Application of bacillus aryabhattai in improving the flavor of aromatic rice

Abstract

The application discloses application of bacillus aryabhattai in improving flavor of fragrant rice, and relates to the field of fragrant rice cultivation; it is found for the first time that bacillus aryabhattai has the effect of improving flavor substances of fragrant rice, and the seedling roots of fragrant rice are soaked with bacillus aryabhattai liquid when the seedlings grow to the stage of 3 leaves and 1 heart; after the seedlings are transplanted to a field, the seedling roots are sprayed with bacillus aryabhattai liquid again, 24 main aroma components in the fragrant rice can be improved by 5.56% to 12036.00% through the method, the content of 2-ethyl-1-hexanol with rose fragrance is improved by more than 120 times, the content of 2-butyl-1-octanol with flower fragrance is improved by 235.71%, the content of 6,10-dimethyl-(E)-5,9-undecadien-2-ketone with fruit fragrance is improved by 132.50%, the contents of limonene, hexanal, 2-heptanone, 2-tridecanone and 2-pentadecanone are all improved by more than 50%, and the content of 2-acetyl-1-pyrroline (2-AP) is improved by more than 30%; the application method is more convenient to operate, and the overall flavor of rice is improved more significantly.

Description

Technical Field

[0001] This invention belongs to the field of fragrant rice cultivation, and in particular relates to a biological fungus and its application in fragrant rice cultivation. Background Technology

[0002] Fragrant rice, characterized by its rich aroma and fragrant cooked rice, has long been favored by consumers, and global consumption of fragrant rice has been increasing year by year. To meet market demand, the content of flavor compounds in fragrant rice has become an important indicator for measuring its quality.

[0003] Currently, using a combination of gas chromatography and mass spectrometry, more than 500 volatile aromatic substances have been detected in rice, including alkanes, acids, alcohols, aldehydes, ketones, esters, phenols, heterocyclic compounds, etc. (References: "Mahattanatawee K, Russell R L. Comparison of aroma active and sulfur volatiles in three fragrant rice cultivars using GC-Olfactometry and GC-PFPD[J]. Food Chemistry, 2014, 154: 1-6."; "He Q, Park Y J. Discovery of a novel fragrant allele and development of functional markers for fragrance in rice[J]. Molecular Breeding, 2015, 35(11): 217"; "Nadaf A, Mathure S, Jawali N. Scented Rice (Oryza sativa L.) Cultivars of India: A Perspective on Quality and Diversity[M]. New Delhi:Springer, 2015:1-142. ;“Lee YS, Oh YJ, Kim TH, Cho Y H. Quantitation of 2-acetyl-1-pyrroline inaseptic-packaged cooked fragrant rice by HS-SPME / GC-MS[J]. Food Science and Nutrition, 2018, 7(1):266-272.”). Alkanes account for the highest proportion in rice, generally 20% to 60%, but their threshold is generally high, giving rice a relatively low aroma value. Aldehydes mainly exhibit fatty, grassy, ​​and fresh aromas. Alcohols present floral, fruity, and balsamic aromas. Ketones present herbal, creamy, and mushroom aromas. Esters present a variety of pleasant fruity flavors. Heterocyclic compounds present roasted nut, popcorn, and baked aromas.

[0004] While rice aroma is genetically controlled, it is closely related to soil and climate conditions. Improving the aroma of fragrant rice through cultivation techniques is a crucial measure for enhancing rice quality. Currently, many researchers utilize cultivation methods such as light, water, fertilizer, exogenous trace elements, proline, ornithine, γ-aminobutyric acid, betaine, gibberellin, 6-benzylaminopurine, and salicylic acid to increase the content of the flavor compound 2-AP (2-acetyl-1-pyrrolidone) in rice. However, rice aroma is not solely composed of 2-AP; rather, it is a unique aroma characteristic of fragrant rice varieties, composed of multiple flavor compounds. Basmati from India and Jasmine from Thailand are world-renowned fragrant rice varieties. Basmati boasts a blend of nutty and floral aromas, while Jasmine is famous worldwide for its unique jasmine fragrance. These aromas cannot be achieved by a single flavor compound but are the result of a complex mixture of various flavor compounds within the rice. Therefore, comprehensively improving the flavor of fragrant rice is an urgent need for the production of high-end fragrant rice.

[0005] As an effective soil conditioner, microorganisms can significantly improve soil physical structure, increase soil organic matter content, and enhance the utilization rate of mineral elements in the soil. They can also produce various bioactive substances that promote crop growth and improve crop resistance. *Bacillus aryepasis*, currently used as a beneficial microbial fertilizer for rice growth regulation, has been reported to promote rice growth, stimulate plant secretion of auxins, increase root length and plant height, and improve seed germination rate. However, current technologies lack microbial agents that can improve rice quality and flavor. Current cultivation techniques for enhancing the aroma of fragrant rice mainly focus on exogenous chemical substances; bioactive microorganisms have not yet been reported. Summary of the Invention

[0006] To address the above problems, this invention provides a cultivation method for enhancing the flavor of fragrant rice using Bacillus aryepasis, the specific scheme of which is as follows:

[0007] First, this application provides an application of Bacillus aryepedae ​​in enhancing the flavor of fragrant rice.

[0008] The above application specifically refers to: when the fragrant rice seedlings reach the 3-leaf-1-heart stage, soaking the seedling roots in a solution of Bacillus aryepasis; after transplanting the seedlings to the field, spraying the soil around the seedling roots again with the Bacillus aryepasis solution to enhance the flavor of the fragrant rice; the aforementioned Bacillus aryepasis solution is a stock solution of Bacillus aryepasis (bacterial content 1×10⁻⁶). 9 -5×10 10 CFU / ml) diluted with water to OD 600 =0.4, the optimal spraying rate is 20L / mu.

[0009] The soaking time for the seedling roots in the bacterial solution is 20-60 seconds, such as 20 seconds, 30 seconds, 40 seconds, 50 seconds, or 60 seconds. The soaking time can be selected according to the actual situation. In one embodiment of this application, the soaking time is preferably 30 seconds.

[0010] The preferred time for spraying the seedling root soil with Bacillus aryepadoides inoculum is 15-25 days after the seedlings are transplanted to the field, such as 15 days, 20 days, or 25 days. In practice, the spraying time can be selected according to the growth of the seedlings. In one embodiment of this application, the preferred spraying time is 20 days after the seedlings are transplanted to the field.

[0011] Furthermore, the specific steps for the above application are as follows:

[0012] 1) Soak the fragrant rice seeds and germinate them until they sprout. Sow the seeds in a 30cm×60cm substrate seedling tray using a seeder, with 100g of seeds per tray.

[0013] 2) Stack the seedling trays, 20 per stack, and keep them in the dark for 2 days at a temperature of 28℃. Regularly spray water on the surface to keep the substrate moist. After the sprouts emerge from the soil, place the trays in the sun in the paddy field and spray water on them daily for cultivation.

[0014] 3) Once the fragrant rice seedlings have reached the 3-leaf, 1-heart stage, immerse the roots of the seedlings in a solution of *Bacillus aryepasis*. Use 10 L of solution to soak 10 seedlings for 30 seconds. The above-mentioned *Bacillus aryepasis* solution is the original *Bacillus aryepasis* solution (bacterial content 1×10⁻⁶). 9 -5×10 10 Dilute with water to a bacterial culture concentration of CFU / ml until the OD value is reached. 600 =0.4.

[0015] 4) After soaking, transplant the seedlings by machine; do not use antibacterial pesticides within 10 days after transplanting, and manage water and fertilizer normally.

[0016] 5) Twenty days after transplanting, spray the soil around the seedling roots with Bacillus aryepasis solution, using 20L of solution per acre. Do not use antibacterial pesticides one week before or after spraying. Use normal tillering and heading fertilizers. Water management follows conventional high-yield management methods: shallow irrigation during the early and mid-tillering stages and from heading to flowering; dry the field during the late tillering stage to jointing stage; and alternate between wet and dry irrigation during the grain-filling stage. The OD of the above Bacillus aryepasis solution... 600 =0.4.

[0017] In this application, the term "fragrant rice" refers to rice varieties with the BADH2 gene, such as Nanjing 46, Nanjing Xiangnuo, Nanjing 9108, and Nanjing 9308. The term "rice flavor" includes flavor compounds such as hexanal, 1-hexanol, 2-acetyl-1-pyrroline (2-AP), 1-octen-3-ol, 2-ethyl-1-hexanol, limonene, (E)-2-octenal, 1-octanol, nonanal, (E)-2-nonenal, 2-methyl-1-undecenol, decanal, 2,3-dihydrobenzofuran, (E)-2-decenal, nonanoic acid, indole, 2-undecanetone, 2-methoxy-4-vinylphenol, 2-butyl-1-octanol, 6,10-dimethyl-(E)-5,9-undecadien-2-one, 2-tetridetone, 2-pentadecanone, 10,14-trimethyl-2-pentadecanone, and 2-heptanetone. "Enhancing rice flavor" includes increasing the content of these flavor compounds.

[0018] The Bacillus aryabhattai strain used in this application is a conventional strain. The strain used in the examples was purchased from the China General Microbiological Culture Collection Center (CGMCC), with accession number 1.914. This strain is in the form of a dry powder. Before use, it is first inoculated onto LB slant agar and activated for 24 hours at 37°C. After bacterial colonies appear, it is inoculated onto LB liquid agar and cultured on a shaker at 37°C for 24 hours, yielding a bacterial count of 1×10⁻⁶. 9 -5×10 10 The stock solution of Bacillus aryepasidia at CFU / ml should be diluted with tap water before use.

[0019] LB liquid culture medium formula: 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract, with the remainder made up with double-distilled water.

[0020] This application is the first to discover the application of *Bacillus aryepasis* in enhancing the flavor of fragrant rice, and based on this discovery, provides a method for cultivating fragrant rice to enhance its flavor. The content of 2-ethyl-1-hexanol (resulting in a rose aroma) in the fragrant rice is increased by more than 120 times, the content of 2-butyl-1-octanol (resulting in a floral aroma) is increased by 235.71%, the content of 6,10-dimethyl-(E)-5,9-undecadien-2-one (resulting in a fruity aroma) is increased by 132.50%, and the contents of limonene, hexanal, 2-heptanone, 2-tetrideone, and 2-pentadecanone are all increased by more than 50%, the content of 2-AP is increased by more than 30%, and the contents of other major substances affecting the flavor of fragrant rice are also increased to varying degrees. Compared with other methods for enhancing the aroma of rice, this method is more convenient to operate and has a more significant effect on improving the overall flavor of rice. Attached Figure Description

[0021] Figure 1The mass spectrometry results of flavor compounds in the CK group are shown in the example.

[0022] Figure 2 The mass spectrometry results of flavor compounds from the AYB microbial community were added to the examples.

[0023] Figure 3 Mass spectrometry results of flavor compounds from group MG2 were added to the examples.

[0024] Figure 4 Mass spectrometry results of flavor compounds from group MG1 were added to the examples.

[0025] Figure 5 BLS group flavor substance mass spectrometry detection results were added to the examples.

[0026] Figure 6 The HQ group flavor substance mass spectrometry detection results were added to the examples.

[0027] Figure 7 BLQ group flavor substance mass spectrometry detection results were added to the examples.

[0028] Figure 8 The LSMU group of flavor substance mass spectrometry detection results were added to the examples.

[0029] Figure 9 The TQ group flavor substance mass spectrometry detection results were added to the examples.

[0030] Figure 10 Example 2 shows the results of an experiment in 2023 on the effect of different microbial agents on the 2-AP content in Nanjing 46 rice.

[0031] Figure 11 The results of the experiment on the effect of different microbial agents on the 2-AP content in Nanjing 46 rice in 2024.

[0032] Figure 12 The results of the 2024 experiment on the effect of AYB inoculant on the 2-AP content in different fragrant rice varieties. Detailed Implementation

[0033] The present invention will be further illustrated by specific implementation examples below.

[0034] Potato dextrose agar (PDA) medium: 200g potato and 20g sucrose per liter, with the remainder made up with double-distilled water.

[0035] LB liquid medium: Each liter contains 10g peptone, 10g NaCl, and 5g yeast extract, with the remainder made up with double-distilled water.

[0036] The Rhizopus oryzae (MG1), Rhizopus oryzae (MG2), Trichoderma viride (LSMU), Aspergillus terreus (TQ), Aspergillus niger (HQ), and Penicillium baicale (BLQ) used in the examples were purchased from the China Agricultural Microbial Culture Collection Center.

[0037] Bacillus lysus (BLS) and Bacillus ayerbuddha (AYB) were purchased from the China General Microbiological Culture Collection Center.

[0038] The fragrant rice varieties involved in the examples, namely Nanjing 46, Nanjing Xiangnuo, Nanjing 9108, and Nanjing 9308, are all conventionally grown rice varieties provided by the Jiangsu Academy of Agricultural Sciences.

[0039] Example 1

[0040] The experiments were conducted in Nandou Village, Liyang District, Nanjing City in 2023 and 2024. A total of nine strains were used in the experiments and applied to Nanjing 46 rice for two consecutive years. Each strain was cultured and propagated under optimal conditions and applied to Nanjing 46 rice. The flavor components of the mature rice were quantitatively determined, and strains with significant effects were screened out. In 2024, the application of the dominant strains was expanded to the fragrant rice varieties Nanjing Xiangnuo, Nanjing 9108, and Nanjing 9308.

[0041] Different fungal species were cultured according to their respective optimal culture media, temperatures, and times. Fungi (ACCC32592 *Rhizopus oryzae*, ACCC31821 *Rhizopus oryzae*, ACCC31988 *Trichoderma viride*, ACCC32543 *Aspergillus terreus*, ACCC32548 *Aspergillus niger*, and *Penicillium baicalensis*) were activated using PDA medium, while bacteria (*Bacillus belye* and *Bacillus aryepedodes*) were activated using LB medium. After the bacterial colonies appeared, they were cultured in their respective liquid media according to their optimal temperatures and times. For fungi, the shaking temperature was 28℃ for 5 days, and for bacteria, the temperature was 37℃ for 24 hours. Due to the formation of sporophytes, fungi needed to be homogenized at low temperature (4-10℃) at 800 rpm for 5 minutes before field use.

[0042] Each bacterial culture was diluted with tap water to OD. 600 Use after =0.4.

[0043] The specific usage method is as follows:

[0044] 1) Soak the fragrant rice seeds and germinate them until they sprout. Sow the seeds in a 30cm×60cm substrate seedling tray using a seeder, with 100g of seeds per tray.

[0045] 2) Stack the seedling trays, 20 per stack, and keep them in the dark for 2 days at a temperature of 28℃. After the sprouts emerge from the soil, place the trays in the sunlight in the paddy field and spray water on them daily for cultivation.

[0046] 3) When the fragrant rice seedlings grow to the 3-leaf and 1-heart stage, soak the roots of the seedlings in the bacterial solution. Dilute the original bacterial solution that has been cultured for 24 hours with 5 times before use. Soak 10 seedlings in 10L of diluted bacterial solution for 30 seconds.

[0047] 4) The seedlings soaked in step 3) are then machine-planted;

[0048] 5) Do not use antibacterial pesticides within 10 days after planting. Perform normal water and fertilizer management. 20 days after planting, spray the soil around the seedling roots with a 5-fold diluted bacterial solution at a rate of 20L per acre. Do not use antibacterial pesticides one week before and after spraying. Use normal tillering fertilizer and heading fertilizer. Water management should follow the high-yield cultivation management model. Irrigate shallowly during transplanting and the early to mid-tillering stages. Irrigate shallowly during the early to mid-tillering stages and from heading to flowering. Dry the field from the late tillering stage to the jointing stage. During the grain-filling stage, perform alternating wet and dry irrigation.

[0049] Group CK: The planting steps are the same as above, except that in step 3), the same amount of tap water is used for soaking, and in step 5), the same amount of tap water is sprayed.

[0050] Following the above implementation steps, during machine planting, each type of bacterial solution was applied in three replicate plots, with each plot area being 90m². 2 Harvesting took place on November 8, 2023, and on November 2, 2024. After threshing and drying, the yield of each plot was recorded. The dried paddy was processed into refined rice, then ground into rice flour, which was passed through a 200-mesh sieve for the determination of rice flavor compounds. Three replicates were performed on each type of fragrant rice used for the microbial strain, with two measurements taken from each replicate plot. The average of the three replicates was used as the result.

[0051] The method for determining flavor compounds was based on the method disclosed in the literature "Nadaf A, Mathure S, Jawali N. Scented Rice (Oryza sativa L.) Cultivars of India: A Perspective on Quality and Diversity [M]. New Delhi: Springer, 2015: 1-142." with slight modifications. Headspace solid-phase microextraction (SPE) was used to extract flavor compounds, and the relative contents of various flavor compounds were determined by gas chromatography-mass spectrometry (GC-MS). TMP was selected as the internal standard. 1g of rice flour was weighed and added to a 20ml headspace vial, along with 100ul of deionized water and 10ul of 2.5ng / ul TMP. The vial was quickly sealed and heated at 80℃ for 30min. A solid-phase microextraction syringe (50 / 30um DVB / CAR / PMDS) was inserted into the headspace vial, and extraction was performed at 80℃ for 30min. The vial was then removed and immediately inserted into the GC-MS inlet for 3min of analysis.

[0052] GC-MS analysis conditions: Gas chromatography conditions: TG-5MS (30m×0.25mm×0.25μm) flexible quartz capillary column; carrier gas: high-purity helium (99.999% purity); carrier gas flow rate: 1.2ml / min; splitless injection; injection port temperature: 260℃; temperature program: initial temperature 50℃, hold for 1 min, then increase to 120℃ at 5℃ / min, then increase to 280℃ at 20℃ / min, hold for 7 min.

[0053] Mass spectrometry conditions: EI source, transfer line temperature: 280℃; ion source temperature: 300℃; electron energy: 70eV; scan range (m / z): 35-400 amu, full scan acquisition mode.

[0054] Qualitative analysis of data processing: The Xcalibur workstation was compared with the standard mass spectra provided by the NIST 17 mass spectrometry library, and the components with a matching degree greater than 800 were used as the identification results.

[0055] Quantitative analysis: The concentration of the compound is calculated using the internal standard TMP, i.e.

[0056] The concentration of the analyte is calculated as the concentration of the internal standard divided by the area of ​​the internal standard * the area of ​​the analyte. The experimental data are then summarized into tables and charts using Excel 2016 software.

[0057] A total of 98 flavor components were detected in Nanjing 46 rice, mainly including 6 esters, 1 carboxylic acid, 12 alcohols, 6 ketones, 54 alkanes, 8 aldehydes, 2 phenols, and 9 aromatic and heterocyclic compounds.

[0058] Because alkanes have high threshold values ​​and impart relatively low aroma values ​​to rice, they are not included in the statistics of flavor compounds. Additionally, some flavor components have unclear aroma descriptions or unpleasant odors; these are also excluded from the statistics. The specific content statistics include substances with accurately described odors internationally and those that significantly contribute to rice flavor, mainly the following 24 substances (Table 1). The odor descriptions of flavor compounds are based on the information published in “VCF online by BeWiDo BV Inc. Volatile compounds in food [DB / OL]. [2021-11-30]. https: / / www.vcf-online.nl / VcfHome.cfm.Gas chromatography-olfactometry (GCO) of natural products sponsored by DATU Inc. Flavornet and human odor space [DB / OL]. [2021-11-30]. http: / / www.flavornet.org / flavornet.html.”

[0059] Table 1. Odor description of main flavor compounds

[0060]

[0061]

[0062] Table 2 Comparison of the content of major flavor compounds after cultivation using Bacillus aryepedae ​​(AYB) agent

[0063]

[0064]

[0065] Figures 1-9The mass spectrometry results for the CK, AYB, MG2, MG1, BLS, HQ, BLQ, LSMU, and TQ treatment groups are shown in order (vertical axis represents relative abundance). Table 2 shows the flavor compound detection results for the AYB treatment group. The above analysis results show that AYB significantly increases the content of flavor compounds in Nanjing 46 rice, but the increase varies for each compound. The compound with the largest increase is 2-ethyl-1-hexanol, which has a rose-like aroma, increasing by more than 120 times. The content of 2-butyl-1-octanol with floral aroma increased by 235.71%, the content of 6,10-dimethyl-(E)-5,9-undecadien-2-one with fruity aroma increased by 132.50%, and the contents of limonene, hexanal, 2-heptanone, 2-tetrideone, and 2-pentadecanone all increased by more than 50%. The content of 2-acetyl-1-pyrroline (2-AP) increased by 34.9%. Although 2-methyl-1-undeceneol and nonanoic acid increased, the difference was not significant. The increase of other flavor substances was significant, which greatly improved the sensory flavor of rice.

[0066] In addition, the embodiments focused on the enhancement of aroma of Nanjing 46 rice by different strains, and conducted research in 2023 (…). Figure 10 ), 2024 Figure 11 The experiment, conducted over two consecutive years, only compared the effects of different bacterial strains on the content of 2-AP, a flavor compound, in Nanjing 46 rice.

[0067] Looking at the results over two years, the 2-AP content in the AYB treatment group increased by 36.11% compared to the control (CK) in 2023 and by 34.9% in 2024. Other treatments that showed increased 2-AP content compared to the control in 2023 included MG1 and BLS, but the increase was less significant than that in the AYB treatment group. In 2024, the AYB treatment group showed an increase compared to the control. Overall, the AYB treatment group showed an increase for two consecutive years.

[0068] In 2024, following the above method, AYB was used to treat Nanjing Xiangnuo, Nanjing 9108, and Nanjing 9308, with 25m² of each variety treated. 2 Set up 3 cells with duplicates, and sample 1 meter from each cell. 2 Rice was processed, and the 2-AP content in rice flour was measured. The average value of three plots was calculated. The test results are as follows: Figure 12 As shown.

[0069] The application results show that after applying AYB, the 2-AP content in Nanjing 9108 rice increased by 41.1%, the 2-AP content in Nanjing 9308 rice increased by 31.1%, and the 2-AP content in Nanjing fragrant glutinous rice increased by 35.3%. This demonstrates that the cultivation method described in the above examples can significantly and efficiently increase the content of 2-AP and other flavor compounds in fragrant rice.

Claims

1. Application of Bacillus aryepasis in enhancing the flavor of fragrant rice cultivation; The application refers to: when the fragrant rice seedlings have grown to the 3-leaf and 1-heart stage, soaking the roots of the seedlings in a solution of Bacillus aryepasis; after the seedlings are transplanted to the field, spraying the soil around the roots of the seedlings with a solution of Bacillus aryepasis again to enhance the flavor of the fragrant rice. The fragrant rice mentioned is a rice variety with the BADH2 gene; The improvement refers to an increase of more than 30% in the 2-AP content; The *Bacillus aryepasidia* mentioned is *Bacillus aryepasidia* with accession number 1.914 from the China General Microbiological Culture Collection Center. Bacillus aryabhattai ).

2. The application as described in claim 1, characterized in that, The OD of the Bacillus aryebuddosi bacterial solution 600 It is 0.

4.

3. The application as described in claim 1, characterized in that, The soaking refers to soaking for 20-60 seconds.

4. The application as described in claim 1, characterized in that, The seedlings being transplanted to the field refer to 15-25 days after transplanting.

5. The application as described in claim 1, characterized in that, The term "spraying" refers to a spraying rate of 20 L / mu.

6. The application as described in any one of claims 1-5, characterized in that, The *Bacillus aryepadoides* bacterial suspension was obtained by the following method: *Bacillus aryepadoides* was inoculated onto LB slant agar for activation. After bacterial colonies appeared, it was inoculated into LB liquid agar and cultured on a shaker until the bacterial count reached 1 × 10⁻⁶. 9 -5×10 10 CFU / ml, after dilution, yields Bacillus aryepadolis bacterial suspension.

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