CN102618469B - Rhodobacter capsulatus and application thereof - Google Patents
Rhodobacter capsulatus and application thereof Download PDFInfo
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- CN102618469B CN102618469B CN2012100920315A CN201210092031A CN102618469B CN 102618469 B CN102618469 B CN 102618469B CN 2012100920315 A CN2012100920315 A CN 2012100920315A CN 201210092031 A CN201210092031 A CN 201210092031A CN 102618469 B CN102618469 B CN 102618469B
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Classifications
-
- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/12—Technologies relating to agriculture, livestock or agroalimentary industries using renewable energies, e.g. solar water pumping
Abstract
The invention discloses rhodobacter capsulatus and application thereof. The collection number of the rhodobacter capsulatus AYGHJ-16 is CCTCC NO: M 2011377; the rhodobacter capsulatus belongs to photosynthetic bacteria; and through the rhodobacter capsulatus, certain organic small molecules (such as organic acid and amino acid) can be converted into growth-stimulating substances and anti-virus substances by using light energy, so that growth of plants is stimulated, and the disease-resistant immunity of the plants is improved. Moreover, through the photosynthetic bacteria, product feedback inhibition of heterotrophic microbes can be released by using organic acid produced by metabolism of the heterotrophic microbes in soil, so that a large quantity of heterotrophic microbes are propagated, and conversion and absorption of certain inorganic substances in the soil are promoted; and the photosynthetic bacteria have strong nitrogen fixation and carbon fixation effects, so more inorganic substances can be provided for plants. The selenium-rich microbial agent can improve the micro-ecological environment of the soil, so that the disease resistance of the plants and internal absorption of various mineral elements and rare elements (such as selenium) are improved, and the selenium-rich requirements of the plants are met.
Description
Technical field
The present invention relates to the red bacterium of a strain pod membrane and application thereof.
Background technology
Selenium is that Sweden chemist Berzelius found in the mine tailing of production sulfuric acid in 1817, is considered at that time a kind of poisonous element.Nineteen fifty-seven, Schwarz etc. prove that first selenium is the essential nutrient element of animal, and people just are familiar with to some extent to the health protection effect of selenium.1973, the discovery such as Rotmck and confirm that selenium is after the moiety of animal and human's body Selenoperoxidase, the importance of selenium has been subject to unprecedented attention.1973, the World Health Organization announced that selenium is the bioelement of needed by human.Medical research in recent years shows, selenium deficiency can cause the generation of 40 various diseases, especially can cause following disease: (1) Keshan disease, Kaschin-Beck disease; (2) various cancers; (3) male infertility and forepart pregnant women hypertension; (4) radiation injury is with old and feeble; (5) immunologic hypofunction and acquired immune deficiency syndrome (AIDS); (6) heavy metal toxicity and occupational illness; (7) cardiovascular diseases; (8) epidemic hemorrhagic fever.Replenish a certain amount of selenium and can prevent well the generation of above-mentioned disease, guarantee HUMAN HEALTH.
Selenium is the skewness weighing apparatus on earth, and known have more than 40 countries and regions to lack selenium.According to incompletely statistics, China approximately is in scarce selenium, low selenium band in 72% area.The deficiency of selenium intake is having a strong impact on the healthy of China's several hundred million populations in the meals, according to Chinese Soclety of Nutrition's report of survey, the selenium intake of China adult every day only is 26.63 μ g, and the daily intaking amount 50 μ g and the 60 μ g that recommend apart from Chinese Soclety of Nutrition and international selenium association differ greatly.Human body mainly is to take in selenium by meals, is used for from the benefit selenium of different shape selenium source to human body, and inorganic selenium has certain toxicity, specific absorption is low again, and by biotransformation inorganic selenium is converted into organoselenium, and can reduce the toxicity of inorganic selenium, improve physiologically active and the specific absorption of selenium.Therefore, all organoselenium is mended selenium at present and be considered as the most effective the safest means.
Plant selenium source in organic selenium source is better than animality selenium source.In the vegetalitas selenium source, tea rich in selenium is one of a kind of desirable benefit selenium source.Tea tree is a kind of natural selenium-rich plant, and the tealeaves that originates in rich selenium district is tea rich in selenium, even the tea tree of planting on low selenium soil also can produce tea rich in selenium by manually increasing the selenium method.Selenium in the tealeaves 80% is the organoselenium to the human body beneficial.Tealeaves is one of the world's three large nourishing drinks, and China people have permanent tea drinking habit, therefore develops tea rich in selenium HUMAN HEALTH is had great significance.Along with the enhancing of people's health care consciousness, people will constantly increase the demand of tea rich in selenium.
China is that low selenium is national, and the place of production of natural selenium-rich tea is fewer, so have the certain significance by the selenium content that manually increases in the selenium approach increase tealeaves.Tealeaves increases the selenium approach to be had a variety of.In the natural tea rich in selenium garden, because the soil selenium-containing amount is high, the selenium content in the middle of the tealeaves is naturally just high.But range of distribution is limited after all for the high soil of selenium content, is difficult to satisfy the large market demand, needs to adopt manually to increase selenium.Manually increase selenium and mainly contain two kinds of approach: the one, soil spraying selenium is fertile, and the 2nd, carry out the foliage spray of tealeaves.After tea garden soil enriched selenium fertilizer, a large amount of selenium can not well be absorbed by tea tree, and the selenium content in the tealeaves increases slowly, and amplification is very little.Behind the foliage-spray selenium fertilizer, can improve rapidly the selenium content in the tealeaves, the tealeaves selenium content is directly proportional with the amount of Spraying Se-fertilizer, and Foliar application of selenium is answered attentional selection suitable spray selenium concentration and rational plucking time especially, be in the exploratory stage, can not guarantee that still tealeaves is to the utilization ratio of selenium element.
Photosynthetic bacterium (Photosynthetic Bacteria, abbreviation PSB) is a kind of in the microorganism of waters, is the prokaryotic organism with original luminous energy synthesis system that occur the earliest on the earth, and it extensively is distributed in the anaerobic layer of Different Waters.Show common approximately 50 genus of 7 jumpbogroups such as obligate aerobic bacteria that known photosynthetic bacterium exists painted Bacteriaceae, outer sulphur Rhodospirillaceae, purple nonsulfur bacteria, green sulfur bacteria, the thread green bacterium of many cells, screw rod Cordycepps, contains bacteriochlorophyll.Photosynthetic bacterium extensively is present in natural paddy field, lake, rivers, ocean, active sludge and the soil as the important microbe groups of occurring in nature, the characteristics such as vitality is extremely strong, nutritional requirement is low, growth and breeding is fast, toxicological harmless.
Summary of the invention
The purpose of this invention is to provide the red bacterium of a strain pod membrane and application thereof.
The red bacterium of pod membrane provided by the invention (Rhodobacter capsulatus) AYGHJ-16 is called for short the red bacterium AYGHJ-16 of pod membrane, is preserved in Chinese Typical Representative culture collection center on November 4th, 2011 and (is called for short CCTCC; Address: Wuhan, China, Wuhan University; Postcode: 430072), deposit number is CCTCC NO:M 2011377.
The red bacterium AYGHJ-16 of described pod membrane can be used for preparing fermented liquid (rich selenium microbial inoculum).
The present invention also protects a kind of method for preparing fermented liquid (rich selenium microbial inoculum), is the red bacterium AYGHJ-16 of the described pod membrane of fermentation, obtains fermented liquid.
The condition of described fermentation specifically can be: 30 ℃, carbonic acid gas ventilation flow velocity is 4L/min, and intensity of illumination is 1000-3000Lux.
The preparation method of the fermention medium that described fermentation is adopted is specific as follows: get 120mg Sodium Selenite, 40g glucose, 7.5g peptone, 9g urea, 5g yeast decoction, 0.4g NaHCO
3, 1g NH
4Cl, 0.2g K
2HPO
4, 2.0CH
3COONa, 0.2g MgSO
47H
2O, 2.0g NaCl and 10mL trace element solution are with water dissolution and be settled to 1000mL; The preparation method of described trace element solution is as follows: get 0.5g H
3BO
3, 400mg MnSO
4, 60mgZnSO
47H
2O, 180mg NaMoO
42H
2O and 10mg Ca (NO
3)
22H
2O is with water dissolution and be settled to 250mL.
The pH of described fermention medium specifically can be 6.7-7.2.
The fermented liquid that above any described method prepares (rich selenium microbial inoculum) also belongs to protection scope of the present invention.
The using method of described fermented liquid is that described fermented liquid is carried out being sprayed on the tea tree blade face after 30-50 doubly dilutes.
Described fermented liquid can be used for producing tealeaves.
Described fermented liquid can be used for cultivating tea tree.
The present invention also protects a kind of method of producing tealeaves, comprises the step of described fermented liquid being carried out being sprayed on after 30-50 doubly dilutes the tea tree blade face.Described tea tree specifically can be golden kwan-yin tea tree.
The present invention also protects a kind of method of cultivating tea tree, comprises the step of described fermented liquid being carried out being sprayed on after 30-50 doubly dilutes the tea tree blade face.Adopt method of the present invention to cultivate tea tree, the selenium content of tealeaves is significantly increased.Described tea tree specifically can be golden kwan-yin tea tree.
The red bacterium AYGHJ-16 of pod membrane provided by the invention, belong to photosynthetic bacterium, can utilize luminous energy, some organic molecules (such as organic acid, amino acid etc.) are converted into growth stimulator and antiviral substance, the stimulating plant growth improves the anti pathologic immunity power of plant.And, photosynthetic bacterium can utilize the organic acid that different oxygen animalcule metabolism produces in the soil, remove the product feedback inhibition of different oxygen animalcule, make its amount reproduction, promoted conversion and the absorption of some inorganic substance in the soil, add photosynthetic bacterium itself and can carry out strong fixed nitrogen and carbon solidification effect, can provide more inorganics for plant.
Rich selenium microbial inoculum provided by the invention can improve soil microenvironment, thereby improves the resistance against diseases of plant and for the interior suction of various mineral elements and rare elements (such as selenium), reach the requirement that improves the rich selenium of plant materials.
Rich selenium microbial inoculum provided by the invention can further improve the se use efficiency of tea tree, improves the rich selenium efficient of tealeaves, and the ecological tea rich in selenium of scale operation satisfies drinking tea according to amount of people and mends the purpose of selenium health care service.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.Gold kwan-yin tea tree: tealeaves institute of Fujian Agricultural research institute.
The acquisition of embodiment 1, strains A YGHJ-16 and preservation
One, the acquisition of strains A YGHJ-16S
Utilize enrichment and select to differentiate culture technique, from West Lake of Fuzhou bed mud soil separation energy enduring high-concentration Sodium Selenite, and can transform the photosynthetic bacterium that inorganic selenium is organoselenium.Obtain a strain bacterial strain, called after strains A YGHJ-16S.The rich selenium amount of strains A YGHJ-16S is 150ug/ml.
Two, the evaluation of strains A YGHJ-16S
1, morphological specificity
Gram-negative bacteria;
Thalline is quarter butt or ovum type, and size is about (0.6-1.2) μ m-(2.0-2.2) μ m;
Bacterium colony is smooth, mellow and full, projection under illumination condition;
Bacterium colony is red under illumination condition, is colourless under dark condition.
2, characterization of molecules
The sequencing result of 16S rDNA shown in the sequence 1 of sequence table, with the sequence similarity of the red bacterium of pod membrane of GENBANK ACCESSION NO.NR043407 be 98%.
Comprehensive morphological feature and characterization of molecules, with reference to " uncle Jie Shi systematic bacteriology handbook (the 9th edition) and eastern elegant pearl etc. write " common bacteria system identification handbook (the 1st edition), strains A YGHJ-16S belongs to the red bacterium genus of pod membrane (Rhodobacter capsulatus).
Three, the acquisition of strains A YGHJ-16
For the bacterium that sets out, by the protoplastis breeding of routine and the high-yield strains of the rich selenium of the further seed selection of DNA shuffling technology, obtain strains A YGHJ-16 with strains A YGHJ-16S.The rich selenium amount of strains A YGHJ-16 is 300ug/ml.
Four, the evaluation preservation of strains A YGHJ-16
The red bacterium of pod membrane (Rhodobacter capsulatus) AYGHJ-16 is called for short the red bacterium AYGHJ-16 of pod membrane, is preserved in Chinese Typical Representative culture collection center on November 4th, 2011 and (is called for short CCTCC; Address: Wuhan, China, Wuhan University; Postcode: 430072), deposit number is CCTCC NO:M 2011377.
The fermented ingredient of embodiment 2, the red bacterium AYGHJ-16 of pod membrane and the optimization of processing condition
By single factor and orthogonal test, determine following substratum composition parameter:
Glucose is the optimum carbon source in the substratum, can obtain high-biomass and selenium content when adding 4-5%;
Peptone and urea are the compound nitrogen source in the substratum, and addition is respectively: peptone 0.5%, urea 0.9%;
Yeast extract paste is the auxiliary nitrogenous source in the substratum, and addition is 0.4%;
Sodium Selenite is that the inorganic selenium source in the substratum is selected, and addition is 120ug/mL.
By the shake flask fermentation experiment of homogeneous design, determine following culture condition parameter:
Adopt the 250mL shaking flask, liquid amount 120mL, 28 ℃ of culture temperature, pH5-6, incubation time 5d.
Adopt above-mentioned preferred parameter, the biomass of the red bacterium AYGHJ-16 of pod membrane reaches 37.4g/L, and the total selenium content of thalline enrichment reaches 36.4mg/L.
Embodiment 3, rich selenium microbial inoculum preparation
One, rich selenium microbial inoculum preparation
The preparation method of fermention medium (pH7.0): get 120mg Sodium Selenite, 40g glucose, 7.5g peptone, 9g urea, 5g yeast decoction, 0.4g NaHCO
3, 1g NH
4Cl, 0.2g K
2HPO
4, 2.0CH
3COONa, 0.2gMgSO
47H
2O, 2.0g NaCl and 10mL trace element solution are with dissolved in distilled water and be settled to 1000mL.The inorganic selenium concentration of fermention medium is 120mg/L.In the practical application, fermention medium adopts the pH of 6.7-7.2 all can.
With fermention medium sterilization 20min under 115 ℃.
The preparation method of trace element solution: get 0.5g H
3BO
3, 400mg MnSO
4, 60mg ZnSO
47H
2O, 180mgNaMoO
42H
2O and 10mg Ca (NO
3)
22H
2O is with dissolved in distilled water and be settled to 250mL.
Adopt the 200L fermentor tank, and fermentor tank is carried out following transformation: (1) installs the underwater lamp (light mainly provides the necessary light source of the growth of photosynthetic bacterium as the energy) that glows in the fermentor tank bottom; (2) the electric heating constant temperature control rod is installed in the fermentor tank bottom; (3) in the fermentor tank bottom two external openings are set, the ventage as supply fermentor tank carbonic acid gas, another is as the opening that adds fermention medium and bacterial classification.
In fermentor tank, add the 160L fermention medium, then inoculate the red bacterium AYGHJ-16 of pod membrane; Fermentation condition: temperature is controlled at 30 ℃, and carbonic acid gas ventilation flow velocity is 4L/min, and intensity of illumination is 1500Lux (1000-3000Lux all can); Bacteria concentration in fermentor tank reaches 5.35 * 10
8During cfu/mL, be warming up to 55 ℃ and process 45min (impelling cytoclasis), room temperature centrifugal (5000g, 15min) is collected supernatant liquor, is rich selenium microbial inoculum, 4 ℃ of preservations.
Two, the mensuration of selenium rich ability
With 10 times of the rich selenium microbial inoculum dilutions behind 4 ℃ of preservation 3d, draw 3mL and water and be settled to 10mL, as solution to be measured.Adopt the methylenum coeruleum reduction method to detect the fading time of solution to be measured.
The methylenum coeruleum reduction method detects principle: under acidic conditions, selenite radical can with the DAB effect, generate yellow complex (Se-DAB), when certain pH, with an organic solvent extraction, according to the degree of organic layer colour developing, can carry out spectrophotometry, calculate the content of inorganic selenium according to inorganic selenium content standard opisometer; Organoselenium can not with the DAB complexing, can not generate Se-DAB.
Sodium Selenite: available from Shanghai epoch bio tech ltd.
Concrete grammar following (carrying out successively following operation):
(1) get the separating funnel of 5 125ml, add respectively 2.0,4.0,6.0,8.0, the sodium selenite aqueous solution (1mg/L) of 10.0ml, then all adding distil water to 40ml;
(2) the EDTA-2Na aqueous solution of every funnel adding 2ml 0.2mol/L uses the first acid for adjusting pH to 2-3 to cover interfering ion;
(3) the DAB aqueous solution of every funnel adding 4ml 5g/L shakes up, the lower 60 ℃ of reaction 20min of dark condition;
(4) regulate pH to 7.0 with dense NaOH, then every funnel adds 4ml toluene, fully shakes up, and leaves standstill the 3-4min layering;
(5) organic layer is filtered by absorbent cotton in the cuvette of 1cm, measured the absorbancy (toluene is as blank) of solution in the 420nm place.Take light absorption value A as X-coordinate, take selenium concentration Y (unit is as mg/L) as ordinate zou drawing standard curve.The typical curve function is as follows: Y=1.8674X+0.0009 (R
2=0.9995).
Rich selenium microbial inoculum with the step 1 preparation replaces sodium selenite aqueous solution to carry out the experiment of above step (1) to (5), and with light absorption value substitution typical curve, the result who obtains is the inorganic selenium concentration in the rich selenium microbial inoculum.
Inorganic selenium concentration in the inorganic selenium concentration of the organoselenium concentration=fermention medium in the rich selenium microbial inoculum-Fu selenium microbial inoculum.
Selenium rich ability (organoselenium transformation efficiency)=(the inorganic selenium concentration of the organoselenium concentration/fermention medium in the rich selenium microbial inoculum) * 100%.
Organoselenium concentration in the rich selenium microbial inoculum is 116.4mg/L, and selenium rich ability is 98.9%.
Three, rich selenium microbial inoculum uses the preparation of liquid
Rich selenium microbial inoculum is diluted with water to 50 times of volumes, is rich selenium microbial inoculum and uses liquid.
The rich selenium test of the tealeaves in embodiment 4, tea place, residential quarter
Experimental period is summer.Adopt at random district's group arrangement design experiment of field, three groups of experiments are set altogether, every group is adopted 3 experiment residential quarters, and the area of every experiment residential quarter is 8m * 2.5m, and every experiment residential quarter is evenly planted and is implanted with 100 strains gold kwan-yin tea tree.Experiment arranges three repetitions, results averaged.
Tested the 1st day, each group is handled as follows respectively:
First group: evenly spray rich selenium microbial inoculum on the tea tree blade face and use liquid, 20L is sprayed in every experiment residential quarter;
Second group: evenly spray clear water on the tea tree blade face, 20L is sprayed in every experiment residential quarter;
The 3rd group: do not do any processing.
Test and gathered tealeaves on the 18th day and carry out selenium content detection (according to the detection method of GB5009.93-2010).Selenium content in first group of tealeaves is 350 μ g/kg, and the selenium content in second group of tealeaves is 75 μ g/kg (please indicate), and the selenium content in the 3rd group of tealeaves is 50 μ g/kg (please indicate).
After summer tea is used rich selenium microbial inoculum use liquid, successful, main manifestations is: 1. improve bud density and the reguarity of summer shoot, increase summer tea output; 2. promote the summer shoot growth, ahead of time the productive life: 3. improve the summer tea quality, optimize the green tea quality biochemical component; 4. reduce the pesticide residue in the tealeaves; 5. increased the selenium content in the tealeaves in the tealeaves.
Claims (9)
1. the red bacterium of pod membrane (Rhodobacter capsulatus) AYGHJ-16 is characterized in that, its deposit number is CCTCC NO:M 2011377.
2. the application of the red bacterium AYGHJ-16 of the described pod membrane of claim 1 in producing rich selenium microbial inoculum.
3. the method for the fermented liquid of the red bacterium AYGHJ-16 of the preparation described pod membrane of claim 1 is characterized in that, the red bacterium AYGHJ-16 of the fermentation described pod membrane of claim 1 obtains fermented liquid.
4. the fermented liquid for preparing of the described method of claim 3.
5. the using method of the described fermented liquid of claim 4 is characterized in that, the method is that described fermented liquid is carried out being sprayed on the tea tree blade face after 30-50 doubly dilutes.
6. the application of the described fermented liquid of claim 4 in producing tealeaves.
7. the application of the described fermented liquid of claim 4 in cultivating tea tree.
8. a method of producing tealeaves is characterized in that, the method comprises: the step of the described fermented liquid of claim 4 being carried out being sprayed on after 30-50 doubly dilutes the tea tree blade face.
9. a method of cultivating tea tree is characterized in that, the method comprises: the step of the described fermented liquid of claim 4 being carried out being sprayed on after 30-50 doubly dilutes the tea tree blade face.
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