WO2023020191A1 - 一株印度微小杆菌及其在合成纳米硒中的应用 - Google Patents

一株印度微小杆菌及其在合成纳米硒中的应用 Download PDF

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WO2023020191A1
WO2023020191A1 PCT/CN2022/106875 CN2022106875W WO2023020191A1 WO 2023020191 A1 WO2023020191 A1 WO 2023020191A1 CN 2022106875 W CN2022106875 W CN 2022106875W WO 2023020191 A1 WO2023020191 A1 WO 2023020191A1
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selenium
nano
fermentation
liquid
medium
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朱燕云
靳红梅
布努埃洛斯·加里
董颐玮
朱宁
余向阳
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江苏省农业科学院
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    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G5/00—Fertilisers characterised by their form
    • C05G5/20—Liquid fertilisers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

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  • the invention belongs to the technical field of microbial technology and biological nano-selenium preparation, and in particular relates to a microbacterium indica and its application in biosynthesizing nano-selenium and improving crop quality.
  • Selenium (Se) is one of the essential trace elements for the growth and metabolism of human and animal organisms, and is the functional core of various selenium-containing enzymes. Selenium deficiency in the human body can easily lead to a variety of diseases. Existing studies have confirmed that cardiovascular and cerebrovascular diseases, diabetes, Keshan disease, Kaschin-Beck disease and reproductive system diseases are all related to selenium deficiency. From a global perspective, there is a selenium deficiency zone above 30° north latitude and above 30° south latitude, involving more than 40 countries. In my country, about 73% of the land is in low (deficiency) selenium areas, and the probability of local people suffering from diseases is much higher than that in other areas.
  • Nano-selenium is a kind of red elemental selenium [Se(0)] with nanometer size, which is easier to be absorbed and utilized by the human body than ordinary elemental selenium, and its toxicity is lower than that of general inorganic selenium (Inorganic selenium) and organic selenium ( Organic selenium), because it is different from inorganic selenium, organic selenium and common elemental selenium, was named nano-selenium (Nano-selenium, Nano-Se) by German scientists earlier. Compared with selenate [Se(VI)], selenite [Se(IV)] and selenoamino acid [Se(-II)], nano-selenium has higher biological activity and safety.
  • the mainstream nano-selenium synthesis methods include chemical synthesis and biosynthesis.
  • nano-selenium synthesized by biological methods is more stable than nano-selenium synthesized by chemical methods, and is more resistant to ambient temperature, acidity, alkalinity, etc. It is also not easy to age into black elemental selenium.
  • microorganisms play an important role in the transformation process of different forms of selenium, and some microorganisms can also convert highly toxic selenite or selenate into red elemental nano-selenium. Compared with the process of chemically synthesizing nano-selenium, microbial synthesis of nano-selenium has low energy consumption, less pollution, and is simple and economical.
  • the bacteria that have been reported to have the ability to synthesize nano-selenium include Enterobacter sp., Rhodobacter sp., Brevibacterium sp., Streptomyces sp. and Lahn Rahnella sp., etc.
  • the habitats of these strains are relatively narrow, which leads to corresponding limitations in the nanosynthesis and application development of the strains.
  • Exiguobacterium sp. belongs to probiotics, is a kind of facultative anaerobic bacteria, can grow in the temperature range of -12 ⁇ 55 °C, and has a broad habitat.
  • researchers at home and abroad have isolated different Exiguobacterium sp. from various environments, which are used in the fields of environmental restoration, plant growth promotion, and pathogen control. So far, there has been no report on the synthesis of nano-selenium using microbacteria.
  • the embodiments of the present application provide a strain of Microbacterium indica and a method for fermenting and synthesizing nano-selenium using the strain.
  • the application provides a strain of Exiguobacterium indicum with the preservation number GDMCC NO.61594, which is now preserved in the Guangdong Microbial Culture Collection Center (GDMCC), with a preservation date of April 2, 2021.
  • GDMCC Guangdong Microbial Culture Collection Center
  • the preservation address is Guangdong Institute of Microbiology, 5th Floor, Compound Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong province, postal code 510070.
  • the Indica microbacterium YAN2 was isolated by the inventor from the soil of Fengsan Town, Kaiyang County, Guiyang City, Guizhou province on October 1, 2020. The colony is round, with regular edges, smooth, moist, yellow, and raised , easy to provoke.
  • the 16SrDNA sequence of the strain was determined, and combined with the results of phylogenetic analysis and physiological and biochemical identification, it was determined to be Exiguobacterium indicum, which the applicant named YAN2.
  • the bacteria can produce spherical biosynthetic nano-selenium (Biosynthetic nano-selenium, Bio-SeNP) in the selenium-containing medium.
  • an embodiment of the present application provides a method for biosynthesizing nano-selenium using Exiguobacterium indica with the deposit number GDMCC NO.61594. The specific steps are as follows:
  • Fermentation medium 5g of yeast extract, 10g of tryptone, 10g of NaCl, supplemented to 1L with deionized water; autoclave at 121°C for 20min; then add 1M sterile selenite solution to make the amount of selenite in the medium The final concentration is 5mM, namely;
  • Fermentation medium yeast extract 5g, tryptone 10g, NaCl 10g, sodium selenite 5mM, deionized water to make up to 1L; autoclave at 121°C for 20min.
  • the obtained nano-selenium can also be further prepared by any conventional method in the art to obtain nano-selenium powder.
  • wash step 3 with sterile physiological saline for 3 to 4 times (sonicate for 10 minutes after each cleaning), and resuspend the precipitate with 1/20 volume of sterile water of the fermentation broth, and freeze-dry the obtained bacterial suspension (– 10 ⁇ –50°C), to obtain nano-selenium dry powder.
  • the nano-selenium obtained in step 3) can be used in the production of liquid/solid biological selenium fertilizers, health care products, selenium-enriched functional foods and pharmaceutical products.
  • an embodiment of the present application also provides a nano-selenium liquid fertilizer, which is prepared by the following method:
  • Selenium-containing TB fermentation medium 24g of yeast extract, 20g of tryptone, 4ml of glycerol, K 2 HPO 4 72mM, KH 2 PO 4 17mM, make up to 1L with deionized water; autoclave at 121°C for 20min, cool to room temperature, Add 5mL of 1M sterile selenite (Na 2 SeO 3 ) mother solution to obtain.
  • the nano-selenium liquid fertilizer can significantly improve the selenium content of crops such as leafy vegetables: the nano-selenium is applied on green vegetables, and when the nano-selenium treatment concentration is 1-10mg/L, the selenium content of green vegetables is compared with The blank increased by 3.2-62.31 times; the Vc content increased by 13.93-45.91% compared with the blank; the total phenol content increased by 12.87-28.59% compared with the blank; the fresh weight of vegetables increased by 6.11-29.35% compared with the blank; the antioxidant activity indicators POD, SOD , GSH-Px content increased by 28.45-32.24%, 38.95-48.07% and 57.53-61.38% respectively compared with the blank.
  • This application first isolates and obtains the microbacterium indica strain YAN2, utilizes the ability of this bacterial strain to tolerate selenite, the characteristics of synthesizing nano-selenium, ferment and synthesize nano-selenium, in one embodiment of the application, applying this nano-selenium can improve the Quality and stress resistance, expand the planting range of selenium-rich vegetables, and increase the planting area of selenium-rich crops.
  • the nano-selenium liquid fertilizer prepared in the embodiment of the present application has the advantages of greenness, safety, and high efficiency. When applied to crops, it has high activity, good safety, and convenient application.
  • Figure 1 is a photograph of the colony morphology of Exiguobacterium indica YAN2 on LB medium.
  • Figure 2 is a schematic diagram of the phylogenetic tree of the YAN216S rRNA gene of Exiguobacterium indica.
  • Fig. 3 is the detection result of the selenium tolerance test of Exiguobacterium indica YAN2.
  • Figure 4 is a schematic diagram of the biosynthesis of nano-selenium by Exiguobacterium indica YAN2;
  • the curve 1 corresponds to the production of nano-selenium at different time points
  • the curve 2 corresponds to the cell protein concentration at the time point.
  • Fig. 5 is a scanning electron microscope image (SEM) of nano-selenium produced by Exiguobacterium indica YAN2 fermented for 120 h.
  • Fig. 6 is the X-ray energy spectrum (EDX) of nano-selenium synthesized by Exiguobacterium indica YAN2.
  • Fig. 7 is the photo of the growth situation of small green vegetables after spraying different nano-selenium liquid fertilizers
  • treatment group 1 used nano-selenium liquid fertilizer concentration is 1mg/L; Treatment group 2, used nano-selenium liquid fertilizer concentration is 10mg/L; Treatment group 3, used nano-selenium liquid fertilizer concentration is 50mg/L; The spraying amount was 60mL/pot; the control group was sprayed with an equal volume of deionized water.
  • SEQ ID NO.1 5'-AGAGTTTGATCMTGGCTCAG-3;
  • SEQ ID NO.2 5'-GGTTACCTTGTTACGACTT-3';
  • Sterile selenite (Na 2 SeO 3 ) mother liquor 8.6g of Na 2 SeO 3 solid powder, 50ml of deionized water, passed through a 0.2 ⁇ m filter membrane into a sterile PE tube to obtain 1M selenite mother liquor, Sealed and stored at 4°C for later use.
  • NA solid medium beef extract 3g, tryptone 5g, glucose 2.5g, agar 18g, make up to 1L with deionized water; autoclave at 121°C for 20min.
  • Selenium-containing NA solid medium 3g of beef extract, 5g of tryptone, 2.5g of glucose, 18g of agar and deionized water to make up to 1L; autoclave at 121°C for 20min, and dry it on an ultra-clean workbench to about 55-65°C. Add 5mL of 1M sterile selenite (Na 2 SeO 3 ) mother solution to obtain.
  • LB solid medium yeast extract 5g, tryptone 10g, NaCl 10g, agar 13g, make up to 1L with deionized water; autoclave at 121°C for 20min.
  • LB liquid medium (fermentation seed medium): 5g of yeast extract, 10g of tryptone, 10g of NaCl, make up to 1L with deionized water; autoclave at 121°C for 20min.
  • Selenium-containing LB liquid medium (fermentation medium): add aseptic selenite mother liquor to the LB liquid medium after autoclaving, so that the final concentration of selenite in the medium is 5mM.
  • Selenium-containing TB fermentation medium 24g of yeast extract, 20g of tryptone, 4ml of glycerol, K 2 HPO 4 72mM, KH 2 PO 4 17mM, make up to 1L with deionized water; autoclave at 121°C for 20min, put in ultra-clean work Cool to room temperature on the bench, add 5 mL of 1M sterile selenite (Na 2 SeO 3 ) mother solution, and obtain.
  • Yeast extract Yeast extract
  • tryptone Tryptone
  • agar Agar
  • beef extract Beef extract
  • selenite specifically refers to sodium selenite (Na 2 SeO 3 ), NaCl, glucose, glycerin, K 2 HPO 4 , KH 2 PO 4 , Na 2 S and other reagents, all purchased from Sinopharm Group Industry Co., Ltd. Ltd.
  • High-pressure steam sterilizer (MLS-3781L-PC, Panasonic), microcentrifuge (5424, Eppendorf), constant temperature culture shaker (HYG-A, Jiangsu Taicang), biochemical incubator (LRH-250, Shanghai Yiheng), Ultra-clean bench (Beijing Yatai Cologne), significant constant temperature water bath (DK-8D, Shanghai Jinghong), vacuum dryer (EM-CPD030, Leica, Germany), scanning electron microscope (S-4800, Japan Hitachi), energy Spectrometer (HT7700, Hitachi, Japan).
  • the 16S rRNA gene of bacterial strain YAN2 was amplified using bacterial universal primers 27F (its nucleotide sequence is shown in SEQ ID NO.1) and 1492R (its nucleotide sequence was shown in SEQ ID NO.2).
  • the PCR reaction system was: template DNA, 1.5 ⁇ L; ddH 2 O, 21.5 ⁇ L; 27F, 1.0 ⁇ L; 1492R, 1.0 ⁇ L; Taq enzyme (Mix), 25 ⁇ L.
  • the PCR reaction conditions were: pre-denaturation at 94°C for 10 min; denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 2 min, a total of 35 cycles; extension at 72°C for 10 min. Centrifuge briefly and store at 4°C for later use.
  • the obtained PCR products were sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing.
  • the colony morphology of strain YAN2 on LB solid medium is shown in Figure 1.
  • the physiological and biochemical properties of the strain are shown in Table 1: the results of catalase, V-P reaction, and nitrate reduction test were all positive, and the results of methyl red test and starch hydrolysis were all negative.
  • strain YAN2 Through the sequence analysis of the 16S rRNA of strain YAN2, after BLAST comparison in NCBI, it has a high homology with Microbacterium indica, with a similarity of 95%.
  • the 16S rDNA evolution tree of strain YAN2 is shown in Figure 2, combined with its appearance, physiology and biochemistry Characteristics, strain YAN2 was preliminarily identified as Exiguobacterium indicum.
  • GDMCC Guangdong Microbial Culture Collection Center
  • the deposit number is GDMCC NO.61594
  • the deposit date is April 2, 2021
  • the deposit address is the compound at No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdongzhou 5th Floor, Laboratory Building, Guangdong Institute of Microbiology, Zip code 510070.
  • Exiguobacterium indica YAN2 into LB medium and culture it on a shaking table at 30°C and 150 rpm.
  • the bacterial solution is serially diluted.
  • the selenium concentration that defines the survival rate in 5% ⁇ SR ⁇ 15% is the minimum sub-inhibitory concentration. From the results in Figure 3, it can be seen that the minimum sub-inhibitory concentration of the bacterial strain YAN2 is 105-120mM, and the concentration of the bacterial strain YAN2 is tolerant to selenite Can reach 120mM.
  • Nano selenium content was determined by Biswas method (Biswas KC, Barton LL, Tsui WL, Shuman K, Gillespie J, Eze CS (2011) A novel method for the measurement of elemental selenium produced by bacterial reduction of selenite.J Microbiol Meth 86:140– 144) determination, the analysis steps are: take fermented liquid, centrifuge at 4 °C, 8000 ⁇ 12000rpm for 10 ⁇ 15min, remove supernatant, wash 3 times with 1M NaCl solution, obtain precipitation; Then add and take fermented liquid volume ratio to be 1: 2 of 1M Na 2 S solution, mix well, react at room temperature for 1 h, centrifuge at 8000-12000 rpm for 5-10 min at 4 °C, take the supernatant and measure its absorbance at 500 nm, with 3 replicates for each sample. According to the standard curve of nano-selenium absorbance, the content of nano-selenium in the fermentation broth at different time points was converted.
  • Cell protein content was determined by the modified Lowry method (Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275), and the analysis steps were as follows: take the fermentation broth, Centrifuge at 4°C, 8000-12000rpm for 10-15min, remove the supernatant, add 0.1M NaOH solution with a volume ratio of 5:8 of the fermented liquid, and mix well; put the sample in a boiling water bath for 10-15min, 8000 ⁇ Centrifuge at 12000rpm for 2-4min; take the supernatant, add 50 times the volume of Coomassie Brilliant Blue to stain for 3-5min, measure the absorbance of the sample at 595nm, and repeat 3 times for each sample.
  • the cell protein content in the fermentation broth at different time points was converted according to the cell protein absorbance standard curve.
  • the activated strain YAN2 was transferred to a sterile selenium-containing LB liquid medium, covered with a sealing film, placed in a shaker, and incubated at a constant temperature of 30°C and 150rpm 120h.
  • YAN2 seed liquid Inoculate Exiguobacterium indica YAN2 in LB liquid medium, culture at 28-35°C, 150-250rpm constant temperature until logarithmic growth phase (OD 600 ⁇ 1.0), collect the bacteria by centrifugation at 4°C, and use 0.86 % sterile saline to resuspend it and place it at 4°C for later use.
  • the basic composition is: yeast extract 24g/L, tryptone 20g/L, glycerin 4
  • Nano-selenium dry powder Put the fermentation liquid into the tank, centrifuge at 10000rpm for 15 minutes to collect the bacterial precipitate, wash the precipitate with sterile physiological saline 3 to 4 times (sonicate for 10 minutes after each cleaning), and use 1/20 volume of the fermentation liquid aseptic The precipitate is resuspended in water, and the obtained bacterial suspension is freeze-dried to obtain biological nano-selenium dry powder.
  • Nano-selenium liquid fertilizer Put the fermentation liquid into the tank, centrifuge at 10,000rpm for 15 minutes, collect the precipitate, wash it with sterile saline for 3 to 4 times, and resuspend the precipitate with 1/20 volume of the fermentation liquid in sterile water to finally obtain concentrated nano-selenium Liquid fertilizer (in which nano-selenium content is about 60mM).
  • Small green cabbage seeds (Brassica chinensis L, Shanghai Qing) were purchased from Tomorrow Seed Industry Company of Jiangsu Academy of Agricultural Sciences.
  • the seeds are sterilized in the following steps: first soak in 70% ethanol for 15 seconds, then soak in 2.5% sodium hypochlorite for 15 minutes, and then rinse with sterile water.
  • the seeds were moved to vermiculite and placed in a light incubator at 24°C for germination.
  • the germinated seeds were sown in the test flower pots, and the light cultivation was continued until 2 true leaves grew, and then the seedlings were thinned, leaving 6 seedlings with uniform growth in each pot.
  • Table 2 The growth-promoting experimental data of the bio-nano-selenium fertilizer obtained from the fermentation of bacterial strain YAN2 on green vegetables
  • treatment group 2 31.58 ⁇ 1.91 28.46 ⁇ 4.94 61.18 ⁇ 3.80 Improve rate 12.80% 29.35% 8.89% treatment group 3 30.50 ⁇ 1.00 31.59 ⁇ 4.10 60.00 ⁇ 5.68 Improve rate 8.93% 43.58% 6.78%
  • treatment 1 the concentration of nano-selenium liquid fertilizer used is 1mg/L; treatment 2, the concentration of nano-selenium liquid fertilizer used is 10mg/L; treatment 3, the concentration of nano-selenium liquid fertilizer used is 50mg/L; the spraying amount of the treatment group 60mL/pot; the control group was sprayed with an equal volume of deionized water.
  • increase rate (plant height of treatment group - plant height of control group)/plant height of control group ⁇ 100%.
  • Example 8 Application of biological nano-selenium fertilizer obtained from the fermentation of Exiguobacterium indica YAN2 in improving the antioxidant performance of leafy vegetables
  • Vegetable sowing and pre-management are as described in Example 7. After 45 days of cultivation, the green vegetable samples were collected for peroxidase (POD), superoxide dismutase (SOD), and glutathione peroxidation. The enzyme (GSH-Px) was analyzed. The POD, SOD, and GSH-Px kits provided by Nanjing Jiancheng Bioengineering Institute were used for determination. The results are shown in Table 3.
  • Example 9 Application of biological nano-selenium fertilizer obtained from fermentation of Exiguobacterium indica YAN2 in improving the quality of leafy vegetables

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Abstract

本申请公开了一株具有耐受高浓度亚硒酸盐特性的印度微小杆菌YAN2(Exiguobacterium indicum YAN2)，其保藏编号为GDMCC NO.61594；以及利用该菌株将无机硒发酵为纳米硒的方法，所获得的纳米硒能够促进叶菜的生长，增加叶绿素、Vc、总酚等含量，降低硝酸盐含量，提升叶菜品质；此外，本申请还提供了利用该菌株发酵制备的纳米硒液态肥。

Description

一株印度微小杆菌及其在合成纳米硒中的应用 技术领域
本发明属于微生物技术及生物纳米硒制备技术领域，特别是涉及一株印度微小杆菌及其在生物合成纳米硒与提高作物品质中的应用。
背景技术
硒(selenium,Se)元素是人和动物机体生长代谢必需的微量元素之一，是多种含硒酶的功能核心。人体缺硒易引发多种疾病，现有研究已明确心脑血管疾病、糖尿病、克山病、大骨节病以及生殖系统疾病等均与缺硒相关。从全球来看，从北纬30°以上和南纬30°以上各有一条缺硒带，其中涉及四十多个国家。在我国，有约73％的国土处在低(缺)硒地区，当地人罹患疾病的几率要远高于其他地区。因此，寻找有效的硒补给手段和方法具有重大意义。目前人们通过外源补充硒的形式，主要是无机态硒，然而硒的有效范围极其狭窄，过量硒容易导致人体或动物体硒中毒，甚至造成死亡。
纳米硒是一种具有纳米级尺寸的红色单质硒[Se(0)]，相对于普通的单质硒更易被人体吸收和利用，并且它的毒性低于一般无机硒(Inorganic selenium)和有机硒(Organic selenium)，因区别于无机硒、有机硒和普通单质硒，早先被德国科学家命名为纳米硒(Nano-selenium,Nano-Se)。纳米硒相对于硒酸盐[Se(VI)]、亚硒酸盐[Se(IV)]和硒氨基酸[Se(-II)]等，具有更高的生物活性和安全性。目前主流的纳米硒合成方法有化学合成和生物合成，一般通过生物方法合成的纳米硒要比化学方法合成的纳米硒更稳定，对环境温度、酸度、碱度等耐受性更强，长期放置也不易陈化为黑色单质硒。
自然界中，微生物在不同形态硒的相关转化过程中扮演着重要角色，部分微生物还可以将高毒性亚硒酸盐或硒酸盐转化为红色单质纳米硒。相对于化学合成纳米硒过程，微生物合成纳米硒能耗低，污染小，简单而经济。
目前已报道的具有纳米硒合成能力的细菌包括肠杆菌属(Enterobacter sp.)、红细菌属(Rhodobacter sp.)、短杆菌属(Brevibacterium sp.)、链霉菌属(Streptomyces sp.)与拉恩氏菌属(Rahnella sp.)等。但是，这些菌株生境较为狭窄，导致菌株在纳米合成及其应用开发方面也相应受限。微小杆菌(Exiguobacterium sp.)属于益生菌，是一 类兼性厌氧菌，在-12～55℃温度范围均可生长，具有广阔的生境。国内外研究人员已从多种环境中分离得到不同的Exiguobacterium sp.，在环境修复、植物促生、病原菌防控等领域微小杆菌均有应用。目前尚未见应用微小杆菌合成纳米硒的报道。
发明内容
针对上述问题，本申请的实施例提供一株印度微小杆菌，以及利用该菌株发酵合成纳米硒的方法。
本申请是采用如下技术方案完成的：
首先，本申请提供了一株保藏编号为GDMCC NO.61594的印度微小杆菌(Exiguobacterium indicum)，该菌株现保藏于广东省微生物菌种保藏中心(GDMCC)，，保藏日期2021年4月2日，保藏地址为广东省广州市越秀区先烈中路100号大院实验大楼5楼，广东省微生物研究所，邮编510070。
该印度微小杆菌YAN2是发明人于2020年10月1日从贵州省贵阳市开阳县冯三镇矿区土壤中分离得到，其菌落呈圆形，边缘规则，表面光滑，湿润，黄色，凸起，易挑起。对菌株的16SrDNA序列进行测定，结合系统发育分析和生理生化鉴定结果，确定为印度微小杆菌(Exiguobacterium indicum)，申请人将其自命名为YAN2。该菌在含硒培养基中，能够生成球形生物纳米硒(Biosynthetic nano-selenium,Bio-SeNP)。
其次，本申请的一个实施例提供了利用保藏编号为GDMCC NO.61594的印度微小杆菌生物合成纳米硒的方法。其具体步骤如下：
1)将保藏编号为GDMCC NO.61594的印度微小杆菌接种至LB液体培养基中，活化培养至OD 600≈1.0，获得活化菌液，备用；
2)将步骤1)获得的活化菌液按照体积比2.5±0.2％(v/v)接菌量接种到含有无机硒的发酵培养基的发酵罐中，发酵温度为30±1℃，搅拌速度为150±5rpm，发酵液体积：每分钟通气体积＝1：0.5，罐压为1.0±0.4F/cm 2，发酵至菌体蛋白含量为0.4～0.6mg/mL(约发酵60～120h)，获得发酵菌液；
发酵培养基：酵母提取物5g，胰蛋白胨10g，NaCl 10g，去离子水补足至1L；121℃高压灭菌20min；然后加入1M无菌亚硒酸盐溶液，使培养基中亚硒酸盐的终浓度为5mM，即得；
3)8000～10000rpm离心发酵菌液10～15min，收集沉淀，即获得纳米硒。
发酵培养基：酵母提取物5g，胰蛋白胨10g，NaCl 10g，亚硒酸钠5mM，去离子水补足至1L；121℃高压灭菌20min。
在实际应用中，所获得的纳米硒也可以采用本领域任何常规方法进一步制备获得纳米硒粉。如用无菌生理盐水清洗步骤3)沉淀3～4遍(每次清洗后均超声处理10min)，并用发酵液1/20体积的无菌水重悬沉淀，所得菌悬液经冷冻干燥(–10～–50℃)，获得纳米硒干粉。
步骤时3)所获得的纳米硒可用于液态/固态生物硒肥、保健品、富硒功能食品及医药产品的生产中。
第三，本申请的一个实施例还提供了一种纳米硒液态肥，该纳米硒液态肥是通过如下方法制备的：
1)将保藏编号为GDMCC NO.61594的印度微小杆菌接种至LB液体培养基中，活化培养至OD 600≈1.0，获得活化菌液；
2)将活化菌液按照2.5±0.2％(v/v)接菌量接种到装有含硒TB发酵培养基(5mM亚硒酸钠)的发酵罐中，使发酵温度为30±1℃，搅拌速度为150±5rpm，发酵液体积：每分钟通气体积＝3：1，罐压为1.2±0.2F/cm 2，发酵至纳米硒合成量达到稳定，(约5～6d)，获得发酵液态肥；
含硒TB发酵培养基：酵母提取物24g，胰蛋白胨20g，甘油4ml，K 2HPO 472mM，KH 2PO 417mM，去离子水补足至1L；121℃高压灭菌20min，置冷却至室温，添加1M的无菌亚硒酸盐(Na 2SeO 3)母液5mL，即得。
3)10000rpm离心15min，收集沉淀，清洗(无菌生理盐水洗3～4遍)后用发酵液态肥1/20体积的无菌水重悬沉淀，得到纳米硒液态肥。
本申请的实施例证明，该纳米硒液态肥能显著提高叶菜等作物的硒含量：将该纳米硒施用在青菜上，纳米硒处理浓度为1～10mg/L时，青菜的硒含量相较空白提高了3.2～62.31倍；Vc含量较空白增加了13.93～45.91％；总酚含量较空白增加了12.87～28.59％；青菜鲜重较空白增加了6.11～29.35％；抗氧化活性指标POD、SOD、GSH-Px含量较空白分别增加了28.45～32.24％、38.95～48.07％和57.53～61.38％。
本申请首次分离获得印度微小杆菌菌株YAN2，利用该菌株耐受亚硒酸盐的能 力、合成纳米硒的特性，发酵合成纳米硒，本申请的一个实施例中，施加该纳米硒可以提高青菜的品质和抗逆性能，扩大富硒蔬菜的种植范围，提高富硒作物的种植面积。同时，本申请实施例制备的纳米硒液态肥具有绿色、安全、高效等优点，施用于作物上，活性高、安全性好、施用方便。
附图说明
图1为印度微小杆菌YAN2在LB培养基上的菌落形态照片。
图2为印度微小杆菌YAN216S rRNA基因进化树示意图。
图3为印度微小杆菌YAN2的耐硒性试验检测结果。
图4为印度微小杆菌YAN2生物合成纳米硒示意图；
其中，曲线1位不同时间点纳米硒产量，曲线2位对应时间点细胞蛋白浓度。
图5为印度微小杆菌YAN2发酵120h产纳米硒的的扫描电镜图像(SEM)。
图6为印度微小杆菌YAN2合成的纳米硒X射线能谱(EDX)。
图7为喷施不同纳米硒液态肥后小青菜的生长情况照片；
其中，处理组1，所用纳米硒液态肥浓度为1mg/L；处理组2，所用纳米硒液态肥浓度为10mg/L；处理组3，所用纳米硒液态肥浓度为50mg/L；处理组的喷施量为60mL/盆；对照组喷施等体积的去离子水。
具体实施方式
下面结合具体实施方式对本发明的技术方案作进一步详细的说明。实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例进行修改，或者对其中部分进行替换；而这些修改或替换，并不使相应的技术方案脱离本发明所要求保护的技术方案的范围。
实施例涉及的序列：
SEQ ID NO.1:5’-AGAGTTTGATCMTGGCTCAG-3；
SEQ ID NO.2:5’-GGTTACCTTGTTACGACTT-3’；
实施例涉及的培养基：
无菌亚硒酸盐(Na 2SeO 3)母液：Na 2SeO 3固体粉末8.6g，去离子水50ml，过0.2μm滤膜至无菌PE管中，即得1M的亚硒酸盐母液，密封4℃储存备用。
NA固体培养基：牛肉浸膏3g，胰蛋白胨5g，葡萄糖2.5g，琼脂18g，去离子水补足至1L；121℃高压灭菌20min。
含硒NA固体培养基：牛肉浸膏3g，胰蛋白胨5g，葡萄糖2.5g，琼脂18g去离子水补足至1L；121℃高压灭菌20min，在超净工作台上晾至55～65℃左右，添加1M的无菌亚硒酸盐(Na 2SeO 3)母液5mL，即得。
LB固体培养基：酵母提取物5g，胰蛋白胨10g，NaCl 10g，琼脂13g，去离子水补足至1L；121℃高压灭菌20min。
LB液体培养基(发酵种子培养基)：酵母提取物5g，胰蛋白胨10g，NaCl 10g，去离子水补足至1L；121℃高压灭菌20min。
含硒LB液体培养基(发酵培养基)：向高压灭菌后的LB液体培养基中加入无菌亚硒酸盐母液，使培养基中亚硒酸盐的终浓度为5mM，即得。
含硒TB发酵培养基：酵母提取物24g，胰蛋白胨20g，甘油4ml，K 2HPO 4 72mM，KH 2PO 4 17mM，去离子水补足至1L；121℃高压灭菌20min，置于超净工作台上冷却至室温，添加1M的无菌亚硒酸盐(Na 2SeO 3)母液5mL，即得。
实施例中涉及的试剂：
酵母提取物(Yeast extract)、胰蛋白胨(Tryptone)、琼脂(Agar)、牛肉浸膏(Beef extract)均购自南京都莱生物技术有限公司；
本申请中，亚硒酸盐特指亚硒酸钠(Na 2SeO 3)、NaCl、葡萄糖、甘油、K 2HPO 4、KH 2PO 4、Na 2S等试剂，均购自国药集团工业股份有限公司。
实施例中涉及的仪器：
高压蒸汽灭菌锅(MLS-3781L-PC,Panasonic)、微型离心机(5424,Eppendorf)、恒温培养摇床(HYG-A,江苏太仓)、生化培养箱(LRH-250,上海一恒)、超净工作台(北京亚泰科隆)、显恒温水浴锅(DK-8D,上海精宏)、真空干燥机(EM-CPD030,Leica,德国)、扫描电镜(S-4800,日本Hitachi)、能谱仪(HT7700,日本Hitachi)。
除非特殊说明，以下实施例中涉及的试剂、材料等均为市售。
实施例1菌株YAN2的分离及筛选
取10g矿区土壤(采自贵州省贵阳市开阳县冯三镇矿区)放入装有90mL无菌生理盐水的锥形瓶中，在25℃、150rpm条件下振荡30min，制成10 -1的土壤悬液， 取10 -1的土壤悬液10mL，加至装有90mL无菌生理盐水的锥形瓶中，制成10 -2的土壤稀释液。以此类推，分别配成10 -3、10 -4、10 -5的土壤稀释液。然后，用移液器分别移取10 -3、10 -4、10 -5的土壤稀释液100μL至含硒牛肉膏蛋白胨(NA)固体培养基上，涂布均匀，每个梯度三个重复。最后，将所有涂布好的平皿倒置于30℃恒温培养箱中静置培养2-4d，每天观察培养基上的变化情况，出现单菌落后，将培养基置于超净台上，用无菌接种环挑取较大且为红色的单菌落转接到不含硒的LB固体培养基上进行纯化。纯化3-5代后，转接至LB液体培养基中，30℃、150rpm恒温培养对数期(OD 600≈1.0)，获得新鲜菌悬液，按照菌悬液：甘油＝3:7比例将菌悬液和无菌甘油混合，保存在–80℃超低温冰箱中，待用。申请人将该菌株自命名为YAN2。
实施例2菌株YAN2的鉴定
采用细菌通用引物27F(其核苷酸序列如SEQ ID NO.1所示)和1492R(其核苷酸序列如SEQ ID NO.2所示)扩增菌株YAN2的16S rRNA基因。
PCR反应体系为：模板DNA,1.5μL；ddH 2O,21.5μL；27F,1.0μL；1492R,1.0μL；Taq酶(Mix),25μL。
PCR反应条件为：94℃预变性10min；94℃变性1min，58℃退火1min，72℃延伸2min，共35个循环；72℃延伸10min。短暂离心，4℃保存备用。
获得的PCR产物送南京擎科生物科技有限公司进行测序。
菌株YAN2在LB固体培养基上的菌落形态外观形态见图1。该菌株的生理生化性质见表1：过氧化氢酶、V-P反应、硝酸盐还原试验结果均为阳性，甲基红试验、淀粉水解结果均为阴性。
表1菌株YAN2的生理生化性质
菌株编号 过氧化氢酶 V-P反应 甲基红试验 硝酸盐还原 明胶水解
YAN2 + + - + -
通过对菌株YAN2的16S rRNA进行序列分析，经NCBI中BLAST比对后与印度微小杆菌同源较高，相似度为95％，YAN2菌株16S rDNA进化树见图2，再结合其外形以及生理生化特性，初步鉴定菌株YAN2为印度微小杆菌(Exiguobacterium indicum)。
申请将该菌株保藏于广东省微生物菌种保藏中心(GDMCC)，其保藏编号为GDMCC NO.61594，保藏日期2021年4月2日，保藏地址为广东省广州市越秀区先烈中路100号大院实验大楼5楼，广东省微生物研究所，邮编510070。
实施例3印度微小杆菌YAN2的耐硒性分析
将印度微小杆菌YAN2接入LB培养基，在30℃、150rpm条件下，摇床培养，当OD 600值为1时，对菌液进行梯度稀释。首先吸取100μL菌液加至900μL无菌水中，得到10-1的稀释液，之后以此类推，获得10 -1～10 -6的YAN2稀释液。用移液器分别移取10 -3、10 -4、10 -5、10 -6的稀释液2.5μL滴加到不同硒浓度的LB固体培养基上，每个梯度4个重复。28～35℃静置培养，观察生长情况，96h后统计菌落个数，计算出存活率(SR)，菌株的耐硒性能见图3。
定义存活率在5％<SR<15％内的硒浓度为最小亚抑制浓度，从图3结果可以看出菌株YAN2的最小亚抑制浓度为105～120mM，菌株YAN2耐受亚硒酸盐的浓度可达到120mM。
实施例4印度微小杆菌YAN2合成生物纳米硒的动态规律
制备纳米硒步骤如下：
1)菌株初活化：从YAN2的LB固体平板上挑取单菌落接种至发酵种子培养基中，30±1℃，150±5rpm，恒温培养至对数期(OD 600≈1.0)；
2)菌株再活化：按照0.5％(v/v)接菌量将上述对数期菌株转接至新的发酵种子培养基中，同等条件下培养至对数生长期，备用；
3)发酵：按照2.5±0.2％(v/v)接菌量，将对数期YAN2菌液转接至含硒TB发酵培养基中，30±1℃，150±5rpm，连续摇培，每隔6h取一次发酵液，测定其中的纳米硒含量及YAN2细胞蛋白含量，直至培养结束。纳米硒含量及细胞蛋白含量测定具体方法如下：
纳米硒含量采用Biswas法(Biswas KC,Barton LL,Tsui WL,Shuman K,Gillespie J,Eze CS(2011)A novel method for the measurement of elemental selenium produced by bacterial reduction of selenite.J Microbiol Meth 86:140–144)测定，分析步骤为：取发酵液，4℃、8000～12000rpm离心10～15min，去除上清，用1M NaCl溶液清洗3次，得到沉淀；然后加入与所取发酵液体积比为1:2的1M Na 2S溶液，混合均匀后 室温反应1h，4℃、8000～12000rpm离心5～10min，取上清测定其在500nm处的吸光值，每个样品3个重复。根据纳米硒吸光度标准曲线换算出不同时间点发酵液中纳米硒的含量。
细胞蛋白含量采用改良的Lowry法(Lowry OH,Rosebrough NJ,Farr AL,Randall RJ(1951)Protein measurement with the folin phenol reagent.J Biol Chem 193:265–275)测定，分析步骤为：取发酵液，4℃、8000～12000rpm离心10～15min，去除上清，加入所取发酵液体积比为5：8的0.1M NaOH溶液，充分混匀；将样品置于沸水浴中处理10-15min，8000～12000rpm离心2-4min；取上清液，加入50倍体积考马斯亮蓝染色3-5min，在595nm处测定样品的吸光度，每个样品3个重复。根据细胞蛋白吸光度标准曲线换算出不同时间点发酵液中细胞蛋白的含量。
菌株YAN2合成生物纳米硒的动态规律及细胞生长动态规律见图4。由图4中的结果可知，菌株YAN2在培养至18h时开始大量合成纳米硒。
实施例5印度微小杆菌合成的纳米硒特征分析
按照2.5±0.2％(v/v)接菌量，将活化好的菌株YAN2转接至无菌含硒LB液态培养基中，盖上封口膜，置于摇床内，30℃，150rpm恒温培养120h。对发酵120h的菌液进行收集，常温10000rpm离心6min，去除上清，向沉淀中加入无菌生理盐水，充分悬浮，同等条件下再离心，收集沉淀，再用生理盐水清洗2次，最后将所得沉淀悬浮于2.5％的戊二醛溶液中，4℃固定12h后用于下一步分析。
扫描电镜观察：将上述固定好的纳米硒与菌的混合液进行前处理(脱水、干燥、喷金)，在扫描电镜(蔡司，EVO-LS10)下观察，并利用能谱分析仪对纳米颗粒进行元素组成分析。
电镜观察结果如图5所示，YAN2细胞上可见球形纳米硒颗粒，颗粒分布于细胞周围的空间及细胞表面，粒径分布范围160～350nm。同时对颗粒进行EDX能谱分析，结果如图6所示，分别在1.37、11.22、12.49keV处观察到了硒元素(Se)的特征峰，进一步说明亚硒酸钠被菌株YAN2转化成的球形纳米颗粒即为纳米硒。
本实施例验证了菌株YAN2可以高效合成球形纳米硒。
实施例6利用印度微小杆菌YAN2生物合成纳米硒液态肥
YAN2种子液制备：将印度微小杆菌YAN2接种于LB液体培养基中，在28～35℃， 150-250rpm恒温培养至对数生长期(OD 600≈1.0)，4℃离心收集菌体，用0.86％的无菌生理盐水将其重新悬浮，置于4℃备用。
发酵罐发酵：采用含硒TB发酵培养基，基本组成为：酵母提取物24g/L，胰蛋白胨20g/L，甘油4ml/L，K 2HPO 4 72mM，KH 2PO 4 17mM，Na 2SeO 3 5mM，pH 7.2±0.2；工作体积为发酵罐体积的60～70％，按照2.5±0.2％(v/v)接种量将YAN2种子液接至发酵罐；发酵温度控制在30±1℃，搅拌速度为150±5rpm，发酵液体积：每分钟通气体积＝3：1，罐压为1.2±0.2F/cm 2；发酵5～6d后下罐，采用Biswas法测定罐内发酵液中的纳米硒含量(～3.1mM)。
纳米硒干粉：发酵液下罐，10000rpm离心15min收集菌体沉淀，将沉淀用无菌生理盐水清洗3～4遍(每次清洗后均超声处理10min)，并用发酵液1/20体积的无菌水重悬沉淀，所得菌悬液经冷冻干燥，即得生物纳米硒干粉。
纳米硒液态肥：发酵液下罐，10000rpm离心15min，收集沉淀，将其用无菌生理盐水洗3～4遍，并用发酵液1/20体积的无菌水重悬沉淀，最终得到浓缩纳米硒液态肥(其中纳米硒含量约为60mM)。
实施例7印度微小杆菌YAN2发酵所得生物纳米硒肥对叶菜的促生作用
小青菜种子(Brassica chinensis L，上海青)购于江苏省农科院明天种业公司。将种子进行消毒，步骤如下：先用70％乙醇浸泡15s，再用2.5％的次氯酸钠浸泡15min，之后用无菌水冲洗。将种子移到蛭石中，放入24℃光照培养箱中催芽。将发芽后的种子播种至供试花盆中，继续光照培养直至长出2片真叶后，进行间苗，每盆留6棵长势均一的苗。将上述制备的纳米硒肥浓缩液用去离子水进行适当稀释，得到浓度为1～50mg/L的纳米硒液态肥，在青菜收获前15d喷施纳米硒肥。在培养45d后(图7)，采集青菜样品对其生长指标(株高、鲜重、根系活力)进行分析。测定结果见表2。
表2菌株YAN2发酵所得生物纳米硒肥对青菜的促生实验数据
组别 株高/cm 鲜重/mg 根系活力
对照组 28.46±1.09 22.67±1.09 56.19±6.25
处理组1 30.92±2.57 23.35±0.95 81.42±1.44
提高率 10.42％ 6.11％ 44.91％
处理组2 31.58±1.91 28.46±4.94 61.18±3.80
提高率 12.80％ 29.35％ 8.89％
处理组3 30.50±1.00 31.59±4.10 60.00±5.68
提高率 8.93％ 43.58％ 6.78％
注：处理1，所用纳米硒液态肥浓度为1mg/L；处理2，所用纳米硒液态肥浓度为10mg/L；处理3，所用纳米硒液态肥浓度为50mg/L；处理组的喷施量为60mL/盆；对照组喷施等体积的去离子水。
以株高为例，提高率＝(处理组株高–对照组株高)/对照组株高×100％。
从表2可以看出在喷施纳米硒肥1mg/L(处理1)、10mg/L(处理2)、50mg/L(处理3)时，均能够促进青菜的生长及根系活力，其中纳米硒肥处理为10mg/L时，促生效果最好，纳米硒肥处理为1mg/L时，根系活力最强。
实施例8印度微小杆菌YAN2发酵所得生物纳米硒肥在提升叶菜抗氧化性能方面的应用
菜播种与前期管理如实施例7中所述，在培养45d后，采集青菜样品对其抗氧化性能指标过氧化物酶(POD)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)进行分析。采用南京建城生物工程研究所提供的POD、SOD、GSH-Px试剂盒测定，测定结果见表3。
表3菌株YAN2发酵所得生物纳米硒肥提升青菜的抗氧化性能实验数据
组别 POD(U/g鲜重) SOD(U/g鲜重) GSH-Px(μmol/g鲜重)
对照组 352.00±6.29 210.62±21.95 455.53±14.14
处理组1 465.48±3.70 292.66±46.72 735.09±14.10
提高率 32.24％ 38.95％ 61.38％
处理组2 452.15±1.85 311.87±21.87 717.54±15.28
提高率 28.45％ 48.07％ 57.53％
处理组3 440.30±9.25 310.39±41.52 665.79±35.12
提高率 25.08％ 47.37％ 46.17％
从表3可以看出在喷施纳米硒肥1mg/L(处理1)、10mg/L(处理2)、50mg/L(处理3)时，均能够提升青菜的抗氧化能力，其中对青菜的GSH-Px提升的最多，其次是SOD。当纳米硒肥处理为1mg/L时，对POD和GSH-Px的提升作用相对于其他两种处理的更强。
实施例9印度微小杆菌YAN2发酵所得生物纳米硒肥在提升叶菜品质方面的应用
青菜播种与前期管理如实施例7中所述，在培养45d后，采集青菜样品对其抗氧化性能指标过氧化物酶(POD)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)进行分析。测定结果见表4。
表4菌株YAN2发酵所得生物纳米硒肥提升青菜品质实验数据
组别 硒含量(mg/kg鲜重) 总酚(mg/g鲜重) Vc含量(mg/100g鲜重)
对照组 0.008±0.001 23.33±0.47 5.94±0.44
处理组1 0.034±0.006 30.00±0.82 8.67±0.89
提高率 325％ 28.59％ 45.91％
处理组2 0.506±0.015 26.33±1.25 6.77±0.58
提高率 6231％ 12.87％ 13.93％
从表4可以看出在喷施纳米硒肥1mg/L(处理1)、10mg/L(处理2)时，均能够提升青菜的硒含量、总酚含量以及Vc含量，其中生物纳米硒肥处理浓度为10mg/L时，青菜中硒的总量提升的最多；1mg/L的生物纳米硒肥处理对青菜的总酚及Vc含量提升的最多。

Claims (7)

  1. 一株保藏号为GDMCC NO.61594的印度微小杆菌(Exiguobacterium indicum)。
  2. 如权利要求1所述印度微小杆菌在合成纳米硒中的应用。
  3. 如权利要求2所述的应用，其特征在于，合成步骤如下：
    1)将印度微小杆菌2接种至LB液体培养基中，活化培养至OD 600≈1.0，获得活化菌液，备用；
    2)将步骤1)获得的活化菌液按照体积比2.5±0.2％的接菌量接种到含发酵培养基的发酵罐中，发酵至菌体蛋白含量为0.4～0.6mg/mL，获得发酵菌液；
    发酵培养基制备方法如下：酵母提取物5g，胰蛋白胨10g，NaCl 10g，去离子水补足至1L；灭菌；然后加入1M亚硒酸盐溶液，使培养基中亚硒酸盐的终浓度为5mM，即得；
    3)离心步骤2)获得的发酵菌液，收集沉淀，即为生物纳米硒。
  4. 如权利要求3所述的应用，其特征在于，步骤2)所述发酵是指，发酵温度为30±1℃，搅拌速度为150±5rpm，发酵液体积：每分钟通气体积＝1：0.5，罐压为1.0±0.4F/cm 2。
  5. 一种纳米硒液态肥，其是通过如下方法制备的：
    1)将印度微小杆菌接种至LB液体培养基中，活化培养至OD 600≈1.0，获得活化菌液；
    2)将活化菌液按照2.5±0.2％接菌量接种到装有含硒TB发酵培养基的发酵罐中，发酵至纳米硒合成量达到稳定，获得发酵液态肥；
    含硒TB发酵培养基配置方法如下：酵母提取物24g，胰蛋白胨20g，甘油4ml，K 2HPO 472mM，KH 2PO 417mM，去离子水补足至1L；121℃高压灭菌20min，置冷却至室温，添加1M的无菌亚硒酸盐(Na 2SeO 3)母液5mL，即得；
    3)离心发酵液态肥，收集沉淀，清洗后加入无菌水，即得到纳米硒液态肥。
  6. 如权利要求5所述的纳米硒液态肥，其特征在于，步骤2)所述发酵是指，发酵温度为30±1℃，搅拌速度为150±5rpm，发酵液体积：每分钟通气体积＝3：1，罐压为1.2±0.2F/cm 2。
  7. 一种合成纳米硒的方法，其特征在于，具体步骤如下：
    1)将保藏号为GDMCC NO.61594的印度微小杆菌接种至LB液体培养基中，
    活化培养至OD 600≈1.0，获得活化菌液，备用；
    2)将步骤1)获得的活化菌液按照体积比2.5±0.2％的接菌量接种到发酵培养基中，发酵至菌体蛋白含量为0.4～0.6mg/mL，获得发酵菌液；
    所述发酵培养基制备方法如下：酵母提取物5g，胰蛋白胨10g，NaCl 10g，去离子水补足至1L；灭菌；然后加入1M亚硒酸盐溶液，使培养基中亚硒酸盐的终浓度为5mM，即得；
    3)离心步骤2)获得的发酵菌液，收集沉淀，即为纳米硒。
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