Mussel mucoprotein-based high-adhesion foliar water-soluble fertilizer as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of a mussel mucoprotein-based high-adhesion foliar water-soluble fertilizer, which comprises the following steps: (1) dissolving mussel mucoprotein and a water-soluble fertilizer in a buffer solution to obtain a mussel mucoprotein-fertilizer solution; dissolving a reducing agent in the buffer solution to obtain a reducing agent solution; and (2) mixing the mussel mucin-fertilizer solution obtained in the step (1) with the reducing agent solution, and uniformly stirring to obtain the mussel mucin-fertilizer. According to the mussel adhesive protein-based high-adhesion foliar water-soluble fertilizer prepared by the invention, long-acting foliar retention of inorganic fertilizers and biostimulants is realized at the same time, and the retention amount is remarkably increased. The preparation method of the mussel mucin-based high-adhesion foliar fertilizer is simple, the required raw materials are less, the use of organic reagents is reduced, and the influence on the environment is less.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural fertilizers, and in particular to a mussel mucin-based high-adhesion foliage water-soluble fertilizer, a preparation method thereof, and an application thereof. Background Art

[0002] Food is essential for human survival. To improve food production and quality, the use of fertilizers has been a constant companion to societal development. Essential plant nutrients are primarily applied to the soil and foliage to maximize economic yield. In theory, foliar fertilization is more environmentally friendly, direct, and targeted than soil fertilization because nutrients can be delivered directly to plant tissues during critical growth stages. Field trials have also demonstrated that foliar fertilizer application increases both cash crop yields and nutrient concentrations in plants. Over the past few decades, foliar fertilizers have garnered increasing attention as they offer the most effective way to address nutrient deficiencies and improve crop yield and product quality. Today, foliar fertilizers have become an integral part of various agricultural production processes. The primary advantage of foliar fertilization is that nutrient absorption occurs directly in the leaves, allowing for their distribution throughout the plant. This helps avoid the adverse effects of soil-based fertilizers, such as phytotoxicity, soil erosion, slow response, and potential contamination. However, foliar fertilizers suffer nearly 60% fertilizer loss due to poor adhesion, climatic factors such as rainfall, and the bouncing of water-soluble fertilizer droplets on leaves, resulting in excessive fertilizer spraying times, which ultimately affects the soil environment and ecosystem.

[0003] Mussel mucin is a protein extracted from the byssus of the marine organism mussel. It has excellent biocompatibility. Its protein molecule carries a large number of dopa groups and positively charged lysine groups, which have multiple benefits, including anti-inflammatory, antioxidant, pigmentation prevention, and wound healing. Furthermore, due to the regular arrangement of lysine and dopa groups in its protein structure, mussel mucin has extremely strong bioadhesive properties. However, mussel mucin can be inactivated by environmental factors during leaf adhesion. Therefore, ensuring the activity of mussel mucin in the complex environment of the leaf surface, and thus ensuring the durability of the adhesion effect, is a difficult problem that needs to be solved in high-adhesion fertilizers. Summary of the Invention

[0004] The object of the present invention is to provide a kind of mussel mucin-based high-adhesion foliage water-soluble fertilizer for the problem that the foliage fertilizer retention amount prepared by prior art is low and the effect is not lasting. In order to ensure the activity of mussel mucin in the fertilizer prepared, so as to achieve long-term adhesion, the present invention is inspired by natural amyloid fibrillation, and induces amyloid aggregation by reducing the internal disulfide bond of mussel mucin, so as to form a phase transition mussel mucin (PTM) with controllable structure and function, and utilizes it to prepare high-adhesion foliage water-soluble fertilizer, realize fertilizer load and foliage long-term attachment by protein self-assembly, significantly improve the retention amount of fertilizer in plant foliage. The preparation method of the present invention is simple, and required raw materials are less, and the use of organic reagents is reduced, and environmental impact is small.

[0005] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0006] A method for preparing a mussel mucin-based high-adhesion foliar water-soluble fertilizer comprises the following steps:

[0007] (1) dissolving mussel mucin and a water-soluble fertilizer in a buffer solution to obtain a mussel mucin-fertilizer solution; dissolving a reducing agent in the buffer solution to obtain a reducing agent solution;

[0008] (2) mixing the mussel mucin-fertilizer solution obtained in step (1) and the reducing agent solution and stirring them evenly to obtain the product.

[0009] In step (1), the mussel mucin is a recombinant mussel byssus protein MFP151, whose amino acid sequence is shown in SEQ ID NO. 1. Specifically, the coding genes of mussel byssus proteins MFP1 and MFP5 from mussels are fused and transformed into E. coli BL21 (DE3) for expression to construct the recombinant mussel mucin MFP151. The detailed construction method of the recombinant mussel mucin MFP151 has been disclosed in the document Practical recombinant hybrid mussel bioadhesive fp-151 (Hwang DS, Gim Y, Yoo HJ, et al. Practical recombinant hybrid mussel bioadhesive fp-151 [J]. Biomaterials, 2007, 28 (24): 3560-3568.). Figure 1 This is the gel electrophoresis diagram of the recombinant mussel mucin MFP151 used in the present invention. Figure 2 Photos of solutions of recombinant mussel mucin MFP151 at different concentrations.

[0010] In step (1), the water-soluble fertilizer is any one of KH2PO4, MgCl2, CuCl2, boric acid, FeSO4, MnSO4, ZnSO4, urea, γ-polyglutamic acid and microbial polysaccharide.

[0011] In step (1), the reducing agent is any one of tris(2-carboxyethyl)phosphine, vitamin C, glutathione and cysteine.

[0012] In step (1), the buffer solution is a Tris-HCl buffer solution with a pH value of 7 to 10.

[0013] In step (1), in the mussel mucin-fertilizer solution, the concentration of the mussel mucin is 0.1 to 10 mg / mL, and the concentration of the water-soluble fertilizer is 0.2 to 10 mg / mL.

[0014] In step (1), the concentration of the reducing agent solution is 1 to 20 mg / mL.

[0015] In step (2), the volume ratio of the mussel mucin-fertilizer solution and the reducing agent solution is 0.2 to 5:1; and the stirring condition is stirring at room temperature for 10 to 30 minutes.

[0016] Inspired by natural amyloid fibrillation, the present invention uses a reducing agent to reduce the internal disulfide bonds of mussel mucin to form β-pleated oligomers, induce amyloid-like aggregation, and thus form a phase transition mussel mucin (PTM) with controllable structure and function, and uses it to prepare a high-adhesion foliar water-soluble fertilizer.

[0017] The present invention also provides a mussel mucin-based high-adhesion foliage water-soluble fertilizer prepared by the preparation method.

[0018] The present invention further provides the use of the mussel mucin-based high-adhesion foliar water-soluble fertilizer as a plant foliar fertilizer.

[0019] The mussel mucin-based high-adhesion foliar water-soluble fertilizer is sprayed on the surface of the plant leaves and dried naturally for at least 30 minutes.

[0020] The mussel mucin-based high-adhesion foliage water-soluble fertilizer of the present invention significantly increases the adhesion and retention amount of fertilizer on plant leaves, and can effectively improve the problem of low fertilizer utilization rate.

[0021] The mussel mucin-based high-adhesion foliar water-soluble fertilizer of the present invention has a high foliar retention effect on inorganic water-soluble fertilizers and water-soluble biostimulants.

[0022] Beneficial effects:

[0023] 1. The present invention is inspired by natural amyloid fibrillation. By reducing the internal disulfide bonds of mussel mucin with a reducing agent, amyloid aggregation of mussel mucin is induced to form a phase-transition mussel mucin with controllable structure and function. The amyloid aggregation of mussel mucin can effectively alleviate the changes in the activity of mussel mucin during the film-forming adhesion process on the leaf surface, thereby achieving long-term adhesion and long-term retention of fertilizer on the leaf surface.

[0024] 2. The mussel mucin-based high-adhesion foliar water-soluble fertilizer prepared by the present invention simultaneously achieves long-term foliar retention of inorganic fertilizers and biostimulants, and the retention amount is significantly improved.

[0025] 3. The preparation method of the mussel mucin-based high-adhesion foliar fertilizer of the present invention is simple, requires fewer raw materials, reduces the use of organic reagents, and has less impact on the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0027] Figure 1 This is the gel electrophoresis image of recombinant mussel mucin MFP151.

[0028] Figure 2 Photos of solutions of recombinant mussel mucin MFP151 at different concentrations.

[0029] Figure 3 This is a diagram showing the wetting effect of pure water and mussel mucin MFP151 solution on plant leaves in Example 1.

[0030] Figure 4 The contact angles of pure water and mussel mucin MFP151 solution on the leaf surface in Example 1.

[0031] Figure 5 Statistical graph of contact angle data of the film-forming PTM prepared with different reducing agents on the surface of the polyvinyl alcohol film in Example 1.

[0032] Figure 6 Statistical graph of contact angle data of the film formation of PTM prepared in Tris-HCl buffer with different pH values on the surface of polyvinyl alcohol film in Example 2.

[0033] Figure 7 This is the foliage retention of different mussel mucin-based high-adhesion foliage inorganic water-soluble fertilizers prepared in Example 4.

[0034] Figure 8 This is the foliar retention of the mussel mucin-based high-adhesion foliar urea fertilizer prepared in Example 4.

[0035] Figure 9This is the foliage retention of the mussel mucin-based high-adhesion foliage γ-polyglutamic acid fertilizer prepared in Example 4.

[0036] Figure 10 This is the foliage retention of the mussel mucin-based high-adhesion foliage Pantoea axialis polysaccharide fertilizer prepared in Example 4. DETAILED DESCRIPTION

[0037] The present invention is further described below with reference to the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, rather than to limit the present invention.

[0038] Example 1

[0039] A 2 mg / mL mussel mucin solution (pH = 9) was prepared using Tris-HCl buffer solution, and a 10 mg / mL vitamin C (VC) solution, a glutathione (GSH) solution, a cysteine (Cys) solution, and a tris(2-carboxyethyl)phosphine (TCEP) solution were prepared using Tris-HCl buffer solution (pH = 9). The mussel mucin solution was mixed with the four reducing agent solutions at a volume ratio of 1:1, stirred at room temperature for 30 min, and then evenly mixed. The mixture was then sprayed evenly on a polyvinyl alcohol film and left at room temperature for 12 h to observe their film-forming properties. The contact angle tester (DSA100, ) were used to test the contact angles of the four mixed solutions on the surface of the polyvinyl alcohol film ( Figure 5 ).

[0040] Pure water and the above 2 mg / mL (0.2%) mussel mucin solution (pH = 9) were sprayed on the leaf surface respectively, and the wetting effect of the two solutions on the leaves was observed ( Figure 3 ), and tested the contact angles of pure water and mussel mucin solution on the leaf surface ( Figure 4 ).

[0041] The above experimental results show that all four reducing agents can induce mussel mucin to form films at the solid-liquid interface, with the film containing the reducing agent GSH exhibiting the highest contact angle. The GSH and Cys groups exhibited yellowing, with yellowing occurring more slowly in a sealed container, suggesting that the reaction process may be related to oxygen content. The secondary structure of Mfp in the GSH, Cys, and TCEP groups underwent significant transformations. Furthermore, the reaction solutions corresponding to these three reducing agents all exhibited a decrease in dynamic surface tension due to rapid aggregation of oligomers at the droplet-air interface, confirming the occurrence of aggregation. VC exhibited a relatively slow aggregation compared to the other three reducing agents. In summary, the film containing the reducing agent GSH exhibited the fastest aggregation process and the highest contact angle, indicating that GSH was the most effective reducing agent.

[0042] Example 2

[0043] Tris-HCl buffer solutions with pH values of 7, 8, 9, and 10 were prepared, and 2 mg / mL mussel mucin solutions and 10 mg / mL glutathione solutions were prepared with these solutions. Mussel mucin solutions and glutathione solutions with the same pH value were mixed at a volume ratio of 1:1, stirred at room temperature for 30 min, and then mixed evenly to obtain four mixed solutions. These solutions were sprayed evenly on polyvinyl alcohol films, and their film-forming properties were observed after standing at room temperature for 12 h. The contact angle tester (DSA100, ) Test the contact angles of the above four mixed solutions on the surface of polyvinyl alcohol film.

[0044] The experimental results are as follows Figure 6 As shown in the figure, the results show that mussel mucin can aggregate and form a uniform film under the action of glutathione at pH 7, 8, 9 and 10. The contact angle is the largest at pH = 9, and the film-forming effect is the best.

[0045] Example 3

[0046] A mixed solution of the mussel mucin solution and glutathione solution (pH 9) prepared in Example 2 was subjected to circular dichroism and infrared spectroscopy, and the coating formed by spraying onto a polyvinyl alcohol film was characterized by atomic force microscopy. Atomic force microscopy results showed that the PTM (phase transition mussel mucin) coating consisted of a dense membrane composed of protein oligomer particles and micron-sized spherical aggregates distributed on its surface. The latter had distinct crystalline regions, which may be related to the regular sequence of MFP151. Circular dichroism and infrared spectra indicated that MFP underwent significant changes in its secondary structure after the amyloid-like phase transition, with a significant increase in the β-sheet content.

[0047] Example 4

[0048] Tris-HCl buffer solution (pH = 9) was used to prepare mixed solutions of mussel mucin and different soluble fertilizers (KH2PO4, MgCl2, CuCl2, boric acid, FeSO4, MnSO4, ZnSO4, urea, γ-polyglutamic acid and camel thorn pantoea polysaccharide), wherein the concentration of mussel mucin was 2 mg / mL, and the concentration of soluble fertilizer was 0.5 mg / mL or 1 mg / mL (the concentration of KH2PO4, MgCl2, CuCl2, boric acid, FeSO4, MnSO4, ZnSO4 was 0.5 mg / mL, and the concentration of urea, γ-polyglutamic acid and camel thorn pantoea polysaccharide was 1 mg / mL), and 10 kinds of mussel mucin-fertilizer solutions were obtained respectively. A 10 mg / mL glutathione / Tris-HCl solution (pH = 9) was prepared using Tris-HCl buffer and mixed with each of the 10 mussel mucin-fertilizer solutions at a 1:1 volume ratio. After stirring for 30 minutes, the mixture was homogeneously mixed to obtain the 10 mussel mucin-based, highly adherent, water-soluble foliar fertilizers (Fertilizer@PTM). 10 mL of each solution was sprayed onto tomato leaves, dried at room temperature, and weighed. The leaves were then rinsed with ultrapure water, dried, and weighed again. The fertilizer retention was calculated by the difference in mass between the two solutions. A pure water solution of soluble fertilizer (Fertilizer@water) and a mixed solution of mussel mucin and soluble fertilizer (Fertilizer@MFP) served as controls. All other experimental procedures were identical to those of the experimental groups.

[0049] The experimental results of soluble fertilizers KH2PO4, MgCl2, CuCl2, boric acid, FeSO4, MnSO4, and ZnSO4 are as follows: Figure 7 The experimental results of urea, γ-polyglutamic acid (PGA) and Pantoea acuminata polysaccharide (PAPS) are shown in Figure 8 、 9 The results show that the mussel mucin-based high-adhesion foliar water-soluble fertilizer prepared in the present invention significantly increases the retention of inorganic fertilizers (KH2PO4, MgCl2, CuCl2, boric acid, FeSO4, MnSO4, ZnSO4, urea) and biostimulants (γ-polyglutamic acid and Pantoea acuminata polysaccharide) on plant leaves.

[0050] The present invention provides a method and concept for preparing a mussel mucin-based, highly adherent, water-soluble foliar fertilizer. Numerous methods and approaches exist for implementing this technical solution. The foregoing description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A method for preparing a mussel mucin-based high-adhesion foliar water-soluble fertilizer, characterized in that: The steps include: (1) dissolving mussel mucin and a water-soluble fertilizer in a buffer solution to obtain a mussel mucin-fertilizer solution; dissolving a reducing agent in the buffer solution to obtain a reducing agent solution; (2) mixing the mussel mucin-fertilizer solution obtained in step (1) and the reducing agent solution and stirring them evenly to obtain the product.

2. The preparation method according to claim 1, characterized in that In step (1), the mussel mucin is recombinant mussel byssal protein MFP151, and its amino acid sequence is shown in SEQ ID NO.

1.

3. The preparation method according to claim 1, characterized in that In step (1), the water-soluble fertilizer is any one of KH2PO4, MgCl2, CuCl2, boric acid, FeSO4, MnSO4, ZnSO4, urea, γ-polyglutamic acid and microbial polysaccharide.

4. The preparation method according to claim 1, characterized in that In step (1), the reducing agent is any one of tris(2-carboxyethyl)phosphine, vitamin C, glutathione and cysteine.

5. The preparation method according to claim 1, characterized in that In step (1), the buffer solution is a Tris-HCl buffer solution with a pH value of 7 to 10.

6. The preparation method according to claim 1, characterized in that In step (1), in the mussel mucin-fertilizer solution, the concentration of the mussel mucin is 0.1-10 mg / mL, the concentration of the water-soluble fertilizer is 0.2-10 mg / mL; and the concentration of the reducing agent solution is 1-20 mg / mL.

7. The preparation method according to claim 1, characterized in that In step (2), the volume ratio of the mussel mucin-fertilizer solution and the reducing agent solution is 0.2 to 5:1; and the stirring condition is stirring at room temperature for 10 to 30 minutes.

8. The mussel mucin-based high-adhesion foliar water-soluble fertilizer prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the mussel mucin-based high-adhesion foliar water-soluble fertilizer according to claim 8 as a plant foliar fertilizer.

10. The use according to claim 9, characterized in that The application method of the mussel mucin-based high-adhesion foliar water-soluble fertilizer is as follows: spraying the mussel mucin-based high-adhesion foliar water-soluble fertilizer on the surface of the plant leaves and allowing it to dry naturally for at least 30 minutes.

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