A composition for zinc-selenium simultaneous biofortification of wheat grain, and applications and methods

Abstract

The present application belongs to the field of genetic breeding and cultivation technology, and particularly relates to a composition for synchronously biofortifying wheat grain zinc-selenium and application and method. The composition is prepared by dissolving zinc sulfate, sodium selenite, potassium dihydrogen phosphate and a surfactant in water; the mass ratio of the zinc sulfate, the sodium selenite and the potassium dihydrogen phosphate is 1:0.00325-0.00425:0.5-0.75; and the surfactant comprises Tween-20. The composition provided by the present application effectively alleviates Zn-Se antagonism, realizes synchronous enrichment of wheat grain Zn-Se and synergistic improvement of nutritional quality, and builds an optimized foliar spraying scheme with clear parameters, safety and controllability and capable of being directly integrated into an existing production system, thereby providing a feasible solution and theoretical support for addressing the global 'invisible hunger' challenge through agronomic measures.

Description

Technical Field

[0001] This invention belongs to the field of genetic breeding and cultivation technology, specifically relating to a composition, application and method for simultaneous zinc-selenium biofortification of wheat grains. Background Technology

[0002] Zinc (Zn) and selenium (Se) are essential micronutrients for the human body, playing crucial roles in immune regulation, antioxidant defense, and nervous system development. Micronutrient deficiencies leading to "hidden hunger" are a global public health issue, particularly prevalent in developing countries and regions where grains are a staple food. Wheat, as a major food crop, contributes approximately 20% of daily energy and protein intake, but its edible portions are generally low in Zn and Se. Studies show that the global average Zn and Se content in wheat grains is 31.84 mg / kg. –1 and 0.06 mg·kg –1 This is far from meeting the recommended target value for biofortification (Zn: 40 mg·kg⁻¹). –1 ~60mg·kg –1 Se: 0.25 mg·kg –1 ~0.30 mg·kg –1 Wheat grains contain insufficient amounts of zinc and selenium to meet the daily nutritional needs of the human body. Therefore, increasing the Zn and selenium content of wheat grains through agronomic measures is of great significance for improving public nutrition and health.

[0003] Foliar application of Zn or Se alone has been proven to stably increase the content of these elements in wheat grains, making it an important measure for cereal biofortification. However, when Zn and Se are applied together for simultaneous Zn-Se biofortification, a significant antagonistic effect exists between them. This is mainly manifested in exogenous Zn inhibiting the absorption and translocation of Se to the grains, resulting in a 25%–86% decrease in grain Se content compared to Se spraying alone. This antagonistic effect has become a key bottleneck restricting the simultaneous enrichment of Zn and Se. Summary of the Invention

[0004] To address the problem that the antagonistic effect of Zn and Se restricts the simultaneous Zn-Se biofortification effect in existing technologies, this invention aims to provide a composition, application, and method for simultaneous Zn-Se biofortification of wheat grains. This composition effectively alleviates Zn-Se antagonism, achieving simultaneous enrichment of Zn and Se in wheat grains and synergistic improvement of nutritional quality. To achieve the above objectives, this invention adopts the following technical solution.

[0005] This invention provides a composition for simultaneous zinc-selenium biofortification of wheat grains, comprising zinc sulfate (ZnSO4·7H2O), sodium selenite (Na2SeO3), potassium dihydrogen phosphate (KH2PO4), and a surfactant dissolved in water; wherein the mass-volume ratio of zinc sulfate, sodium selenite, potassium dihydrogen phosphate, and surfactant is 1:0.00325~0.00425:0.5~0.75:0.025~0.05, and the solvent is water.

[0006] The surfactant includes Tween-20.

[0007] In actual production, to simplify operations and reduce costs, a "one-spray-multiple-protection" strategy is often adopted, mixing various micronutrients with potassium dihydrogen phosphate (KH2PO4) or pesticides for spraying. Phosphorus (P) is an essential macronutrient for plants, participating in important physiological processes such as energy metabolism, membrane structure construction, and signal transduction. Foliar spraying of KH2PO4 in the early grain-filling stage can enhance crop resistance to stress (such as drought resistance and resistance to hot, dry winds), thereby stabilizing yield. However, complex interactions may occur when multiple chemical substances are mixed, affecting the final enhancement effect. Currently, there is still a lack of systematic field trial evidence regarding the regulatory effect of P on Zn-Se antagonism when Zn, Se, and P are sprayed together in wheat. Based on this, the present invention solves the zinc-selenium antagonism problem in the prior art by precisely controlling the zinc-selenium ratio and the combination of components: a specific concentration combination of 0.4% zinc sulfate and 0.0013%~0.0017% sodium selenite is used to control the zinc-selenium molar ratio in the range of about 100:1 to 230:1, avoiding the competitive inhibition of selenium absorption by high concentrations of zinc; at the same time, 0.2%~0.3% potassium dihydrogen phosphate is introduced to provide more binding sites for the storage of zinc and selenium in the grain by optimizing the amino acid composition of the grain, and surfactants are used to improve the leaf surface adhesion and penetration efficiency, thereby achieving simultaneous biofortification of zinc and selenium in the grain.

[0008] The present invention also provides the use of the composition in improving the nutritional quality of wheat grains.

[0009] According to the latest recommendations from the Chinese Nutrition Society, the recommended daily intake (RNI) of zinc for adults is 12.0 mg / day for men. –1 8.5 mg / day for women –1 The tolerable upper intake level (UL) is 40 mg / day. –1 The RNI for Se is 60 μg / day for adults. –1Its UL has been based on the Se intake at which plasma selenoprotein P (SELENOP) is saturated, from 400 μg·d –1 Reduced to 255 μg·d –1 This indicates that the safe intake range for selenium (Se) has further narrowed. Furthermore, zinc (Zn) is unevenly distributed in wheat grains, with significant loss after refining into flour; while selenium (Se) is relatively evenly distributed. Therefore, assessing the intake of Zn and Se from the edible portion for different dietary groups (whole wheat or refined flour) is crucial to ensuring the safety of nutritional fortification. The composition provided by this invention enables simultaneous zinc-selenium biofortification of wheat grains, improving the nutritional quality of wheat grains while considering the retention characteristics of trace elements under different processing methods, providing an effective way to supplement dietary zinc and selenium intake.

[0010] Furthermore, the composition is applied to wheat to increase the selenium content of wheat grains, thereby improving the nutritional quality of wheat grains.

[0011] Furthermore, the composition is applied to wheat by spraying.

[0012] Furthermore, the composition is sprayed onto the wheat leaves and ears.

[0013] Furthermore, the composition is sprayed during the wheat flowering period or the early grain-filling stage of wheat.

[0014] Furthermore, the composition is sprayed 2 to 3 times, with each application amount being 1000 L·ha. –1 ~1250L·ha –1 .

[0015] Furthermore, the amount of nitrogen fertilizer applied to the soil before wheat sowing, after spraying the composition, was 120 kg N·ha. –1 ~150kg N·ha –1Nitrogen (N) fertilizer management regulates crop accumulation of Zn and Se by influencing nitrogen metabolism and protein synthesis. In wheat grains, especially the endosperm, Zn primarily binds to small, sulfur-containing proteins rich in cysteine. Se, on the other hand, can participate in the assimilation pathway of its sulfur analogues, being converted into organic forms such as selenomethionine (SeMet), selenocysteine ​​(SeCys2), and selenomethylcysteine ​​(SeMeCys), which are then integrated into protein structures. This indicates that the storage of Zn and Se in grains is closely related to protein and amino acid metabolism. Notably, organic Se has higher bioavailability and better safety, and its absorption is less affected by the organism's Se nutritional status. In actual production, excessive application of N fertilizer is common; therefore, clarifying the differences in the effects of simultaneous Zn-Se biofortification under different N levels is crucial for optimizing spraying strategies.

[0016] Furthermore, the composition improves the nutritional quality of wheat grains by mitigating the antagonistic effect of zinc-selenium synergistic spraying, thereby increasing the selenium content of wheat grains.

[0017] Furthermore, the composition is used to increase the total amino acid content of wheat grains, increase the proportion of selenomethionine (SeMet), promote the conversion of inorganic selenium to organic form, and thus improve the nutritional quality of wheat grains.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a composition for simultaneous zinc-selenium biofortification of wheat grains, comprising ZnSO4·7H2O, Na2SeO3, KH2PO4, and a surfactant dissolved in water. After spraying, KH2PO4 regulates the transport and redistribution of Se after absorption by wheat leaves, promoting Se transport and accumulation in the grains by modulating the expression of phosphorus transport proteins. This solves the problem in existing technologies where combined Zn-Se spraying leads to a significant decrease in grain Se content, and Zn-Se antagonism results in grain Se content failing to meet Se-enrichment standards.

[0019] Zinc (Zn) and selenium (Se) are essential trace elements for the human body. Foliar spraying is an effective way to achieve Zn and Se biofortification in wheat grains. However, the antagonistic effect of the two when sprayed together severely restricts the synchronous fortification effect. This invention investigates the role of phosphorus (P) in alleviating antagonism and achieving simultaneous and efficient enrichment of Zn and Se by spraying Zn-Se together. Two years of field trials showed that under conventional and high-level nitrogen (N) application conditions, the total Zn content in the grains consistently reached the fortification target (40 mg·kg⁻¹) after Zn spraying. –1 ~60mg·kg –1Furthermore, the Zn enrichment effect was not affected by the combined application of Se or P. The combined Zn-Se application significantly reduced the Se content in various grain tissues compared to Se spraying alone, confirming the existence of Zn-Se antagonism. However, the addition of P restored the grain Se content to a level comparable to that of Se spraying alone at the same concentration, effectively compensating for the Se loss caused by antagonism. The combined application of Zn, Se, and P significantly increased the total amino acid and multiple amino acid content in flour, and increased the proportion of selenomethionine (SeMet), promoting the conversion of inorganic Se into more bioavailable organic forms and significantly improving the nutritional quality of the grains. Dietary risk assessment showed that the daily Zn and Se intake of all treated whole grains and flours was below the tolerable upper intake level, with a target hazard index far less than 1, indicating no health risk. Considering both enrichment objectives and food safety, this invention proposes an optimized foliar spraying scheme suitable for conventional nitrogen application conditions: 0.4% ZnSO4·7H2O + 0.0013%~0.0017% Na2SeO3 + 0.2%~0.3% KH2PO4. This scheme can simultaneously meet the Zn-Se nutritional needs of populations with different dietary structures while ensuring dietary safety. In summary, the addition of phosphorus effectively alleviates Zn-Se antagonism, achieving simultaneous enrichment of Zn-Se in wheat grains and synergistic improvement in nutritional quality. This invention constructs an optimized foliar spraying scheme with clearly defined parameters, safe and controllable operation, and direct integration into existing production systems, providing a practical solution and theoretical support for addressing the global challenge of "hidden hunger" through agronomic measures. Attached Figure Description

[0020] Figure 1 Meteorological data for the growing season from 2021 to 2023 in this invention; wherein: (A) represents the monthly rainfall and average temperature during the wheat growing season from 2021 to 2023; (B) represents the total rainfall during the entire wheat growing season and after flowering; (C) represents the average daily temperature after wheat flowering; (D) represents the average daily temperature within 10 days after the first (1st spray) and second (2nd spray) foliar spraying.

[0021] Note: The solid lines and diamonds inside the boxes represent the median and mean of all data, respectively; the top and bottom edges of the boxes represent 75% and 25% of all data, respectively, and the horizontal lines at the top and bottom represent 95% and 5% of all data, respectively. Student t-test: *** P <0.001; ns, no significant difference.

[0022] Figure 2 This invention relates to the effects of foliar and soil application of nitrogen (N) on grain yield, Zn carryover from grain, and Se carryover from grain during the growing season from 2021 to 2023; wherein: (0) represents grain yield; (0)(B) represents the amount of Zn carried out by the grains; (0)(C) represents the amount of Se carried out by the grains; (0) Note: An asterisk indicates that there is a significant difference between different N levels within the same year: ***, P <0.001;**, P <0.01;*, P <0.05; Different uppercase letters indicate significant differences in foliar spraying of phosphorus at the same nitrogen application rate, while different lowercase letters indicate significant differences among different foliar spraying treatments at the same nitrogen application rate in the same year. P <0.05; error bars indicate standard error (n=4). Treatment: N1 / N2, 120 / 240kg N·ha –1 ; Zn, 0.4% (w / v) ZnSO4·7H2O; Se1 / Se2 / Se3, 0.0010% / 0.0015% / 0.002% (w / v) Na2SeO3; P, 0.2% (w / v) KH2PO4.

[0023] Figure 3 This invention relates to the effects of foliar and soil application of nitrogen (N) during the growing season from 2021 to 2023 on the (A-C) Zn and (D-F) Se contents in whole grains, flour, and bran; wherein: (A)~(C) represent the Zn content of the grains; (D)~(F) represent the Se content of the grains; Note: An asterisk indicates that there are significant differences between different N application levels within the same year: *** P <0.001;** ，P <0.01;*, P <0.05; different uppercase letters indicate significant differences in foliar spraying of phosphorus at the same nitrogen application rate, and different lowercase letters indicate significant differences among different foliar spraying treatments at the same nitrogen application rate in the same year. P <0.05); Error bars indicate standard error (n=4); Treatment: N1 / N2, 120 / 240 kg N·ha –1 ; Zn, 0.4% (w / v) ZnSO4·7H2O; Se1 / Se2 / Se3, 0.0010% / 0.0015% / 0.002% (w / v) Na2SeO3; P, 0.2% (w / v) KH2PO4.

[0024] Figure 4 This refers to the estimated daily (A–B) Zn and (C–D) Se intake in whole grains and flour during the growing season of 2021–2023 in this invention; wherein: (A)–(B) represent the estimated daily Zn intake from grains; (C)–(D) represent the estimated daily Se intake from grains; Note: An asterisk indicates that there are significant differences between different N application levels within the same year: *** P <0.001;**, P <0.01;*, P <0.05; different uppercase letters indicate significant differences in foliar spraying of phosphorus at the same nitrogen application rate, and different lowercase letters indicate significant differences among different foliar spraying treatments at the same nitrogen application rate in the same year. P <0.05); Error bars indicate standard error (n=4); Treatment: N1 / N2, 120 / 240 kg N·ha –1 ; Zn, 0.4% (w / v) ZnSO4·7H2O; Se1 / Se2 / Se3, 0.0010% / 0.0015% / 0.002% (w / v) Na2SeO3; P, 0.2% (w / v) KH2PO4.

[0025] Figure 5 This invention presents the effects of foliar and soil application of nitrogen (N) during the growing season of 2021–2023 on (A~B) crude protein content and (C) protein component content (albumin, globulin, prolamins, glutenin) in whole grains and flour; and (D) a Pearson correlation coefficient heatmap of Zn content, Se content, crude protein content, and protein component content in flour. Wherein: (A)–(B) represent the crude protein content of the grains; (C) represents the content of seed protein components; (D) is the Pearson correlation coefficient between Zn content, Se content, crude protein content and protein component content; Note: (A~C) asterisks indicate significant differences between different N application levels within the same year: *** P <0.001;*, P <0.05. Different uppercase letters indicate significant differences in foliar spraying of phosphorus at the same nitrogen application rate, while different lowercase letters indicate significant differences between different foliar spraying treatments at the same nitrogen application rate in the same year. P <0.05). Error bars represent standard errors (n=4). Treatment: N1 / N2, 120 / 240kg N·ha –1 ; Zn, 0.4% (w / v) ZnSO4·7H2O; Se1 / Se2 / Se3, 0.0010% / 0.0015% / 0.002% (w / v) Na2SeO3; P, 0.2% (w / v) KH2PO4.

[0026] Figure 6This invention relates to the effects of foliar and soil application of nitrogen (N) on the amino acid content of flour during the growing season from 2021 to 2023; wherein: (A) is the amino acid profile of wheat flour from the 2022-2023 growing season; (B) is a heatmap of amino acid content (data standardized to the range of 0–1). (C) is a heatmap of Zn content, Se content, total amino acid content and Pearson correlation coefficients among the contents of each amino acid in flour (data from ZnSe2 and ZnSe2P treatments at N1 level and ZnSe1P and ZnSe2P treatments at N2 level during the 2022-2023 growing season). Note: Different lowercase letters indicate significant differences in total amino acid content among treatments. P <0.05). Error bars represent standard errors (n=3). Treatment: N1 / N2, 120 / 240 kg N·ha –1 ; Zn, 0.4% (w / v) ZnSO4·7H2O; Se1 / Se2 / Se3, 0.0010% / 0.0015% / 0.002% (w / v) Na2SeO3; P, 0.2% (w / v) KH2PO4.

[0027] Figure 7 This study investigates the effects of foliar and soil application of nitrogen (N) during the 2022-2023 growing season on the distribution of selenium (Se) speciation in wheat flour. Note: Different lowercase letters indicate that the same Se morphology differs significantly between different treatments. P <0.05). Error bars represent standard errors (n=3). Treatment: N1 / N2, 120 / 240 kg N·ha –1 ; Zn, 0.4% (w / v) ZnSO4·7H2O; Se1 / Se2 / Se3, 0.0010% / 0.0015% / 0.002% (w / v) Na2SeO3; P, 0.2% (w / v) KH2PO4.

[0028] Figure 8 Specific data for the recommended implementation scheme in this invention; wherein: (A)~(D) represent the standard nitrogen application level (120 kg N ha). –1 The linear relationship between Se spraying amount and flour Se content under the following conditions (data from the ZnSe1P treatment in the 2021-2022 growing season, and the ZnSe1P, ZnSe2P and ZnSe3P treatments in the 2022-2023 growing season). (E) is a partial least squares path model (PLS-PM) analysis, showing the relationships between environmental factors (post-flowering rainfall and daily average temperature), agronomical measures (foliar application of Se, Zn, and P, and soil application of N), crude protein content, protein component content (albumin, globulin, prolamins, glutenin), flour Zn content, and flour Se content; path coefficients are represented by line width and color; red and blue indicate significance, respectively. P <0.05) indicates positive and negative correlations; gray dashed lines indicate insignificant relationships. Gof, goodness of fit; R², explained variance;

[0029] (F) represents the total effect of each variable on the Se content of flour (the sum of direct and indirect effects). Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0031] Example I. Materials and Methods 1. Overview of the test site The field experiment was conducted from October 2021 to June 2023 at the Northwest A&F University Crop Station 1 (34°17′56″N, 108°4′7″E) in Yangling, Shaanxi Province, China. The area has an altitude of 525 m, is a semi-humid but prone-to-arid region, with an average annual temperature of 13℃ and an average annual precipitation of approximately 600 mm. The soil belongs to the loess parent material brown soil class, loess subclass, red oil soil genus, and is a type of brown soil developed from loess parent material (soil from artificial dryland farming). A winter wheat-summer fallow planting system was adopted. The basic physicochemical properties of the topsoil (0–20 cm) before the start of the experiment are shown in Table 1. The average monthly temperature and precipitation for the two wheat growing seasons are shown in Table 2. Figure 1 (A) in the middle.

[0032] Table 1. Basic characteristics of the test soils (n=4) Note: "-" indicates no data.

[0033] 2. Experimental Design This invention includes two field trial schemes.

[0034] A composition for simultaneous zinc-selenium biofortification of wheat grains is prepared by dissolving the following components in water: 0.4% by weight (v / v) zinc sulfate (ZnSO4·7H2O), 0.0013%~0.0017% by weight (v / v) sodium selenite (Na2SeO3), 0.2% by weight (v / v) potassium dihydrogen phosphate (KH2PO4), and 0.02% by weight (v / v) Tween-20, with water added to bring the total volume to 100%. The composition for simultaneous zinc-selenium biofortification of wheat grains is simply referred to as the composition, and is also known as a mixed spray solution or foliar spray solution of zinc sulfate (ZnSO4·7H2O), sodium selenite (Na2SeO3), potassium dihydrogen phosphate (KH2PO4), and Tween-20.

[0035] (1) Experiment 1: Four factors were set for the concentrations of zinc sulfate (ZnSO4·7H2O), sodium selenite (Na2SeO3), and potassium dihydrogen phosphate (KH2PO4) in the mixed spray solution of soil-applied nitrogen (N) fertilizer and foliar spray. Two levels were set for soil-applied N fertilizer: 120 kg N·ha –1 (Standard N application rate, N derived from nitrogen fertilizer), 240 kg N·ha –1 (High nitrogen application rate); Concentration of ZnSO4·7H2O in the mixed spray solution: 0, 0.4% (w / v); Concentration of Na2SeO3 in the mixed spray solution: 0, 0.0010% (w / v) in 2021-2022, and 0, 0.0010%, 0.0015% (w / v) in 2022-2023; Concentration of KH2PO4 in the mixed spray solution: 0, 0.2% (w / v). An incomplete randomized block design was adopted, with 4 blocks. There were 8 treatments in 2021-2022 and 12 treatments in 2022-2023. Details of the treatments are shown in Table 2.

[0036] Among them, soil-applied nitrogen (N) fertilizer refers to the nitrogen fertilizer applied to the soil in the experimental site. The nitrogen fertilizer was purchased from Shaanxi Shanhua Coal Chemical Group Co., Ltd.

[0037] The preparation method for foliar spray solution is as follows: add zinc sulfate (ZnSO4·7H2O) to water until completely dissolved, then add sodium selenite (Na2SeO3) until completely dissolved, then add potassium dihydrogen phosphate (KH2PO4) until completely dissolved, and finally add the surfactant Tween-20 to obtain a mixed solution; mix the mixed solution by sonication at room temperature for 30 minutes (40kHz, 320W) to obtain the final solution.

[0038] (2) Experiment 2: To establish the quantitative relationship between the Se concentration of the sprayed fertilizer and the Se content of the grain when Zn-Se-P are sprayed together (mixed spray solution) under conventional N application conditions, 12 kg N·ha of nitrogen (N) fertilizer was applied to the soil in 2022-2023. –1Under the given conditions, three foliar spraying treatments were set up: ZnSe1P, ZnSe2P, and ZnSe3P. Zn was sprayed with 0.4% (w / v) ZnSO4·7H2O; Se1, Se2, and Se3 were sprayed with 0.0010%, 0.0015%, and 0.0020% (w / v) Na2SeO3, respectively; and P was sprayed with 0.2% (w / v) KH2PO4.

[0039] The wheat variety tested was "Xiaoyan 22" ( Triticumaestium L.), sown on October 21, 2021 and October 18, 2022 respectively, at a sowing rate of 210 kg·ha. –1 ~225kg·ha –1 Nitrogen fertilizer (urea) and phosphorus fertilizer (superphosphate, P2O5 100 kg·ha) –1 All fertilizer was applied evenly as base fertilizer before sowing, followed by tilling with a rotary tiller. All foliar spray solutions were applied evenly to the ears and leaves in the evening on the 7th and 14th day after wheat flowering (specifically April 30th and May 7th, 2022, and April 27th and May 4th, 2023, respectively) using a 330mL manual sprayer (8cm x 22cm). Each spraying micro-area was 2m². 2 (1.45m × 1.45m), the amount of solution sprayed each time is 250mL, which is equivalent to a spraying volume of 1250L·ha. –1 All chemical reagents used in the spraying were of analytical grade, and other field management practices were the same as those used in the local area.

[0040] Table 2 Test Treatment 3. Plant sample collection and testing 3.1 Sample Collection and Pretreatment At wheat maturity, all plants in each microplot were harvested, threshed manually, and the yield was calculated. The grains were mixed evenly, and a portion was ground using a high-throughput tissue grinder (TL2020, DHS Life Science and Technology Co., LTD, Beijing, China) and stored as a whole grain sample; another 100g of grains was ground into flour and bran using a small experimental mill (Quadrumat Junior mill, Brabender, Duisburg, Germany), with a flour yield of approximately 60%.

[0041] 3.2 Determination of Zn, Se, Fe, Mn, and Cu content Dry ashing-atomic absorption spectrometry was used. 0.5 g of sample (whole grains, flour, and bran) was weighed and ashed in a muffle furnace at 600℃ for 6 h. 5 mL of 1:1 (v / v) HNO3 was added to dissolve the ash, and the volume was brought to 50 mL. Elemental content was determined using an atomic absorption spectrometer (AAS; PE-PinAAcle 900F, PerkinElmer, Waltham, MA, USA). A blank control was included for each batch of samples, and the national standard reference material (Zn = 41.1 mg·kg⁻¹) was used. –1 ±2.0 mg·kg –1 The Zn recovery rate was between 95% and 105% when the sample was tested at (GWB08503c, Academy of State Administration of Grain, Beijing, China).

[0042] The blank control is an experimental treatment in which no sample is added, but all other operating procedures are the same.

[0043] 3.3 Se content determination Wet digestion-hydride generation atomic fluorescence spectrometry (HFF-AFS) was employed. 0.5 g of sample (whole grains, flour, and bran) was weighed and digested in HNO3:HClO4 (4:1, v / v) at 165 °C until clear. The solution was then diluted to 25 mL with ultrapure water. Before analysis, 5 mL of the digest was taken and 5 mL of 1:1 HCl was added, followed by reduction in a 95 °C water bath for 30 min. The Se content was determined using a hydride generation-atomic fluorescence spectrometer (LC-AFS8530, Beijing Haiguang Instrument Co., Ltd., Beijing, China). A blank control was included for each batch of samples, and the national standard reference material (Se = 0.439 ± 0.046 mg·kg⁻¹) was used. –1 The quality control of the product (MCS-10294, Beijing Micro Standard Material Technology Co., Ltd., Beijing, China) is carried out, and the Se recovery rate is between 95% and 105%.

[0044] The blank control is an experimental treatment in which no sample is added, but all other operating procedures are the same.

[0045] 3.4 Determination of crude protein, phosphorus, and potassium content Weigh 0.2g of sample (whole grain, flour, and bran), add 5mL of H2SO4 and digest until the solution turns completely brownish-black. Add 8 drops of H2O2, heat again, and repeat 3 times until the solution is clear. N and P contents were determined using a continuous flow analyzer (Auto Analyzer 3-AA3, SEAL Instruments, Maqueen, Wisconsin, USA), and K contents were determined using an AAS (Alternating Current Analysis). Crude protein content was calculated by multiplying the N content by the corresponding conversion factor: 5.83 for whole grain, 5.7 for flour, and 6.31 for bran.

[0046] 3.5 Protein component determination Weigh 0.5g of sample (whole grains and flour), add 5mL of ultrapure water, shake at room temperature, centrifuge at 3500g for 15min, repeat twice, and obtain 10mL of supernatant (albumin); add 5mL of 5% (w / v) NaCl to the precipitate, shake at room temperature, centrifuge at 3500g for 15min, repeat twice, and obtain 10mL of supernatant (globulin); add 5mL of 75% ethanol to the precipitate, shake at room temperature, centrifuge at 3500g for 15min, repeat twice, and obtain 10mL of supernatant (albumin); add 5mL of 0.2% (w / v) NaOH to the precipitate, shake at room temperature, centrifuge at 3500g for 15min, repeat three times, and obtain 15mL of supernatant (glutenin). The nitrogen content in the extracts of each protein component was determined by the semi-micro Kjeldahl method (K9860, Jinan Haineng Instrument Co., Ltd., Jinan, China), and multiplied by the corresponding conversion factor to calculate the protein content (Osborne and Voorhees 1894).

[0047] 3.6 Selenium speciation determination Weigh 0.5g of flour sample, add 60mg of protease XIV, 30mg of lipase and 10mL of Tris-HCl buffer, sonicate for 30min, and then shake in a 37℃ constant temperature water bath at 150r·min in the dark. –1 Enzymatic hydrolysis was performed for 20 h. The hydrolysate was centrifuged at 10,000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm aqueous filter membrane and stored at -20 °C. 5 mL of hydrolysate containing 100 mg of the destructive enzyme was added to the residue, and the above steps were repeated. The supernatants from both hydrolysis attempts were combined. Se speciation was analyzed using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS).

[0048] The liquid chromatography conditions used in the analysis were as follows: Column: Hamilton PRP X-100 analytical column (Hamilton Laboratory Equipment Co., Ltd., Shanghai, China); Guard column: Hamilton PRP X-100 guard column (Hamilton Laboratory Equipment Co., Ltd., Shanghai, China); Mobile phase: 5 mmol·L⁻¹ methanol containing 2% (v / v) –1 Citric acid solution (pH 5.3); flow rate: 1.0 ml / min –1 Injection volume: 100 µL. ICP-MS (Thermo Fisher Scientific, Waltham, Massachusetts, USA) operating parameters: RF power: 1400 W; Nebulizer gas flow rate: 1.05 L / min –1 Helium flow rate: 1.0 L / min –1 Monitoring element: 77Se; Integration mode: Peak area.

[0049] 3.7 Amino Acid Determination Weigh 50 mg of flour sample into a hydrolysis tube, add 10 mL of 1:1 (v / v) hydrochloric acid, purge with nitrogen for 30 seconds, seal, and hydrolyze in an oil bath at 110 °C for 24 h. After cooling, filter through a 0.45 μm filter membrane and bring the volume to 50 mL. Take 2 mL of the solution and deacidify twice on an 85 °C hot plate until a small amount of solid or residue remains at the bottom of the flask. Redissolve in 1 mL of sodium citrate buffer, filter through a 0.22 μm filter membrane, and analyze using an amino acid analyzer (Biochrom 30). + Tested by Biocom, Cambridge, UK.

[0050] 4. Calculation formula Zn / Se carried out by grains (g·ha) –1 The calculation is as follows: Zn / Se carry-out amount of grain = Zn / Se content in whole grain × grain yield (1); In the above formula, the unit for the Zn / Se carried out by the grain is g·ha. –1 ; Daily Zn / Se intake (EDI, μg / person) –1 ·day –1 The estimated value is calculated according to (Di et al. 2023) as follows: EDI Zn / Se =C 籽粒 ×W 籽粒摄入 (2); In the above formula, EDIZn / Se (μg·person) –1 ·day –1 ( ) represents the estimated daily Zn / Se intake; C 籽粒 (μg·kg) –1 The Zn / Se ratio in wheat grains is denoted as W. 籽粒摄入 (kg·person) –1 ·day –1 The average daily intake of wheat flour and its products by adults is 0.1732 kg per person. –1 ·day –1 ); The dietary non-carcinogenic risk of Zn / Se was assessed using the target hazard quotient (THQ) (Ismail et al. 2024): THQ= (3); In the above formula, C Zn / Se Zn / Se content in wheat grains; CF is the dry / fresh weight conversion factor (0.162); DI is the average daily intake of wheat flour and its products for adults (0.1732 kg / person). – 1 day –1 BW represents the average adult body weight (70 kg); RFD is the oral reference dose, Zn is 0.57 mg / kg. –1 ·day –1 Se was 3.64 μg·kg –1 ·day –1 Based on the upper limit of daily intake of 40 mg Zn·d for a 70 kg adult. –1 and 255μg Se·day –1 The calculations show that THQ < 1 indicates no significant health risk to the exposed population, while THQ ≥ 1 indicates a possible health risk.

[0051] 5. Statistical Analysis Microsoft Excel 2021 (Microsoft Corp., Redmond, WA, USA), IBM SPSS Statistics 26 (IBM Corp., Armonk, NY, USA), and Origin 2021 (OriginLab Corp., Northampton, MA, USA) were used for data processing, statistical analysis, and chart creation. Two-way ANOVA was used to test the main effects and interaction between soil nitrogen application and foliar spraying treatments. Multiple comparisons were performed using the least significant difference (LSD) test at a significance level of [missing value]. P <0.05. Data in the figure are expressed as mean ± standard error (Mean ± SE). Pearson correlation analysis was used to assess the correlation between grain Zn and Se content and crude protein, protein composition, and amino acid content. A partial least squares path model (PLS-PM) was constructed using the "plspm" package in R 4.3.2 (R core development team, Vienna, Austria) to comprehensively analyze the association pathways between environmental factors (post-flowering rainfall and post-flowering mean temperature), agronomical measures (spraying Se, Zn, and P concentrations, soil-applied N level), flour crude protein content, flour protein composition content, flour Zn content, and flour Se content. Model reliability was assessed by explaining the variance (R0.05). 2 The results were evaluated using the goodness of fit (Gof).

[0052] II. Results 1. Environmental conditions, yield, and zinc and selenium carryover The climatic conditions differed between the two growing seasons. Compared to 2021-2022, the total rainfall during the entire wheat growing season and the rainfall after flowering were approximately 29% and 120% higher in 2022-2023, respectively, while the average daily temperature after flowering was significantly lower. Figure 1 (B) ~ Figure 2 (D) in the middle.

[0053] In both growing seasons, compared with conventional nitrogen (N) application (120 kg N·ha) –1 Compared to high-volume N application (240 kg N ha), –1 This significantly increased wheat grain yield. Figure 2 (A)). Among different foliar spraying treatments, the treatment with the combined application of Zn-Se-P had a relatively higher yield. Soil application of high N also significantly increased the amount of Zn and Se carried out by the grains. Figure 2 (B) and Figure 2(C)). Spraying Zn significantly increased the amount of Zn carried out of the grains. When Se was sprayed in combination with P, the amount of Se carried out of the grains reached its maximum, indicating that when Se supply is sufficient, the addition of P can further promote the accumulation of Se in the grains.

[0054] 2. Effects of combined foliar spraying on zinc and selenium content in wheat grains High nitrogen application significantly increased the Zn and Se content in whole wheat grains, flour, and bran. Figure 3 (Table 3).

[0055] At both N levels, all treatments involving Zn spraying significantly increased the Zn content in all grain tissues. Figure 3 (A)A~ Figure 3 (C) ensures that the total Zn content of the whole grains consistently reaches the target range for biofortification (40 mg·kg⁻¹). –1 ~60mg·kg –1 (Cakmak 2008). Except for the reduction of Zn content in bran by combined spraying of Zn and P, different concentrations of Se or P combined with Zn did not affect the enrichment effect of Zn. The distribution of Zn in different components of the grain was uneven, and the Zn content of flour was significantly lower than that of whole grain.

[0056] Compared with Se spraying alone, Zn-Se combined spraying significantly reduced the Se content in various tissues of the grain, proving the existence of Zn-Se antagonism. Figure 3 (D) ~ Figure 3 (F)). However, the addition of P overcame this antagonistic inhibition of grain Se content. Compared with spraying Se or Zn-Se alone, the addition of P significantly increased grain Se content. Se distribution in grains was relatively uniform, with no significant difference in Se content between whole grains and flour. Regardless of the soil N application level, the grain Se content of the Zn-Se-P co-application treatment reached a level comparable to that of spraying the same concentration of Se alone, compensating for the Se loss caused by Zn-Se antagonism.

[0057] Table 3. Two-way ANOVA analysis of the significance levels of soil-applied nitrogen (N), foliar spraying (F), and their interaction (N×F) on grain yield at maturity, and the Zn, Se, or crude protein content in whole grains, flour, and bran. P -values) 3. The effect of combined spraying on dietary zinc and selenium intake. Based on the estimated daily consumption of flour products by Chinese adults (173.2g) (Chinese Center for Disease Control and Prevention), the trend of daily dietary intake (EDI) of flour is consistent with the changes in Zn and Se content. Figure 4Spraying Zn increased the EDI Zn content of whole grains and flour by 61% and 70%, respectively. Figure 4 (A) ~ Figure 4 (B)). When Zn-Se-P are sprayed together, the addition of P effectively alleviates Zn-Se antagonism, allowing the EDI Se of whole grains and flour to reach the same level as that of Se sprayed alone at the same concentration. Figure 4 (C) ~ Figure 4 (D)). The target hazard quotients (THQ) for Zn and Se content in all treated whole grains and flours were well below 1 (Table 4), indicating that the non-carcinogenic risk of consuming fortified wheat products is within a safe range (Ismail et al. 2024).

[0058] Table 4. Effects of foliar and soil application of nitrogen on the target hazard quotient (THQ) of whole grain and flour during the 2021-2023 growing season. 4. Effects of combined spraying on crude protein, protein composition and amino acid profile of wheat grains The application of high-N soil significantly increased the content of crude protein, various protein components (albumin, globulin, prolamins, and glutenin) and total amino acids in whole grains and flour. Figure 5 (A) ~ Figure 5 (C) and Table 3). Foliar spraying treatments did not show a consistent pattern in the protein component content. Correlation analysis showed that the Zn content of flour was significantly positively correlated with the content of prolamins and glutenins, the Se content was positively correlated with the content of globulins, and the crude protein content was significantly positively correlated with the content of all protein components. Figure 5 (D) in the middle.

[0059] Compared with spraying Se alone or in combination with Zn-Se, spraying Zn-Se-P together significantly increased the total amino acid content and the content of most individual amino acids. Figure 6 (A) ~ Figure 6 (B)). In all treatments, glutamic acid (Glu) was the most abundant amino acid, accounting for 36%–40% of the total amino acids, followed by leucine (Leu). Under conditions of sufficient exogenous Zn and Se supply, the Zn content and crude protein content of flour were significantly positively correlated with all amino acids except cysteine ​​(Cys) and lysine (Lys), the Se content of flour was significantly positively correlated with all amino acids except aspartic acid (Asp), alanine (Ala), Cys, methionine (Met), and arginine (Arg), and the total amino acid content was significantly positively correlated with Zn, Se, and crude protein content. Figure 6 (C) in the middle.

[0060] 5. Effects of combined spraying on selenium form in flour Se(IV) was not detected in mature wheat flour. Figure 7 Se in flour exists primarily in organic form, with selenomethionine (SeMet) accounting for the largest proportion, exceeding 65% of the total Se, followed by selenocysteine ​​(SeCys) and selenomethylcysteine ​​(SeMeCys). Compared to other treatments, the combined application of Zn, Se, and P significantly increased the relative proportion of SeMet. This result indicates that inorganic Se applied via foliar spraying is effectively assimilated into a more bioavailable and safer organic form within the plant and stored in the grain.

[0061] 6. Effects of combined foliar spraying on other nutrients in various tissues of wheat grains Soil application of nitrogen fertilizer and foliar spraying did not negatively affect the contents of Fe, Mn, Cu, P, and K in any of the grain tissues (whole grain, flour, and bran) (Tables 5-9). High-level nitrogen application significantly increased the Fe content in whole grain, flour, and bran. These results indicate that the Zn-Se simultaneous fortification strategy adopted in this invention, while increasing the content of the target micronutrients, did not interfere with the normal accumulation of other essential mineral elements in crops.

[0062] Table 5. Content of Fe, Mn, Cu, P, and K in whole wheat grains, flour, and bran. Note: "-" indicates no data.

[0063] Table 6 Appendix 1 of Table 5 Note: "-" indicates no data.

[0064] Table 7 Appendix 2 of Table 5 Note: "-" indicates no data.

[0065] Table 8 Appendix 3 of Table 5 Note: "-" indicates no data.

[0066] Table 9 Appendix 4 of Table 5 Note: "-" indicates no data.

[0067] 7. Zn-Se simultaneous and enhanced spraying scheme based on dietary safety A partial least squares path model (PLS-PM) was used to comprehensively evaluate the direct and indirect effects of environmental factors, agronomic practices, and protein traits on Zn and Se accumulation in grains. Although the Zn and Se content in flour was significantly positively correlated with the crude protein and protein component content (…),… Figure 5 (D) suggests that proteins may be a potential reservoir of elements. However, under comprehensive regulation, the direct effect of foliar spraying (Zn, Se, P) on the Zn and Se content of flour is far greater than that of environmental factors and protein components. Figure 8 (E) in Figure 8 (F)). This result further confirms that human-controlled agronomic measures are the main driving factor for the simultaneous enhancement of Zn-Se, indicating that a stable simultaneous enhancement effect of Zn-Se can be achieved under different environmental conditions by precisely optimizing the foliar spraying program.

[0068] This invention, under conventional nitrogen application levels, achieves a comprehensive Se enrichment target value (0.25 mg·kg⁻¹). –1 ~0.30 mg·kg –1 Se toxicity threshold (1.0 mg·kg) –1 Based on multiple criteria including RNI, UL, and THQ, a safe spraying scheme for simultaneous bio-enhanced Zn-Se was constructed. Figure 8 When Zn-Se-P are sprayed together, for people whose staple food is whole wheat, the appropriate Se spraying concentration is 0.0013%~0.0017% and below 0.0038%; for people whose staple food is flour, the appropriate Se spraying concentration is 0.0011%~0.0013% and below 0.0026%. Figure 8 (A) ~ Figure 8 (D) Considering the needs of populations with different dietary structures, it is recommended that the concentration of Se sprayed in production applications be controlled within the range of 0.0013%~0.0017%, and sprayed in combination with 0.4% ZnSO4·7H2O and 0.2% KH2PO4. This scheme can meet the Zn-Se nutritional needs of populations whose staple food is whole wheat or flour while ensuring dietary safety, thus achieving simultaneous Zn-Se biofortification.

[0069] The results above show that (1) spraying Zn-Se with an appropriate concentration of KH2PO4 during the wheat grain-filling stage can effectively compensate for the Se loss caused by Zn-Se antagonism and achieve simultaneous Zn-Se fortification in wheat. The combined spraying of Zn-Se-P restores the Se content in various tissues of the grain to a level comparable to that of spraying with the same concentration of Se alone, while ensuring that the Zn content stably reaches the biofortification target (40 mg·kg). –1 ~60mg·kg –1Furthermore, this compensatory effect was consistent across two growing seasons with significantly different climatic conditions and under different nitrogen application levels, indicating that it has good environmental adaptability.

[0070] (2) This strategy further improved the nutritional quality of the grains. After fortification, Se in the flour mainly existed in a bioavailable and safe organic form, and the total amino acid and multiple amino acid content in the flour were significantly increased. Nitrogen fertilizer management provided more potential binding sites for Zn and Se by increasing the protein reserves in the grains, while the addition of phosphorus promoted element accumulation by optimizing the amino acid composition. Dietary risk assessment showed that the intake of Zn and Se from whole grains and flour was lower than the tolerable upper intake level, and the target hazard coefficient was far less than 1, indicating no health risk.

[0071] (3) Based on the above findings, this study proposes an optimized spraying scheme suitable for conventional N application levels: 0.4% ZnSO4·7H2O + 0.0013%–0.0017% Na2SeO3 + 0.2% KH2PO4. This scheme can meet the Zn-Se nutritional needs of different dietary groups while ensuring dietary safety.

[0072] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0073] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A composition for simultaneous zinc-selenium biofortification of wheat grains, characterized in that, It is prepared by dissolving zinc sulfate, sodium selenite, potassium dihydrogen phosphate, and a surfactant in water; the mass ratio of zinc sulfate, sodium selenite, and potassium dihydrogen phosphate is 1:0.00325~0.00425:0.5~0.

75. The surfactant includes Tween-20.

2. The use of the composition according to claim 1 in improving the nutritional quality of wheat grains.

3. The application according to claim 2, characterized in that, The composition is applied to wheat to increase the selenium content of wheat grains, thereby improving the nutritional quality of wheat grains.

4. The application according to claim 3, characterized in that, The composition is applied to wheat by spraying.

5. The application according to claim 4, characterized in that, The composition is sprayed onto wheat leaves and ears.

6. The application according to claim 5, characterized in that, The composition is sprayed during the wheat flowering period or the early grain-filling stage of wheat.

7. The application according to claim 6, characterized in that, The composition is sprayed 2 to 3 times, with each application amount being 1000 L·ha. –1 ~1250L·ha –1 .

8. The application according to claim 7, characterized in that, The amount of nitrogen fertilizer applied to the soil before wheat sowing, after spraying with the composition, was 120 kg N·ha. –1 ~150kg N·ha –1 .

9. The application according to claim 3, characterized in that, The composition improves the selenium content of wheat grains and thus enhances their nutritional quality by mitigating the antagonistic effect of zinc-selenium synergistic spraying.

10. The application according to claim 3, characterized in that, The composition is used to increase the total amino acid content of wheat grains, increase the proportion of selenomethionine, promote the conversion of inorganic selenium to organic form, and thus improve the nutritional quality of wheat grains.

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