CN105777283A - Water-soluble compound biological fertilizer and preparation thereof - Google Patents
Water-soluble compound biological fertilizer and preparation thereof Download PDFInfo
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- CN105777283A CN105777283A CN201610244982.8A CN201610244982A CN105777283A CN 105777283 A CN105777283 A CN 105777283A CN 201610244982 A CN201610244982 A CN 201610244982A CN 105777283 A CN105777283 A CN 105777283A
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
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Abstract
The invention discloses a water-soluble compound biological fertilizer. The fertilizer is prepared from, by weight, 10-25 parts of aspergillus niger cultures, 3-8 parts of phosphorus-dissolving bacillus megaterium, 5-25 parts of bacillus subtilis, 10-25 parts of lactobacillus plantarum CGMCC NO.9405 and 3-5 parts of polyglutamic acid, wherein the polyglutamic acid is gamma-polyglutamic acid. By means of the water-soluble compound biological fertilizer, the grain yield can be increased by 20%-40%, the vegetable yield is increased by 20%-40%, and the quality and flavor of vegetables and grains can be improved.
Description
Technical field:
Invention relates to the fertilizer in agricultural technology, relates to a kind of novel bio-fertilizer and production thereof.
Background technology:
The class that refers to bio-feritlizer contains the special fertilizer of a large amount of microorganism lived.In this kind of incorporation of fertilizerin the soil, a large amount of microorganisms lived under optimum conditions can be actively movable: have can around root of the crop amount reproduction, performance is from growing nitrogen-fixing or associative nitrogen fixation；Go back decomposable asymmetric choice net phosphorus, potassium mineral element supply Crop or the secretion growth hormone that have stimulate plant growth.As can be seen here, bio-feritlizer is not to directly feed the nutrient substance that crop needs, but the nutrient substance or the generation hormone that provide crop to need by a large amount of microorganisms lived positive activity in soil stimulate plant growth, these are the most different from the effect of other fertilizer and chemical fertilizer.
Now popularization and application mainly have root nodule mushroom fertilizer, Azobacter fertilizer, dissolving phosphor and dissolving potassium mushroom fertilizer, antibiosis mushroom fertilizer and Mycophyta fertilizer etc..These bio-feritlizers have plenty of the goods containing single effective bacterium, also have plenty of compound goods nitrogen-fixing bacteria, dissolving phosphor and dissolving potassium bacterium compound made, on market in addition to the minority fertilizer product such as root nodule mushroom are containing single effective bacterium, most of goods are all compound bio-feritlizers.Various nitrogen-fixing bacteria fertilizer, can increase the sources of nitrogen in soil, and the phosphorus of slightly solubility, potassium in soil can be dissolved out by dissolving phosphor and dissolving potassium bacterial manure material, increase Soil Phosphorus (P), the source of potassium (K) element.It addition, bio-feritlizer can also promote the growth of crop, improve the quality of agricultural product.During various bio-fertilizers are manured into soil, different growth hormone can be produced, stimulate the growth of crop, such as " 5406 " actinomycetes bio-fertilizer, not only have antagonism pathogen diseases prevention to strengthen the effect of bacterium, moreover it is possible to secretory cell mitogen promotes the growth of crop.The bio-fertilizer of Mycophyta not only has the strongest effect in terms of assisting the mineral elements such as Crop phosphorus, zinc and copper, also has and strengthens the water suction of crop, water conservation to improve the effect of crop drought resistance ability.Owing to bio-feritlizer can manufacture and assist Crop to utilize various nutrient elements, therefore the quality of agricultural product is had greatly improved, thus it is possible to vary the present situation of " melon is the most fragrant, the most sweetless; tea is tasteless " because producing to fertilize, make agricultural product indices reach the standard of pollution-free food.
A kind of good bio-feritlizer has strict requirements at aspects such as living bacteria count, water content, pH value, absorbent particles fineness, the content of organic matter, miscellaneous bacteria rate and shelf lifes.According to China's NY227-94 agricultural industry criteria regulation, liquid bio-fertilizer every milliliter should contain effective bacterium of 500,000,000～1,500,000,000 work.Solid biologic fertilizer every gram is 100,000,000～300,000,000 containing the effective bacterium lived, and water content 20%～35% is advisable, and adsorbent fineness is in 0.18 millimeter, and effective bacterium that the fineness of adsorbent is the most carefully adsorbed is the most.PH5.5～7.5, miscellaneous bacteria rate is less than 15%～20%, and without pathogenic bacterium and parasite, shelf life is not less than 6 months.
The bacillus subtilis of Thermostable α-Amylase is produced in one strain, application number: 201310566374.5, the invention discloses a strain and produce the bacillus subtilis of Thermostable α-Amylase, described bacillus subtilis (Bacillussubtilis) Li-2013-02 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on July 15th, 2013, and preserving number is CGMCC No.7926.This bacterial strain produces the bacillus subtilis Li-2010 of Thermostable α-Amylase and obtains through UV-LiCl-dithyl sulfate Mutation screening, and this bacterial strain has the most acidproof, thermostability, produces the feature that enzyme activity is high, and application prospect is boundless.
Currently, along with the development of bio-feritlizer, few places occurs in that when producing bio-feritlizer and falls behind, with not carrying out, by the strain identified, phenomenons such as producing, effective bacterial content is low, miscellaneous bacteria exceeds standard without production licence, production technology.Also some producer adds an inappropriate amount of chemical fertilizer or other additive in bio-feritlizer, makes that the nitrogen of chemical fertilizer in fertilizer, phosphorus, potassium content are too high causes effective bacterium dead, and loses the effect of bio-fertilizer.Therefore when selecting bio-feritlizer it is noted that product quality, counterfeit and shoddy goods are prevented, to avoid bringing unnecessary loss to agricultural production.
Summary of the invention:
The goal of the invention of the present invention is to provide a kind of can develop a kind of Water Soluble Compound bio-fertilizer for the basic condition of China's soil fertility.
The technical solution of the present invention is:
A kind of Water Soluble Compound bio-fertilizer, is made up of amino acid composition, phosphorus decomposing Bacillus megaterium, bacillus subtilis and mixed plant lactobacillus microbial inoculum；The parts by weight of Water Soluble Compound bio-fertilizer consist of: amino acid composition 2-4, phosphorus decomposing Bacillus megaterium 4-8, bacillus subtilis 6-10, mixed plant lactobacillus microbial inoculum 10-20.
Mixed plant lactobacillus microbial inoculum is made up of Lactobacillus plantarum CGMCC NO.9405 and Lactobacillus plantarum CGMCC NO.11763.
The parts by weight of mixed plant lactobacillus microbial inoculum consist of: Lactobacillus plantarum CGMCC NO.9405 1-3, and Lactobacillus plantarum CGMCC NO.11763 7-10 forms.
Amino acid composition consists of lysine, methionine and polyglutamic acid.Amino acid composition parts by weight consist of lysine 2-5, methionine 1-2, polyglutamic acid 2-4；
Described polyglutamic acid is gamma-polyglutamic acid-.
The preparation method of a kind of composite bio-fertilizer, is mixed in proportion compacting pelletize by amino acid composition, phosphorus decomposing Bacillus megaterium, bacillus subtilis and mixed plant lactobacillus microbial inoculum.
Phosphorus decomposing Bacillus megaterium is a kind in Bacillus (Bacillus).Motion, forms pod membrane, aerobic.Produce acid from glucose, also often produce acid from arabinose and mannitol.Hydrolysis starch, does not produce lecithinase, and VP is negative.The organophosphor in soil can be decomposed and become the available rapid available phosphorus of plant.
Phosphorus decomposing bacillus megaterium agent is provided by Cangzhou prosperous generation thing technical research institute, address: liberation West Road, Hebei China Cangzhou City Canal Zone chin or cheek and Building A 1, international business affairs center district 807-812.
In the present invention, Lactobacillus plantarum CGMCC9405 bacterial strain feature is as follows: examine under a microscope, and this bacterial strain is rod-short, and Gram’s staining is positive, atrichia, does not produce spore；On solid medium, this bacterium bacterium colony is white, and smooth surface is fine and close, and form is circular, and edge is more neat.
Physicochemical characteristics is: catalase (-), gelatin liquefaction (-), indole experiment (+), mobility (-), fermentation gas (-), nitrate reductase (-), fermentation gas (-), product hydrogen sulfide gas (-), pH4.0MRS culture medium grows (+).
Lactobacillus plantarum of the present invention uses following flow process to carry out selection-breeding:
The original strain that sets out → test tube activation → dithyl sulfate (DES) mutation → nitrosoguanidine (NTG) mutation → plasma mutation → flat board primary dcreening operation → shaking flask sieves → mitotic stability test again.
Starting strain of the present invention is in MRS dextrose culture-medium, and the throughput rate of its lactic acid is 1.5g/L/d, almost stops growing when medium pH is 3.5, and the decomposition rate to sodium nitrite is 0.34mg/h/kg Chinese cabbage.Starting strain is the greenfeed that Li Zheng is collected in Fattening Sheep field, Yanchi county Ningxia, acquisition time JIUYUE in 2013 15 days.
In order to improve its production of lactic acid speed, acid-fast ability and the decomposition rate of nitrite, use DES and NTG technology that this strain is carried out mutation successively, after mutation, bacterial strain uses MRS calcium carbonate flat board to carry out primary dcreening operation, then 500mL shake flask fermentation is used, biosensor analysis instrument carries out multiple sieve to Producing Strain, the lactobacillus plantarum strain that selection-breeding is excellent, then does passage assays, evaluates its hereditary stability.
Lactobacillus plantarum tlj-2014 hereditary stability result shows: through continuous passage ten times, property indices is the most more stable, and heritability is preferable, and character is not replied, the purpose bacterial strain therefore Lactobacillus plantarum tlj-2014 obtained as selection-breeding.
Empirical tests finds: the production of lactic acid speed of this mutagenic strain can reach 35g/L/d, and this bacterial strain lactic acid concn after fermentation in 71 hours reaches 95g/L；Can survive under conditions of pH is 1.80.Degrading nitrite speed is fast, and capacity of decomposition reaches 9.8mg/h/kg (speed of natural fermentation process nitrite accumulation is about 1.1mg/h/kg), it is possible to resistance to 1% cholate.
Therefore using this strain to produce Pickles, whole sweat nitrite concentration is at below 5mg/kg, far below the content (20mg/kg) of regulation in standard GB/T 2714-2003.
Lactobacillus plantarum (Lactobacillus plantarum) tlj-2014, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 2nd, 2014 and (is called for short CGMCC, address is: North Star West Road 1, Chaoyang District, city of BeiJing, China institute 3, postcode: 100101), preserving number is CGMCC NO.9405.
1.DES mutagenic and breeding
1) on super-clean bench, take Lactobacillus plantarum L mono-ring on test tube slant, access in the 250mL triangular flask equipped with 50mL culture medium MRS (without agar, glucose 20g/L) culture medium, 200rpm, cultivate about 12h, make thalline be in logarithmic growth early stage for 37 DEG C.
2) taking 5mL bacterium solution, 5000rpm is centrifuged 10min and collects thalline, with brine 2 times.
3) it is diluted to 10 with pH7.0 phosphate buffer7Individual/mL bacteria suspension.
4) take the kaliumphosphate buffer of 32mL pH7.0,8mL bacteria suspension, 0.4mL DES are sufficiently mixed in the 150mL triangular flask be placed in advance in rotor, and making DES ultimate density is 1% (v/v).
5) in 37 DEG C of shaking tables, 150rpm reacts 30min, takes 1mL mixed liquor, adds 0.5mL 25%Na2S2O3Solution stopped reaction.
6) suitably dilute, take last dilution bacterium solution 0.2mL, coat in calcium carbonate screening culture medium (the calcium carbonate MRS culture medium containing 100g/L glucose) plate.Cultivating after 2～3 days at 37 DEG C, use photolithography to be transferred to by the bacterial strain of this screening flat board LPHMRS culture medium (low ph value modification MRS culture medium) that pH is 1.5,1.8 and 2.0 is upper and in the sodium nitrite screening culture medium modified MRS screening culture medium of 2g/L sodium nitrite (the single nitrogen source be).
7), after cultivating 2～3 days at 37 DEG C, choosing colony is relatively big, can grow and in calcium carbonate screening culture medium respectively in LPHMRS culture medium, sodium nitrite screening culture medium.Through Preliminary screening, the bacterium colony named Lactobacillus plantarum L1 that picking goes out.
2. nitrosoguanidine mutagenesis
1) on super-clean bench, take Lactobacillus plantarum L1 mono-ring on test tube slant, access in the 250mL triangular flask equipped with 50mL culture medium MRS (without agar) culture medium (concentration of glucose is 60g/L), 200rpm, cultivate about 12h, make thalline be in logarithmic growth early stage for 37 DEG C.
2) take 5mL bacterium solution 5000rpm to be centrifuged 10min and collect thalline, with brine 2 times.
3) it is diluted to 10 with pH6.0 phosphate buffer7Individual/mL bacteria suspension.
4) take 10mL bacteria suspension to be transferred in 100mL triangular flask, add the NTG of 10mg, be configured to the NTG solution of final concentration of 10mg/mL, and add 4-5 and drip acetone, be beneficial to NTG and dissolve.
5) at 37 DEG C, 200rpm oscillating reactions 30min, 5000rpm are centrifuged 10min and collect thalline, wash for several times with physiological saline solution, stopped reaction.
6) suitably dilute, take last dilution bacterium solution 0.2mL, coat in calcium carbonate screening culture medium (the calcium carbonate MRS culture medium containing 100g/L glucose) plate.Cultivating after 2～3 days at 37 DEG C, use photolithography to be transferred to by the bacterial strain of this screening flat board LPHMRS culture medium (low ph value modification MRS culture medium) that pH is 1.5,1.8 and 2.0 is upper and in the sodium nitrite screening culture medium modified MRS screening culture medium of 2g/L sodium nitrite (the single nitrogen source be).
7) bacterial strain method is selected: choosing colony is relatively big, can grow and in calcium carbonate screening culture medium respectively in LPHMRS culture medium, sodium nitrite screening culture medium.Through Preliminary screening, 100 bacterium colonies meeting conditions above of picking.
3. shaking flask is sieved again
1) on super-clean bench, take Lactobacillus plantarum one ring on each test tube slant respectively, access in the 250mL triangular flask equipped with 50mL culture medium MRS (without agar) culture medium (concentration of glucose is 100g/L), 200rpm, cultivate about 15h, make thalline be in mid log phase for 37 DEG C.
2) take 5mL bacterium solution respectively, access equipped with in 50mL calcium carbonate screening fluid medium (the calcium carbonate MRS culture medium containing 250g/L glucose) plate, pH be 1.5,1.8 and 2.0 LPHMRS fluid medium (low ph value modification MRS culture medium) and the sodium nitrite liquid screening medium modified MRS screening culture medium of 2g/L sodium nitrite (the single nitrogen source be) upper (note: employing 250mL triangular flask).200rpm, cultivates 3-4 days for 37 DEG C, detects Pfansteihl in calcium carbonate screening fluid medium every day respectively and produces the Biomass in speed, LPHMRS fluid medium and the wear rate of sodium nitrite liquid screening medium nitrite.After fermentation ends, compare Pfansteihl in the calcium carbonate screening fluid medium of 100 strain strains and produce the Biomass in speed, LPHMRS fluid medium and the wear rate of sodium nitrite liquid screening medium nitrite.
3) select to have high Pfansteihl concurrently to produce speed, tolerate the bacterial strain that the wear rate of low pH (this strain is only capable of in the culture medium of minimum pH1.8 growth) and nitrite is high, by its named L2 bacterium.
4. hereditary stability test
L2 bacterium is passed on for continuous ten times on inclined-plane, and the method sieved again by shaking flask detects the fermentation situation after every time passing on.Experiment finds, passing on for continuous ten times on inclined-plane, this strain character does not has significant change, and property indices is all normal, illustrates that the hereditary stability of this strain is stronger.Strain Designation is Lactobacillus plantarum (Lactobacillus plantarum) tlj-2014.
5.5L fermentation tank is tested
1) Lactobacillus plantarum L2 mono-ring on inclined-plane is taken, access in the 250mL triangular flask equipped with 50mL culture medium MRS (without agar) (concentration of glucose is 150g/L) culture medium, 200rpm, cultivates about 12h, makes thalline be in mid log phase for 37 DEG C.
2) strain of logarithmic (log) phase is accessed in the 5L fermentation tank equipped with 3L MRS fluid medium (initial glucose is 150g/L).Inoculum concentration is 10%, and at 37 DEG C, 100rpm cultivates 8 hours, and logarithm early stage dissolved oxygen controls 10% (ventilation 0.5L/min), later stage Anaerobic culturel 63 hours.After fermentation ends, the lactic acid production of Lactobacillus plantarum L2 reaches 95g/L.Such lactic acid producing speed is beneficial to the rapid fermentation of Pickles.
3) strain of logarithmic (log) phase is accessed equipped with in the 5L fermentation tank of the LPHMRS fluid medium (initial glucose is 50g/L) that 3L pH is 1.8.Inoculum concentration is 10%, and at 37 DEG C, 100rpm cultivates 8 hours, and logarithm early stage dissolved oxygen controls 10% (ventilation 0.5L/min), and later stage anaerobism, fermentation liquid pH is controlled 1.8 by the sodium hydroxide of whole process 0.5mol/L, and total incubation time is 48 hours.After fermentation ends, the Biomass of detection Lactobacillus plantarum L2 is 2.5g/L, illustrates that Lactobacillus plantarum L2 can survive in the environment of pH1.8.
4) strain of logarithmic (log) phase is accessed in the 5L fermentation tank equipped with the 3L sodium nitrite liquid screening medium modified MRS screening culture medium of 2g/L sodium nitrite (the single nitrogen source be).Inoculum concentration is 10%, and at 37 DEG C, 100rpm cultivates 8 hours, and logarithm early stage dissolved oxygen controls 10% (ventilation 0.5L/min), and later stage anaerobism, sweat adds the sodium nitrite solution of 20g/L according to the wear rate stream of nitrite, cultivates 2-3 days.After fermentation ends, calculate the sweat Lactobacillus plantarum L2 degradation rate to sodium nitrite.Found that: under this condition, L2 can reach 563mg/h/L to the degradation rate of sodium nitrite.
The strain 10mL of logarithmic (log) phase is accessed equipped with in Chinese cabbage pretreated for 2kg, is processed according to conventional Kimchi method, the content of nitrite measured in Pickles for every 12 hours.It was found that in whole sweat, L2 bacterium is 9.8mg/h/kg Chinese cabbage to the decomposition rate of sodium nitrite.Content of sodium nitrite in Pickles is consistently lower than 5mg/kg, far below the content (20mg/kg) of regulation in standard GB/T 2714-2003.
Beneficial effect:
Experiment shows, uses this product can improve grain yield 20-40%, improves yield of vegetables 20-40%, also can improve vegetable, the quality of grain and local flavor.The amount of the fertilizer of every mu of land use present invention is 50-100 gram, is the most domestic and international less water soluble fertilizer of usage amount.
Detailed description of the invention:
The following examples can make those skilled in the art that the present invention is more fully understood, but limits the present invention never in any form.1, microbial inoculum is cultivated and is prepared:
Preparation cultivated by bacillus subtilis microbial inoculum: prepared by bacillus subtilis microbial agent: from inclined-plane, bacillus subtilis is cultivated in switching, and the seed liquor after spreading cultivation step by step is transferred in fermentation tank, and complete centrifugation of fermenting obtains wet thallus and fermentation liquid；The complete fermentation liquor low-temperature negative-pressure that ferments is concentrated in vacuo to the 40-50% of original volume, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixed, mix homogeneously；Concentrated solution is 0.5:1 with the weight ratio of carrier, and vehicle group becomes: CaCO320-40 part, dextrin 10-20 part.Fluid bed drying, baking temperature 50 DEG C.
The bacterial strain producing Thermostable α-Amylase that the present invention provides is specially bacillus subtilis (Bacillus subtilis) Li-2013-02.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC, address is: North Star West Road 1, Chaoyang District, city of BeiJing, China institute 3) on July 15th, 2013, and preserving number is CGMCC No.7926.
Described bacterial strain feature is as follows:
Described bacterial strain colony colour on solid plate is milky, and dry tack free is opaque, neat in edge, for having the aerobic bacteria of mobility.Microscopy is elongated rod shape, and Gram’s staining is positive.This bacterium may utilize citrate, and nitrate reductase, V-P test into the positive.
The bacillus subtilis Li-2013 producing Thermostable α-Amylase that described bacillus subtilis (Bacillus subtilis) Li-2013-02 is preserved in this laboratory by a strain obtains through UV-LiCl-dithyl sulfate Mutation screening, and concrete screening step is as follows:
(1) preparation of bacteria suspension
The mono-bacterium colony of Li-2013 that grow after plate streaking separates is accessed in seed culture medium, 100r/min, after 40 DEG C are cultivated 12h, take after 1mL medium centrifugal with brine twice, and resuspended with in 9mL normal saline.
(2) UV-LiCl-dithyl sulfate complex mutation
Being placed in by bacteria suspension in aseptic flat board, be 30cm in distance, under the uviol lamp of power 15w, 100s is irradiated in stirring.Bacterium solution through irradiating is coated after gradient dilution lithium chloride flat board, and to compare without the bacterium solution of ultra-vioket radiation dilution painting flat board.By uniform for above-mentioned coating flat board, cloth or newspaper with black are wrapped, put 40 DEG C and cultivate 48h, the flat board grow bacterium colony filters out hydrolysis circle choose to inclined-plane preservation with colony diameter ratio the maximum, it is configured to bacteria suspension after purification, it is sufficiently mixed with dithyl sulfate stock solution after gradient dilution, and processes 40min in 40 DEG C of concussions, the bacterium solution processed is coated lithium chloride flat board after gradient dilution.
(3) primary dcreening operation of high-yield strains
By uniform for above-mentioned coating flat board, putting 40 DEG C and cultivate 48h, on the flat board grow bacterium colony, primary dcreening operation goes out hydrolysis circle and colony diameter ratio the greater is chosen and preserved to inclined-plane, obtains three strain bacterium Li-2013-01, Li-2013-02, Li-2013-03 after purification
(4) shake flask fermentation sieves again
The three strain bacterium Li-2013-01 that will obtain, Li-2013-02, Li-2013-03 carry out shake flask fermentation, seed inoculum concentration 10% (V/V) in the 250mL shaking flask containing 30mL fermentation medium, 40 DEG C, 100r/min cultivate 72h, centrifuging and taking fermented supernatant fluid prepares crude enzyme liquid.
(5) enzyme activity determination
Enzyme is lived the definition of unit: 1mL crude enzyme liquid, in 105 DEG C, under the conditions of pH 4.2,1min liquefies 1mg soluble starch, is 1 enzyme activity unit, represents with U/mL.
After measured, bacterial strain Li-2013-02, for stable superior strain, and enzyme is lived and is reached 30000U/mL, lives than original strain enzyme and improves 1.6 times.
Described lithium chloride flat board: starch 1%, peptone 1%, (NH)2SO40.4%, K2HPO40.8%, CaCl20.2%, lithium chloride 0.9%, agar 2%.
Described seed culture medium: yeast powder 0.5%, peptone 1%, soluble starch 1%, NaCl 1%.
Described fermentation medium: Semen Maydis powder 5%～15%, soybean cake powder 4%～10%, (NH) 2SO40.4%, K2HPO40.8%, CaCl20.2%.
Described shake flask culture conditions: this bacterium in the 250mL shaking flask containing 30mL fermentation medium, inoculum concentration 10% (V/V), 100r/min, 40 DEG C of fermentation culture 72h.
Being obtained a kind of high-temperature resistant alpha-amylase by bacterial strain Li-2013-02 fermentation, its zymologic property is as follows:
(1) this enzyme temperature adaptation wider range, optimum temperature is between 105-115 DEG C, and the temperature stability preserved below 110 DEG C is preferable, and more than 115 DEG C to preserve long-time temperature stability poor.
(2) this enzyme optimal reaction pH value is 4.2.High enzyme vigor is all had, the enzyme complete stability alive when pH value is 3.0 between pH value 3.0-7.0.
(3) enzymatic activity: by mutant Li-2013-02 provided by the present invention, the Thermostable α-Amylase enzyme activity of preparation is 30000-35000U/ml.
1, the present invention uses the method for UV-LiCl-dithyl sulfate complex mutation to obtain a plant height and produces the bacillus subtilis Li-2013-02 of Thermostable α-Amylase, and this bacterial strain has the most acidproof, thermostability, produces the feature that enzyme activity is high.
2, the Thermostable α-Amylase enzyme activity having this bacterial strain to produce gained is up to 30000-35000u/ml,；Applicable temperature scope is 25-115 DEG C, optimal reactive temperature 110 DEG C, at 110 DEG C of enzymes complete stability alive；Being suitable for pH value in reaction scope is 3.0-7.0, the enzyme complete stability alive when pH value is 3.0, optimal reaction pH value is 4.2, higher than existing Thermostable α-Amylase enzyme activity, enzyme effect optimum pH wide scope, resistance to temperature is high, is particularly suitable for reaction temperature height, liquefaction process and Mashing process the industrialization demand deposited.
Invent described Lactobacillus plantarum (Lactobacillus plantarum) XH and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on November 30th, 2015, preserving number is CGMCC NO.11763, preservation address is: North Star West Road 1, Chaoyang District, city of BeiJing, China institute 3, Institute of Microorganism, Academia Sinica, postcode: 100101..
Lactobacillus plantarum probiotic properties is as follows:
Lactobacillus plantarum CGMCC NO.11763 provided by the present invention is found under conditions of pH is 1.50 survive, still in existing state after 1% cholate is cultivated 4 hours through experiment；Lactobacillus plantarum CGMCC NO.11763 degrading nitrite speed is fast, and capacity of decomposition reaches 10.9mg/h/kg, and this strain is when producing Pickles, and whole sweat nitrite concentration is at below 4.8mg/kg；CGMCC NO.11763, after fermentation 60h hour, can reach 64.76% to degrading rate of cholesterol.CGMCC NO.11763 Adhering capacity measure from coagulation rate be 95.71%.
CGMCC NO.11763 is to cholesterol degradation capability study and mensuration:
Take 1ml CGMCC NO.11763 mother solution and be inoculated in MRS cholesterol fluid medium (the cholesterol level 0.1mg/ml of 10mL, pH 6.2) in, the constant temperature of 37 DEG C stands and cultivates 20h respectively, 40h, 60h is standby, with access 1mL sterilized water MRS cholesterol culture medium for comparison, take bacteria liquid sample and the comparison each 1ml of liquid of above cultivation different time, 9000r/min, centrifugal 10min at 4 DEG C, obtain fermented supernatant fluid, in o-phthalaldehyde method mensuration supernatant, cholesterol level is (particularly as follows: take each supernatant 0.1ml in corresponding test tube, add glacial acetic acid 0.3ml, o-phthalaldehyde(OPA) 0.15ml of 1mg/ml, it is slowly added into concentrated sulphuric acid 1.0ml, mix homogeneously.Room temperature stands 10min, surveys light absorption value under 550nm).Each process 3 repetition, in kind makes cholesterol standard curve, calculates cholesterol level and degradation rate in supernatant, the results are shown in Table 1.Understanding, CGMCC NO.11763 has good Degradation to cholesterol, and after fermentation 60h hour, degradation rate can reach 64.76%.
The table 1 degraded situation to cholesterol
The bile tolerance test of CGMCC NO.11763 bacterial strain:
Take CGMCCNO.11763 bacterium solution 1mL inoculation strain in 10mL MRS fluid medium (PH=6.4) containing different cholate (concentration gradients is 0.0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%), be placed at 37 DEG C and cultivate 0 respectively, 2,4h, each process 3 repetition.Respectively take 1ml sample bacterium solution to mix in 9ml normal saline, prepare dilution factor solution, take 0.1ml diluent to be coated with in MRS, be inverted in 37 DEG C of biochemical cultivation cases and cultivate 48 hours (each dilution factor do 3 parallel) record and calculate the several number of bacterium on flat board.The results are shown in Table 2.Understand this bacterium increment of bacterium after gallbladder salinity is 1% process 4h and still reach 0.59 ± 0.92 × 107(cfu/ml), there is good bile tolerance ability.
Table 2 bile tolerance ability detection [(± s) × 107cfu/ml]
The acid resistance test of CGMCC NO.11763 bacterial strain
Take CGMCC NO.11763 mother solution by 1ml inoculation strain in the 10mL MRS fluid medium of different pH value (pH gradient is 1.5,2.0,2.5,3.0,3.5,4.0), be placed at 37 DEG C and cultivate 0 respectively, 2,4h, each process 3 repetition.Respectively take 1ml sample bacterium solution to mix in 9ml normal saline, prepare dilute solution, take 0.1ml diluent and be coated with in MRS, in 37 DEG C of biochemical cultivation cases, be inverted the bacterium colony number cultivated on 48 hours (each dilution factor do 3 parallel) record flat board.The results are shown in Table 3.Illustrate that this bacterium has the strongest acid-fast ability.
Table 3 acid-fast ability detection [(± s) × 107cfu/ml]
The Adhering capacity of CGMCC NO.11763 bacterial strain measures
Cultivate CGMCC NO.11763 (MRS fluid medium), bacillus coli DH 5 alpha (LB fluid medium) 24h obtains fermentation liquid, it is respectively placed in 3000r/min, centrifugal 10min at 4 DEG C, collect bacterium mud, (in bacterium colony, PBS is i.e. added 2 times respectively with sterile phosphate buffer (PBS) the washing bacterium mud of pH=7.0, after concussion mix homogeneously, it is placed in 3000r/min, centrifugal 10min at 4 DEG C, collects thalline).From coagulation rate (%): with aseptic PBS, bacterium mud CGMCC NO.11763 is formed in suspension bacteria liquid and the bacteria suspension that the light absorption value at wavelength 600nm is 0.4 ± 0.1 (A 0), measure light absorption value A 24 after standing 24h, be (A 0 A 24)/A 0 from coagulation rate (%) formula.；His coagulation rate (%): the outstanding bacterium solution of CGMCC NO.11763 and bacillus coli DH 5 alpha is adjusted to the mix suspending bacterium solution that the light absorption value at wavelength 600nm is 0.6 ± 0.1 (A 0).Measuring light absorption value A 24 after standing 24H, his coagulation rate (%) formula is (A 0 A 24)/A 0.Measurement result is shown in Table 4, it is known that CGMCC NO.11763 is 95.71% from coagulation rate, has the strongest Adhering capacity.
Table 4 Adhering capacity table
Bacterial strain physiological property
Described Lactobacillus plantarum (Lactobacillus plantarum) XH is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on November 30th, 2015, preserving number is CGMCC NO.11763, preservation address is: North Star West Road 1, Chaoyang District, city of BeiJing, China institute 3, Institute of Microorganism, Academia Sinica, postcode: 100101.
This bacterial strain feature is as follows: examine under a microscope, and this bacterial strain is rod-short, and Gram’s staining is positive, atrichia, does not produce spore；On solid medium, this bacterium bacterium colony is white, and smooth surface is fine and close, and form is circular, and edge is more neat.
Physicochemical characteristics is: catalase (-), gelatin liquefaction (-), indole experiment (+), mobility (-), fermentation gas (-), nitrate reductase (-), fermentation gas (-), product hydrogen sulfide gas (-), pH4.0MRS culture medium grows (+).It is accredited as Lactobacillus plantarum (Lactobacillus plantarum), named Lactobacillus plantarum (Lactobacillus plantarum) XH through Physiology and biochemistry.
Bacterial strain can at 57 DEG C well-grown, glucose tolerance is 275g/L.
Lactobacillus plantarum of the present invention by gathering people Li Jianshu, isolated in Yoghourt from Xinjiang Uygur fellow-villager family, acquisition time on June 2nd, 2015.
5L fermentation tank is tested
(1) Lactobacillus plantarum CGMCC NO.11763 mono-ring on inclined-plane is taken, access in the 250mL triangular flask equipped with 50mL culture medium MRS (without agar) (concentration of glucose is 150g/L) culture medium, 200rpm, cultivate about 12h, make thalline be in mid log phase for 37 DEG C.
(2) strain of logarithmic (log) phase is accessed in the 5L fermentation tank equipped with 3L MRS fluid medium (initial glucose is 150g/L).Inoculum concentration is 10%, and at 37 DEG C, 100rpm cultivates 8 hours, and logarithm early stage dissolved oxygen controls 10% (ventilation 0.5L/min), later stage Anaerobic culturel 63 hours.After fermentation ends, the lactic acid production of Lactobacillus plantarum CGMCC NO.11763 reaches 110g/L.Such lactic acid producing speed is beneficial to the rapid fermentation of Pickles.
(4) strain of logarithmic (log) phase is accessed in the 5L fermentation tank equipped with the 3L sodium nitrite liquid screening medium modified MRS screening culture medium of 2g/L sodium nitrite (the single nitrogen source be).Inoculum concentration is 10%, and at 37 DEG C, 100rpm cultivates 8 hours, and logarithm early stage dissolved oxygen controls 10% (ventilation 0.5L/min), and later stage anaerobism, sweat adds the sodium nitrite solution of 20g/L according to the wear rate stream of nitrite, cultivates 2-3 days.After fermentation ends, calculate the sweat Lactobacillus plantarum CGMCC NO.11763 degradation rate to sodium nitrite.Found that: under this condition, XH can reach 653mg/h/L to the degradation rate of sodium nitrite.
(5) the strain 10mL of logarithmic (log) phase is accessed equipped with in Chinese cabbage pretreated for 2kg, be processed according to conventional Kimchi method, the content of nitrite measured in Pickles for every 12 hours.It was found that in whole sweat, XH bacterium is 10.9mg/h/kg Chinese cabbage to the decomposition rate of sodium nitrite.Content of sodium nitrite in Pickles is consistently lower than 4.8mg/kg, far below the content (20mg/kg) of regulation in standard GB/T 2714-2003.
Embodiment 1
A kind of Water Soluble Compound bio-fertilizer, is made up of amino acid composition, phosphorus decomposing Bacillus megaterium, bacillus subtilis and mixed plant lactobacillus microbial inoculum；The parts by weight of Water Soluble Compound bio-fertilizer consist of: amino acid composition 3, phosphorus decomposing Bacillus megaterium 6, bacillus subtilis 8, mixed plant lactobacillus microbial inoculum 16.
Mixed plant lactobacillus microbial inoculum is made up of Lactobacillus plantarum CGMCC NO.9405 and Lactobacillus plantarum CGMCC NO.11763.
The parts by weight of mixed plant lactobacillus microbial inoculum consist of: Lactobacillus plantarum CGMCC NO.9405 1 part, Lactobacillus plantarum CGMCC NO.11763 8 parts composition.
Amino acid composition consists of lysine, methionine and polyglutamic acid.Amino acid composition parts by weight consist of lysine 3, methionine 1, polyglutamic acid 3；
Described polyglutamic acid is gamma-polyglutamic acid-.Bacillus subtilis preserving number is CGMCC No.7926.
The preparation method of a kind of composite bio-fertilizer, is mixed in proportion compacting pelletize by amino acid composition, phosphorus decomposing Bacillus megaterium, bacillus subtilis and mixed plant lactobacillus microbial inoculum.
Embodiment 2
A kind of Water Soluble Compound bio-fertilizer, is made up of amino acid composition, phosphorus decomposing Bacillus megaterium, bacillus subtilis and mixed plant lactobacillus microbial inoculum；The parts by weight of Water Soluble Compound bio-fertilizer consist of: amino acid composition 2, phosphorus decomposing Bacillus megaterium 8, bacillus subtilis 6, mixed plant lactobacillus microbial inoculum 20.
Mixed plant lactobacillus microbial inoculum is made up of Lactobacillus plantarum CGMCC NO.9405 and Lactobacillus plantarum CGMCC NO.11763.
The parts by weight of mixed plant lactobacillus microbial inoculum consist of: Lactobacillus plantarum CGMCC NO.9405 3, and Lactobacillus plantarum CGMCC NO.11763 7 forms.
Amino acid composition consists of lysine, methionine and polyglutamic acid.Amino acid composition parts by weight consist of lysine 5, methionine 1, polyglutamic acid 4；
Described polyglutamic acid is gamma-polyglutamic acid-.Bacillus subtilis preserving number is CGMCC No.7926.
Embodiment 3
A kind of Water Soluble Compound bio-fertilizer, is made up of amino acid composition, phosphorus decomposing Bacillus megaterium, bacillus subtilis and mixed plant lactobacillus microbial inoculum；The parts by weight of Water Soluble Compound bio-fertilizer consist of: amino acid composition 4, phosphorus decomposing Bacillus megaterium 4, bacillus subtilis 6, mixed plant lactobacillus microbial inoculum 10.
Mixed plant lactobacillus microbial inoculum is made up of Lactobacillus plantarum CGMCC NO.9405 and Lactobacillus plantarum CGMCC NO.11763.
The parts by weight of mixed plant lactobacillus microbial inoculum consist of: Lactobacillus plantarum CGMCC NO.9405 1, and Lactobacillus plantarum CGMCC NO.11763 10 forms.
Amino acid composition consists of lysine, methionine and polyglutamic acid.Amino acid composition parts by weight consist of lysine 3, methionine 1, polyglutamic acid 4；
Described polyglutamic acid is gamma-polyglutamic acid-.
Area, Wuzhong, Ningxia is selected to test；Test nonirrigated farmland maize planting 10 mu, uses product of the present invention 0.2 kilogram every mu when plantation respectively, and emerging about 1 month to be sprayed by blade face side uses invention product 0.3 kilogram, and matched group uses the similar bio-feritlizer of market sale.
Invention product uses corn on dry land yield to reach 390 kilograms, and matched group reaches 130 kilograms, saves irrigation water 16% than matched group.
Claims (8)
1. a Water Soluble Compound bio-fertilizer, by amino acid composition, phosphorus decomposing Bacillus megaterium, bacillus subtilis and mixed plant
Lactobacillus microbial inoculum forms；The parts by weight of Water Soluble Compound bio-fertilizer consist of: amino acid composition 2-4, the huge bud of phosphorus decomposing are embraced
Bacillus 4-8, bacillus subtilis 6-10, mixed plant lactobacillus microbial inoculum 10-20.
2. a kind of Water Soluble Compound bio-fertilizer, it is characterised in that described mixed plant lactobacillus microbial inoculum is by plant
Lactobacillus CGMCC NO.9405 and Lactobacillus plantarum CGMCC NO.11763 composition.
3. a kind of Water Soluble Compound bio-fertilizer, it is characterised in that the weight of described mixed plant lactobacillus microbial inoculum
Number consists of: Lactobacillus plantarum CGMCC NO.9405 1-3, and Lactobacillus plantarum CGMCC NO.11763 7-10 forms.
4. as claimed in claim 1 a kind of Water Soluble Compound bio-fertilizer, it is characterised in that described amino acid composition consist of lysine,
Methionine and polyglutamic acid.Amino acid composition parts by weight consist of lysine 2-5, methionine 1-2, polyglutamic acid 2-4.
5. a kind of Water Soluble Compound bio-fertilizer as described in claim 1-4 is arbitrary, by amino acid composition, phosphorus decomposing Bacillus megaterium,
Bacillus subtilis and mixed plant lactobacillus microbial inoculum composition；The parts by weight of Water Soluble Compound bio-fertilizer consist of: aminoacid group
Compound 3, phosphorus decomposing Bacillus megaterium 6, bacillus subtilis 8, mixed plant lactobacillus microbial inoculum 16.
6. a kind of Water Soluble Compound bio-fertilizer as described in claim 5 is arbitrary, mixed plant lactobacillus microbial inoculum is by Lactobacillus plantarum CGMCC
NO.9405 and Lactobacillus plantarum CGMCC NO.11763 composition；The parts by weight of mixed plant lactobacillus microbial inoculum consist of: plant
Lactobacillus CGMCC NO.9405 1 part, Lactobacillus plantarum CGMCC NO.11763 8 parts composition.
7. a kind of Water Soluble Compound bio-fertilizer as described in claim 1-4 is arbitrary, amino acid composition consist of lysine, methionine and
Polyglutamic acid.Amino acid composition parts by weight consist of lysine 3, methionine 1, polyglutamic acid 3.
8. the preparation method of a kind of Water Soluble Compound bio-fertilizer as described in claim 1-7 is arbitrary, by amino acid composition, phosphorus decomposing Bacillus megaterium, withered
Grass bacillus cereus and mixed plant lactobacillus microbial inoculum are mixed in proportion compacting pelletize.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107602256A (en) * | 2017-10-30 | 2018-01-19 | 成都云图控股股份有限公司 | A kind of Water soluble fertilizer of the lysine containing L and preparation method thereof |
| SE1951269A1 (en) * | 2019-11-06 | 2021-05-07 | Arevo Ab | Preparations for enhanced biocontrol |
| CN112759458A (en) * | 2021-01-28 | 2021-05-07 | 西安众民生物化工有限公司 | Bactericidal regulation agricultural fertilizer containing polyglutamic acid and chitosan |
| WO2021091463A1 (en) * | 2019-11-06 | 2021-05-14 | Arevo Ab | Preparations for enhanced biocontrol |
-
2016
- 2016-04-19 CN CN201610244982.8A patent/CN105777283A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107602256A (en) * | 2017-10-30 | 2018-01-19 | 成都云图控股股份有限公司 | A kind of Water soluble fertilizer of the lysine containing L and preparation method thereof |
| SE1951269A1 (en) * | 2019-11-06 | 2021-05-07 | Arevo Ab | Preparations for enhanced biocontrol |
| WO2021091463A1 (en) * | 2019-11-06 | 2021-05-14 | Arevo Ab | Preparations for enhanced biocontrol |
| SE545713C2 (en) * | 2019-11-06 | 2023-12-19 | Arevo Ab | Preparations for enhanced biocontrol |
| CN112759458A (en) * | 2021-01-28 | 2021-05-07 | 西安众民生物化工有限公司 | Bactericidal regulation agricultural fertilizer containing polyglutamic acid and chitosan |
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Application publication date: 20160720 |