Multi-mineral selenium-rich rice and preparation method thereof

Abstract

The invention discloses multi-mineral selenium-enriched rice and a preparation method thereof, and belongs to the technical field of agricultural microorganism technology and functional agricultural product processing. Based on the requirement for increasing the selenium content and the mineral content in rice, a novel selenium transport / binding protein capable of being secreted by a saccharomyces cerevisiae strain CICC 1389 is found, the protein is good in heat resistance and capable of remarkably promoting accumulation of selenium elements, and it is found for the first time that the protein can activate plant glutamate decarboxylase and promote synthesis of gamma-aminobutyric acid (GABA). When the selenium-enriched GABA is prepared into a microbial agent to be applied to rice planting, efficient accumulation of selenium, GABA and multiple minerals in grains can be achieved at the same time, the technical span from single nutrient enrichment to multiple nutrient enrichment is achieved, and a new technical approach is provided for development of functional agricultural products.

Description

Technical Field

[0001] This invention relates to the fields of agricultural microbial technology and functional agricultural product processing technology, and more specifically to multi-mineral selenium-enriched rice and its preparation method. Background Technology

[0002] Selenium is an essential trace element for the human body, participating in the formation of various selenoproteins and selenoenases, and playing a crucial role in antioxidation, immune regulation, and thyroid hormone metabolism. Selenium deficiency in the human body is associated with a variety of diseases, including Keshan disease, Kashin-Beck disease, thyroid dysfunction, and weakened immunity. Producing selenium-enriched agricultural products is an effective way to address insufficient selenium intake in the human body.

[0003] Currently, the production of selenium-enriched rice mainly relies on the application of inorganic selenium fertilizers (such as sodium selenite and sodium selenate) to the soil or leaves. However, this method has many drawbacks: First, the absorption and conversion efficiency of inorganic selenium by plants is limited, usually not exceeding 30%, and most of the selenium is fixed or lost in the soil, which not only wastes resources but also poses potential environmental risks; second, the proportion of inorganic selenium residue in the product is relatively high, and its bioavailability and safety are lower than those of organic selenium forms (such as selenomethionine); third, this method has a single function, only increasing the selenium content and failing to simultaneously improve other nutritional qualities of rice.

[0004] Utilizing microorganisms to regulate selenium metabolism in rice is an emerging research direction. Selenium transporter / binding proteins (SelPs) play a crucial role in selenium transport and assimilation. However, current research on SelPs largely focuses on animals and certain microorganisms, and the discovery of efficient and stable SelP sources from Saccharomyces cerevisiae remains a gap. Even with existing technologies that utilize microorganisms for selenium fortification, the following technical problems are commonly encountered: Poor protein stability: It is especially sensitive to temperature and is easily deactivated in the granulation process of fertilizer production or in high-temperature field environments; Limited Function: The known functions of SelP are limited to the binding and transport of selenium, and it cannot produce other beneficial physiological regulation in crops.

[0005] Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter with various physiological functions, including lowering blood pressure, reducing anxiety, and improving sleep. In recent years, functional foods rich in GABA have received increasing attention. Anaerobic and fermentation treatments can increase the GABA content in rice, but these methods are usually independent of selenium enrichment treatments, making it difficult to achieve synergistic enhancement of multiple functional components in the same product.

[0006] Therefore, there is an urgent need in this field for a novel microbial resource that can produce high-yield, stable, and multifunctional SelP proteins, and for developing its application technology in the production of high-quality selenium-enriched agricultural products to achieve synergistic enhancement of multiple nutrients. This is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides multi-mineral selenium-enriched rice and its preparation method. In order to increase the selenium content in rice, this application uses brewing yeast CICC 1389 under specific conditions for fermentation. Proteins with selenium transport / binding functions were found in the fermentation products, which can effectively increase the selenium content in rice.

[0008] To achieve the above objectives, this application adopts the following technical solution: The primary objective of this application is to provide a selenium transporter / binding protein produced by Saccharomyces cerevisiae CICC1389, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] As a preferred technical solution, the protein has the following characteristics: (1) After being treated in a 75℃ water bath for 30 minutes, the retention rate of its selenate binding activity was over 85%; (2) It can activate plant-derived glutamate decarboxylase and promote the synthesis of γ-aminobutyric acid.

[0010] As a preferred technical solution, the selenium transporter / binding protein is derived from Saccharomyces cerevisiae CICC 1389.

[0011] Another object of this application is to provide: a method for producing the said selenium transporter / binding protein, comprising the following steps: (1) The brewing yeast CICC 1389 was inoculated into seed culture medium and cultured at 28-30℃ and 180-220rpm for 20-28 hours; (2) Transfer to fermentation medium at an inoculum rate of 5-10%, wherein the fermentation medium contains: glucose 20-40 g / L, peptone 10-20 g / L, yeast extract 5-15 g / L, sodium selenite 50-200 μM, potassium dihydrogen phosphate 1-3 g / L, magnesium sulfate 0.5-1.5 g / L, pH 5.5-6.5; (3) Ferment and culture at 28-30℃ and 150-250rpm for 48-72 hours; (4) Centrifuge and collect the supernatant to obtain a crude enzyme solution containing the selenium transporter / binding protein.

[0012] As a preferred technical solution, the fermentation medium further contains 0.1-0.5 mM of glutathione.

[0013] Another object of this application is to provide a microbial inoculum comprising the aforementioned Saccharomyces cerevisiae CICC 1389 and / or fermentation broth.

[0014] As a preferred technical solution, the microbial inoculant also includes an agriculturally acceptable carrier, which is selected from one or more of peat moss, vermiculite, bentonite, and diatomaceous earth.

[0015] Another object of this application is to provide the application of the selenium transporter / binding protein or the selenium transporter / binding protein prepared by the method or the microbial agent in increasing the selenium content of plants.

[0016] Another object of this application is to provide a method for preparing multi-mineral selenium-enriched rice, comprising the following steps: applying the selenium transporter / binding protein or the microbial agent to rice during the tillering stage, booting stage and / or grain-filling stage.

[0017] Another object of this application is to provide a multi-mineral selenium-enriched rice prepared by the method.

[0018] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: A discovery was made that Saccharomyces cerevisiae CICC 1389 can produce a protein with selenium transport / binding function. Its heat-resistant properties allow it to withstand the high-temperature granulation process in microbial fertilizer production, ensuring product activity. Furthermore, it possesses a novel function of promoting GABA accumulation, achieving a leap from "single-nutrient selenium enrichment" to "dual-nutrient fortification with selenium and GABA," resulting in a qualitative leap in technological effectiveness.

[0019] Synergistic effects enhance the overall quality of rice: The final rice produced is not only a highly effective source of selenium but also a natural source of GABA. This synergistic enhancement effect of "one method, two high-value nutrients" was something that those skilled in the art could not have foreseen before the application date.

[0020] Environmental friendliness and sustainability: This invention improves the efficiency of plant utilization of selenium in the soil through microbial enhancement, reduces dependence on inorganic selenium fertilizers, reduces environmental risks, and is in line with the development direction of green agriculture. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] The brewing yeast CICC 1389 used in the embodiments of this application was purchased from the China Industrial Microbial Culture Collection Center.

[0023] Example 1

[0024] Preparation, extraction and purification of selenium transporter / binding proteins

[0025] The fermentation medium was optimized through single-factor and response surface methodology experiments, and the optimal formulation was finally determined to be: glucose 30 g / L, peptone 15 g / L, yeast extract 10 g / L, sodium selenite 150 μM, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 1 g / L, glutathione 0.3 mM, pH 6.0.

[0026] Fermentation conditions: Saccharomyces cerevisiae CICC 1389 was inoculated into seed medium (YPD medium) and cultured at 30℃ and 200 rpm for 24 hours. Then, it was transferred to fermentation medium at an 8% inoculation rate and fermented at 30℃ and 200 rpm for 60 hours.

[0027] Protein purification: After fermentation, centrifuge (8000 rpm, 15 min) to collect the supernatant, which is the crude enzyme solution. The following steps are used to purify the SelP protein: 1) Pretreatment and concentration: The supernatant was filtered through a 0.45 μm filter membrane to remove residual cell debris.

[0028] Ultrafiltration was performed using an ultrafiltration tube with a molecular weight cutoff of 10 kDa to concentrate the material to 1 / 5 of its original volume, thereby reducing the volume required for subsequent purification.

[0029] 2) Ammonium sulfate fractionation precipitation: Slowly add solid ammonium sulfate to the supernatant until it reaches 40% saturation, let it stand at 4°C for 3 hours, and then centrifuge (10,000 rpm, 20 min) to collect the precipitate.

[0030] Anion exchange chromatography: Load the resuspended solution onto a pre-equilibrated DEAE Sepharose Fast Flow column (50 mL column volume) and wash with 20 mM Tris-HCl buffer (pH 8.0) until baseline is stable.

[0031] Elution was performed using a linear gradient containing 0-0.5M NaCl (instead of the original 0-1M NaCl), and the active peak was collected (detection wavelength 280nm).

[0032] Gel filtration chromatography: Concentrate the active peak to 5 mL, load it onto a Sephacryl S-300 column (column volume 100 mL), elute isocratically with 20 mM Tris-HCl buffer (containing 150 mM NaCl, pH 8.0) at a flow rate of 0.5 mL / min, and collect the single protein peak.

[0033] (6) Purity verification: Take the final merged purified sample and perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to verify the purity of the final product. After Coomassie brilliant blue staining, only a single, clear band appears at the position of molecular weight of about 55 kDa (its apparent molecular weight is consistent with the theoretical molecular weight calculated based on the amino acid sequence), which proves that electrophoretically pure selenium transporter / binding protein has been obtained. The purified enzyme solution is aliquoted, 50% glycerol is added, and it is frozen at -20℃ or freeze-dried to make dry powder for storage.

[0034] The amino acid sequence of the selenium transporter / binding protein was analyzed, and its amino acid sequence is as follows: , SEQ ID NO.1.

[0035] Example 2

[0036] Performance determination of selenium transporter / binding protein (SelP) Heat resistance test:

[0037] I. Sample Preparation 1) Protein sample preparation: The SelP protein of this invention was prepared, purified, and extracted according to Example 1. Commercial control protein: Yeast glutathione peroxidase (GSH-Px) produced by Sigma-Aldrich, catalog number: G6137, source: recombinant expression of Saccharomyces cerevisiae, activity: ≥100 units / mg protein (GSH-Px is a selenium-containing enzyme from yeast, with a similar biological origin to SelP of this invention. As a commercial protein, its quality and activity are guaranteed, making it suitable as a control. The thermal stability of this protein has been reported in the literature, and its activity decreases significantly at 65°C. Its molecular weight (approximately 55 kDa) is similar to SelP of this invention, making it somewhat comparable).

[0038] 2) Buffer solution preparation: 20 mM Tris-HCl buffer (pH 7.5), Tris-base 2.42 g, ultrapure water 800 mL, adjust pH to 7.5 with HCl and bring volume to 1 L. Store at 4°C.

[0039] 3) Selenate substrate solution: 1 mM Na2SeO4 solution (prepared with ultrapure water).

[0040] II. Experimental Procedure 1) Sample pretreatment: The SelP protein of this invention and commercial GSH-Px were diluted to 0.5 mg / mL with 20 mM Tris-HCl buffer. The protein concentration was accurately determined by the BCA method to ensure that the concentrations of the two groups were consistent. Each sample was aliquoted into sterile PCR tubes in the form of 200 μL.

[0041] 2) Heat treatment process: Set the water bath temperature gradient: 40℃, 55℃, 65℃, 75℃, 85℃; set 3 replicate samples at each temperature point; place the sample tubes in the water bath at the corresponding temperature and time precisely for 30 minutes; use a thermometer to monitor the water bath temperature in real time to ensure that the temperature fluctuation is within ±0.5℃; 3) Termination of the reaction: After heat treatment, immediately transfer the sample tube to an ice-water bath (0-4℃); let it stand on ice for 10 minutes to allow the sample to cool rapidly; briefly centrifuge to collect the condensate from the tube wall; 4) Activity assay: Selenate binding activity was determined by atomic fluorescence spectrometry (AFS). The reaction system consisted of: protein sample 50 μL, 1 mM Na₂SeO₄ 50 μL, and reaction buffer 400 μL. After incubation at 37°C for 30 minutes, 100 μL of 10% TCA was added to terminate the reaction. The mixture was centrifuged at 12000 rpm for 15 minutes, and the supernatant was used to determine the selenium content.

[0042] Standardization of activity assay: A standard curve (0-100 μg / L selenium standard solution) is set for each assay. The measured values ​​of the samples must be within the linear range of the standard curve (R² > 0.995); three technical replicates should be set up for each sample.

[0043] Data recording: Record the raw fluorescence values ​​and calculate the relative activity; use the activity of the untreated sample as 100%; perform statistical analysis using GraphPad Prism software.

[0044] The experimental results are shown in Table 1.

[0045] Table 1. Activity assay of the control protein in Example 1 Heat treatment temperature (°C) Protein activity retention rate (%) of this invention Commercial control protein activity retention rate (%) 25 (Unprocessed) 100.0 ± 2.5 100.0 ± 3.1 40 99.5 ± 2.8 95.2 ± 3.5 55 98.1 ± 3.0 75.4 ± 4.2 65 92.3 ± 2.7 39.8 ± 5.1 75 85.7 ± 3.5 15.3 ± 3.8 85 58.9 ± 4.8 5.2 ± 2.1 Results Analysis: At 65°C, the activity retention rate of the SelP protein of this invention remained as high as 92.3%, while the activity of the commercial control protein was rapidly inactivated, remaining at only 39.8%. This indicates that the protein of this invention has a more stable conformation at high temperatures. Under the harsh conditions of 75°C, the SelP protein of this invention still maintained a high activity of 85.7%, fully demonstrating its excellent heat resistance. In contrast, the control protein was essentially inactivated at this temperature (activity retention rate of only 15.3%).

[0046] Overall trend: As the temperature increases, the activity of both proteins decreases, but the activity decrease curve of SelP in this invention is more gradual, and the activity retention rate at each temperature point (especially above 55°C) is significantly higher than that of the control protein.

[0047] This data fully demonstrates that the SelP protein provided by this invention has unexpectedly superior thermal stability compared to existing technologies (commercial control proteins), a characteristic that gives it significant advantages in processing, storage, and practical applications.

[0048] Functional validation that promotes GABA accumulation:

[0049] I. In vitro experiments: Crude glutamate decarboxylase (GAD) solution was extracted from rice leaves.

[0050] The reaction system (1 mL) contains: 50 mM phosphate buffer (pH 5.8), 5 mM sodium glutamate, 1 mM PLP, and: Group A: Add 50 μg of the SelP protein of this invention; Group B: Add 50 μg of the heat-inactivated SelP protein of this invention; Group C: No protein added.

[0051] After reacting at 37℃ for 1 hour, the content of generated GABA was determined by HPLC. The experimental results are shown in Table 2.

[0052] Table 2. GABA production levels in different groups Experimental Groups Processing instructions GABA production (μM, Mean ± SD) Statistical significance (compared to group A) Group A (Experimental Group) Added active SelP protein 92.3 ± 4.1 - Group B (Control Group 1) Adding heat-inactivated SelP protein 47.2 ± 2.5 p < 0.01 Group C (Control Group 2) No additional protein added 44.5 ± 2.1 p < 0.01 Results Analysis: As shown in the table, group A, which only added active SelP protein, had a significantly higher GABA production than groups B and C. The GABA production in groups B and C was at a low level with no significant difference, ruling out the possibility that other components in the experimental system or SelP protein itself, as a physical additive, promoted GABA synthesis.

[0053] This result strongly demonstrates that the SelP protein of the present invention can specifically activate the activity of rice GAD enzyme, and that this function is strictly dependent on its own biological activity.

[0054] II. Plant in vivo experiments: Rice seedlings with uniform growth were selected and randomly divided into three groups. During the seedling stage, the following treatments were applied to the leaves of each group: Treatment I: A solution containing the SelP protein of this invention (50 μg / mL); Treatment II: Solution containing heat-inactivated SelP protein (50 μg / mL); Treatment III: Equal volume of buffer solution (control).

[0055] After culturing for another 24 hours under the same environmental conditions, leaf samples were collected from each group of rice plants. The GABA content in the collected leaf samples was measured, and the data from the three groups were statistically analyzed. The experimental results are shown in Table 3.

[0056] Table 3. GABA content in leaf samples from different groups Experimental Groups Processing instructions GABA content (μmol / g FW, Mean ± SD) Statistical significance (compared to treatment I) Treatment I (Experimental Group) Spraying active SelP protein 4.35 ± 0.28 - Treatment II (Control Group 1) Spraying heat-inactivated SelP protein 2.18 ± 0.16 p < 0.01 Treatment III (Control Group 2) Spray blank buffer solution 2.12 ± 0.14 p < 0.01 Results Analysis: As shown in the table, only treatment I, which involved spraying with active SelP protein, showed a significant doubling of GABA content in the leaves. Treatments II (heat-inactivated protein) and III (blank buffer) showed similarly low GABA levels with no significant difference. This result rules out the possibility that the increased GABA content was solely due to a stress response from proteins or other components in the solution.

[0057] These in vivo experimental data strongly demonstrate that the SelP protein of this invention can cross leaf tissue and effectively activate the GABA synthesis pathway in plants, and that this function is strictly dependent on its own biological activity. This provides crucial evidence for its application as a biostimulant in agricultural production.

[0058] Example 3 A method for preparing selenium-enriched rice with multiple minerals Preparation of microbial inoculants:

[0059] Preparation of fermentation broth: Saccharomyces cerevisiae CICC 1389 was inoculated into seed culture medium (YPD medium) and cultured at 30℃ and 200 rpm for 24 hours. Then, at an 8% inoculum size, it was transferred to a 50L fermenter containing fermentation medium (30 g / L glucose, 15 g / L peptone, 10 g / L yeast extract, 150 μM sodium selenite, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 0.3 mM glutathione, pH 6.0) and fermented at 30℃ and 200 rpm for 60 hours to obtain the fermentation broth. Pretreatment and separation of fermentation broth Cooling: The fermentation broth is rapidly cooled to 4-10℃ through a heat exchanger to reduce cell metabolism and protein degradation.

[0060] Separation: The fermentation broth was separated into bacterial sludge and supernatant rich in SelP protein by centrifugation at 4°C and 8000 rpm using a continuous flow centrifuge.

[0061] Separate treatment of bacterial cells and proteins Cell preservation: Slowly mix the collected wet bacterial sludge with an equal weight of a sterile, low-temperature 10% trehalose preservation agent solution and store at 4°C for later use. Trehalose can effectively protect the cell membrane of bacteria during subsequent drying, improving the survival rate.

[0062] Protein Concentration and Stabilization: The supernatant was sterilized by filtration through a 0.22 μm filter membrane.

[0063] The supernatant was concentrated to about 2L at 4°C using an ultrafiltration system (molecular weight cutoff of 10 kDa). This step can simultaneously remove some impurities and salts.

[0064] Add stabilizers to the concentrated protein solution to achieve a final concentration of 5% (w / v) trehalose and 1% (w / v) glycine. Mix well and store at 4°C.

[0065] Carrier preparation and compounding Carrier selection and sterilization: A mixture of peat moss and vermiculite with strong adsorption and high porosity in a ratio of 3:1 (w / w) was selected as the composite carrier. It was autoclaved at 121°C for 60 minutes and then spread out to cool to room temperature.

[0066] Mineral Addition: The following mineral mixture (by weight of carrier) is added to the carrier: Zinc sulfate: 0.5% (w / w), provides elemental zinc. Magnesium sulfate: 1.0% (w / w), provides magnesium. Calcium gluconate: 0.8% (w / w), provides elemental calcium. Ammonium molybdate: 0.1% (w / w), provides molybdenum. Mixed adsorption: First, the concentrated protein stabilizer solution is evenly sprayed onto the sterilized carrier and thoroughly mixed to allow the carrier to fully adsorb the protein. Then, the protected wet bacterial sludge is evenly mixed with the protein-adsorbed carrier. The final ratio is controlled as follows: bacterial cells : carrier = 1 : 2 (w / w).

[0067] Moisture adjustment: Spray sterile deionized water into the mixture while stirring, and finally adjust the moisture content of the material to 30-35% (the texture should be "clumps together when squeezed in the hand, and crumbles when touched").

[0068] Packaging and post-ripening Repackaging: The mixed wet solid microbial agent is repackaged into breathable aseptic bags, 1-2 kg per bag.

[0069] Post-ripening: Place the packaged inoculum in a constant temperature environment of 20-25℃ and 50-60% humidity for 24-48 hours. This process helps the microorganisms recover from the fermentation state and initially colonize the carrier surface.

[0070] The viable bacteria concentration of the prepared bacterial agent was determined to be ≥ 5 × 10⁻⁶. 9 CFU / g; Protein activity: Selenate binding activity retention of SelP protein ≥ 85% (compared to fermentation broth supernatant); Moisture content: 30-35%; Contamination rate: < 1%.

[0071] Field trials:

[0072] Experimental location: a neutral soil paddy field (soil selenium background value 0.12 mg / kg).

[0073] Experimental design: There are 3 treatments, and each treatment is repeated 3 times.

[0074] Treatment T1 (this invention): Apply 1.5 kg of solid microbial agent per mu each time during the tillering, booting, and grain-filling stages of rice; Treatment CK1 (positive control): At the same time, a sodium selenite aqueous solution was sprayed on the leaves. Each time, 7 grams of sodium selenite, the active ingredient, was applied per acre, diluted in 50 liters of water. Treatment of CK2 (blank control): No treatment was performed.

[0075] After the rice from different treatment groups matured, the indicators in the polished rice were measured, and the experimental results are shown in Table 4.

[0076] Table 4. Selenium and GABA content in different groups Processing group Total selenium content (mg / kg) Organic selenium content (%) GABA content (mg / 100g) Zinc content (mg / kg) Magnesium content (mg / kg) Calcium content (mg / kg) T1 0.55±0.05 94.2±1.5 46.8±3.2 28.5±2.1 285.6±15.3 125.3±8.7 CK1 0.35±0.03 70.5±3.8 18.2±1.1 15.2±1.5 195.3±12.6 85.6±6.9 CK2 0.10±0.02 62.3±4.5 16.5±1.3 12.8±1.2 168.5±10.8 72.3±5.8 Results Analysis: As shown in Table 4, the treatment of this invention significantly increases the selenium and GABA content in rice, and the proportion of organic selenium is significantly increased. In addition, through the addition of multi-mineral components to the inoculant, the zinc, magnesium, and calcium content in rice is also significantly increased, by 87.5%, 46.3%, and 46.4% respectively compared with the control group, truly achieving the goal of "multi-mineral selenium enrichment".

[0077] Example 4

[0078] Cooking characteristics experiment

[0079] To evaluate the retention rate of the core functional components (GABA and selenium) of the selenium-enriched, high-GABA rice produced by this invention under conventional cooking conditions, and to demonstrate the stability of the product's edible value, the following experiments were conducted: Experimental group: Rice produced by the method of the present invention (obtained from Example 3).

[0080] Control group 1: Commercially available ordinary selenium-enriched rice (prepared by foliar spraying of sodium selenite).

[0081] Control group 2: Ordinary white rice.

[0082] Cooking process: Accurately weigh 50.0 g of rice sample from each group. Add boiled ultrapure water at a rice:water ratio of 1:1.5, cook using the standard rice cooking mode on a rice cooker for 20 minutes, then let it sit for 5 minutes.

[0083] Composition analysis: GABA content: Rice samples before and after cooking were determined by HPLC.

[0084] Total selenium content: The total selenium content of rice samples before and after cooking was determined by atomic fluorescence spectrometry.

[0085] Retention rate calculation: (content after cooking / content before cooking) × 100%.

[0086] Table 5 Retention rate of functional components after cooking Experimental Groups Processing instructions GABA retention rate (%) Selenium retention rate (%) Zinc retention rate (%) Magnesium retention rate (%) Calcium retention rate (%) experimental group The rice produced by this invention 90.2±2.1 96.5±1.5 95.8±1.8 97.2±1.2 96.9±1.4 Control group 1 Commercially available selenium-enriched rice 81.5±3.5 88.3±2.8 85.6±2.5 89.1±2.1 87.8±2.3 Control group 2 ordinary white rice 83.8±2.9 - 84.2±2.8 86.7±2.4 85.3±2.6

[0087] Results analysis: After undergoing a standard cooking process, the rice produced by this invention exhibited significantly higher retention rates of its core functional components GABA and selenium compared to commercially available control products. In particular, the selenium retention rate was extremely high, indicating that the selenium form (mainly organic selenium) in the product was very stable, with minimal cooking loss, thus ensuring the nutritional efficacy of the final product.

[0088] Storage stability test

[0089] To evaluate the changes in the functional components of the rice of the present invention over time under normal storage conditions, in order to determine the product's shelf life and storage stability.

[0090] Rice produced by the method of this invention (obtained from Example 3) was stored at room temperature (25±3℃) and normal humidity (relative humidity 60±10%) in ordinary food-grade polyethylene bags, simulating commercial sales, for 6 months. Samples were taken at the beginning of storage (0 months), 3 months, and 6 months. The GABA content and total selenium content were determined, and the rate of change in content was calculated.

[0091] Table 6 Changes in functional components during room temperature storage Storage time GABA content (mg / 100g) GABA rate of change Total selenium content (mg / kg) Selenium change rate 0 months 42.5 ± 2.8 (benchmark) - 0.52 ± 0.04 (benchmark) - 3 months 40.1 ± 2.5 -5.6% 0.52 ± 0.03 ±0.0% 6 months 38.4 ± 2.2 -9.5% 0.52 ± 0.04 ±0.0%

[0092] Results Analysis: GABA Stability: After 6 months of storage at room temperature, the GABA content decreased by about 9.5%, indicating that the GABA in the product has good storage stability and most of its functions are retained.

[0093] Selenium stability: The total selenium content remained almost unchanged over 6 months, further demonstrating the extreme stability of the organic selenium form in the product, which will not be lost over time.

[0094] The rice produced by this invention maintains stable main functional components under normal storage conditions, exhibiting excellent shelf-life characteristics and fully meeting the needs of commercial distribution.

[0095] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0096] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A selenium transporter / binding protein produced by Saccharomyces cerevisiae CICC1389, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

1.

2. The selenium transporter / binding protein according to claim 1, characterized in that, The protein has the following characteristics: (1) After being treated in a 75℃ water bath for 30 minutes, the retention rate of its selenate binding activity was over 85%; (2) It can activate plant-derived glutamate decarboxylase and promote the synthesis of γ-aminobutyric acid.

3. A method for producing the selenium transporter / binding protein of claim 1 or 2, characterized in that, Includes the following steps: (1) The brewing yeast CICC 1389 of claim 1 is inoculated into seed culture medium and cultured at 28-30℃ and 180-220rpm for 20-28 hours; (2) Transfer to fermentation medium at an inoculum of 5-10%, wherein the fermentation medium contains: glucose 20-40 g / L, peptone 10-20 g / L, yeast extract 5-15 g / L, sodium selenite 50-200 μM, potassium dihydrogen phosphate 1-3 g / L, magnesium sulfate 0.5-1.5 g / L, pH 5.5-6.5; (3) Ferment and culture at 28-30℃ and 150-250rpm for 48-72 hours; (4) Centrifuge and collect the supernatant to obtain a crude enzyme solution containing the selenium transporter / binding protein.

4. The method according to claim 3, characterized in that, The fermentation medium also contains 0.1-0.5 mM of glutathione.

5. A microbial inoculant, characterized in that, It contains the brewing yeast CICC 1389 as described in claim 1 and / or the fermentation broth.

6. The microbial agent according to claim 5, characterized in that, It also includes agriculturally acceptable carriers selected from one or more of peat, vermiculite, bentonite, and diatomaceous earth.

7. The application of the selenium transporter / binding protein according to any one of claims 1-2, or the selenium transporter / binding protein prepared by the method according to any one of claims 3-4, or the microbial agent according to any one of claims 5-6 in increasing the selenium content of plants.

8. A method for preparing selenium-enriched rice with multiple minerals, characterized in that, Includes the following steps: During the tillering, booting, and / or grain-filling stages of rice, the selenium transporter / binding protein of claim 1 or the microbial agent of claim 5 or 6 is applied to the rice.

9. A multi-mineral selenium-enriched rice prepared by the method of claim 8.