloride-urea solution is 1g:35mL. After adding hawthorn powder, the pH of the extract is measured to be 1.5. The extraction temperature is controlled at 80℃ and the extraction time is controlled at 80min. After the extraction is completed, wait for the extract to cool to room temperature, centrifuge at 5000r / min for 15min and collect the supernatant, which is the hawthorn pectin solution.

[0041] a2. Add 95% ethanol to the hawthorn pectin solution obtained in step a1. Stir at 500 rpm for 20 min using a magnetic stirrer to precipitate the hawthorn pectin while washing away impurities such as hawthorn pigment. Then centrifuge at 5000 rpm for 10 min, discard the supernatant, and collect the precipitate. Repeat the above operation three times with 95% ethanol to ensure relatively pure pectin. Allow the obtained pectin precipitate to stand to allow the ethanol to evaporate, then dissolve it with a small amount of distilled water. Place it in a 3500 Da dialysis bag and dialyze with distilled water as the dialysate for 24 h, changing the dialysate every 4 h to obtain purified hawthorn pectin. Freeze-dry for 24 h for later use.

[0042] Step b: Modify hawthorn pectin to obtain a pectin adsorbent that has advantages in the adsorption of heavy metals.

[0043] b1. The pectinase-producing Aspergillus niger strain (purchased from ATCC Company, strain name ATCC16404, isolated from blueberries in North Carolina) was inoculated into potato dextrose agar (PDA) slant medium and cultured in a constant temperature incubator at 27°C for 24 hours.

[0044] Under aseptic conditions, the strain activated by slant culture was inoculated into 50 mL of liquid seed medium, potato broth (PDB), for seed culture at 28 °C and shaking at 200 r / min for 72 h.

[0045] Under aseptic conditions, the seed culture was inoculated into the fermentation medium. 25 mL of fermentation medium was placed in a 250 mL Erlenmeyer flask. The seed culture inoculation volume was 6%. The rotary shaker was set at 200 r / min and 30 °C for 72 h.

[0046] Fermentation medium formula: 3g orange peel powder, 2g (NH4)2SO4, 12g wheat bran, 0.075g urea, 0.03g CaCl2, 3.8g KH2PO4, 0.2g K2HPO4·3H2O, 15ml ethylene glycol, 100mL water, pH 6.0;

[0047] After the culture was completed, the fermentation broth was centrifuged at 4℃ and 8000r / min for 10min and the supernatant was taken to obtain the Aspergillus niger fermentation broth. The enzyme activity was 7500U / mL when determined by the DNS method.

[0048] b2. Take the hawthorn pectin prepared in step a2 and dissolve it in a pH 5.0 disodium hydrogen phosphate-citric acid buffer solution. After complete dissolution, the concentration of hawthorn pectin in the solution is 25 mg / mL.

[0049] Take 100-200 mL of the dissolved hawthorn pectin solution, add Aspergillus niger fermentation broth at a concentration of 200 U / mL, react in a water bath at 45°C for 3 hours, then remove the solution, inactivate the enzyme in a boiling water bath for 5 minutes, and cool to room temperature of 26°C.

[0050] The cooled pectin solution was dialyzed with distilled water, and the molecular weight cutoff was 3500 Da. After vacuum freeze-drying for 48 hours, hawthorn modified pectin I was obtained, which was a light yellow powder.

[0051] In the above-mentioned extraction conditions of hawthorn pectin, the ratio of extractant, the ratio of hawthorn powder to extractant, pH, extraction temperature, extraction time, centrifugation speed, centrifugation time, etc. can all be adjusted within the appropriate range given in the invention.

[0052] In the modification conditions of hawthorn pectin, the activation of the strain used in the preparation of Aspergillus niger fermentation broth, seed culture, fermentation culture temperature and time, enzyme addition amount, enzymatic hydrolysis temperature and time, etc., can all be adjusted within the appropriate range given in the invention.

[0053] Comparative Example 1

[0054] The hawthorn-modified pectin in this comparative example was prepared by modifying commercial pectin (purchased from Sigma, CAS No.: 9000-69-5) with commercial pectinase. The specific preparation method includes the following steps:

[0055] Step a: Dissolve the commercial pectin in a disodium hydrogen phosphate-citric acid buffer solution. The concentration of hawthorn pectin in the solution is 25 mg / mL, and the pH of the disodium hydrogen phosphate-citric acid buffer solution is 5.0.

[0056] Step b: Take 100-200 mL of the dissolved pectin solution and add commercial pectinase. The amount of commercial pectinase added is 200 U / mL. After reacting in a water bath at a constant temperature of 45℃ for 3 hours, take it out and then inactivate the enzyme in a boiling water bath for 5 minutes. Cool to room temperature of 26℃.

[0057] The cooled pectin solution was dialyzed with distilled water, and the molecular weight cutoff was 3500 Da. After vacuum freeze-drying for 48 h, hawthorn modified pectin II was obtained as a white powder.

[0058] Comparative Example 2

[0059] The hawthorn-modified pectin in this comparative example was prepared by modifying commercial pectin with the fermentation broth of Aspergillus niger, which produces pectinase. The specific preparation method includes the following steps:

[0060] Step a: Prepare the dissolved pectin solution, just like step a in Comparative Example 1;

[0061] Step b: Prepare Aspergillus niger fermentation broth, as in step b1 of Example 1;

[0062] Take 100-200 mL of the dissolved pectin solution, add Aspergillus niger fermentation broth (the amount of Aspergillus niger fermentation broth added is 200 U / mL), react in a water bath at a constant temperature of 45℃ for 3 hours, then remove it, inactivate the enzyme in a boiling water bath for 5 minutes, and cool to room temperature of 26℃.

[0063] The cooled pectin solution was dialyzed with distilled water, and the molecular weight cutoff was 3500 Da. After vacuum freeze-drying for 48 h, hawthorn modified pectin III was obtained as a light yellow powder.

[0064] Comparative Example 3

[0065] The main difference between the hawthorn-modified pectin in this comparative example and the hawthorn-modified pectin in the examples is that, for the modification of the extracted pectin, a purified enzyme obtained from the Aspergillus niger fermentation broth is used instead of the Aspergillus niger fermentation broth itself. The specific preparation method includes the following steps:

[0066] Step a: Extract hawthorn pectin from dried hawthorn, following the same procedure as in Example a;

[0067] Step b involves modifying hawthorn pectin.

[0068] b1. The pectinase-producing Aspergillus niger strain was activated, seed cultured, and fermented to obtain Aspergillus niger fermentation broth. The specific operation was the same as step b1 in the example.

[0069] The Aspergillus niger fermentation broth was placed in an ice-water bath, and ground ammonium sulfate powder was slowly added until the final concentration of ammonium sulfate was 80% (w / w). After precipitation in a refrigerator at 4°C overnight, the precipitated protein was obtained by centrifugation at 4°C and 10000 r / min for 10 min.

[0070] The protein was reconstituted with a pH 5.0 disodium hydrogen phosphate-citric acid buffer solution, dialyzed with deionized water at 4°C, with a molecular weight cutoff of 8000 Da, and desalted for 12 hours (with the dialysate deionized water replaced every 4 hours). The desalted Aspergillus niger fermentation broth was then concentrated with polyethylene glycol 20000 using an embedding method to obtain a purified enzyme solution. The enzyme activity was determined to be 9000 U / mL using the DNS method.

[0071] b2. Take the hawthorn pectin extracted in step a and dissolve it in a pH 5.0 disodium hydrogen phosphate-citric acid buffer solution. After complete dissolution, the concentration of hawthorn pectin in the solution is 25 mg / mL.

[0072] Take 100-200 mL of the dissolved hawthorn pectin solution, add the purified enzyme solution, and add 200 U / mL of Aspergillus niger fermentation broth. After reacting in a water bath at a constant temperature of 45℃ for 3 hours, take it out, then inactivate the enzyme in a boiling water bath for 5 minutes, and cool it to room temperature of 26℃.

[0073] The cooled pectin solution was dialyzed with distilled water, and the molecular weight cutoff was 3500 Da. After vacuum freeze-drying for 48 h, hawthorn modified pectin IV was obtained.

[0074] Comparative Example 4

[0075] This comparative example involves the extraction of hawthorn pectin using different eutectic solvents as extractants. The difference from the example is the use of different extractants, as detailed in Table 1 below.

[0076]

[0077]

[0078] Comparative Example 5

[0079] This comparative example involves the extraction of hawthorn pectin using different extraction methods, as detailed in Table 2 below.

[0080]

[0081] The results of the above embodiments and comparative examples were measured and analyzed.

[0082] The measured physicochemical properties are as follows:

[0083] 1. Determination of extraction rate

[0084] Pectin was quantified according to the People's Republic of China Agricultural Industry Standard NY / 2016-2011. Using galacturonic acid solution concentration as the x-axis and absorbance as the y-axis, the best-fit linear equation, y = 0.052x + 0.004, was obtained after three experiments, with a correlation coefficient of R0.052. 2 =0.996, where: y represents the measured absorbance, and x represents the mass concentration of galacturonic acid (μg / mL).

[0085] The pectin extraction yield Y is calculated according to formula (1).

[0086] Y = (N × V × A) / (m × 10) 6 )×100 (1)

[0087] In the formula: pectin yield is calculated as galacturonic acid produced after hydrolysis, %; N represents the mass concentration of galacturonic acid obtained from the standard curve, μg / mL; V represents the volume of the extract, mL; m represents the mass of hawthorn powder weighed, g; A represents the dilution factor of the extract.

[0088] 2. Determination of degree of esterification

[0089] The degree of esterification in pectin was determined by titration.

[0090] Dissolve 50 mg of dried hawthorn modified pectin in 100 mL of deionized water containing 2 mL of anhydrous ethanol. Add 5 drops of phenolphthalein and titrate the pectin solution with sodium hydroxide solution (0.05 mol / L). Record the titration volume when the solution turns red as the initial titration V1. Then add 10 mL of sodium hydroxide solution (0.05 mol / L), shake the sample vigorously, and let it stand at (20 ± 1) °C for 15 min. Add hydrochloric acid solution (1.5 mol / L) and shake the sample until the red color fades. Add 5 drops of phenolphthalein and titrate the sample with sodium hydroxide solution (0.05 mol / L). Record the titration volume when the solution turns red again as the final titration V2. The degree of esterification is calculated using the following formula.

[0091] Degree of esterification / % = V2 / (V1 + V2) × 100

[0092] In the formula: V1 is the initial titer of the solution / mL; V2 is the final titer of the solution / mL.

[0093] 3. Determination of molecular weight

[0094] Three columns were used in series: a TSK guard column (40 mm × 46 mm id), a TSKgel G3000PWxl column (300 mm × 7.8 mm i.d.), and a TSKgel G4000PWxl column (300 mm × 7.8 mm id). 100 mM NaNO3 containing 0.05% NaN3 was used as the mobile phase. A 2 mg / mL pectin sample was prepared using this mobile phase and filtered through a 0.45 μm aqueous filter. The injection volume was 20 μL, the column temperature was 40 °C, and the elution flow rate was 0.4 mL / min. The molecular weight of the sample (y = -0.00036x) was calculated using a standard curve. 3 +0.0413x 2 -1.703x + 29.37, R 2 =0.9997). The molecular weight standard is P-82 pullulanose, with molecular weights of 1080, 6100, 9600, 21100, 47100, 107000, 194000, 337000 and 708000 Da.

[0095] 4. Scanning electron microscope

[0096] A thin layer of gold was coated onto the crude polysaccharide fraction extracted via MAE after vacuum drying at 75°C for 24 hours using a benchtop sputtering system. Images were then captured using a field emission scanning electron microscope at a scale bar of 50 μm and an accelerating voltage of 15 kV.

[0097] 5. Infrared Spectroscopy

[0098] The dried pectin powder and KBr powder were ground together and pressed into a thin sheet approximately 1 mm thick. FTIR spectroscopy was performed using an air background. Scanning was conducted in absorption mode with a resolution of 4 cm⁻¹. -1 The frequency range is 4000-800cm. -1 The total number of scans was 32, and the absorption spectrum was obtained after denoising and baseline correction.

[0099] 6. Measurement of intrinsic viscosity (η)

[0100] The intrinsic viscosity of pectin samples was determined at 25℃ using the Ubbelohde viscometer method. Homogeneous solutions of different mass concentrations (1.0, 0.9, 0.8, 0.7, 0.6 mg / mL) were prepared by dissolving the pectin samples in distilled water. The outflow times of the solvent and the pectin solutions of different concentrations through a capillary tube (0.52 mm in diameter) were accurately recorded. The [η] value was calculated using the Huggins-Kramer equation.

[0101]

[0102]

[0103] In the formula, c is the concentration of the pectin solution, and η sp and η rel These are specific viscosity and relative viscosity, respectively.

[0104] 7.Pb 2+ Adsorption capacity

[0105] Weigh out Pb(NO3)2 to prepare a Pb solution with a concentration of 20 mg / L. 2+ Solution; use 0.1 mol / L HNO3 or NaOH to dissolve Pb 2+ The pH of the solution was adjusted to 5.0. At room temperature (25±1℃), 20 mg of modified pectin was added to 20 mL of the prepared Pb solution. 2+ The solution was then subjected to adsorption by shaking at 120 rpm for 4 hours in a constant temperature water bath shaker, followed by centrifugation at 12000 rpm for 10 minutes. The supernatant was then used to determine Pb using an atomic absorption spectrophotometer (AAS). 2+ The concentration of pectin for Pb. 2+ Adsorption amount q e The calculation formula (mg / g) is as follows:

[0106]

[0107] In the formula, C0 represents Pb. 2+ Initial solution concentration (mg / L); C e Pb after adsorption 2+ Solution concentration (mg / L); V represents the amount of Pb added. 2+ The volume of the solution (L); m represents the amount of pectin used (g).

[0108] The degree of esterification, molecular weight, intrinsic viscosity, and Pb of the hawthorn modified pectin obtained in Examples 1-3 and Comparative Examples 1-3 were analyzed. 2+ The adsorption capacity was determined, and the results are shown in Table 3 below.

[0109]

[0110] The structure of pectin is mainly composed of three domains: galacturonic acid polysaccharide (HG) in the smooth region, and rhamnose galacturonic acid polysaccharide type I (RG-I) and type II (RG-II) in the hair region. The ability of pectin to bind cations is mainly related to the HG in the pectin structure, while the degree of esterification (DE) refers to the percentage of methylated galacturonic acid in HG out of the total galacturonic acid, which has a very important influence on the functional properties of pectin.

[0111] Intrinsic viscosity ([η]) is a key parameter reflecting the hydrodynamic volume occupied by polymer molecules in a given solvent. A higher η value indicates that the polymer has more extended molecular chains. The modified pectin I obtained in the examples has a higher [η] value, which may be because modified pectin I contains more side chains, resulting in more extended chains than the modified pectin II to IV obtained in Comparative Examples 1 to 3.

[0112] Differences in the physicochemical properties of pectin, such as its degree of esterification, molecular weight distribution, and viscosity, all lead to variations in its ability to bind to metals. The modified pectin I in this embodiment of the invention has a low degree of esterification (only 26.8%), a high intrinsic viscosity of 421.2 mL / g, and a low molecular weight of 3.59 kDa, demonstrating good binding affinity to Pb. 2+ It has the strongest adsorption capacity.

[0113] Analysis suggests that the modified pectin I of this invention exhibits a low degree of esterification, small molecular weight, and high intrinsic viscosity, possibly due to the presence of a high amount of pectin methyl esterase and polygalacturonide endopeptidase in the Aspergillus niger fermentation broth. Pectin methyl esterase acts on the methyl ester bonds of pectin, reducing the degree of esterification and causing deesterification of the protopectin, thus reducing the COO content in the pectin. - Increase, available for Pb 2+ The binding sites are "exposed." Polygalacturonidase mainly acts on the α-1,4 glycosidic bonds of protopectin, causing them to break. The broken glycosidic bonds lead to shorter polymer chains and may also cause a decrease in the degree of polymerization of the pectin chain. Both of these results in a decrease in the molecular weight of the pectin. Pectins with lower molecular weights have a large number of short chains, increasing their intrinsic viscosity, allowing almost all active sites to participate in the binding with Pb. 2+ It comes into combination with the short-chain pectin molecules, and because the distance between the chains is shortened, it is easier to form Pb. 2+ The "binding region" enables modified pectin I to bind to Pb. 2+ The adsorption is stronger.

[0114] In addition, the effect of hawthorn pectin extraction on the modification was further analyzed.

[0115] The purified hawthorn pectin obtained in step a2 of Example 1 was compared with the hawthorn pectin obtained in Comparative Examples 4 and 5 in terms of extraction rate and degree of esterification. The results are shown in Table 4 below.

[0116]

[0117] As shown in Table 4, the hawthorn pectin extracted in the Examples and Comparative Examples 4 and 5 are all high-ester pectin (HMP), and there is a significant difference (P < 0.05), indicating that the extraction method affects the degree of esterification of pectin.

[0118] The degree of esterification of hawthorn pectin extracted in the examples was approximately 60%. The esterification degrees of hawthorn pectin extracted in Comparative Examples 4-1, 4-2, 5-1, and 5-2 ranged from 63% to 79%, while those in Comparative Examples 4-3 and 4-4 ranged from 50% to 57%. These data indicate that the extracted pectin molecules are relatively large, which is not conducive to the adsorption of heavy metals, necessitating modification of the hawthorn pectin. Analysis revealed that if the esterification degree is too high, achieving a specific range conducive to metal binding requires a large amount of enzyme solution and a long enzymatic hydrolysis period, increasing costs and introducing more uncontrollable factors that directly affect the modification results. Conversely, if the esterification degree is too low, enzymatic hydrolysis further reduces it, potentially damaging the pectin's original structure and failing to achieve the desired adsorption effect. The hawthorn pectin extraction method described in this invention is more suitable for subsequent modification of the Aspergillus niger fermentation broth, achieving optimal adsorption performance with heavy metal ions. Moreover, the high extraction rate of the embodiments is more conducive to reducing costs in industrial production.

[0119] By comparing the hawthorn pectin extracted in the examples with that extracted in comparative examples 4 and 5 using scanning electron microscopy, the microstructure of hawthorn pectin was studied. This further verifies that the extraction of hawthorn pectin in the examples of this invention is more suitable for the subsequent modification treatment of Aspergillus niger fermentation broth, and can obtain the conclusion that it has the best adsorption performance with heavy metal ions.

[0120] Figure 1 The image shown is a scanning electron microscope (SEM) image of modified pectin I obtained in an embodiment of the present invention. Figure 1 It can be seen that modified pectin I exhibits a loose structure at 2000x magnification, and a porous structure is observed on its surface at 5000x to 20000x magnification. This means that during the adsorption of heavy metal ions, it can increase the contact area with the heavy metal solution and provide more active sites for adsorbing heavy metal ions.

[0121] Figure 2The images show scanning electron microscope (SEM) images of hawthorn pectin from Comparative Examples 4-1 to 4-4. The images reveal that the hawthorn pectin extracted from Comparative Example 4-1 is pale yellow. At 100x magnification, its surface is slightly rough, exhibiting a sheet-like structure. At 500x to 1000x magnification, its surface appears relatively smooth. The hawthorn pectin extracted from Comparative Examples 4-2 and 4-3 exhibits a pale yellow, reticular structure, which does not meet the standard requirements for pectin texture in GB 25533-2010 National Food Safety Standard for Food Additives (Pectin). At 100x magnification, both Comparative Examples 4-2 and 4-3 show a filamentous structure. At 500x to 1000x magnification, the surface of Comparative Example 4-2 appears relatively rough, while the surface of Comparative Example 4-3 appears smoother at 500x to 1000x magnification. The hawthorn pectin extracted in Comparative Example 4-4 was reddish-brown, which did not meet the standard requirements for pectin color in the "National Food Safety Standard for Food Additives Pectin" (GB 25533-2010). Under 100x magnification, its surface was slightly rough and had a sheet-like structure. Under 500x to 1000x magnification, its surface was relatively smooth and its edges were thin.

[0122] Comparative Examples 4-1 to 4-4 exhibit a surface morphology of either a monolithic sheet-like structure or a network structure, neither of which is conducive to subsequent enzymatic modification and the adsorption of heavy metal ions. While the monolithic sheet-like structures of Comparative Examples 4-1 and 4-4 can be enzymatically modified, the number of exposed active sites after modification will be limited. Similarly, the network structures of Comparative Examples 4-2 and 4-3, when magnified, also exhibit a monolithic sheet-like surface, which is also unfavorable for subsequent enzymatic modification and the adsorption of heavy metal ions. Furthermore, their appearance does not meet the standard requirements for pectin texture in GB 25533-2010 National Food Safety Standard for Food Additives Pectin.

[0123] Figure 3 The images show SEM images of hawthorn pectin extracted in the examples and those extracted in Comparative Examples 5-1 and 5-2. The microstructure of the hawthorn pectin extracted in the examples differs significantly from that of Comparative Examples 5-1 and 5-2. Comparative Example 5-1 appears as a relatively smooth, solid sheet structure with thicker edges under low magnification, but under high magnification, its surface is rough and contains some protruding particles. Comparative Example 5-2 appears similar to Comparative Example 5-1 under low magnification, but its lamellar structure has obvious cavitation and thinner edges; under high magnification, many small air bubbles can be observed on its surface (possibly due to cavitation from ultrasound). The hawthorn pectin extracted in the examples has a fragmented structure under low magnification, with very thin edges in its lamellar structure, and many protruding small particles on its surface under high magnification.

[0124] Depend on Figure 3 The scanning electron microscope results show that the hawthorn pectin extracted in the example not only meets the standard requirements of "GB 25533-2010 National Food Safety Standard for Food Additives Pectin", but also has a fragmented structure on its surface with many small protruding particles. After subsequent enzymatic hydrolysis modification, more active sites can be exposed, which is more conducive to the adsorption of heavy metal ions. Moreover, this fragmented structure can also increase the contact area with the solvent.

[0125] The hawthorn pectin extracted in the previous examples and the hawthorn pectin extracted in comparative examples 4 and 5 were then analyzed by infrared spectroscopy. Figure 4 The infrared spectra of comparative examples 4-1 to 4-4 are shown below. Figure 5 Infrared spectra of the embodiments shown and comparative examples 5-1 and 5-2.

[0126] The hawthorn pectin extracted in the examples showed similar infrared spectral curves and possessed the same characteristic peaks as the hawthorn pectin extracted in Comparative Examples 4 and 5, but the peak intensities differed. (3600–3000 cm⁻¹) -1 Within this region, all pectins exhibit a broad peak, attributed to OH stretching vibrations and related to intramolecular and intermolecular hydrogen bonds in the Gal A polymer. This peak is observed in the 3000–2900 cm⁻¹ range. -1 The weak peak in the region corresponds to the stretching vibration of the CH bond. At 1735 cm⁻¹ -1 and 1604cm -1 The two nearby peaks are caused by the asymmetric stretching vibrations of the esterified carboxyl group (CO) and the free carboxyl group (C=O), respectively. (1200 cm⁻¹) -1 ~1400cm -1 The spectral bands in the region correspond to CH2 bends, OH bends, and -CH3CO. - Stretching. 1200cm -1 ~800cm -1 The bands between these bands are considered "fingerprint" regions of carbohydrates, reflecting variations in monosaccharide composition. At 10¹⁰ cm⁻¹... -1 ~1150cm -1 The bands between these bands indicate that the pectin sample contains pyranose. At 1102 cm⁻¹ -1 and 1019cm -1 The peaks at the points are attributed to the asymmetric stretching vibrations of the COC bond and the stretching vibrations of the CO and COH bonds, respectively.

[0127] In addition, it can be seen that the hawthorn pectin extracted in the example was at 1735 cm⁻¹. -1The peak area of ​​the extract is slightly larger than that of comparative examples 5-1 and 5-2, indicating that the hawthorn pectin extracted in this example contains a higher concentration of esterified carboxyl groups (CO). The pectin extracted in this example has a peak area of ​​1610 cm⁻¹. -1 The smaller peak area in the vicinity compared to Comparative Example 5-1 indicates that the hawthorn pectin extracted in this example contains fewer free carboxyl groups (C=O). This unique structure provides a basis for obtaining more active sites through subsequent enzyme modification. Furthermore, the hawthorn pectin extracted in this example has a peak area of ​​1102 cm⁻¹. -1 and 1019cm -1 The large peak indicates that it has more COC, CO, and COH bonds, meaning that its structure has more branches. This provides the possibility of exposing more active sites after subsequent enzyme modification treatment, so as to bind heavy metal ions and increase the adsorption capacity.

[0128] Based on the above data, it can be concluded that the hawthorn modified pectin obtained by this invention conforms to the relevant provisions for pectin in the "GB25533-2010 National Food Safety Standard for Food Additives Pectin". Under a microscopic scale, it has a loose and porous structure, which can increase the contact area with heavy metal ion solution. It contains more pectin branches and presents more active sites, which is conducive to contact with heavy metal ions, thereby significantly improving the adsorption effect of heavy metal ions.

[0129] In summary, this invention extracts hawthorn pectin using a choline chloride-urea solution, and then modifies the extracted pectin using Aspergillus niger fermentation broth obtained from Aspergillus niger fermentation. By altering the molecular structure of the pectin, it endows hawthorn pectin with excellent functional activity in adsorbing heavy metal ions, thereby obtaining a pectin product that can significantly improve the heavy metal adsorption rate. The development of this technology can effectively avoid the waste of hawthorn resources and is conducive to environmentally friendly and efficient solutions to heavy metal pollution problems. Furthermore, the pectin preparation adopts green production technology, which has extremely significant application significance and value for the remediation of non-renewable resources and the purification of food.

Claims

1. A modified pectin for adsorbing heavy metals, characterized in that: The modified hawthorn pectin is obtained by extracting hawthorn powder with choline chloride-urea solution, followed by modification treatment with fermentation broth of Aspergillus niger producing pectinase. The supernatant of the Aspergillus niger fermentation broth is obtained by centrifuging a pectinase-producing Aspergillus niger strain at 4-6℃ after activation at 25-30℃ and expansion culture at 25-35℃.

2. The modified pectin for adsorbing heavy metals according to claim 1, characterized in that: The modification process involves dissolving hawthorn pectin in a disodium hydrogen phosphate-citric acid buffer solution, adding Aspergillus niger fermentation broth, and reacting in a constant temperature water bath at 40-50°C.

3. The modified pectin for adsorbing heavy metals according to claim 1, characterized in that: The choline chloride-urea solution is mainly obtained by mixing choline chloride, urea and water. The molar ratio of choline chloride to urea in the solution is 1:3~4, and the water content of the solution is 30~40%.

4. The modified pectin for adsorbing heavy metals according to claim 1, characterized in that: The modified hawthorn pectin is a light yellow powder with a molecular weight of 3.5~4.5kDa and a degree of esterification of 25%~35%.

5. A method for preparing modified pectin for adsorbing heavy metals as described in any one of claims 1 to 4, characterized in that, The preparation method includes the following steps: a. Extraction of hawthorn pectin: Hawthorn powder was heated and extracted using choline chloride-urea solution. After cooling and centrifugation, the supernatant was collected to obtain hawthorn pectin solution. Ethanol was added to the hawthorn pectin solution to precipitate the pectin, which was then washed, purified, and dried to obtain hawthorn pectin. b. Modification of hawthorn pectin: The hawthorn pectin prepared in step a was dissolved in disodium hydrogen phosphate-citric acid buffer solution, and the Aspergillus niger fermentation broth was added for constant temperature water bath reaction. Then, it was treated with boiling water bath, cooled to room temperature, and finally purified and dried to obtain modified hawthorn pectin.

6. The method for preparing modified pectin for adsorbing heavy metals according to claim 5, characterized in that, The extraction process of hawthorn pectin includes: a1. Use choline chloride-urea solution to heat and extract the sieved hawthorn powder. The ratio of hawthorn powder to choline chloride-urea solution is 1g:30~40mL. The extraction temperature is 70~80℃ and the extraction time is 60~80min. After the extraction is completed, let the extract cool to room temperature, centrifuge and collect the supernatant, which is the hawthorn pectin solution. a2. Add 85-95% ethanol to the hawthorn pectin solution obtained in step a1 to precipitate the pectin and wash out the impurities by stirring thoroughly. After centrifugation, discard the supernatant and collect the precipitate. Wash it again with ethanol 1-4 times. After the ethanol evaporates, dissolve the obtained pectin precipitate in distilled water, put it into a dialysis bag, and dialyze it with distilled water as the dialysis solution for purification. Freeze-dry to obtain hawthorn pectin for later use.

7. The method for preparing modified pectin for adsorbing heavy metals according to claim 5, characterized in that, The modification process for hawthorn pectin includes: b1. After activating and scaling up the pectinase-producing Aspergillus niger strain, the fermentation broth is centrifuged at 4-6℃, and the supernatant is collected to obtain Aspergillus niger fermentation broth; the activation conditions are 25-30℃ and 24-48h activation culture; the scaling up culture conditions are 2-8% inoculum, 25-35℃ and 36-72h fermentation culture. b2. Take the hawthorn pectin prepared in step a2 and dissolve it in a pH 4-5 disodium hydrogen phosphate-citric acid buffer solution to make the concentration of hawthorn pectin 25-30 mg / mL; add Aspergillus niger fermentation broth, react in a constant temperature water bath at 40-50℃ for 2-4 hours, then treat with a boiling water bath, cool to room temperature, dissolve in distilled water, put into a dialysis bag, dialyze and purify with distilled water as the dialysate, freeze dry to obtain modified hawthorn pectin.

8. The method for preparing modified pectin for adsorbing heavy metals according to claim 7, characterized in that: In step b2, the amount of Aspergillus niger fermentation broth added is 10~200 U / mL; and the mixture is treated in a boiling water bath for 5 min.

9. The method for preparing modified pectin for adsorbing heavy metals according to claim 6 or 7, characterized in that: In steps a2 and b2, the dialysis bag should be 2000~8000 Da, the dialysis time should be 24~48 hours and the dialysis fluid should be changed every 2~6 hours, and the freeze-drying time should be 24~48 hours.

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