A attapulgite / antibacterial lipopeptide composite material and its preparation method and application

Abstract

The present invention discloses a composite material of attapulgite / antimicrobial lipopeptide, a preparation method and an application thereof. Natural attapulgite is modified by sulfuric acid, and the modified attapulgite is used to load the antimicrobial lipopeptide bacillusin D. Then, a single factor test is used to optimize the conditions for loading bacillusin D on the modified attapulgite, and the prepared composite material is characterized by attenuated total reflection technology and X-ray diffractometer technology. Under unit conditions, the modified attapulgite can load more than 367.97 mg / L of bacillusin D at 2h and 40°C, and the loading rate is as high as 93.13%. At the same time, the composite material under unit conditions can remove 11.08 ppb of aflatoxin B at 2h and 50°C. 1 The composite material prepared by the present invention can not only resist bacteria but also remove fungal toxins, and has good stability and a wide range of applications, thus solving the problems of poor antibacterial performance of inorganic materials and poor stability, acid and alkali resistance, and easy oxidation of organic materials.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to an attapulgite / antibacterial lipopeptide composite material and a preparation method and application thereof. Background Art

[0002] As people's living standards improve and they pay more attention to healthy living, their demands for living environment and health care are also increasing. Especially in terms of food safety, how to eat safely and at ease has become the primary issue that most people consider. The ubiquitous harmful fungi are the main cause of food safety hazards. Therefore, the research and development of antimicrobial agents has received more and more attention.

[0003] Bacillomycin D (BD) is a natural antibacterial lipopeptide produced by the secondary metabolism of Bacillus. It is a cyclic lipopeptide composed of a hydrophilic heptapeptide and a hydrophobic β-amino fatty acid chain (Anti-toxicogenic fungi and toxin-reducing effects of bacillomycin D in combination with fungicides. Toxicon, 2022, 216, 107-113.). Bacillomycin D has good antifungal and antitumor biological activities (Biological activity of lipopeptides from Bacillus. Applied Microbiology and Biotechnology, 2017, 101(15): 5951-5960), which makes it have broad application prospects in food preservation, grain storage and biomedicine (Effect of combined Bacillomycin Dand chitosan on growth of Rhizopus stolonifer and Botrytis cinerea and cherry tomato preservation. Journal of the Science of Food and Agriculture, 2020, 101(1): 229-239; Microbial lipopeptide supramolecular self-assemblies as amethuosis-like cell death inducer with in vivo antitumor activity. Small, 2021, 18(3): e2104034).

[0004] Aflatoxins are secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus, which are highly toxic and carcinogenic (Relationship between drought and preharvest aflatoxin contamination in groundnut (Arachis hypogaea L.). World Mycotoxin Journal, 2018, 11: 187-199). These fungi can infect crops, including peanuts and corn, during growth, harvesting, drying, storage and other processes (Worldwide aflatoxin contamination of agricultural products and foods: From occurrence to control. Comprehensive Reviews in Food Science and Food Safety, 2021, 20: 2332-2381.). In recent years, many research hotspots have focused on preventing and controlling the growth of fungi and mycotoxins.

[0005] Attapulgite (ATP, Jiangsu Jiuchuan Nanomaterial Technology Co., Ltd.) is a crystalline hydrated magnesium aluminum silicate clay mineral with a layered chain fibrous structure. It is widely found all over the world and is inexpensive (Traditional and new applications for kaolin, smectite, and palygorskite: a general overview. Applied Clay Science, 2000, 17(5): 207-221). It consists of two continuous tetrahedral layers and a 2:1 band-shaped discontinuous octahedral layer. The interior of the crystal is loose and porous, the outer surface is concave and convex, and the edge has multiple Si-OH bonds (Effect of pH, ionic strength, temperature and humic substances on the sorption of Ni(II) to Na-attapulgite. Chemical Engineering Journal, 2008, 150(1): 188-195). The basic structure of attapulgite makes it a natural nanomaterial that exhibits various excellent physical and chemical properties, such as adsorption and rheological properties (Preparation of well-defined starpolymer from hyperbranched macroinitiator based attapulgite by surface-initiated atom transfer radical polymerization technique. Industrial & Engineering Chemistry Research, 2007, 46(1): 97-102). Its adsorption performance depends on its large specific surface area, surface physicochemical structure and its ionic state. Its adsorption effects include physical adsorption, chemical adsorption and ion exchange adsorption (Magnetic gamma-Fe2O3-loaded attapulgite sorbent for Hg-0removal incoal-fired flue gas. Energy & Fuel, 2019, 33(8): 7522-7533).Therefore, attapulgite is widely used in chemical industry, light industry, agriculture, textile, building materials, geological exploration, casting, silicate industry, atomic energy industry, environmental protection and pharmaceutical fields. It is acid and alkali resistant and has good stability. It is an excellent material carrier. Secondly, attapulgite has been listed as a food processing aid allowed for use in food in GB 29225-2012. In addition to the adsorption and removal of heavy metal ions in the environment, attapulgite is also gradually added to food. However, its antibacterial performance is poor (Characterization of a newantibiotic of iturin group: bacillomycin DJ Antibiot (Tokyo). 1980; 33 (10): 1146-9.), which is also a more important problem.

[0006] Bacillomycin D has good antifungal and antitumor biological activities (Bacillomycin D: An iturin with antifungal activity against Aspergillus flavus. Journal of Applied Microbiology, 2001, 90(4): 622-629.), which makes it have broad application prospects in food preservation, grain preservation and biomedicine (Effect of combined Bacillomycin D and chitosan on growth of Rhizopus stolonifer and Botrytis cinerea and cherry tomato preservation. Journal of the Science of Food and Agriculture, 2020, 101(1): 229-239; Microbial lipopeptide supramolecular self-assemblies as a methuosis-like cell death inducer with in vivo antitumor activity. Small, 2021, 18(3): e2104034).

[0007] Attapulgite is approved by the USDA, recognized by AAFCO, and has passed the USDA review. It is approved by the FDA and can be used in animal feed. The basic structure of attapulgite makes it a natural nanomaterial, thus showing various excellent physical and chemical properties, such as adsorption, rheology, etc. It is widely used in chemical industry, light industry, agriculture, textile, building materials, geological exploration, casting, silicate industry, atomic energy industry, environmental protection and pharmaceutical fields.

[0008] In the prior art, the application of fungal toxins such as bacillusmycin D is mainly direct application or direct fermentation of bacillusmycin D-producing strains to achieve antibacterial effects. Due to the defects of bacillusmycin D such as easy oxidation and poor stability, this may cause a certain loss of the antibacterial effect of bacillusmycin D. At present, few scholars have studied materials that can load bacillusmycin D to improve its shortcomings such as poor stability. Summary of the invention

[0009] In view of the problems mentioned in the background technology, the present invention provides a composite material of attapulgite / antimicrobial lipopeptide and a preparation method thereof, wherein natural attapulgite is modified by sulfuric acid, and then the modified attapulgite is used to load fungal toxins such as bacillus mycin D, and the modified attapulgite is firmly bound to the surface by covalent bonds to prepare a new type of composite material, which is recorded as H-ATP-BD. The attapulgite / antimicrobial lipopeptide composite material prepared by the present invention has good antibacterial performance and good stability, and solves the problems of poor water solubility, alkali resistance, easy oxidation, and poor stability of bacillus mycin D, so that it can be better applied to food preservation, grain storage and other fields.

[0010] The above technical objectives of the present invention are achieved through the following technical solutions:

[0011] A method for preparing a attapulgite / antimicrobial lipopeptide composite material, characterized in that it comprises the following steps:

[0012] Step (1): Preparation of bacillus mycin D: extracting bacillus mycin D, diluting bacillus mycin D with deionized water, and freeze-drying;

[0013] Step (2) preparing modified attapulgite with an acidic solution;

[0014] Step (3) Loading of modified attapulgite on bacillusmycin D: Take the modified attapulgite in step (2), add the bacillusmycin D aqueous solution, load for a period of time, and then perform ultrasonic treatment to obtain a attapulgite / antibacterial lipopeptide composite material, recorded as H-ATP-BD.

[0015] Preferably, in step (1), the dilution ratio of bacillusin D to deionized water is 1:30-1:50 (v:v). The dilution ratio of bacillusin D to deionized water is any value within the range of 1:30-1:50, such as 1:30, 1:35, 1:40, 1:45, 1:50.

[0016] In any of the above schemes, preferably, in step (2), the acidic solution is sulfuric acid, and the sulfuric acid concentration is 0.5-4 mol / L. The sulfuric acid concentration is any value within the range of 0.5-4 mol / L, such as 0.5 mol / L, 1 mol / L, 2 mol / L, 3 mol / L, 4 mol / L.

[0017] Preferably, in any of the above schemes, in step (3), during the process of loading the modified attapulgite with bacillusmycin D, the amount of modified attapulgite added is 0.1%-0.5wt%, the loading temperature is 30-70°C, the stirring rate is 300-700r / min, and the loading time is 0-300min. Specifically, the addition amount of modified attapulgite can be any value in the range of 0.1%-0.5wt%, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%; the load temperature is any value in the range of 30-70℃, such as 30℃, 35℃, 40℃, 45℃, 50℃, 60℃, 70℃; the stirring rate is any value in the range of 300-700r / min, such as 300r / min, 400r / min, 450r / min, 500r / min, 550r / min, 600r / min, 700r / min; the load time is 0-300min, such as 1min, 5min, 50min, 100min, 150min, 200min, 300min.

[0018] Preferably, in any of the above schemes, in step (3), during the process of loading the modified attapulgite with bacillusmycin D, the amount of modified attapulgite added is 0.2-0.4wt%, the loading temperature is 35-45°C, the stirring rate is 450-550r / min, and the loading time is 100-150min.

[0019] Preferably in any of the above schemes, in step (3), the ultrasonic treatment condition is 40 kHz for 30 min.

[0020] The invention also discloses an attapulgite / antibacterial lipopeptide composite material, which is prepared by the above-mentioned preparation method.

[0021] The invention also discloses an application of the attapulgite / antimicrobial lipopeptide composite material in removing fungal toxins.

[0022] Preferably, the fungal toxins include at least one or more of aflatoxin and / or aspergillus niger and / or bacillus mycin.

[0023] In any of the above schemes, preferably, the aflatoxin is aflatoxin B1, and the bacillus mycin is bacillus mycin D.

[0024] Preferably, the method of using the attapulgite / antimicrobial lipopeptide composite material for removing aflatoxin and / or Aspergillus niger is as follows: prepare in advance a mother solution of removing aflatoxin and / or Aspergillus niger, and add the aflatoxin and / or Aspergillus niger solution and the attapulgite / antimicrobial lipopeptide composite material prepared by the above-mentioned preparation method into a beaker, respectively.

[0025] Aflatoxin is specifically aflatoxin B1. The present application studies the removal effect of the composite material on aflatoxin B1 at different initial concentrations of aflatoxin B1, different amounts of composite material added, different removal times, and different removal temperatures.

[0026] The invention discloses a preparation method and application of a attapulgite / antimicrobial lipopeptide composite material. Natural attapulgite is modified by sulfuric acid, and the modified attapulgite (inorganic) is used to load the antimicrobial lipopeptide bacillusin D (organic). Then, a single factor experiment is used to optimize the conditions of the modified attapulgite loading bacillusin D, and the prepared composite material is characterized by attenuated total reflection technology and X-ray diffractometer technology.

[0027] Beneficial Effects

[0028] 1. The present invention adopts sulfuric acid to modify natural attapulgite, removes some carbonate impurities it contains, increases its surface adsorption sites, and efficiently improves the loading effect. Then, the modified attapulgite is used to load bacillusin D, and it is firmly bound to the surface by covalent bonds to prepare a new type of composite material (recorded as H-ATP-BD). Under unit conditions, the modified attapulgite can load more than 367.97 mg / L of bacillusin D under 2h and 40°C, with a loading rate of up to 93.13%, and there is no significant difference in the inhibitory effect of the prepared composite material and bacillusin D on mold. Secondly, bacillusin D is fixed to the surface of the modified attapulgite by covalent bonding, and has a strong anti-desorption ability. At the same time, the composite material under unit conditions can remove 11.08 ppb of aflatoxin B1 in 2 hours and 50°C, and the removal rate can reach more than 80%. The composite material prepared by the present invention has good antibacterial performance and good stability, and solves the shortcomings of bacillus D such as poor water solubility, alkali resistance, easy oxidation, and poor stability, so that it can be better used in food preservation, grain storage and other fields.

[0029] 2. The present invention uses natural attapulgite as an excellent carrier, and uses sulfuric acid to modify it and efficiently load bacillus mycin D to prepare a composite material that has good antifungal activity and can remove mycotoxins, providing a practical technology for solving the problem of food contaminated by mycotoxins.

[0030] 3. The modified attapulgite prepared by the method of the present invention has a good loading effect on bacillusin D. The maximum loading amount of bacillusin D per unit of modified attapulgite can reach 367.97 mg / L, and the loading rate can reach 93.13%. The loading mode of modified attapulgite on bacillusin D is chemical adsorption through covalent bond binding. Compared with physical adsorption, its loading effect is firm and stable, reducing the risk of falling off after loading.

[0031] 4. The composite material prepared by the method of the present invention also has a good removal effect on aflatoxin B1. The removal amount of aflatoxin B1 per unit composite material can reach 11.08 ppb, and the removal rate is more than 80%. In addition, the composite material is green and easy to remove.

[0032] 5. The preparation method of the present invention is an organic (bacitracin D) / inorganic (modified attapulgite) composite method, which can make the advantages of organic materials and inorganic materials complement each other, and the prepared composite material has the advantages of both attapulgite and bacillus D.

[0033] 6. The method for preparing the modified attapulgite capable of loading bacillusin D of the present invention has low cost, simple preparation method, and will not cause environmental pollution and waste of resources. Moreover, attapulgite mineral resources are extremely abundant and are already food-grade additives, which can be directly applied to grain storage, fruit and vegetable preservation, animal feed and biomedicine.

[0034] 7. The composite material prepared by the present invention can be both antibacterial and fungal toxin-removing, and has good stability and a wide range of applications, solving the limitations of poor antibacterial performance of inorganic materials and poor stability, acid and alkali resistance, and easy oxidation of organic materials. Secondly, the composite material prepared by the preparation method of the present invention is inexpensive, does not use organic solvents, is not easy to cause secondary pollution, and does not pollute the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a flow chart of the preparation method of the attapulgite / antimicrobial lipopeptide composite material of the present invention;

[0036] Figure 2 : ATR graph of modified attapulgite prepared in the present invention; A is the ATR graph of natural attapulgite and modified attapulgite, and B is the ATR graph of attapulgite loaded with bacillusin D;

[0037] Figure 3 The SEM images of the modified attapulgite prepared in the present invention; A is natural attapulgite, B is attapulgite modified with 0.5 mol / L sulfuric acid, C is attapulgite modified with 1 mol / L sulfuric acid, D is attapulgite modified with 2 mol / L sulfuric acid, and E is attapulgite modified with 4 mol / L sulfuric acid;

[0038] Figure 4 Figure 1 is a trend chart of the change of modified attapulgite loaded with bacillusmycin D in different single factor experiments in the present invention; Figure A shows the effect of different concentrations of sulfuric acid-modified attapulgite on the loaded bacillusmycin D; Figure B shows the effect of different amounts of modified attapulgite added on the loaded bacillusmycin D; Figure C shows the effect of different temperatures on the loaded bacillusmycin D; Figure D shows the effect of different loading times on the loaded bacillusmycin D; Figure E shows the effect of different initial concentrations of bacillusmycin D on the load.

[0039] Figure 5 The XRD diagram of the composite material obtained by single factor optimization of the present invention;

[0040] Figure 6 This is an antibacterial test of the composite material prepared by the present invention after single factor optimization on Aspergillus flavus;

[0041] Figure 7 This is an antibacterial test of the composite material prepared by the present invention after single factor optimization on Aspergillus niger;

[0042] Figure 8 This is an experiment on the removal of aflatoxin B1 by the composite material prepared after single factor optimization of the present invention; Figure A shows the effect of different aflatoxin concentrations on the removal; Figure B shows the effect of different amounts of composite material added on the aflatoxin removal; Figure C shows the effect of different temperatures on the aflatoxin removal; Figure D shows the effect of different removal times on the aflatoxin removal. DETAILED DESCRIPTION

[0043] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only those obtained by the technicians of the present invention without creative work. All other embodiments belong to the scope of protection of the present invention. Unless otherwise specified, the relevant materials appearing in the subsequent embodiments are prepared by the previous embodiments.

[0044] Example 1

[0045] A method for preparing a attapulgite / antibacterial lipopeptide composite material, the preparation flow chart is as follows Figure 1 As shown, specifically including:

[0046] Modification of attapulgite: Take 10g of natural attapulgite (Jiangsu Jiuchuan Nanomaterial Technology Co., Ltd., 200 mesh), add 50mL of sulfuric acid with a concentration of 0.5-4.0mol / L, vortex mix for 30s, stir in a 90℃ water bath for 3h, filter while hot, wash with ultrapure water several times until neutral, put it in an electric blast drying oven at 45℃ and dry it for 24h, grind it, pass it through a 100-mesh sieve, and obtain attapulgite modified with different acids, which are recorded as 0.5MH-ATP, 1M-H-ATP, 2M-H-ATP, and 4M-H-ATP respectively;

[0047] Figure 3 The SEM images of the modified attapulgite prepared in the present invention; A is natural attapulgite, B is attapulgite modified with 0.5 mol / L sulfuric acid, C is attapulgite modified with 1 mol / L sulfuric acid, D is attapulgite modified with 2 mol / L sulfuric acid, and E is attapulgite modified with 4 mol / L sulfuric acid;

[0048] Preparation of bacillomycin D: The method of Gong Qingwei et al. (Identification of bacillomycin D from Bacillus subtilis fmbJ and its inhibition effects against Aspergillus flavus. Food Control 2014; 36(1): 8-14) was used to extract bacillomycin D. Bacillomycin D was diluted with ultrapure water at 1:30 (v / v) and then freeze-dried; the freeze-dried sample was reconstituted with ultrapure water to obtain the bacillomycin D solution required for the experiment.

[0049] Example 2

[0050] 20 mg of the attapulgite before modification and the modified attapulgite in Example 1 were added to 5 mL of bacillus mycin D solution (prepared in Example 1), vortex mixed for 30 seconds, stirred at 500 r / min for 2 hours at room temperature, and ultrasonicated for 30 minutes to obtain a attapulgite / antibacterial lipopeptide composite material loaded with bacillus mycin D.

[0051] Example 3

[0052] Take 1 mL of modified attapulgite loaded with bacillomycin D (prepared in Example 2), centrifuge at 4°C and 8000 r / min for 4 min; take the supernatant and filter it through a 0.45 μm inorganic filter membrane, and use the method of Gong Qingwei et al. (Identification of bacillomycin D from Bacillus subtilis fmbJ and its inhibition effects against Aspergillus flavus. Food Control 2014; 36(1): 8-14) to determine the concentration of bacillomycin D using a high performance liquid chromatograph, and calculate its loading rate. The loading rate calculation formula is as follows:

[0053]

[0054] Where: c0 is the initial concentration of bacillus spp. D (mg / L); c e is the concentration of bacillus D when the load reaches equilibrium (mg / L).

[0055] Example 4

[0056] Take 20 mg of attapulgite modified with different acid concentrations, add it to 5 mL of bacillus mycin D solution (prepared in Example 1), vortex mix for 30 seconds, stir at 500 r / min for 2 hours at room temperature, and ultrasonicate for 30 minutes. Centrifuge the attapulgite solution loaded with bacillus mycin D at 4°C and 8000 r / min for 4 minutes; take the supernatant and filter it through a 0.45 μm inorganic filter membrane, and use the method of Gong Qingwei et al. to determine the concentration of bacillus mycin D using a high performance liquid chromatograph, and calculate its loading rate. Among them, if Figure 4 As shown in A, a sulfuric acid concentration of 1 mol / L is preferably selected to modify attapulgite, and the loading rate can reach 90.30%.

[0057] Example 5

[0058] Take 10-50 mg of 1 mol / L sulfuric acid-modified attapulgite, add it to 5 mL of bacillus mycin D solution, vortex mix for 30 seconds, stir at 500 r / min for 2 hours at room temperature, and ultrasonicate for 30 minutes. Centrifuge the attapulgite solution loaded with bacillus mycin D at 4°C and 8000 r / min for 4 minutes; take the supernatant and filter it through a 0.45 μm inorganic filter membrane, and use the method of Gong Qingwei et al. to determine the concentration of bacillus mycin D using a high performance liquid chromatograph, and calculate its loading rate. Among them, if Figure 4 As shown in B, 30 mg of acid-modified attapulgite is preferably selected to load bacillusin D, and the loading rate can reach 92.51%.

[0059] Example 6

[0060] Take 30 mg of 1 mol / L sulfuric acid-modified attapulgite and add it to 5 mL of bacillus mycin D solution. Vortex mix for 30 seconds, stir at 500 r / min for 2 hours at 30-70°C, and ultrasonicate for 30 minutes. Centrifuge the attapulgite solution loaded with bacillus mycin D at 4°C and 8000 r / min for 4 minutes; take the supernatant and filter it through a 0.45 μm inorganic filter membrane. Use the method of Gong Qingwei et al. to determine the concentration of bacillus mycin D using a high performance liquid chromatograph and calculate its loading rate. Among them, if Figure 4 As shown in Figure C, the acid-modified attapulgite is preferably selected to load bacillusin D at 40°C, and the loading rate can reach 92.46%.

[0061] Example 7

[0062] Take 30 mg of 1 mol / L sulfuric acid-modified attapulgite and add it to 5 mL of bacillus mycin D solution. Vortex mix for 30 seconds, stir at 500 r / min for 0-360 minutes at 40°C, and ultrasonicate for 30 minutes. Centrifuge the attapulgite solution loaded with bacillus mycin D at 4°C and 8000 r / min for 4 minutes; take the supernatant and filter it through a 0.45 μm inorganic filter membrane. Use the method of Gong Qingwei et al. to determine the concentration of bacillus mycin D using a high performance liquid chromatograph, and calculate its loading rate. Among them, if Figure 4 As shown in D, the acid-modified attapulgite is preferably selected to load bacillusin D under the condition of stirring at 400 r / min for 240 min, and the loading rate can reach 93.13%.

[0063] Example 8

[0064] Characterization experiments such as Fourier transform infrared spectroscopy, scanning electron microscopy and X-ray diffractometer proved that bacillusmycin D was loaded on the surface of attapulgite through chemical reaction, which better protected the biological activity of bacillusmycin D and did not reduce the antibacterial properties of bacillusmycin D.

[0065] The Fourier transform infrared spectroscopy results show that the attapulgite material of the present invention removes some carbonate impurities carried by itself after acid modification, which is beneficial to the subsequent loading of bacillus mycin D, such as Figure 2 A. The infrared spectrum of modified attapulgite loaded with bacillus mycin D has an additional peak at 1535 cm -1 The stretching vibration absorption peak of the aromatic carbon skeleton appeared near the surface, indicating that bacillusmycin D was firmly bound to the surface of the modified attapulgite by covalent bonds. Figure 2 As shown in B.

[0066] Scanning electron microscopy shows that the attapulgite material used in the present invention is micron-sized. And as sulfuric acid modifies it, its surface pore structure shows a trend of increasing first and then decreasing, indicating that too high acid concentration may damage its crystal structure and reduce its surface adsorption active sites. It shows that selecting a good sulfuric acid concentration is more conducive to loading bacillus D on the modified attapulgite. Figure 5 shown.

[0067] The X-ray diffraction results show that the modified attapulgite loaded with bacillusin D has significantly more crystal characteristic peaks of bacillusin D located at 2θ=31.50° and 45.27°, indicating that bacillusin D has been successfully loaded on the surface of attapulgite. Figure 5 shown.

[0068] Example 9

[0069] The results of the antibacterial experiment show that the prepared composite material has a significant inhibitory effect on Aspergillus niger (A. niger, MK779059.1) and Aspergillus flavus (A. oryzae, MTO93257.1), which are common in food. Among them, the modified attapulgite has no antibacterial effect on Aspergillus flavus and Aspergillus niger, the inhibition zone size of bacillus mycin D on Aspergillus flavus is (20.95±0.56) mm, and the inhibition zone size of the composite material prepared by the present invention on Aspergillus flavus is (20.30±0.21) mm. Figure 6 As shown in FIG. 1 , the size of the inhibition zone of bacillus mycin D against Aspergillus niger is (20.22±0.02) mm, and the size of the inhibition zone of the composite material prepared by the present invention against Aspergillus niger is 19.70±0.36) mm. Figure 7 As shown, there was no significant difference, indicating that the prepared composite material has good antifungal activity.

[0070] Example 10

[0071] Take 20 mg of the composite material prepared by the optimal conditions of the above embodiment, add it to 5 mL of aflatoxin B1 solution of different concentrations, vortex mix for 30 seconds, and stir at 500 r / min for 2 hours at room temperature. Centrifuge the mixed solution at 4°C and 6000 r / min for 10 minutes; take the supernatant and pass it through a 0.22 μm organic filter membrane, and use the method of Wang Shaoying (Isotope internal standard-ultra-performance liquid chromatography-tandem mass spectrometry to detect aflatoxin B1 in peanuts and its products and its bioavailability. Journal of Food Safety and Quality, 2021, 12(22): 8643-8650.) et al. to determine the concentration of aflatoxin B1 by ultra-performance liquid chromatography-tandem mass spectrometry, and calculate its removal efficiency. The removal rate calculation formula is as follows:

[0072]

[0073] Where: C0 is the initial concentration of aflatoxin B1 (ppb); C e Aflatoxin B1 concentration at which removal reaches equilibrium (ppb)

[0074] Among them, Figure 8 As shown in Figure A, as the initial concentration of aflatoxin B1 increases, the removal efficiency of the composite material prepared by the present invention for aflatoxin B1 first increases and then decreases. This is because during the initial removal, the composite material surface has more removal sites, so the removal efficiency is very high. As the concentration of aflatoxin B1 increases, the removal sites of the composite material are gradually filled, resulting in a decrease in the removal efficiency. Preferably, the concentration of aflatoxin B1 used in subsequent experiments is about 30 ppb.

[0075] Embodiment 11

[0076] Take 10-50 mg of the composite material, add it to 5 mL, 36.40 ppb aflatoxin B1 solution, vortex mix for 30 seconds, and stir at 500 r / min for 2 hours at room temperature. Centrifuge the mixture at 4°C and 6000 r / min for 10 minutes; take the supernatant and filter it through a 0.22 μm organic filter membrane. Use the method of Wang Shaoying et al. to determine the concentration of aflatoxin B1 by ultra-high performance liquid chromatography-tandem mass spectrometry, and calculate its removal efficiency. Among them, if Figure 8 As shown in B, 30 mg of the composite material is preferably selected to remove aflatoxin B1, and the removal rate can reach 52.66%.

[0077] Example 12

[0078] Take 30 mg of the composite material, add it to 5 mL, 44.15 ppb aflatoxin B solution, vortex mix for 30 seconds, and stir at 500 r / min for 0-360 minutes at 50°C. Centrifuge the mixture at 4°C and 6000 r / min for 10 minutes; take the supernatant and filter it through a 0.22 μm organic filter membrane. Use the method of Wang Shaoying et al. to determine the concentration of aflatoxin B1 by ultra-high performance liquid chromatography-tandem mass spectrometry, and calculate its removal efficiency. Among them, if Figure 8 As shown in C, the time for the optimal composite material to remove aflatoxin B1 is 120 minutes, and the removal rate can reach 66.49%.

[0079] Embodiment 13

[0080] Take 30 mg of the composite material, add it to 5 mL, 44.20 ppb aflatoxin B1 solution, vortex mix for 30 seconds, and stir at 500 r / min for 2 hours at 20-70°C. Centrifuge the mixture at 4°C and 6000 r / min for 10 minutes; take the supernatant and filter it through a 0.22 μm organic filter membrane. Use the method of Wang Shaoying et al. to determine the concentration of aflatoxin B1 by ultra-high performance liquid chromatography-tandem mass spectrometry, and calculate its removal efficiency. Among them, if Figure 8 As shown in D, the composite material is preferably selected to remove aflatoxin at 50°C, and the removal rate can reach 89.06%.

[0081] The present invention provides an attapulgite / antimicrobial lipopeptide composite material and a preparation method thereof. The prepared composite material can be well applied in grain storage, fruit and vegetable preservation and animal feed.

[0082] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a attapulgite / antimicrobial lipopeptide composite material, characterized in that: The following steps are involved: Step (1): Preparation of bacillus mycin D: extracting bacillus mycin D, diluting bacillus mycin D with deionized water, and freeze-drying; Step (2): preparing modified attapulgite with an acidic solution, the acidic solution is sulfuric acid, the sulfuric acid concentration is 0.5-4 mol / L, taking natural attapulgite, adding it to sulfuric acid, vortex mixing for 30 seconds, stirring in a 90°C water bath for 3 hours, filtering while hot, washing with ultrapure water for multiple times until neutral, placing it in an electric blast drying oven at 45°C for 24 hours, grinding it, and passing it through a 100-mesh sieve to obtain acid-modified attapulgite; Step (3): Loading of bacillus mycin D on modified attapulgite: Take the modified attapulgite in step (2), add the bacillus mycin D aqueous solution, load for a period of time, and then perform ultrasonic treatment to obtain a attapulgite / antibacterial lipopeptide composite material; the added amount of modified attapulgite is 0.1%-0.5wt%, the loading temperature is 30-70°C, the stirring rate is 300-700r / min, and the loading time is 5-300min.

2. The method for preparing a attapulgite / antimicrobial lipopeptide composite material according to claim 1, characterized in that: In step (1), the dilution ratio of bacillus mycin D to deionized water is 1:30-1:50 (v:v).

3. The method for preparing a attapulgite / antimicrobial lipopeptide composite material according to claim 1, characterized in that: In step (3), during the process of loading bacillus mycin D on the modified attapulgite, the addition amount of the modified attapulgite is 0.2-0.4wt%, the loading temperature is 35-45°C, the stirring rate is 450-550r / min, and the loading time is 100-150 min.

4. The method for preparing a attapulgite / antimicrobial lipopeptide composite material according to claim 1, characterized in that: In step (3), the ultrasonic treatment condition is 40 kHz for 30 min.

5. An attapulgite / antibacterial lipopeptide composite material, prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the attapulgite / antimicrobial lipopeptide composite material according to claim 5 in removing aflatoxin B1 and / or Aspergillus niger.

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