A yeast-loaded modified montmorillonite, its preparation method and application

Abstract

This invention relates to the field of mycotoxin adsorbent preparation technology. More specifically, it relates to a yeast-loaded modified montmorillonite, its preparation method, and its application. The preparation method of the yeast-loaded modified montmorillonite includes the following steps: S1, converting calcium-based montmorillonite into sodium-based montmorillonite via a sodium-modification method, followed by thermal activation treatment; S2, mixing and grinding the thermally activated sodium-based montmorillonite with a carbon precursor, and calcining under an inert atmosphere to obtain carbon-based montmorillonite; S3, adsorbing and coupling the carbon-based montmorillonite with activated yeast to obtain yeast-loaded modified montmorillonite. This invention utilizes the organic groups modified on the surface of the modified montmorillonite to form a specific interaction with yeast, achieving directional and stable loading of yeast, enabling it to stably perform its metabolic function during mycotoxin removal.

Description

Technical Field

[0001] This invention relates to the field of mycotoxin adsorbent preparation technology. More specifically, it relates to a yeast-loaded modified montmorillonite, its preparation method, and its applications. Background Technology

[0002] Statistics show that approximately 25% of global grains are contaminated with mycotoxins. Mycotoxin contamination not only leads to a decline in agricultural product quality and causes livestock diseases, but can also harm human health through the food chain. Mycotoxins have a stable structure and are difficult to remove effectively during conventional processing and distribution. Each year, about 2% of grains become inedible due to severe contamination. Even raw materials that meet national standards may accumulate toxins during deep processing due to the accumulation of byproducts. Currently, over 400 types of mycotoxins are known to be present in grains and oils, among which aflatoxin, vomitoxin, and zearalenone are the most harmful. Among existing removal technologies, chemical methods, biodegradation methods, and photocatalysis still face many challenges in large-scale application, while adsorption methods have become mainstream due to their simplicity and practicality. Commercially available adsorbents mainly include aluminosilicates, carbon materials, and organic polymers. Attapulgite, montmorillonite, kaolin, and zeolite aluminosilicate raw materials are inexpensive and widely used to adsorb aflatoxin with polar groups, but their adsorption capacity for weakly polar zearalenone and vomitoxin is relatively weak. Improving the adsorption performance of aluminosilicate materials through modification has become an important research direction. Existing detoxification technologies generally suffer from the following limitations: poor adsorbent targeting, low removal efficiency, and easy loss of nutrients; microbial agents, on the other hand, suffer from weak stability, easy inactivation in practical applications, and significant susceptibility to environmental factors. While montmorillonite, as a natural adsorbent, possesses a certain adsorption capacity, its unmodified form exhibits poor selectivity and may inhibit bacterial activity when combined with microorganisms, making it difficult to achieve the ideal effect of synergistic removal of mycotoxins through "adsorption + degradation." Therefore, developing a mycotoxin removal material with high stability, good removal efficiency, and suitability for practical applications has become a critical issue that urgently needs to be addressed. Summary of the Invention

[0003] Based on the above-mentioned shortcomings, the first objective of this invention is to provide a method for preparing modified montmorillonite loaded with yeast.

[0004] A second objective of this invention is to provide a modified montmorillonite loaded with yeast prepared by the preparation method described above.

[0005] A third objective of this invention is to provide an application of modified montmorillonite loaded with yeast as described above in the removal of mycotoxins.

[0006] To achieve the first objective mentioned above, the present invention adopts the following technical solution: This invention discloses a method for preparing yeast-loaded modified montmorillonite, comprising the following steps: S1. Calcium-based montmorillonite is converted into sodium-based montmorillonite by sodium conversion and then subjected to thermal activation treatment; S2. The thermally activated sodium-based montmorillonite is mixed with the carbon precursor and ground, and then calcined under an inert atmosphere to obtain carbon-based montmorillonite. S3. Based on the confined synthesis method, carbon-based montmorillonite is adsorbed and coupled with activated yeast to obtain modified montmorillonite loaded with yeast.

[0007] In this invention, calcium-based montmorillonite is converted into sodium-based montmorillonite using a sodium-based conversion method, which significantly improves the cation exchange capacity, interlayer expansion performance, and dispersion stability of the material. This creates better surface active sites and reaction conditions for the subsequent construction of carbon composite structures and the bioloading process of yeast. By co-calcining the carbon precursor with sodium-based montmorillonite, a dense carbon layer or nano-carbon composite structure can be formed between the carbon precursor and the montmorillonite layers and on the surface. This not only effectively improves the chemical stability and mechanical strength of the modified montmorillonite material, but also further enhances the adsorption performance of the material itself and provides a protective microenvironment for the immobilization of yeast, preventing the yeast from being inactivated by the external environment. By utilizing the specific electrostatic interactions, hydrogen bonding, or functional group coupling between the organic groups rich in the surface of carbon-based montmorillonite and the components of the yeast cell wall, the yeast is oriented and stably adsorbed on the surface of carbon-based montmorillonite, effectively reducing the randomness and instability of traditional physical adsorption methods and improving the uniformity and stability of the loading system.

[0008] The loading of yeast in this invention is a key step in upgrading the technical solution from a "high-efficiency adsorbent" to a "smart adsorption-bioreactor." By employing adsorption to combine yeast with modified montmorillonite, without the need for stringent chemical cross-linking or encapsulation processes, the integrity and biological activity of the yeast cells can be preserved to the greatest extent, ensuring its stable metabolic function during mycotoxin removal. The synergistic effect of the combination enables this composite material to significantly surpass traditional adsorbents in adsorption efficiency when dealing with difficult-to-treat, weakly polar mycotoxins such as vomitoxin. Furthermore, it achieves a qualitative leap in the fundamental, thorough, and long-lasting nature of detoxification, providing a highly promising innovative strategy for solving mycotoxin contamination problems in feed and agricultural products.

[0009] Furthermore, the carbon precursor is selected from one or more of coal tar pitch, cocamidopropyl betaine, coconut shell powder, and rice husk.

[0010] Furthermore, the sodium-based montmorillonite is mixed with the carbon precursor for 3-18 hours.

[0011] Furthermore, the mass ratio of sodium-based montmorillonite to carbon precursor is 1:1-30; for example, the mass ratio of sodium-based montmorillonite to carbon precursor can be 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, etc.

[0012] Furthermore, the yeast is selected from Pichia pastoris and / or Saccharomyces cerevisiae.

[0013] Furthermore, the temperature of the thermal activation treatment is 300-500℃, and the thermal activation treatment time is 1-5h.

[0014] Furthermore, the calcination temperature is 200-500℃, preferably 400℃, and the calcination time is 0.5-6 h, preferably 2 h.

[0015] Further, step S3 further includes: adding the activated yeast culture to the carbon-based montmorillonite suspension, stirring in a water bath at 37°C for 4-6 hours, centrifuging, washing, and drying to obtain modified montmorillonite loaded with yeast.

[0016] Furthermore, the mass concentration of the carbon-based montmorillonite suspension is 0.005-0.1 g / mL. Exemplarily, the mass concentration of the carbon-based montmorillonite suspension can be 0.005 g / mL, 0.01 g / mL, 0.02 g / mL, 0.03 g / mL, 0.04 g / mL, 0.05 g / mL, 0.06 g / mL, 0.07 g / mL, 0.08 g / mL, 0.09 g / mL, 0.1 g / mL, etc.; the viable cell count of the yeast culture is 1×10⁻⁶. 8 -1×10 9 cfu / mL.

[0017] Furthermore, the volume ratio of the carbon-based montmorillonite suspension to the yeast culture is 10:1.

[0018] To achieve the second objective mentioned above, the present invention adopts the following technical solution: This invention discloses a modified montmorillonite loaded with yeast prepared by the preparation method described above.

[0019] To achieve the second objective mentioned above, the present invention adopts the following technical solution: This invention discloses the application of modified montmorillonite loaded with yeast as described above in the removal of mycotoxins.

[0020] Furthermore, the application further includes: Modified montmorillonite loaded with yeast was added to a mycotoxin solution and adsorbed at a constant temperature under light-protected conditions.

[0021] Furthermore, the concentration of the mycotoxin solution is 1-50 mg / L; exemplaryly, the concentration of the mycotoxin solution may also be 1-3 mg / L, 1-5 mg / L, 1-7 mg / L, 1-10 mg / L, 2-5 mg / L, 2-7 mg / L, 2-10 mg / L, 5-7 mg / L, 5-10 mg / L, 7-10 mg / L, 5-50 mg / L, 10-50 mg / L, 20-50 mg / L, 30-50 mg / L, 40-50 mg / L, 10-20 mg / L, 10-30 mg / L, 10-40 mg / L, 20-40 mg / L, 30-40 mg / L, etc.

[0022] Furthermore, the mass concentration of the modified montmorillonite loaded with yeast in the mycotoxin solution is 1-5 wt‰; exemplaryly, the mass concentration of the modified montmorillonite can also be any range between 1, 1.25 wt‰, 1.5 wt‰, 1.75 wt‰, 2 wt‰, 2.25 wt‰, 2.5 wt‰, 2.75 wt‰, 3 wt‰, 3.25 wt‰, 3.5 wt‰, 3.75 wt‰, 4 wt‰, 4.25 wt‰, 4.5 wt‰, 4.75 wt‰, and 5 wt‰.

[0023] Furthermore, the isothermal adsorption conditions are 37-45℃ for 2-6 hours.

[0024] Furthermore, the mycotoxins include vomitoxin, zearalenone, or aflatoxin. In this invention, modified montmorillonite can not only adsorb aflatoxin with polar groups, but also exhibits good adsorption properties for weakly polar zearalenone and vomitoxin.

[0025] The beneficial effects of this invention are as follows: In this invention, the cation exchange capacity of montmorillonite is enhanced by sodium treatment. A carbon precursor is thermally swelled with montmorillonite, followed by high-temperature calcination under anaerobic conditions to obtain carbon-based montmorillonite. The carbon-based montmorillonite is then adsorbed onto the organic groups on the carbon-based surface layer to obtain a carbon-based montmorillonite / yeast composite. This invention utilizes adsorption to achieve a composite combination of yeast and carbon-based montmorillonite, eliminating the need for stringent chemical cross-linking or encapsulation processes. This maximizes the preservation of yeast cell integrity and biological activity, ensuring stable metabolic function during mycotoxin removal. The synergistic effect of the yeast and carbon-based montmorillonite results in a composite material with significantly higher adsorption efficiency than traditional adsorbents when treating weakly polar mycotoxins such as vomitoxin, which are difficult to remove. Furthermore, it significantly improves the thoroughness and long-term effectiveness of detoxification.

[0026] This invention fully utilizes the structural characteristics of montmorillonite, a two-dimensional layered material, including its large specific surface area, ultra-thin lamellar structure, abundant adsorption active sites, and high proportion of coordination-unsaturated surface sites, to significantly improve the adsorption performance and detoxification efficiency of carbon-based montmorillonite / yeast composite materials for mycotoxins. In this work, considering both cost and dosage, when the carbon-based montmorillonite / yeast composite material contains 5 mg, the adsorption efficiencies for zearalenone and vomitoxin are increased by 86% and 92%, respectively, compared to calcium-based montmorillonite. This invention features a simple preparation process, low cost, and suitability for large-scale production. Attached Figure Description

[0027] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0028] Figure 1 The XRD pattern of C-MMT / yeast-400-1 prepared in Example 1 and the raw material calcium-based montmorillonite confirms that carbon is inserted into the interlayer.

[0029] Figure 2 SEM images of carbon-based montmorillonite and C-MMT / yeast-300 prepared in Example 1, wherein... Figure 2 In the diagram, a represents carbon-based montmorillonite, and b represents C-MMT / yeast-300. Detailed Implementation

[0030] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0031] It should be noted that the needle filter selected in this invention does not adsorb mycotoxins.

[0032] Example 1 This example provides a method for preparing yeast-loaded modified montmorillonite, comprising the following steps: 1. Sodium-based montmorillonite was subjected to sodium-based montmorillonite by ion exchange to obtain sodium-based montmorillonite, which was then dried. 2. Weigh 1g of sodium-based montmorillonite and 5g of rice husk carbon precursor and mix them thoroughly. Place the mixture in a tube furnace and calcine it at 300℃ under an inert atmosphere for 1 hour to obtain carbon-based montmorillonite.

[0033] 3. Weigh 1 g of carbon-based montmorillonite powder and dissolve it in 100 mL of aqueous solution to obtain a carbon-based montmorillonite suspension. Add 10 mL of 1×10⁻⁶ ppm of the solution. 8CFU / mL Pichia pastoris was stirred in a water bath at 37°C for 6 hours, centrifuged, washed multiple times to remove free bacteria, and dried to obtain solid montmorillonite, which is the yeast-loaded modified montmorillonite, denoted as C-MMT / yeast-300.

[0034] Figure 2 SEM images of carbon-based montmorillonite and C-MMT / yeast-300 prepared in Example 1. The carbon-based montmorillonite nanosheets have smooth surfaces. After reaction with yeast, it was confirmed that yeast was attached to the surface of the nanosheets (the small red circles indicate yeast).

[0035] Adsorption performance The mycotoxin adsorption experiment was conducted using the aqueous solution method. A needle filter, which has no adsorption effect on mycotoxins, was selected for the experiment. The steps were as follows: 5 mg of C-MMT / yeast-300 was weighed and added to 4 mL of mycotoxin solution (2 mg / L vomitoxin or 2 mg / L zearalenone solution). The solution was placed at 37℃ and magnetically stirred for adsorption reaction for 3 hours. After adsorption, 1 mL of the solution was drawn up with a syringe and filtered through the needle filter into a liquid chromatography vial. The content of vomitoxin or zearalenone in the solution was determined according to the recommended method of the national standard for feed hygiene (GB / T 30956-2014), and the adsorption rate was calculated. The results are shown in Table 1.

[0036] Example 2 This example provides a method for preparing yeast-loaded modified montmorillonite, comprising the following steps: 1. Sodium-based montmorillonite was subjected to sodium-based montmorillonite by ion exchange to obtain sodium-based montmorillonite, which was then dried. 2. Weigh 1g of sodium-based montmorillonite and 5g of rice husk carbon precursor and mix them thoroughly. Place the mixture in a tube furnace and calcine it at 400℃ under an inert atmosphere for 1 hour to obtain carbon-based montmorillonite.

[0037] 3. Weigh 1 g of carbon-based montmorillonite powder and dissolve it in 100 mL of aqueous solution to obtain a carbon-based montmorillonite suspension. Add 10 mL of 1×10⁻⁶ ppm of the solution. 8 CFU / mL Pichia pastoris was stirred in a water bath at 37°C for 6 hours, centrifuged, washed multiple times to remove free bacteria, and dried to obtain solid montmorillonite, which is the yeast-loaded modified montmorillonite, denoted as C-MMT / yeast-400-1.

[0038] Figure 1 The image shows a comparison of the XRD patterns of C-MMT / yeast-400-1 prepared in Example 1 and the raw material calcium-based montmorillonite. The comparison shows that the carbon layer is inserted into the interlayer of montmorillonite, resulting in a smaller interlayer structure.

[0039] The adsorption performance was studied in the same manner as in Example 1, and the results are shown in Table 1.

[0040] Example 3 This example provides a method for preparing yeast-loaded modified montmorillonite, comprising the following steps: 1. Sodium-based montmorillonite was subjected to sodium-based montmorillonite by ion exchange to obtain sodium-based montmorillonite, which was then dried. 2. Weigh 1g of sodium-based montmorillonite and 5g of rice husk carbon precursor and mix them thoroughly. Place the mixture in a tube furnace and calcine it at 500℃ under an inert atmosphere for 1 hour to obtain carbon-based montmorillonite.

[0041] 3. Weigh 1 g of carbon-based montmorillonite powder and dissolve it in 100 mL of aqueous solution to obtain a carbon-based montmorillonite suspension. Add 10 mL of 1×10⁻⁶ ppm of the solution. 8 CFU / mL Pichia pastoris was stirred in a water bath at 37°C for 6 hours, centrifuged, washed multiple times to remove free bacteria, and dried to obtain solid montmorillonite, which is the yeast-loaded modified montmorillonite, denoted as C-MMT / yeast-500.

[0042] Example 4 This example provides a method for preparing yeast-loaded modified montmorillonite, comprising the following steps: 1. Sodium-based montmorillonite was subjected to sodium-based montmorillonite by ion exchange to obtain sodium-based montmorillonite, which was then dried. 2. Weigh 1g of sodium-based montmorillonite and 3g of coconut shell powder precursor and mix them thoroughly. Place the mixture in a tube furnace and calcine it at 400℃ under an inert atmosphere for 1 hour to obtain carbon-based montmorillonite.

[0043] 3. Weigh 1 g of carbon-based montmorillonite powder and dissolve it in 100 mL of aqueous solution to obtain a carbon-based montmorillonite suspension. Add 10 mL of 1×10⁻⁶ ppm of the solution. 8 CFU / mL Pichia pastoris was stirred in a water bath at 37°C for 6 hours, centrifuged, washed multiple times to remove free bacteria, and dried to obtain solid montmorillonite, which is the yeast-loaded modified montmorillonite, denoted as C-MMT / yeast-400-2.

[0044] The adsorption performance was studied in the same manner as in Example 1, and the results are shown in Table 1.

[0045] Comparative Example 1 This example provides a method for preparing modified montmorillonite without yeast loading, comprising the following steps: 1. Sodium-based montmorillonite was subjected to sodium-based montmorillonite by ion exchange to obtain sodium-based montmorillonite, which was then dried. 2. Weigh out sodium-based montmorillonite and rice husk carbon precursor, mix them thoroughly, place them in a tube furnace, and calcine them at 300°C under an inert atmosphere for 1 hour to obtain carbon-based montmorillonite, denoted as C-MMT.

[0046] The adsorption performance was studied in the same manner as in Example 1, and the results are shown in Table 1.

[0047] Comparative Example 2 This example provides a method for preparing modified montmorillonite without yeast loading, comprising the following steps: 1. Sodium-based montmorillonite was subjected to sodium-based montmorillonite by ion exchange to obtain sodium-based montmorillonite, which was then dried. 2. Weigh 1g of sodium-based montmorillonite and 5g of rice husk carbon precursor and mix them thoroughly. Place the mixture in a tube furnace and calcine it at 400℃ under an inert atmosphere for 1 hour to obtain carbon-based montmorillonite, denoted as C-MMT.

[0048] 3. Weigh 1 g of carbon-based montmorillonite powder and dissolve it in 100 mL of aqueous solution to obtain a carbon-based montmorillonite suspension. Add 10 mL of 1×10⁻⁶ ppm of the solution. 8 CFU / mL Pichia pastoris was stirred in a water bath at 37°C for 6 hours, centrifuged, washed multiple times to remove free bacteria, and dried to obtain solid montmorillonite, which is the yeast-loaded modified montmorillonite, denoted as C-MMT / yeast-400.

[0049] 4. Place C-MMT / Yeast-400 at 120°C for 1 hour to inactivate the yeast.

[0050] The adsorption performance was studied in the same manner as in Example 1, and the results are shown in Table 1.

[0051] Table 1

[0052] Note: Calcium-based montmorillonite and Pichia pastoris were used as control groups in the adsorption performance study.

[0053] Table 1 shows the adsorption rates of zearalenone and vomitoxin for the samples prepared in each example and comparative example. The yeast-loaded modified montmorillonite prepared in Examples 1-3 all have high adsorption capacity for zearalenone and vomitoxin, which is better than that of unloaded modified montmorillonite and pure yeast, and significantly better than that of calcium-based montmorillonite. Furthermore, the sample prepared in Example 2 has the strongest adsorption capacity for zearalenone and vomitoxin, indicating that the preparation method provided by the present invention can effectively improve the adsorption capacity of montmorillonite for zearalenone and vomitoxin.

[0054] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing yeast-loaded modified montmorillonite, characterized in that, Includes the following steps: S1. Calcium-based montmorillonite is converted into sodium-based montmorillonite by sodium conversion and then subjected to thermal activation treatment; S2. The thermally activated sodium-based montmorillonite is mixed with the carbon precursor and ground, and then calcined under an inert atmosphere to obtain carbon-based montmorillonite. S3. Carbon-based montmorillonite is adsorbed and coupled with activated yeast to obtain modified montmorillonite loaded with yeast.

2. The preparation method according to claim 1, characterized in that, The carbon precursor is selected from one or more of coal tar pitch, cocamidopropyl betaine, coconut shell powder, and rice husk. Preferably, the mass ratio of sodium-based montmorillonite to carbon precursor is 1:1-30.

3. The preparation method according to claim 1, characterized in that, The yeast is selected from Pichia pastoris and / or Saccharomyces cerevisiae.

4. The preparation method according to claim 1, characterized in that, The temperature of the thermal activation treatment is 300-500℃, and the thermal activation treatment time is 1-5h; Preferably, the calcination temperature is 200-500℃ and the calcination time is 0.5-6 h.

5. The preparation method according to claim 1, characterized in that, Step S3 further includes: adding the activated yeast culture to the carbon-based montmorillonite suspension, stirring in a water bath at 37°C for 4-6 hours, centrifuging, washing, and drying to obtain modified montmorillonite loaded with yeast.

6. The preparation method according to claim 5, characterized in that, The carbon-based montmorillonite suspension had a mass concentration of 0.005-0.1 g / mL; the viable cell count of the yeast culture was 1×10⁻⁶. 8 -1×10 9 cfu / mL; Preferably, the volume ratio of the carbon-based montmorillonite suspension to the yeast culture is 10:

1.

7. A modified montmorillonite loaded with yeast, characterized in that, It is prepared by the preparation method described in any one of claims 1-6.

8. The application of yeast-loaded modified montmorillonite as described in claim 7 in the removal of mycotoxins.

9. The application according to claim 8, characterized in that, The application further includes: Modified montmorillonite loaded with yeast was added to a mycotoxin solution and adsorbed at a constant temperature under light-protected conditions.

10. The application according to claim 9, characterized in that, The concentration of the mycotoxin solution is 1-50 mg / L; The modified montmorillonite loaded with yeast has a mass concentration of 1-5 wt‰ in the mycotoxin solution. Preferably, the isothermal adsorption conditions are 37-45℃ for 2-6 hours; Preferably, the mycotoxin includes vomitoxin, zearalenone, or aflatoxin.

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