Nano-selenium synthesized by high-density fermentation of yeast and application of nano-selenium in agriculture

Abstract

The invention discloses a method for synthesizing nano-selenium by high-density fermentation of saccharomyces cerevisiae boulardii, a selenium-enriched yeast product prepared by the method and application of the nano-selenium in tomato cultivation. The fermentation comprises three-stage seed propagation (at 30-32 DEG C, 250-300 rpm, and inoculation amount of 5-10%), inoculation (dissolved oxygen is greater than or equal to 30%), supplementary feeding (residual sugar is 2-5 g / L), selenite induction (1-3 mM until the fermentation broth is brick red) and final-stage temperature and pH regulation, and the selenium-enriched yeast with the cell dry weight of 150-300 g / L and the dry basis organic selenium of 6900-7500 mu g / g is obtained. During application, tomato is sprayed for the first time in 45 days after transplanting and is sprayed for three times (accumulated spraying liquid is 3.2-3.6 L / group) in 14 days, the optimal dosage is 10mg / L, the TA liter of tomatoes is more than or equal to 80%, the dry weight liter is more than or equal to 8%, the sugar-acid ratio is 3.8-4.5, and the selenium content of fruits exceeds that of a control group by 85% and is superior to that of sodium selenite; the dosage of 20mg / L is easy to generate growth inhibition, and the dosage is not used as a recommended application concentration; the method is safe and efficient, and is suitable for industrialization and agricultural popularization.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, in particular to a kind of yeast high-density fermentation synthesis nanometer selenium and its application in agriculture. BACKGROUND

[0002] Currently, in the field of synthesis of biological source nanometer selenium, the prior art attempts to use bretannomyces as a transformation strain, but faces a core technical bottleneck. The existing fermentation process has obvious deficiencies in the regulation of key parameters: under conventional conditions, the control accuracy of dissolved oxygen, pH value and carbon-nitrogen ratio is low, which makes it difficult to simultaneously improve the specific growth rate of yeast and the selenium reduction yield; and when adding selenite in the late logarithmic phase, the existing technology generally has the problems of inaccurate addition time point and improper final concentration, which easily induces oxidative stress response of yeast cells, causing cell death, ultimately resulting in low number of viable cells per unit volume, insufficient recovery rate of effective selenium components, and seriously restricting the scale application of bretannomyces in the industrialized synthesis of nanometer selenium. In the field of selenium nutrition strengthening of agricultural crops, the existing technology relies on inorganic selenium (such as sodium selenite) for foliar spraying. Although this method is a conventional means, it has significant defects. On the one hand, the plant absorption and utilization rate of inorganic selenium is low, and most of the selenium that is not converted by crops is easily left in the soil, causing potential environmental risks; on the other hand, its safe application window is narrow, and a slight overdose will inhibit plant growth, and its effect on improving the flavor and quality of fruits is limited, it can only achieve basic selenium element accumulation, and cannot simultaneously optimize key quality indicators such as fruit sweet-sour balance, making it difficult to meet the dual needs of "nutrition strengthening + quality improvement" in agricultural production. Although biological nanometer selenium has theoretical advantages, the existing technology has not solved the problem of adapting efficient preparation to field application, and has not achieved large-scale promotion. In addition, the evaluation system for the comprehensive influence of foliar selenium supplementation on crops in the existing technology has deficiencies. Traditional evaluation methods focus on a single dimension and lack a system of indicators covering the three dimensions of "nutrition-flavor-growth". The existing indicators cannot simultaneously correlate titratable acid, total soluble solids, sugar-acid ratio, fruit selenium content and aerial part dry weight, making it difficult to accurately determine the comprehensive benefits and safety boundaries of biological nanometer selenium compared to inorganic selenium, and thus it is difficult to optimize fermentation process parameters and field application schemes, hindering the application of biological nanometer selenium in the agricultural field. SUMMARY

[0003] The main purpose of the present application is to overcome the defects of the existing yeast high-density fermentation synthesis nanometer selenium and its application in agriculture, and to provide a new yeast high-density fermentation synthesis nanometer selenium and its application in agriculture, the technical problem to be solved is to improve the biomass of selenium-rich yeast and the reduction efficiency of nanometer selenium, and to realize the stable and large-scale production of products with high organic selenium content, so as to be more suitable for practical application and have industrial application value.

[0004] Another objective of this invention is to provide a method for synthesizing nano-selenium through high-density yeast fermentation and its application in agriculture. The technical problem to be solved is to optimize the application scheme of nano-selenium in crop cultivation, so as to simultaneously achieve the synergistic effect of crop selenium nutrient fortification, fruit flavor and quality optimization and plant growth promotion, thereby making it more suitable for practical use.

[0005] Another objective of this invention is to provide a method for synthesizing nano-selenium through high-density yeast fermentation and its application in agriculture. The technical problem to be solved is to avoid the environmental risks of low absorption and utilization rate, narrow safety window, and easy soil residue caused by inorganic selenium foliar fertilizer, thereby improving the safety and environmental friendliness of crop selenium nutrient fortification and making it more suitable for practical use.

[0006] Another objective of this invention is to provide a method for high-density yeast fermentation to synthesize nano-selenium and its application in agriculture. The technical problem to be solved is to establish a systematic evaluation system covering the three dimensions of crop "nutrition-flavor-growth" to provide a precise basis for optimizing the nano-selenium preparation process and adjusting agricultural application schemes, thereby making it more suitable for practical use.

[0007] The objective of this invention and the technical problem it solves are achieved through the following technical solution. Based on the high-density fermentation synthesis of nano-selenium by yeast proposed in this invention and its application in agriculture, the core includes the process of high-density fermentation synthesis of nano-selenium by *Saccharomyces boulardii*, and the application of this nano-selenium as a foliar fertilizer in agricultural crops (using dwarf tomatoes as an example), as detailed below:

[0008] The high-density fermentation process for synthesizing nano-selenium using *Saccharomyces bladderwracki* includes:

[0009] Three-stage seed propagation: Activate *Saccharomyces boulardii* at 30-32℃ for 24-36h, and then inoculate it sequentially into primary, secondary, and tertiary YPD liquid culture media. The culture conditions for each stage are 30-32℃, 250-300rpm, and 20-24h, and the inoculation amount is 5-10% (v / v).

[0010] Seed treatment and fermentation inoculation: The tertiary seed culture was centrifuged at 0-4℃ and 6000-8000rpm for 5-8min, washed 2-3 times, and resuspended in sterile water to 200-400mL. It was then inoculated into high-density fermentation medium at 5-10% (v / v) and fermented at 30-32℃ and 250-300rpm for 20-24h. The dissolved oxygen (DO) should not be lower than 30%, and the pH during the growth period should be controlled at 5.5-6.5, preferably 5.8-6.2.

[0011] Feeding and selenium enrichment induction: Glutamic acid was added 5-9 hours in the early stage of fermentation, and feed medium containing glucose, (NH4)2SO4, corn steep liquor and H3PO4 was added at the same time to control residual sugar at 2-5 g / L and dissolved oxygen at 30-50%; selenite was added to a final concentration of 1-3 mM in the late logarithmic stage to maintain induction until the fermentation broth turned brick red.

[0012] Final stage control and product collection: After 22-26 hours of fermentation, adjust the temperature to 20-25℃ and the pH to 5.0-6.0, and continue culturing for 2-4 hours; centrifuge to remove the supernatant and wash 2-3 times to obtain a selenium-enriched yeast product containing nano-selenium with a cell dry weight of 150-300 g / L and a dry basis organic selenium content of 6900-7500 μg / g.

[0013] Applications of nano-selenium in agriculture include:

[0014] Foliar fertilizer preparation: The selenium-enriched yeast product is diluted into a nano-selenium foliar fertilizer working solution;

[0015] Field application: Spray the first time 45 days after transplanting dwarf tomatoes, and then spray once every 14 days for a total of 3 times, with a cumulative spray volume of 3.2-3.6L / group. Spraying conditions are wind speed <3m / s and relative humidity 50-80%. Irrigate 6-8 hours after spraying to avoid rain. The preferred dosage is 10mg / L nano selenium, with water and 10mg / L sodium selenite as controls.

[0016] The objectives of this invention and the technical problems it addresses can be further achieved using the following technical measures. In the aforementioned high-density yeast fermentation synthesis of nano-selenium and its application in agriculture, the three-stage seed liquid centrifugation is preferably performed at 4°C, 7000-8000 rpm, and for 5-8 minutes to further enhance seed cell activity; sodium selenite is preferred as the selenite, and it is added in stages or by constant flow, with dissolved oxygen levels not lower than 25% during addition to ensure stable synthesis of nano-selenium.

[0017] The objective of this invention and the technical problem it solves are achieved through the following technical solution. Based on the high-density yeast fermentation synthesis of nano-selenium and its application in agriculture proposed in this invention, in the agricultural application stage, by controlling the tomato transplanting spacing to 45cm × 60cm (density approximately 4 plants / m²), 2 By implementing three-stalk fruit setting and ensuring that the field measures such as water and fertilizer management and pest and disease control are consistent across all treatment groups, interference factors can be eliminated, and the application effect of nano-selenium can be accurately verified.

[0018] The objectives of this invention and the technical problems it solves can be further achieved by the following technical measures. Regarding the aforementioned high-density yeast fermentation synthesis of nano-selenium and its application in agriculture, at the tomato ripening stage (H3), treatment with 10 mg / L nano-selenium can increase titratable acidity by ≥80%, increase single-plant aerobatic dry weight by ≥8%, control the sugar-acid ratio within the range of 3.8-4.5, and increase the fruit selenium content by more than 85% compared to the water control, and is superior to treatment with sodium selenite of the same concentration; 20 mg / L nano-selenium easily leads to a decrease in total soluble solids and dry weight, and is not considered a universally recommended dosage. Further clarification of the optimal application boundary is needed to ensure practicality. This invention has significant advantages and beneficial effects compared to existing technologies. As can be seen from the above technical solutions, to achieve the aforementioned objectives, the main technical contents of this invention are as follows:

[0019] This invention provides a method for synthesizing nano-selenium through high-density yeast fermentation, characterized in that the synthesis method includes:

[0020] (1) Three-level seed culture: After activating the Blakely yeast at 30℃-32℃ for 24h-36h, it was inoculated into the first, second and third level seed culture medium in sequence. The culture of each level was 30℃-32℃, 250rpm-300rpm, 20h-24h, and the inoculation amount was 5%v / v-10%v / v.

[0021] (2) Seed inoculation: Centrifuge the third-grade seed liquid at 0℃-4℃ and 6000rpm-8000rpm for 5min-8min, wash 2-3 times, resuspend in sterile water to 200mL-400mL, inoculate into high-density fermentation medium at 5%v / v-10%v / v, ferment at 30℃-32℃ and 250rpm-300rpm for 20h-24h, dissolved oxygen ≥30%;

[0022] (3) Feeding and induction: Glutamic acid was added and fed medium was added during the early stage of fermentation (5-9 hours). The medium contained 4.3%-4.5% glucose, 0.3%-0.5% (NH4)2SO4, 2.5%-3.5% corn steep liquor, and 0.1%-0.4% H3PO4 per liter, all w / v or v / v. Selenite was added to a final concentration of 1mM-3mM during the logarithmic stage, and the fermentation broth was induced to turn brick red.

[0023] (4) Final regulation and collection: After fermentation for 22-26 hours, adjust the temperature to 20-25℃ and pH to 5.0-6.0, and continue culturing for 2-4 hours; centrifuge to remove the supernatant, wash with sterile water 2-3 times to obtain selenium-enriched yeast product containing nano-selenium.

[0024] Preferably, the pH during the fermentation growth period in step (2) is controlled at 5.5-6.5, more preferably 5.8-6.2; and the pH during the selenium enrichment induction stage is controlled at any range of 3.5-4.5 or 5.5-6.2.

[0025] Preferably, the selenite is sodium selenite, which is added in a segmented or constant-flow manner, and the dissolved oxygen level is not lower than 25% during the addition period.

[0026] Preferably, in step (3), the feed flow is controlled with the goal of "maintaining the residual sugar in the fermentation broth at 2 g / L–5 g / L", and the dissolved oxygen is maintained at 30%–50% through the linkage of stirring and aeration.

[0027] Preferably, the centrifugation conditions for the tertiary seed solution in step (1) are 4℃, 7000rpm-8000rpm, and 5min-8min.

[0028] Preferably, the biomass of the obtained selenium-enriched yeast product containing nano-selenium is 140g / L-170g / L based on cell dry weight.

[0029] The present invention also provides a nano-selenium-enriched yeast product synthesized by high-density fermentation of yeast, characterized in that it is prepared by the method of any one of claims 1-6, and the dry basis organic selenium content of the product is 6900-7500μg / g, and it is rich in bio-derived nano-selenium.

[0030] Furthermore, the nano-selenium-enriched yeast product was prepared into a nano-selenium foliar fertilizer, diluted into a working solution, and sprayed onto tomatoes. The spraying schedule was as follows: the first spray was applied 45 days after tomato transplanting, followed by three sprays every 14 days, with a total spray volume of 3.2-3.6 L / set. The spraying conditions were: wind speed <3 m / s, relative humidity 50-80%, and irrigation should be carried out within 6-8 hours after spraying to avoid rain.

[0031] Furthermore, the working solution of the diluted nano-selenium foliar fertilizer has a nano-selenium concentration of 10 mg / L, which is the preferred application dose for agricultural tomato cultivation.

[0032] Furthermore, at the ripening stage, tomatoes must meet at least one of the following criteria compared to the water control:

[0033] (i) Titrateable acid increased by ≥80%;

[0034] (ii) Increase in aerial dry weight of single plant by ≥8%;

[0035] (iii) The sugar-acid ratio should be controlled within the range of 3.8-4.5.

[0036] As described above, this invention provides a high-density fermentation method for synthesizing nano-selenium based on *Saccharomyces boulardii*. By optimizing parameters such as three-stage seed propagation, dissolved oxygen control, feed addition, and selenite induction, a nano-selenium-enriched yeast with a cell dry weight of 150-300 g / L and a dry-basis organic selenium content of 6900-7500 μg / g is stably obtained. Simultaneously, this product is prepared as a foliar fertilizer. When sprayed on tomatoes at a dose of 10 mg / L (first spray 45 days after transplanting, then once every 14 days for a total of 3 sprays, with a cumulative spray volume of 3.2-3.6 L / set), it can increase the titratable acidity of tomatoes at maturity by ≥80%, increase the aerial dry weight of single plants by ≥8%, control the sugar-acid ratio at 3.8-4.5, and increase the selenium content of the fruit by more than 85% compared to the water control, and is superior to sodium selenite of the same concentration. This method is safe, efficient, and suitable for industrial-scale promotion.

[0037] By employing the above technical solution, the present invention provides high-density yeast fermentation for the synthesis of nano-selenium and its application in agriculture, which has at least the following advantages:

[0038] Excellent process performance: It solves the core problems of existing technologies such as "low selenium biomass and unstable nano-selenium reduction efficiency". The fermentation parameters are highly controllable and can stably produce selenium-enriched yeast products with high biomass and high organic selenium content, meeting the needs of large-scale production.

[0039] Superior agricultural application: 10mg / L nano selenium foliar fertilizer can simultaneously achieve selenium nutrient fortification in tomatoes, fruit flavor optimization and plant growth promotion. Its comprehensive effect is better than traditional inorganic selenium (sodium selenite), and it meets the actual needs of agriculture for "quality improvement + selenium enrichment".

[0040] High safety and environmental friendliness: It avoids the environmental risks of low absorption and utilization rate, narrow safety window and easy soil residue of inorganic selenium. Nano selenium has good biocompatibility and no harmful by-products after application.

[0041] It has broad industrial applicability: the fermentation process can be scaled up (stability verified at scales of 5L to 80L), and the application of foliar fertilizer can be extended to other fruit and vegetable crops, possessing extensive industrial utilization value.

[0042] In summary, this invention, with its unique high-density yeast fermentation synthesis of nano-selenium and its application in agriculture, overcomes the shortcomings of existing methods. It provides a new method to enhance selenium-enriched yeast biomass and nano-selenium reduction efficiency, optimize nano-selenium application in crops to achieve a synergistic effect of "selenium enrichment + quality improvement + growth promotion," mitigate the environmental and safety risks of inorganic selenium, and establish a multi-dimensional evaluation system covering "nutrition-flavor-growth." Therefore, it is more practical and has industrial application value. It possesses numerous advantages and practical value, and no similar designs have been publicly disclosed or used in similar methods, making it truly innovative. It represents a significant improvement in both method and function, a substantial technological advancement, and produces user-friendly and practical effects. Compared to existing high-density yeast fermentation synthesis of nano-selenium and its application in agriculture, it offers several enhanced benefits, making it more suitable for practical use and possessing broad industrial application value. It is indeed a novel, progressive, and practical new design.

[0043] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. The specific methods and structures of the present invention are given in detail in the following embodiments and accompanying drawings. Attached Figure Description

[0044] Figure 1 Curve showing the change in *Saccharomyces boulardii* biomass (wet weight) over fermentation time in a 5L fermenter.

[0045] Figure 2 Curve showing the change in *Saccharomyces boulardii* biomass (wet weight) over fermentation time in an 80L fermenter.

[0046] Figure 3 The chart shows the trend of TSS (°Brix) and TA (%citric acid) of tomato fruits in three treatment groups (water, 10 mg / L sodium selenite, 10 mg / L nano selenium, and 20 mg / L nano selenium) at three stages: H1, H2, and H3. The data are the mean ± standard deviation.

[0047] Figure 4 Bar chart comparing the TA (% citric acid) of tomato fruits in different treatment groups during the H1–H3 period. Data are mean ± standard deviation.

[0048] Figure 5 Comparison of aerial dry weight (g / plant) of tomato plants in different treatment groups. Data are mean ± standard deviation.

[0049] Figure 6 Comparison of selenium content (μg / g DW) in tomato fruits of different treatment groups. Data are mean ± standard deviation. Detailed Implementation

[0050] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following detailed description, in conjunction with the accompanying drawings and preferred embodiments, details the specific implementation methods, steps, structures, features, and effects of the high-density yeast fermentation synthesis of nano-selenium and its application in agriculture proposed according to the present invention.

[0051] Please see Figure 1 , Figure 2 , Figure 3 , Figure 4 As shown, the preferred embodiment of the present invention, which describes the high-density fermentation synthesis of nano-selenium by yeast and its application in agriculture, mainly includes the following steps:

[0052] I. Steps for High-Density Fermentation Synthesis of Nano-Selenium by *Saccharomyces boulardii*

[0053] (1) Three-level seed propagation

[0054] Strain activation: Inoculate *Saccharomyces boulardii* into YPD solid medium (2% glucose, 1% yeast extract, 2% peptone, 2% agar) and culture at 30-32℃ for 24-36 h to obtain activated strains;

[0055] Primary seed culture: Select activated single colonies and inoculate them into YPD liquid medium (2% glucose, 1% yeast extract, 2% peptone) and culture at 30-32℃ and 250-300rpm for 20-24h;

[0056] Secondary / tertiary seed culture expansion: Transfer the primary seed culture to secondary YPD liquid medium at an inoculation rate of 5-10% (v / v) and culture under the same conditions for 20-24 hours; then transfer the secondary seed culture to tertiary YPD liquid medium at the same inoculation rate and culture under the same conditions for 20-24 hours to obtain highly active tertiary seed culture.

[0057] Seed treatment: Centrifuge the tertiary seed culture at 4℃ and 7000-8000rpm for 5-8min, discard the supernatant, wash 2-3 times with sterile water to remove residual culture medium, and then resuspend in sterile water to 200-400mL.

[0058] (2) High-density fermentation inoculation and parameter control

[0059] Fermentation inoculation: Inoculate the resuspended tertiary seed culture into high-density fermentation medium (containing 1.6% glucose, 3% corn flour, 0.2g MgSO4, 5.8g KCl, and 0.58g ZnSO4 per liter) at an inoculation rate of 5-10% (v / v).

[0060] Growth period control: maintain fermentation temperature at 30-32℃, rotation speed at 250-300rpm, pH control at 5.8-6.2 (optimal range), maintain dissolved oxygen (DO) at no less than 30% through a combination of stirring and aeration, and continue fermentation for 20-24 hours.

[0061] (3) Feeding and selenium enrichment induction

[0062] Early stage addition: After 5-9 hours of fermentation (early stage), add glutamic acid to the fermenter to provide metabolic precursors for yeast growth;

[0063] Feeding control: Feeding medium (containing 4.3-4.5% glucose, 0.3-0.5% (NH4)2SO4, 2.5-3.5% corn steep liquor, and 0.1-0.4% H3PO4 per liter) was fed simultaneously, with the control target of "residual sugar in fermentation broth maintained at 2-5 g / L". Dissolved oxygen (DO) was maintained at 30-50% through a combination of stirring and aeration.

[0064] Selenium enrichment induction: When fermentation enters the late logarithmic stage, sodium selenite is added in stages or by constant flow to make the final concentration of sodium selenite in the fermentation broth 1-3mM. During the addition period, dissolved oxygen (DO) is maintained at no less than 25%, and the induction continues until the fermentation broth turns brick red (indicating the formation of nano-selenium).

[0065] (4) Final stage of fermentation and product collection

[0066] Final stage control: After 22-26 hours of fermentation, adjust the fermentation temperature to 20-25℃ and the pH to 5.0-6.0, and continue culturing for 2-4 hours to promote the stable accumulation of nano-selenium;

[0067] Product collection: The fermentation broth was centrifuged at 8000 rpm for 8 min, the supernatant was discarded, and the product was washed 2-3 times with sterile water to obtain selenium-enriched yeast product containing nano-selenium. After drying at 105℃ to constant weight, the dry weight of the product cells can reach 140-170 g / L (preferably about 154 g / L), and the dry basis organic selenium content is 6900-7500 μg / g.

[0068] II. Application Steps of Nano-Selenium in Dwarf Tomato Cultivation

[0069] (1) Preparation of foliar fertilizer

[0070] The above-mentioned selenium-enriched yeast product was pulverized and diluted in proportion to form a nano-selenium foliar fertilizer mother liquor. Before use, it was further diluted to a working solution of a specified concentration (10 mg / L, 20 mg / L).

[0071] (2) Tomato Cultivation and Experimental Design

[0072] Transplanting and planting: Select dwarf tomato seedlings with 4-5 true leaves, and plant them at a spacing of 45cm × 60cm (density approximately 4 plants / m²). 2 Transplanted to 100m 2 The plot of land is managed with three ears of fruit setting.

[0073] Treatment setup: Four treatment groups were set up, with 25 replicates in each group. A buffer zone was set up between the groups. Field water and fertilizer management, pest and disease control, weeding and pruning measures were kept consistent across all treatments.

[0074] Control group: Water;

[0075] Inorganic selenium group: 10 mg / L sodium selenite;

[0076] Low-dose nano-selenium group: 10 mg / L nano-selenium (optimal dose);

[0077] High-dose nano-selenium group: 20 mg / L nano-selenium.

[0078] (3) Foliar spraying and effect verification

[0079] Spraying regimen: The first spray should be given 45 days after tomato transplanting, followed by spraying once every 14 days for a total of 3 sprays. The cumulative spray volume for each group is 3.2-3.6L.

[0080] Spraying conditions: Choose a time when the wind speed is <3m / s and the relative humidity is 50-80% for spraying. Irrigate within 6-8 hours after spraying to ensure that the nano selenium is fully absorbed by the leaves.

[0081] Effect test: During the ripening period (H3 stage) of tomatoes, the 10 mg / L nano selenium treatment group achieved the following: titratable acid (TA) increased by ≥80% compared with the water control, single plant aerial dry weight increased by ≥8%, sugar-acid ratio controlled at 3.8-4.5, and fruit selenium content increased by more than 85% compared with the water control, which was better than the 10 mg / L sodium selenite treatment group.

[0082] The experimental materials required for this invention are as follows:

[0083] Strain: Saccharomyces boulardii;

[0084] Instruments: pH meter (PHS-3C), atomic fluorescence spectrometer (AF-640A), benchtop high-speed centrifuge (H1650-W), 5L / 80L fermenter;

[0085] The culture medium required for this invention is as follows:

[0086] YPD solid medium: 2% glucose, 1% yeast extract, 2% peptone, 2% agar, sterilized at 115℃ for 20 min;

[0087] Primary / Secondary / Third-level seed culture medium (YPD liquid medium): 2% glucose, 1% yeast extract, 2% peptone, sterilized at 115℃ for 20 min;

[0088] High-density fermentation medium (for nano-selenium synthesis): per liter contains 1.6% glucose, 3% corn flour, 0.2g MgSO4, 5.8g KCl, and 0.58g ZnSO4; glucose is sterilized at 115℃ for 20 min, and other components are filtered and sterilized (using a 0.22μm filter membrane) before mixing.

[0089] Feed medium (for nano-selenium synthesis feed): Each liter contains 14% glucose, 0.5% (NH4)2SO4, 3% corn steep liquor, and 0.2% H3PO4; glucose is sterilized at 115℃ for 20 min, and other components are filtered and sterilized before mixing.

[0090] Example 1:

[0091] Preparation of high-density fermentation medium:

[0092] The high-density fermentation medium, using sterile deionized water as the solvent, contains 1.6% glucose, 3% corn flour, 0.2 g / L MgSO4, 5.8 g / L KCl, and 0.58 g / L ZnSO4 per liter. The glucose is dissolved in deionized water and sterilized at 115°C for 20 minutes. Other nutrients are prepared with sterile deionized water and filtered through a 0.22 μm filter for sterilization. Under aseptic conditions, the sterilized glucose solution is added to prepare the culture medium.

[0093] Preparation of supplemental culture medium:

[0094] Weigh 140g of glucose, dissolve it in 1000mL of deionized water, autoclave at 115℃ for 20min, and cool to room temperature;

[0095] Weigh 5g (NH4)2SO4, 30mL corn steep liquor, and 2mL H3PO4, dilute with 500mL deionized water, and filter through a 0.22μm filter membrane for sterilization;

[0096] Under aseptic conditions, the filtered and sterilized mixture is poured into the sterilized glucose solution, and the volume is adjusted to 1000 mL. The mixture is then shaken well and set aside to provide a carbon and nitrogen source for the continuous synthesis of nano-selenium in the later stages of fermentation.

[0097] Example 2:

[0098] Seed propagation: Following the "three-level seed propagation" steps, obtain the third-level seed solution, centrifuge at 4℃ and 7000rpm for 6min, wash twice, and resuspend in sterile water to 300mL;

[0099] Fermentation inoculation: 300 mL of resuspended seed culture was inoculated into a 5 L fermenter (3 L of liquid volume) at 6% (v / v). The temperature was set at 31℃, the rotation speed at 280 rpm, and the pH at 6.0. The dissolved oxygen (DO) was maintained at 35% by aeration (1.5 vvm) and stirring.

[0100] Fed feeding and nano-selenium induction: Glutamic acid (final concentration 0.2%) was added at 7h of fermentation, and fed feeding was started at the same time, with residual sugar controlled at 3g / L; Sodium selenite (final concentration 2mM) was added at a constant flow rate at 18h of fermentation (late logarithmic stage), with DO maintained at ≥25% during the addition period to induce nano-selenium formation;

[0101] Final stage control and termination: After 24 hours of fermentation, the temperature was adjusted to 22℃ and pH to 5.5, and culture was continued for another 3 hours. Fermentation was terminated after 27 hours. The fermentation broth was centrifuged at 10,000 rpm for 1 minute, and the wet weight of the yeast was measured to be 154 g / L. After drying, the dry basis organic selenium content was measured to be 7021 μg / g, and the fermentation broth was brick red, proving that the nano-selenium was successfully synthesized. The wet weight of the yeast broth was measured to be 154 g / L. Based on a wet weight:dry weight ratio of approximately 4:1, the corresponding dry cell weight is estimated to be approximately 38.5 g / L. After optimization and scale-up, the target range of 150–300 g / L can be achieved.

[0102] Example 3:

[0103] Seed propagation: Same as in Example 2, resuspend the third-stage seed culture to 400 mL;

[0104] Fermentation inoculation: Inoculate 8% (v / v) into an 80L fermenter (50L liquid volume), set the temperature to 31℃, the rotation speed to 260rpm, the pH to 6.0, and maintain the dissolved oxygen (DO) at 35% through aeration (1.2vvm) and stirring.

[0105] Fed feeding and nano-selenium induction: Glutamic acid (final concentration 0.2%) was added after 8 hours of fermentation, and the fed feeding was started with residual sugar controlled at 3 g / L; Sodium selenite (final concentration 2 mM) was added at a constant flow rate after 19 hours of fermentation, and DO was maintained at ≥25% to induce nano-selenium formation;

[0106] Final stage control and termination: After 25 hours of fermentation, the temperature was adjusted to 23℃ and pH to 5.5, and cultured for another 3 hours. Fermentation was terminated after 28 hours, and the wet weight of yeast cells was measured to be 273 g / L after centrifugation. After drying, the dry basis organic selenium content was measured to be 7156 μg / g, and the fermentation broth was brick red, proving that the nano-selenium synthesis process has good scale-up stability. The wet weight of yeast broth was measured to be 273 g / L. Based on a wet weight:dry weight ratio of approximately 4:1, the corresponding dry cell weight is estimated to be approximately 68.3 g / L. After optimization and scale-up, the target range of 150–300 g / L can be achieved.

[0107] Example 4:

[0108] Sample preparation: Take the selenium-enriched yeast containing nano-selenium after fermentation is terminated, centrifuge at 8000 rpm for 8 min, wash 3 times with sterile water, dry at 105℃ to constant weight, and weigh 0.1 g of dry sample into a 50 mL acid-resistant conical flask.

[0109] Digestion treatment: Add 10 mL of nitric acid-perchloric acid mixture (HNO3:HClO4 = 9:1, v / v), 5 acid-washed glass beads, cover the petri dish, and nitrate at room temperature for 12 h; pre-digest at 120℃ for 1 h, oxidize at 180℃ until the solution is clear and emits white fumes; after cooling, add 5 mL of 6 mol / L hydrochloric acid, concentrate at 150℃ until white fumes are emitted, make up to 25 mL, dilute 10,000 times with 20% hydrochloric acid, and filter through a 0.22 μm filter membrane;

[0110] Instrumental determination: Atomic fluorescence spectrometry (300V negative voltage, 80mA lamp current, 700mL / min carrier gas flow rate) was used for determination, and the results were calculated after blank correction.

[0111] The formula for calculating organic selenium content is as follows:

[0112] Organic selenium content (μg / g) = (N – N0) × V × dilution factor / M1

[0113] In the formula:

[0114] N—Concentration of the sample digest solution (μg / mL);

[0115] N0—Concentration of blank digest solution (μg / mL);

[0116] V—Volume at constant volume (mL);

[0117] M1 — Mass of the sample (g).

[0118] Calculations show that the dry basis organic selenium content of the selenium-enriched yeast is 7088.9 μg / g, which falls within the range of 6900–7500 μg / g.

[0119] Results: The organic selenium content was 7088.9 μg / g, which meets the index requirement of 6900-7500 μg / g, indirectly reflecting that the amount of nano-selenium synthesized meets the standard.

[0120] Example 5:

[0121] Under the same cultivation and spraying conditions as described above, field verification was carried out on dwarf tomatoes. Experimental design: dwarf tomato varieties were selected for agricultural tomato cultivation. After transplanting, four treatment groups were set up (water control, 10 mg / L sodium selenite, 10 mg / L nano selenium, and 20 mg / L nano selenium), with 25 plants replicated in each group. The plant spacing was 45 cm × 60 cm, and three fruit clusters were set.

[0122] Spraying implementation: First spray on the 45th day after transplanting, once every 14 days, for a total of 3 times, with a cumulative spray volume of 3.4L / set; wind speed of 2.5m / s and relative humidity of 65% during spraying, and no rainfall 7 hours after spraying, which meets the requirements for agricultural field spraying;

[0123] Indicator Measurement:

[0124] TSS: Measured with a handheld saccharimeter, the TSS of the 10 mg / L nano selenium group in phase H3 was 3.17°Brix;

[0125] TA: NaOH potentiometric titration (endpoint pH 8.1), the TA in the 10 mg / L nano selenium group in phase H3 was 0.783%, which was 90% higher than the control;

[0126] Sugar-acid ratio: 4.05 for the 10mg / L nano selenium group, which is within the optimal flavor range of 3.8-4.5 for agricultural tomato cultivation;

[0127] Dry weight per plant: 20.61 g / plant in the 10 mg / L nano selenium group, which was 9.3% higher than the control, demonstrating a growth-promoting effect;

[0128] Selenium content in fruit: Atomic fluorescence spectrometry showed that the 10 mg / L nano selenium group had a selenium content of 104.58 μg / g DW, which was more than 85% higher than the control and better than the 10 mg / L sodium selenite group (90.99 μg / g DW), demonstrating the nutritional fortification advantages of nano selenium in agricultural tomato cultivation.

[0129] Conclusion: The 10 mg / L nano selenium treatment showed the best overall effect in agricultural tomato cultivation. The 20 mg / L nano selenium group had a single plant dry weight of only 15.24 g / plant, which inhibited growth and is not suitable for agricultural tomato cultivation.

[0130] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for high-density fermentation synthesis of nano-selenium using yeast, characterized in that, Synthesis methods include: (1) Three-level seed culture: After activating the Blakely yeast at 30℃-32℃ for 24h-36h, it was inoculated into the first, second and third level seed culture medium in sequence. The culture of each level was 30℃-32℃, 250rpm-300rpm, 20h-24h, and the inoculation amount was 5%v / v-10%v / v. (2) Seed inoculation: Centrifuge the third-grade seed liquid at 0℃-4℃ and 6000rpm-8000rpm for 5min-8min, wash 2-3 times, resuspend in sterile water to 200mL-400mL, inoculate into high-density fermentation medium at 5%v / v-10%v / v, ferment at 30℃-32℃ and 250rpm-300rpm for 20h-24h, dissolved oxygen ≥30%; (3) Feeding and induction: Glutamic acid was added and fed medium was added during the early stage of fermentation (5-9 hours). The medium contained 4.3%-4.5% glucose, 0.3%-0.5% (NH4)2SO4, 2.5%-3.5% corn steep liquor, and 0.1%-0.4% H3PO4 per liter, all w / v or v / v. Selenite was added to a final concentration of 1mM-3mM during the logarithmic stage, and the fermentation broth was induced to turn brick red. (4) Final regulation and collection: After fermentation for 22-26 hours, adjust the temperature to 20-25℃ and pH to 5.0-6.0, and continue culturing for 2-4 hours; centrifuge to remove the supernatant, wash with sterile water 2-3 times to obtain selenium-enriched yeast product containing nano-selenium.

2. The method according to claim 1, characterized in that, In step (2), the pH during the fermentation growth period is controlled at 5.5-6.5, preferably 5.8-6.2; the pH during the selenium enrichment induction stage is controlled at any range of 3.5-4.5 or 5.5-6.

2.

3. The method according to claim 1, characterized in that, The selenite is sodium selenite, which is added in stages or by constant flow, and the dissolved oxygen level is not lower than 25% during the addition period.

4. The method according to claim 1, characterized in that, In step (3), the feed flow is controlled with the goal of "maintaining the residual sugar in the fermentation broth at 2 g / L–5 g / L", and the dissolved oxygen is maintained at 30%–50% through the linkage of stirring and aeration.

5. The method according to claim 1, characterized in that, In step (1), the centrifugation conditions for the third-stage seed solution are 4℃, 7000rpm-8000rpm, and 5min-8min.

6. The method according to claim 1, characterized in that, The biomass of the obtained selenium-enriched yeast product containing nano-selenium was 140 g / L-170 g / L based on cell dry weight.

7. A nano-selenium-enriched yeast product synthesized through high-density yeast fermentation, characterized in that, The product is prepared by the method of any one of claims 1-6, and the dry basis organic selenium content is 6900μg / g-7500μg / g, and it is rich in bio-derived nano-selenium.

8. An application of nano-selenium synthesized by high-density yeast fermentation in agriculture, characterized in that: The nano-selenium-enriched yeast product was prepared as a nano-selenium foliar fertilizer, diluted into a working solution, and sprayed on tomatoes. The spraying regimen was to first spray on the 45th day after tomato transplanting, and then spray once every 14 days for a total of 3 times, with a cumulative spray volume of 3.2-3.6L / set. The spraying conditions were wind speed <3m / s, relative humidity 50%-80%, and irrigation within 6-8 hours after spraying to avoid rain.

9. The application of nano-selenium synthesized by high-density yeast fermentation according to claim 8 in agriculture, characterized in that, The working solution of the diluted nano-selenium foliar fertilizer has a nano-selenium concentration of 10 mg / L, which is the preferred application dose for agricultural tomato cultivation.

10. The application of nano-selenium synthesized by high-density yeast fermentation according to claim 8 in agriculture, characterized in that, At the ripening stage of tomatoes, compared with the water control, at least one of the following must be met: (i) titratable acidity is increased by ≥80%; (ii) Increase in aerial dry weight of single plant by ≥8%; (iii) The sugar-acid ratio should be controlled within the range of 3.8-4.5.

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