Preparation method and application of attapulgite composite enzyme decolorizing agent

Abstract

The invention discloses a preparation method and application of a attapulgite composite enzyme decolorizing agent, and belongs to the technical field of oil processing. The pore structure of the attapulgite of the invention is destroyed during high-temperature carbonization, and the surface area and total pore volume of the attapulgite are increased. The biological carbon source increases the surface functional groups of the attapulgite, and also significantly increases the micropore surface area and micropore volume of the attapulgite, so that the adsorption points and pore volume of the attapulgite are increased, and the decolorizing ability of the attapulgite is enhanced. At the same time, more binding sites are provided, which is conducive to the immobilization of laccase. Laccase can convert oxidized phenolic substances into polyphenol oxides, and then the polyphenol oxides themselves polymerize to form large particles, which are filtered out, and effectively remove harmful polycyclic aromatic hydrocarbons such as benzopyrene. The attapulgite composite enzyme decolorizing agent of the invention can not only remove pigments in crude oil, but also remove byproducts in oil and fat and harmful substances that are not conducive to food safety, such as benzopyrene, and can also retain the flavor of oil to the greatest extent.

Description

Technical Field

[0001] The invention relates to a preparation method and application of an attapulgite composite enzyme decolorizing agent, and belongs to the technical field of oil processing. Background Art

[0002] In order to ensure the quality of edible oil, the oil mill will refine it during the processing to remove various impurities and harmful substances in the oil and ensure that it meets national standards. In the refining process, bleaching is one of the main steps. The main purpose of edible oil bleaching is to reduce the color of the oil and make it light or natural. However, today's edible oil bleaching is not only to remove pigments and improve the color of oil, but more importantly to remove possible by-products and harmful substances that are not conducive to food safety in the oil, such as polycyclic aromatic hydrocarbons caused by pollution, pesticide residues, oxidative deterioration, etc., while retaining the original flavor to the greatest extent.

[0003] At present, adsorption decolorization is the most widely used decolorization method in oil processing. Among them, attapulgite has very strong adsorption performance, and compared with activated clay, it uses less amount during decolorization, has less oil loss, and is cheap. The structure of attapulgite contains structural water, crystal water and adsorbed water. This is very advantageous for enzyme carriers, as it can provide a certain amount of water for the enzyme and maintain the integrity of the enzyme structure. Laccase is a copper-rich protein, which consists of three main parts: peptide chain, sugar ligand and Cu 2+ . Usually, a peptide chain contains 500 to 550 amino acids, and its molecular weight is about 60 to 70 kDa. As a biocatalyst, the degradation process of laccase has the characteristics of mild reaction conditions and green and pollution-free. Studies have shown that it can catalyze the oxidation of more than 200 different types of substances. The characteristics of laccase have something in common with oxidized phenolic substances, so laccase can convert oxidized phenolic substances into polyphenol oxides, and then the polyphenol oxides themselves polymerize to form large particles, which can be filtered out. In addition, laccase can also degrade dyes such as anthraquinone, azo and triphenylmethane, and has a high decolorization rate, long-lasting activity and high utilization rate.

[0004] At present, the research on composite adsorption materials for oil decolorization mainly focuses on the ratio compounding and process research of materials such as activated carbon, activated clay, and attapulgite. These materials can remove pigments in oils and improve the color of oils, but they cannot achieve the purpose of removing harmful substances in oils and fats, and in the process of adsorbing pigments in oils and fats, the flavor is also greatly lost. This patent uses cellulose as a biomass carbon source, which can not only improve the adsorption performance of attapulgite and enhance the decolorization ability, but also provide more binding sites on the surface of attapulgite to assist the enzyme to bind to attapulgite and retain the original flavor to the greatest extent while decolorizing and removing harmful substances, thereby improving the quality of oil. The invention patent application with publication number CN 114231521 A discloses a method for preparing a modified attapulgite-immobilized enzyme and its application, which first modifies the modified attapulgite and then immobilizes the erythromycin degrading enzyme, ignoring the application of biomass carbon sources in improving the performance of the carrier and the modified attapulgite-immobilized enzyme is suitable for the purification of environmentally polluted water bodies but cannot be used in the decolorization and refining of oils and fats. Summary of the invention

[0005] In view of the shortcomings of the related art, the present invention provides a preparation method and application of a attapulgite composite enzyme decolorizing agent, wherein the attapulgite composite enzyme decolorizing agent uses a attapulgite-carbon composite as a carrier, on which the enzyme is immobilized, which can not only achieve the goal of oil decolorization but also further remove harmful substances such as benzopyrene.

[0006] The first technical solution provided by the present invention is a method for preparing a attapulgite composite enzyme decolorizing agent, comprising the following steps:

[0007] S1, using attapulgite powder and biological carbon source as raw materials, preparing attapulgite-carbon composite carrier by hydrothermal carbonization method;

[0008] S2, using the attapulgite-carbon composite carrier in step S1 to immobilize laccase to obtain an attapulgite composite enzyme decolorizing agent.

[0009] In certain embodiments, in step S1, the hydrothermal carbonization method is as follows: attapulgite powder and a biological carbon source are added to water to form a suspension, and then ammonium ferrous sulfate hexahydrate is added and stirred to form a mixed solution, followed by carbonization. After carbonization, the precipitate is collected by centrifugation and washed.

[0010] In certain embodiments, in step S1, the biological carbon source is one of starch, cellulose, and chitosan.

[0011] In certain embodiments, in step S1, the amount of the biocarbon source added to the suspension is 1-5% of the mass of the attapulgite powder.

[0012] In certain embodiments, in step S1, the amount of ammonium ferrous sulfate hexahydrate added is 3.00-3.50 g.

[0013] In certain embodiments, in step S1, the carbonization temperature is 200-280°C, and the carbonization time is 12-48 hours.

[0014] In certain embodiments, in step S2, the mass ratio of the attapulgite-carbon composite carrier to the enzyme is (8-12):1.

[0015] The second technical solution provided by the present invention is a attapulgite composite enzyme decolorizing agent, which is prepared by the method described in the first technical solution.

[0016] The third technical solution provided by the present invention is a method for decolorizing oils and fats, wherein the attapulgite composite enzyme decolorizing agent described in the second technical solution is added to the oils and fats for decolorization.

[0017] In certain embodiments, the decolorizing agent is added in an amount of 3-8% of the weight of the oil, and the decolorizing time is 20-40 minutes.

[0018] The fourth technical solution provided by the present invention is the method described in the first technical solution, or the attapulgite composite enzyme decolorizing agent described in the second technical solution, or the method described in the third technical solution applied in the decolorization and refining process of oils and fats.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] Constructing attapulgite-biocarbon composite materials can significantly improve the performance of various parts of the material. Cellulose is a green renewable resource in nature. In the process of forming a composite material with attapulgite, a very small amount of cellulose is hydrolyzed into glucose at the microscopic level, dehydrated to form 5-methylfurfural, and further polymerized-condensed to form a polyfuran structure, forming carbon particles with oxygen-containing functional groups on the surface; the other part undergoes intramolecular rearrangement, condensation, dehydration and decarbonation to form carbon with aromatic and oxygen-containing functional groups on the surface, which can enhance the adsorption sites of attapulgite. At the macroscopic level, the pore structure of attapulgite is destroyed during high-temperature carbonization, and the surface area and total pore volume of attapulgite increase. While increasing the surface functional groups of attapulgite, cellulose also significantly increases the micropore surface area and micropore volume of attapulgite, which increases the adsorption sites and pore volume of attapulgite and enhances the decolorization ability of attapulgite. The structure of attapulgite contains structural water, crystal water and adsorbed water, and the combination of cellulose and attapulgite also provides more binding sites, which is conducive to the immobilization of laccase. Laccase has the characteristics of mild reaction conditions, green and pollution-free in the degradation process. Laccase can convert oxidized phenolic substances into polyphenol oxides, and then the polyphenol oxides themselves polymerize to form large particles, which can be filtered out, and effectively remove harmful polycyclic aromatic hydrocarbons such as benzopyrene. In summary, the attapulgite composite enzyme decolorizer can not only remove the pigment in the crude oil, and the original chromaticity is reduced from yellow 48 and red 9.5 to below yellow 30 and below red 3, but also remove the by-products in the oil and harmful substances that are not conducive to food safety, such as the removal rate of benzopyrene can reach 92%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a flavor comparison chart of rapeseed crude oil and Examples 1, 2, and 3. DETAILED DESCRIPTION

[0022] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0023] Test method:

[0024] 1. PAHs detection method: The PAHs content of rapeseed crude oil was determined by reverse HPLC. Weigh 0.5g of rapeseed crude oil sample, accurate to 0.001g, place it in a 10mL centrifuge tube, add 2ml of petroleum ether and vortex for 1min to dissolve the oil sample for purification. The sample was treated by MIP (molecular imprinting polymer) solid phase extraction method, and the MIP-PAHs polycyclic aromatic hydrocarbons-specific SPE column was inserted into the CNW12-position solid phase extraction vacuum device for activation. 5ml of ethyl acetate and 5mL of petroleum ether were added to the SPE column in turn for activation. After activation, 2mL of sample was added to the column, and then 3mL of n-hexane was used to rinse the 10mL centrifuge tube and load it to the column. The loading speed was controlled to be less than 1mL / min. After loading, 5mL of petroleum ether was added to rinse the SPE column to further remove impurities. After elution, add 10mL of ethyl acetate to elute, collect the eluate in a 10mL centrifuge tube, blow dry with nitrogen, dissolve with acetonitrile, ultrasonicate for 1min and dilute to 1mL. Pass through a 0.22μm filter for injection. Chromatographic conditions: LC-PAH column (250mm×4.6mm×5μm); injection volume: 20μL; temperature: 25℃; mobile phase: A water, B acetonitrile; gradient elution program: 0-5min, 50% A; 5-30min, 50% A rises uniformly to 100% A; 30-45min, 100% A. The detector is a fluorescence detector with an excitation wavelength of 260nm and an emission wavelength of 420nm.

[0025] Benzo[a]pyrene removal rate = [benzo[a]pyrene content in crude oil - benzo[a]pyrene content after bleaching and refining] / benzo[a]pyrene content in crude oil

[0026] 2. Chroma: The chroma of rapeseed oil was determined using the method of GB / T 22460-2008 “Determination of the color of Lovibond in animal and vegetable oils and fats”.

[0027] 3. Aroma profile analysis: 12 sensory assessors who have undergone more than half a year of aroma description training, are familiar with rapeseed oil samples, and have good aroma description and differentiation capabilities were selected to conduct free description analysis of rapeseed oil, and the descriptive words were unified through literature research and group discussion. Finally, seven aroma descriptors of rapeseed oil were determined, including cabbage aroma, oil aroma, caramel aroma, nut aroma, spicy aroma, roasted aroma, and caramel aroma. The intensity of the aroma is rated on a 3-point scale (scale 0.5), with 0 points representing imperceptible and 3 points representing strong perceptibility.

[0028] The raw materials used in the embodiment:

[0029] 1. Attapulgite powder is made of attapulgite clay powder;

[0030] 2. Laccase (CAS: 80498-15-3), purchased from source leaves, activity 120 U / g, 1 U corresponds to the amount of enzyme that converts 1 U MOL of catechol per minute at pH 5.0 and 25°C.

[0031] Example 1

[0032] A method for preparing a attapulgite composite enzyme decolorizing agent comprises the following steps:

[0033] S1, preparation of attapulgite-carbon composite carrier: specifically as follows: 3g attapulgite clay powder and a certain mass of biological carbon source are mixed with 100ml distilled water to form a suspension, 3.25g of ammonium ferrous sulfate hexahydrate is added, stirred at room temperature for two hours, and then the mixture is placed in a polytetraoxyethylene hydrothermal autoclave, the filling rate is maintained at 80%, after carbonization, the product is naturally cooled to room temperature, centrifuged at 8000rpm / min, washed three times with distilled water and ethanol, and dried at 60°C. Among them, cellulose is used as the biological carbon source, the addition amount is 2% (0.06g), the carbonization temperature is 240°C, and the carbonization time is 36h.

[0034] S2, immobilizing laccase with the attapulgite-carbon composite carrier in step S1, wherein the mass ratio of the carrier to the enzyme is 10:1, to obtain an attapulgite composite enzyme decolorizing agent.

[0035] The above 3.06 g of attapulgite composite enzyme decolorizing agent was added to about 61.2 g of rapeseed crude oil, stirred and mixed, and after decolorization for 30 minutes, the decolorizing agent and the oil phase were separated by filtration, the decolorizing agent was recovered, and the oil phase was used for subsequent detection.

[0036] Example 2

[0037] A method for preparing a attapulgite composite enzyme decolorizing agent comprises the following steps:

[0038] S1, preparation of attapulgite-carbon composite carrier: specifically as follows: 3g attapulgite clay powder and a certain mass of biological carbon source are mixed with 100ml distilled water to form a suspension, 3.25g of ammonium ferrous sulfate hexahydrate is added, stirred at room temperature for two hours, and then the mixture is placed in a polytetraoxyethylene hydrothermal autoclave, the filling rate is maintained at 80%, after carbonization, the product is naturally cooled to room temperature, centrifuged at 8000rpm / min, washed three times with distilled water and ethanol, and dried at 60°C. Among them, starch is used as the biological carbon source, the addition amount is 3% (0.09g), the carbonization temperature is 220°C, and the carbonization time is 12h.

[0039] S2, immobilizing laccase with the attapulgite-carbon composite carrier in step S1, wherein the mass ratio of the carrier to the enzyme is 8:1, to obtain an attapulgite composite enzyme decolorizing agent.

[0040] The above 3.06 g of attapulgite composite enzyme decolorizing agent was added to about 61.2 g of rapeseed crude oil, stirred and mixed, and after decolorization for 30 minutes, the decolorizing agent and the oil phase were separated by filtration, the decolorizing agent was recovered, and the oil phase was used for subsequent detection.

[0041] Embodiment 3:

[0042] A preparation method and application of a attapulgite composite enzyme decolorizing agent, comprising the following steps:

[0043] S1, preparation of attapulgite carbon composite carrier: specifically as follows: 3g attapulgite clay powder and a certain mass of biological carbon source are mixed with 100ml distilled water to form a suspension, 3.25g of ammonium ferrous sulfate hexahydrate is added, stirred at room temperature for two hours, and then the mixture is placed in a polytetraoxyethylene hydrothermal autoclave, the filling rate is maintained at 80%, after carbonization, the product is naturally cooled to room temperature, centrifuged at 8000rpm / min, washed three times with distilled water and ethanol, and dried at 60℃. Among them, chitosan is used as the biological carbon source, the addition amount is 4% (0.12g), the carbonization temperature is 260℃, and the carbonization time is 48h.

[0044] S2, immobilizing laccase with the attapulgite-carbon composite carrier in step S1, wherein the mass ratio of the carrier to the enzyme is 12:1, to obtain an attapulgite composite enzyme decolorizing agent.

[0045] The above 3.06 g of attapulgite composite enzyme decolorizing agent was added to about 61.2 g of rapeseed crude oil, stirred and mixed, and after decolorization for 30 minutes, the decolorizing agent and the oil phase were separated by filtration, the decolorizing agent was recovered, and the oil phase was used for subsequent detection.

[0046] The reaction conditions, chromaticity and benzo[a]pyrene removal rate of Examples 1-3 are shown in Table 1. The chromaticity of crude rapeseed oil before bleaching was yellow 48 and red 9.5 (Lovibond colorimetric trough 25.4 mm); the benzo[a]pyrene detection value was 8.54 μg / kg.

[0047] Table 1

[0048]

[0049] As can be seen from Table 1, cellulose, starch and chitosan have good decolorization ability under their respective optimal conditions, and can effectively remove harmful substances such as benzo[a]pyrene. Among them, under the conditions of Example 1, the color of the bleached rapeseed oil is light yellow to light yellow, which meets the color requirements of first-grade oil in rapeseed oil GB / T 1536-2021, and the removal rate of benzo[a]pyrene is higher than that of Examples 2 and 3. Figure 1It can be seen that the loss of flavor substances in the decolorized rapeseed oil of Example 1 is also the least, so the conditions of Example 1 are optimal in terms of decolorization ability, benzo[a]pyrene removal rate and flavor substance retention.

[0050] Example 4

[0051] Referring to Example 1, the amount of cellulose added was changed, carbonization was carried out at 240°C for 48 hours, and the other steps remained unchanged. The chromaticity after decolorization and the benzo[a]pyrene removal rate were measured, as shown in Table 2.

[0052] Table 2

[0053]

[0054] As can be seen from Table 2, with the increase of cellulose addition, the removal rate of benzo[a]pyrene shows a trend of first increasing and then decreasing. When cellulose is added at 2%, the removal rate of benzo[a]pyrene is the highest, and the chromaticity also meets the requirements of rapeseed oil GB / T 1536-2021 for first-grade oil. When cellulose is used as the carbon source, the carbon in the product is composited with attapulgite, and the product contains more CH organic functional groups, so the carrier has the highest removal rate of benzo[a]pyrene.

[0055] Embodiment 5:

[0056] Referring to Example 1, the carbonization temperature was changed and the carbonization was carried out for 48 hours. The other steps remained unchanged. The chromaticity after decolorization and the removal rate of benzo[a]pyrene were measured, as shown in Table 3.

[0057] Table 3

[0058]

[0059] It can be seen from Table 3 that with the increase of carbonization temperature, the removal rate of benzo[a]pyrene shows a trend of first increasing and then decreasing. When the carbonization temperature is 240°C, the removal rate of benzo[a]pyrene is the highest and has good decolorization ability.

[0060] Embodiment 6:

[0061] Referring to Example 1, the carbonization time was changed, and the other steps remained unchanged, and the chromaticity after decolorization and the benzo[a]pyrene removal rate were measured.

[0062] Table 4

[0063]

[0064] It can be seen from Table 4 that with the increase of carbonization time, the removal rate of benzo[a]pyrene shows a trend of first increasing and then decreasing. When the carbonization time is 36h, the removal rate of benzo[a]pyrene is the highest, and it has good decolorization ability, which meets the color requirements of the first-grade oil in rapeseed oil GB / T1536-2021. After the carbonization time is greater than 36h, the chromaticity deteriorates and the removal rate of benzo[a]pyrene decreases. This may be because the internal structure of the carrier is destroyed by carbonization for too long, the activity of the functional groups on the surface of the carrier is lost, and the adsorption performance is reduced.

[0065] Embodiment 7:

[0066] Referring to Example 1, the ratio of the carrier to the enzyme was changed, and the other steps remained unchanged. The chromaticity after decolorization and the removal rate of benzo[a]pyrene were measured, as shown in Table 5.

[0067] Table 5

[0068]

[0069] It can be seen from Table 5 that with the increase in the ratio of carrier to enzyme, the removal rate of benzo[a]pyrene shows a trend of first increasing and then remaining almost unchanged. When the ratio of carrier to enzyme reaches 10:1, the removal rate of benzo[a]pyrene is the highest and has good decolorization ability. Increasing the ratio of carrier to enzyme has almost no effect on the results, and the binding of the enzyme to the active site has reached saturation.

[0070] Comparative Example 1

[0071] Referring to Example 1, no biomass carbon source was added, and the other steps remained unchanged, and the chromaticity after decolorization and the benzo[a]pyrene removal rate were measured.

[0072] Table 6

[0073]

[0074] It can be seen from Table 6 that compared with Example 1, without adding biomass carbon source cellulose, the chromaticity removal ability of the attapulgite-immobilized enzyme composite carrier decreased and the removal rate of benzo[a]pyrene also decreased significantly. This may be because the addition of cellulose helps to improve the adsorption performance of the carrier and can also better fix the enzyme.

[0075] Comparative Example 2

[0076] Referring to Example 1, the biomass carbon source was replaced with activated carbon, and the other steps remained unchanged, and the chromaticity after decolorization and the benzo[a]pyrene removal rate were measured.

[0077] Table 7

[0078]

[0079] It can be seen from Table 7 that in the case of adding activated carbon, the attapulgite-immobilized enzyme composite carrier has a certain decolorization and removal ability of benzopyrene [a], but there is still a gap compared with the effect of adding cellulose biomass carbon source in Example 1. This may be because cellulose can combine with the surface of attapulgite by chemical reaction, which is more firmly than physical adsorption, thereby helping to improve the adsorption performance of the carrier and can also better fix the enzyme.

[0080] Comparative Example 3

[0081] The crude rapeseed oil was decolorized using the attapulgite carbon composite carrier prepared in Example 1, and the other steps remained unchanged. The chromaticity after decolorization and the removal rate of benzo[a]pyrene were measured.

[0082] Table 8

[0083]

[0084] It can be seen from Table 8 that compared with Example 1, without the addition of laccase, the chromaticity removal ability of the attapulgite-immobilized enzyme composite carrier decreased and the removal rate of benzo[a]pyrene also decreased significantly, indicating that laccase played a huge role in the removal of benzo[a]pyrene.

[0085] Comparative Example 4

[0086] Rapeseed crude oil was decolorized by laccase, and the other steps remained unchanged. The chromaticity after decolorization and the removal rate of benzo[a]pyrene were measured.

[0087] Table 9

[0088]

[0089]

[0090] It can be seen from Table 9 that when only laccase is used for decolorization, the chromaticity removal ability of the attapulgite-immobilized enzyme composite carrier is greatly reduced compared with Example 1, indicating that the attapulgite-immobilized enzyme composite carrier has a significant effect in decolorization.

[0091] In summary: the carrier of the attapulgite composite enzyme decolorizer uses cellulose as the carbon source, with an addition amount of 2%. The attapulgite-carbon composite carrier is prepared by hydrothermal carbonization, the carbonization temperature is 240°C, and the carbonization time is 36h. The laccase is immobilized on the attapulgite-carbon composite as a carrier, wherein the mass ratio of the carrier to the enzyme is 10:1. After decolorization of the crude rapeseed oil, the chromaticity values ​​are yellow 21 and red 1.9, which meet the color requirements of the first-grade oil in rapeseed oil GB / T 1536-2021. The removal rate of benzo[a]pyrene is 92%, achieving the purpose of effectively removing harmful substances.

[0092] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a attapulgite composite enzyme decolorizing agent, characterized in that: The steps include: S1, using attapulgite powder and cellulose as raw materials, preparing an attapulgite-carbon composite carrier by a hydrothermal carbonization method, wherein the amount of cellulose added is 2% of the mass of the attapulgite powder, the carbonization temperature is 200-280° C., and the carbonization time is 12-48 hours; S2, using the attapulgite-carbon composite carrier in step S1 to immobilize laccase, the mass ratio of the attapulgite-carbon composite carrier to the enzyme is (10-12):1, and obtaining an attapulgite composite enzyme decolorizing agent.

2. The method according to claim 1, characterized in that In step S1, the hydrothermal carbonization method is specifically as follows: attapulgite powder and cellulose are added to water to form a suspension, and then ammonium ferrous sulfate hexahydrate is added and stirred to form a mixed solution, followed by carbonization. After carbonization, the precipitate is collected by centrifugation and washed.

3. A attapulgite composite enzyme decolorizing agent, characterized in that: A decolorizing agent prepared by the method of claim 1 or 2.

4. A method for decolorizing oil, characterized in that: The attapulgite composite enzyme decolorizing agent according to claim 3 is added into oils and fats for decolorization.

5. The method according to claim 4, characterized in that The amount of the decolorizing agent added is 3-8% of the weight of the oil, and the decolorizing time is 20-40 minutes.

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