CN108676722B - Improve the store method of nitragin survival rate - Google Patents
Improve the store method of nitragin survival rate Download PDFInfo
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Abstract
The present invention relates to field of biotechnology.For the survival rate for improving rhizobium, the invention discloses a kind of store methods for improving nitragin survival rate, include the following steps: breeding rhizobium strain: rhizobium strain is obtained the rhizobium strain of breeding through culture and breeding；The rhizobium strain of the breeding is fermented；Adsorbent will be used as after the sterilizing of soybean wire-drawing protein；Rhizobium strain after fermentation is added in the adsorbent, cool place saves.The method of the present invention substantially increases the survival rate of rhizobium.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of store method for improving nitragin survival rate.
Background technique
The present invention belongs to the relevant technologies related to the present invention for the description of background technique, be only used for explanation and just
In understanding the contents of the present invention, should not be construed as applicant be specifically identified to or estimate applicant be considered the present invention be put forward for the first time
The prior art of the applying date of application.
Leguminous plant, in occupation of very important effect, has greatly economic and social value in China plant library.
As a kind of gram-negative brevibacterium, thallus can be formed rhizobium after entering the root system of leguminous plant with legume symbiosis
Root nodule.Leguminous plant and rhizobium symbiosis have the advantages that nitrogen fixing capacity is strong, amount of nitrogen fixation is big, resistance is strong, are in biological nitrogen fixation
Most efficient system, amount of nitrogen fixation account for about the 65% of biological nitrogen fixation total amount.Rhizobium fertilizer can make dross, carry out biological nitrogen fixation, no
But good application effect, and production cost is low, can be reduced the fertilizer application amount in production after application, improves the yield and quality, it is right
Nuisance free crops production and reduction peasant throw people, and protection environment etc. has a very important role.Existed for the first time from 1895
Since the rhizobium inoculant with " Nitragin " for trade name occurs in Germany, the former Soviet Union, Britain, the U.S. and France etc. are successive
It realizes the industrialized production of nitragin and is widely applied.Currently, developing China in the developed countries such as the U.S. and Brazil etc.
Family, Rhizobium Inoculation area generally all account for the 30%~50% of sown area, both improve soil, protect environment, and advantageous
In maintenance Nitrogen balance.The height of nitragin quality is always an outstanding problem of its application effect.
Summary of the invention
The purpose of the embodiment of the present invention is that a kind of store method for improving nitragin survival rate, the method for the present invention can be big
The big survival rate for improving rhizobium.
The purpose of the embodiment of the present invention is that be achieved through the following technical solutions:
The embodiment of the invention provides a kind of store methods for improving nitragin survival rate, include the following steps:
Breeding rhizobium strain: rhizobium strain is obtained into the rhizobium strain of breeding through culture and breeding；
The rhizobium strain of the breeding is fermented；
Adsorbent will be used as after the purification of soybean wire-drawing protein, dry sterilization；
Rhizobium strain after fermentation is added in the adsorbent, cool place saves.
Further, the culture includes the following steps:
It after rhizobium are activated, is inoculated in rhizobium YMA fluid nutrient medium, is cultivated with shaking culture at 28 DEG C
Under the conditions of after culture to fermentation liquid cell density is 8,000,000,000/ml or more in 180rpm shaking table, be placed in cryo-conservation in 4 DEG C of refrigerator
It is spare.
Further, the parameter that the fermentation uses are as follows:
Above-mentioned activated spawn is inoculated into seed fermentation tank and carries out three grade fermemtation, the parameter setting of fermentor is revolving speed
220rpm, tank press 0.06rpm, and cultivation temperature is 28 DEG C, and in 7.0-7.4, fermentation is completed for the pH control of culture medium in fermentation process
Every milliliter containing rhizobium 80-100 hundred million living.
Further, the purification uses physical purification method: the soybean wire-drawing protein being impregnated with pure water, is gone
It is drawn except impurity, grease and water-soluble substance, then by soybean wire-drawing protein by the soybean that centrifugal separation method obtains purity is high
Silk-fibroin.
Further, the drying uses low temperature drying seasoning: the soybean wire-drawing protein after the purification is thrown
Enter dryer, 60 DEG C of low temperature dryings, water content 2% after drying.The molecule that fineness is 70 mesh is ground into after drying again.
Further, the sterilizing of the soybean wire-drawing protein uses cobalt source radiation sterilization method, takes soybean wire-drawing protein point
Loaded in polypropylene plastics pocket, i.e., rolling the above-mentioned polypropylene plastics pocket folding for installing soybean wire-drawing protein can play very well
Sealing prevent miscellaneous bacteria effect, be placed in radiation sterilization in cobalt source room.
Further, the absorption is adsorbed using sterile spray, and its step are as follows:
Soybean wire-drawing protein after the sterilizing-drying is transferred to sterile spray inoculation tank, the rhizobium are fermented
Liquid is linked by sterile pipes and inoculation tank, is inoculated into soybean wire-drawing protein with spraying agitating mode, inoculum concentration 20%, is stirred
Device revolving speed 60rpm, spray flow 0.06m3/h.
Further, the preservation uses aseptic packaging, is stored at room temperature, its step are as follows: will be big after the inoculation
Beans wire-drawing protein rhizobium are transferred to aseptic packaging workshop, are vacuum-packed using automatic packaging machine, are stored at room temperature at the drying of cool place.
Further, the soybean wire-drawing protein carries out perforation processing before it is dried, forms honeycomb structure.
According to the above aspect of the present invention, the control device and method of the display device of that present invention at least has the following beneficial effects:
The present invention, as adsorbent, substantially increases the survival during rhizobium save by using soybean wire-drawing protein
Rate.
Detailed description of the invention
Fig. 1 is that the present invention improves wire-drawing protein schematic diagram in the store method embodiment of nitragin survival rate；
Fig. 2 is that the present invention improves carrier adsorption microbial inoculum ability column in the store method embodiment of nitragin survival rate
Figure；
Fig. 3 is the microbial inoculum survival rate in the store method embodiment of present invention raising nitragin survival rate on absorption carrier
Histogram；
Fig. 4 is that the present invention improves bacterium number histogram of surviving after absorption in the store method embodiment of nitragin survival rate.
Specific embodiment
The present invention is further discussed in detail with reference to the accompanying drawings and examples, it should be understood that attached drawing and implementation
Example is technical solution of the present invention to be easier to understand for those skilled in the art, and cannot function as the limit of the scope of the present invention
It is fixed.
In following introductions, term " first ", " second " only for descriptive purposes, and should not be understood as instruction or dark
Show relative importance.Following introductions provide multiple embodiments of the invention, can replace or merge between different embodiments
Combination, therefore the present invention is it is also contemplated that all possible combinations comprising documented identical and/or different embodiments.Thus, such as
Fruit one embodiment include feature A, B, C, another embodiment include feature B, D, then the present invention also should be regarded as include containing
A, the every other possible combined embodiment of one or more of B, C, D, although the embodiment may be in the following contents
In have specific literature record.
The embodiment of the invention provides a kind of store methods for improving nitragin survival rate, include the following steps:
Breeding rhizobium strain: rhizobium strain is obtained into the rhizobium strain of breeding through culture and breeding；
The rhizobium strain of the breeding is fermented；
Adsorbent will be used as after the purification of soybean wire-drawing protein, dry sterilization；
Rhizobium strain after fermentation is added in the adsorbent, cool place saves.
The embodiment of the present invention uses soybean wire-drawing protein for adsorbent, substantially increases the survival during rhizobium save
Rate, above scheme may be implemented the present invention improve rhizobium survival rate purpose, herein below on the basis of provide preferred side
Case:
In some embodiments of the invention, the culture is used with shaking culture under 28 DEG C of condition of culture
After culture to fermentation liquid cell density is 8,000,000,000/ml or more in 180rpm shaking table, it is spare to be placed in cryo-conservation in 4 DEG C of refrigerator
To note here is that: 28 DEG C be rhizobium optimum culturing temperature, shaking culture controllability is strong, condition of culture
Stablize, fermentation liquid cell density can be improved.
The present invention to the specific method of culture without limitation, as long as can complete to rihizobium japonicum Spawn incubation.
In other embodiments of the invention, the fermentation of rhizobium carries out three grade fermemtation using fermentor, fermentor
Parameter setting is revolving speed 220rpm, and tank presses 0.06rpm, and cultivation temperature is 28 DEG C, and the pH control of culture medium exists in fermentation process
7.0-7.4, fermentation complete every milliliter containing rhizobium 80-100 hundred million living.
To note here is that: three grade fermemtation can effectively reduce the probability that miscellaneous bacteria is infected in fermentation process, improve fermentation liquid
Bacteria containing amount, the growth of 28 DEG C of most suitable rhizobium.
In other embodiments of the invention, the purification of soybean wire-drawing protein uses physical purification method: will be described big
Beans wire-drawing protein is impregnated with pure water to be filtered, removal impurity, grease and water-soluble substance, then by soybean wire-drawing protein by from
Heart separation method obtains the soybean wire-drawing protein of purity is high.
To note here is that: soybean wire-drawing protein is not soluble in water, can remove with pure water immersion filtering and is dissolved in the miscellaneous of water
Matter, grease and other substances and will not with soybean wire-drawing protein generate chemical reaction, soybean wire-drawing protein by pure water immersion, meeting
What is become is looser, is beneficial to the preservation of strain.
The present invention to the specific method of purification without limitation, as long as the purification to soybean wire-drawing protein can be completed.
In other embodiments of the invention, the drying of soybean wire-drawing protein uses low temperature drying seasoning: will be described
Purification after soybean wire-drawing protein, put into dryer, 60 DEG C of low temperature dryings, it is dry after water content 2%.It is ground into again after drying
Fineness is the molecule of 70 mesh.
To note here is that: dry pair of low temperature drying, very small to the qualitative effects of soybean wire-drawing protein, drying is thorough
Bottom, water content can≤2%, by soybean wire-drawing protein be ground into fineness be 70 mesh little particle, the surface area of adsorbent can be increased,
Increase porosity, improves the adsorbance of unit adsorbent.
The present invention to dry specific method without limitation, as long as the drying to soybean wire-drawing protein can be completed.
In other embodiments of the invention, the sterilizing of soybean wire-drawing protein uses cobalt source radiation sterilization method, takes soybean
Wire-drawing protein is sub-packed in polypropylene plastics pocket, i.e., rolling the above-mentioned polypropylene plastics pocket folding for installing soybean wire-drawing protein can
Miscellaneous bacteria effect is prevented to play sealing well, is placed in radiation sterilization in cobalt source room.
To note here is that: the sterilizing of cobalt source irradiation thoroughly, facilitates operation, at low cost.
The present invention to the specific method of sterilizing without limitation, as long as the sterilizing to soybean wire-drawing protein can be completed.
The soybean wire-drawing protein carries out perforation processing before it is dried, forms honeycomb structure, increases specific surface area.
To note here is that: rhizobium activation, can also be using following special culture medium in addition to using YMA medium
It is activated:
The high-concentration culturing base of superelevation thalline quantity: beef extract 2g is prepared by formula as below；Peptone 90g；Capacitive forms sediment
Powder 66g；Glucose 50g；Sucrose 5g；Agar 10g；NaCl 9g；KNO33g；Distilled water 1000ml.
The content of peptone is very high in activation medium used in the embodiment of the present invention, has accounted for 7.29%, gold therein
Belong to ion and only have two kinds of strong base ions of sodium and potassium, osmotic effect is good, obtains after culture medium activation used in the embodiment of the present invention
Rhizobium are conducive to and the combination of soybean wire-drawing protein, use 28 DEG C of CMC models to concentration for 8,000,000,000/ml in the method for the present invention
After save, it is ensured that effective rhizobium quantity under Unit Weight or volume conditions in obtained product after the absorption of soybean wire-drawing protein
Increase, and applicant warp a large number of studies show that, compared with the rhizobium of low concentration, the rhizobium of larger concentration are helped to improve
Survival rate when preservation, especially after saving the long period, effect is become apparent from.
The detailed process that each parameter determines in this programme is given below:
The breeding of 1 rhizobium strain:
Bacterial strain: rhizobium strain
Culture medium: YMA medium
Drug and equipment:
Drug: mannitol, sucrose, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, yeast powder, calcium sulfate boric acid, sodium molybdate.Fine jade
Rouge is produced by three factory of Tianjin chemical reagent.
Required equipment is as shown in table 1:
Table 1
Method:
It after rhizobium are activated, is inoculated in rhizobium fluid nutrient medium, with shaking culture in 28 DEG C of culture items
After culture to fermentation liquid cell density is 8,000,000,000/ml or more in 180rpm shaking table under part, it is standby to be placed in cryo-conservation in 4 DEG C of refrigerator
With.
Living bacteria count measuring method
Blood cell plate counting method combination plating dilutions viable bacteria counting method calculates rhizobium quantity, Gram's staining, normal dyeing
Carry out rhizobial growth condition detection and qualitative analysis.
2 rhizobium fermentation manufacturing techniques
Instrument and equipment is as shown in table 2
Table 2
| Title | Model | The place of production |
| Steam generator | YN18-0.7-D | Shanghai |
| Biochemical cultivation case | MJP-150 | Beijing Road is uncommon |
| Isothermal vibration incubator | ZD-85 | Granary |
| Microscope | XSP-44X.9 | Shanghai |
| PH meter | EC.TDS | Tianjin |
| Superclean bench | DL-CJ-2ND | Beijing |
| Refrigerator | SC-287NE | Beijing |
| Pressure cooker | LDZF-50KB | Shanghai |
| Automatic fermenter | HND-BJ-50L-500L | Shanghai |
| Vacuum sealer | V1 | Jiangsu |
Method:
1) preparation of single colonie strain: crossing on above-mentioned YMA medium solid plate respectively, and preparation rhizobium strain is solid
Body plate single colonie strain.
2) seed activation: eugonic single colonie is respectively connected to above-mentioned Y MA liquid training on picking solid plate plate
It supports in base the culture solution of 250ml (in 500ml triangular flask fill), it is spare after shake culture in 28 DEG C, the shaking table of 180rmp.
3) strain fermentation: will cultivate each strain seed liquor of 48h, is respectively connected to above-mentioned YMA by 10% inoculum concentration and cultivates
In the fermentation medium culture solution of based formulas, 28 DEG C, shake culture in 180rpm shaking table.
4) strain in the shaking flask of culture medium prescription will the measurement of rhizobial growth situation: be inoculated in 28 DEG C, 180rpm
Under the conditions of shaking table in cultivate, sampled respectively in incubation time section, dyeing microscopic examination detects its thalli growth situation, use blood count
Plate microscopy counting method combination viable plate count method measures cell density and its thallus under each time conditions in fermentation liquid and becomes
Change situation.
Best fermentation manufacturing technique parameter determines
Method:
1) YMA root nodule bacterium culture medium is prepared, is added in full-automatic three grade fermemtation tank, high temperature and pressure is gone out under the conditions of 121 DEG C
Bacterium 30min, culture medium be cooled to 30 DEG C it is spare.
2) the rhizobium seed liquor prepared is linked into culture medium by 10% inoculum concentration, carries out full-automatic fermentation training
It supports.
3) technological parameter is set: for the parameter setting of fermentor as revolving speed 200rpm, tank presses 0.06rpm, and cultivation temperature is
28 DEG C, the pH value of culture medium is controlled in 7.0-7.4 in fermentation process.When the culture medium in fermentation process, pH value, temperature, tank pressure
Equal technical parameters it is preferable to determine rear, the oxygen demand of the thallus in thallus fermentation process in fermentation liquid for incubation time just
It is a variable, and is a particularly important parameter, directly affects the growth and development of thallus in fermentation liquid.What is fermented is first
Phase, the cell density in fermentation liquid is smaller, and the consumption of the oxygen in fermentor is little, at this moment if being passed through a large amount of air,
Although dissolved oxygen improves, but thallus does not need so high dissolved oxygen, wastes the energy and machinery equipment.With culture
The increase of time, thalli growth breeding progress into logarithmic growth phase, and the speed of growth of thallus is constantly accelerated, oxygen demand
It is continuously increased, at this moment if dissolved oxygen content is low, just will affect the growth of thallus, influence product quality.We pass through in thallus
When growing into logarithmic growth phase, increases and enter the filtrated air intake of tank body to ventilating the 40% of total amount, and from pair of thallus
In number growth period, this level of ventilation is maintained always, until fermentation terminates, so that the content of dissolved oxygen maintains in fermentation liquid
In a higher level, meets the needs of thalli growth is to oxygen.
Fermentation results analysis:
Carbon source and nitrogen source are the material bases for constituting phage structure, are the important elements of constitutive protein matter and nucleic acid, simultaneously
Also main energy sources are provided for microorganism growth.Selection suitable nitrogen source and carbon source, are conducive to the growth and development and breeding of microorganism.
Different nitrogen sources and carbon source influences the biological characters such as fermentation liquid cell density and thalli morphology very big.This patent passes through not
The quantity variation of rhizobium thallus, establishes the optimal medium of rhizobium, on this basis to culture medium in same culture medium
In be added to suitable carbon-nitrogen ratio ingredient, i.e., the additional amount of optimal wheat bran and beancake powder has reached and has promoted rhizobial growth
Purpose, to increase thalline quantity and metabolite in fermentation liquid.
Influence micropopulation growth and breeding principal element have the ingredient of culture medium, in fermentation liquid dissolved oxygen concentration height
The temperature of culture solution in low, fermentation liquid value, fermentation process, concentration of solvable nutriment etc. in culture medium.When culture medium at
Point and the optimized determination of technical parameters such as value, temperature after, the height of dissolved oxygen concentration then becomes influence carefully in fluid nutrient medium
An important factor for quality and quantity of bacterium growth and development, metabolisming way and product.Oxygen demand is opposite in thallus fermentation process cultivates
Time is a variable, is fermented initial stage, although thallus is in the vigorous metabolism growth phase, because its total number is few, therefore oxygen demand
Also few.With the passage of incubation time, thallus breeding enters logarithmic growth phase, and bacterial number is increased at this time with geometry step velocity,
Thallus is in high-speed rapid growth animated period, and oxygen demand is continuously increased, and at this moment will correspondingly increase containing for the dissolved oxygen in fermentation liquid
Amount, to meet the needs of rhizobial growth development.We when studying zymotechnique, thallus culture initial stage by dissolved oxygen control
System when thalli growth arrives logarithmic growth phase, by increasing the filtrated air intake into tank body, is risen to 20%
30%, then it is increased to 40%, and since the logarithmic growth phase of thallus, maintaining ventilatory capacity 40% always, this is horizontal, until hair
Ferment terminates, and obtains the Rhizobium Inoculant that cell density is 80,000,000,000/ml or more.
The source of 3 raw materials and processing compare
For the absorption carrier of examination
Careless carbon: Heilongjiang Province's Qitaihe City, rich reserves；
Perlite: Xinyang City, Henan Province perlite factory；
Vermiculite: Nanyang City, Henan Province Guang Yi mineral products Co., Ltd；
Soy meal: Anyang get Tian Li Food Co., Ltd.
The sterilizing of adsorbent
Microbial bacterial agent is broadly divided into liquid bacterial agent, solid fungicide two types.Liquid bacterial agent is easy putrid and deteriorated, it is necessary to
Addition preservative could save the long period.The addition of preservative is influenced on microbial metabolic products product as main component
It is smaller, but to being the product of quality standard because strong acid is to the lethal effect and inhibiting effect of microorganism using living bacteria count, seriously
Influence the bioactivity of microorganism.At home and abroad, to the probiotics based on effective bacterium number, multiselect is inhaled with solid material
Attached mode makes liquid be changed into solid, i.e., is adsorbed onto bacteria suspension on certain sterilant carriers, solid fertilizer is made.Carrier
Type it is very much, mainly have careless carbon, frog stone, lignite, perlite, rice husk, in swelling and talcum etc..We according to cheap and
It draws materials convenient principle, high spot reviews turf, perlite, four kinds of carriers of vermiculite and soybean wire-drawing protein, using pasteurization
Three kinds of method, high temperature and pressure moist hear heat test and a ray sterilizing method methods, sterilize to above-mentioned carrier, determine optimal load
Body and sterilizing methods.
Instrument and equipment is as shown in table 3
Table 3
| Title | Model | The place of production |
| Isothermal vibration incubator | ZD-85 | Granary |
| Superclean bench | DL-CJ-2ND | Beijing |
| Pressure cooker | LDZF-50KB | Shanghai |
| Ray cobalt source | —— | Beijing |
Sterilizing methods
1) pasteurization: taking above-mentioned adsorbent in polypropylene plastics pocket respectively, i.e., installs poly- the third of carrier for above-mentioned
The folding of alkene plastics bag, which is rolled to play to seal well, prevents miscellaneous bacteria effect, is inserted among the bag with fertilizer of sterilizing, uses Pasteur
Sterilization sterilizes.Specific sterilising conditions and method are that every cubic metre of a pile is made in heap under natural conditions, and outsourcing polybag is protected
Wet covering woven straw heat preservation, artificial heating when necessary, stacking time is to bank up 7d, 14d, 21d respectively, is observed and recorded therebetween each
The temperature change of test group pays attention to observing whether temperature gradually mildly increases from room temperature, rise to about 70 DEG C or so and gradually fall after rise to
Room temperature.
2) high temperature and pressure moist hear heat test takes four kinds of above-mentioned adsorbents to be sub-packed in polypropylene plastics pocket respectively, i.e., will be upper
State install carrier polypropylene plastics pocket folding roll can play well sealing prevent miscellaneous bacteria effect, it is wet to be placed in Large-scale High-Pressure
In heat sterilization pot, 30min is sterilized separately under the conditions of 121 DEG C.
3) ray sterilizing: take four kinds of above-mentioned adsorbents in polypropylene plastics pocket respectively, i.e., by above-mentioned dress
The polypropylene plastics pocket folding of good vector, which is rolled to play to seal well, prevents miscellaneous bacteria effect, sets ray brill
Radiation sterilization in source chamber.
Sterilizing test
If containing miscellaneous bacteria in absorption carrier selected by rhizobium microbial bacterial agent, the matter of rhizobium will be directly affected
Amount.Therefore it sterilizes to absorption carrier, so that the beneficial microbe in rihizobium japonicum fertilizer be allow to inhale to the maximum extent
It is attached on carrier and is colonized and breeds, competitive inhibitory effect of original miscellaneous bacteria to the thallus in fertilizer in reduction absorption carrier.
We study the sterilizing methods and sterilising conditions of absorption carrier, use three kinds of sterilizing methods, go out respectively in difference
The sampling of bacterium period is detected in different time periods under every kind of sterilising conditions with Microscope examination combination viable plate counts
Viable bacteria survival condition determines optimal sterilizing methods and sterilising conditions.
Adsorbent selection is compared
Method
1) adsorbance detection of the rhizobium in careless carbon, perlite, vermiculite, soybean wire-drawing protein difference absorption carrier is super
Root nodule bacteria culture fluid is added in batches in the absorption carrier of above-mentioned four kinds of sterilizings in net workbench, each additional amount is 5ml, with
The bacterium solution amount that 100g carrier can be added is carrier adsorption amount.Survival rate measurement of the rhizobium in different absorption carriers will absorption
The carrier placement of microbial inoculum saves in the cool, and 10g sample is taken out after 48h and is added in the sterile physiological saline of 100mL,
After cultivating 1h in shaking table under 200r/min, the viable count in liquid is detected.
2) Stability Determination of the rhizobium in careless carbon, perlite, vermiculite, wire-drawing protein difference absorption carrier will be placed on
Microbial inoculum at room temperature takes l0g sample to be added in 100mL physiological saline respectively in 2d, 12d, 21d, 45d and 120d,
After cultivating 1h under 200r/min, the viable count discharged in different time sections carrier is detected.
As a result with analysis
Adsorption rate and survival rate of the rhizobium in different absorption carriers
Compare the ability and microbial inoculum survival rate of the load microbial inoculum of soybean wire-drawing protein (Fig. 1) and current common carrier.We
Detection finds the absorption microbial inoculum ability of shown 4 kinds of absorption carriers successively are as follows: vermiculite > soybean wire-drawing protein > turf > perlite (figure
2).Survival rate of the rhizobium thallus as shown in Figure 3 on 4 kinds of absorption carriers is successively are as follows: soybean wire-drawing protein > vermiculite > turf > treasure
Zhu Yan；Although the adsorption rate of soybean wire-drawing protein slightly below vermiculite, the survival rate of rhizobium on it reaches 95.4%.?
Survival rate of the rhizobium on absorption carrier when absorption carrier is selected to should be used as key index, so that it is more to survive in fertilizer
Rhizobium, play bigger fertilizer efficiency.
Stability of the rhizobium in different absorption carriers
Every milliliter of viable count is 2,500,000,000 in root nodule bacterium solution used, and soybean wire-drawing protein, vermiculite, turf, perlite are inhaled
Attached living bacteria count is respectively 5.65 hundred million/g, 2.85 hundred million/g, 2.12 hundred million/g, 1.85 hundred million/g (Fig. 4), with the holding time
Increase and in different carriers is in reduced trend.The rhizobium survival number put in upper wire-drawing protein in different times respectively is bright
It is aobvious to be higher than other adsorbents.We have discovered that in 4 kinds of absorption carriers, powder is best absorption carrier, followed by vermiculite, good
In turf and perlite.
Conclusion
The preservation of microbial inoculum is extremely important for microbial inoculum production.The length of shelf-life is directly related to microbial inoculum
Transport and using effect, and then it is related to the profit and loss of manufacturer.Therefore, good method for preserving is one after microbial inoculum preparation
The important work of item.For the preservation of microbial inoculum, solid fungicide mainly takes preservation under room temperature method, we have discovered that utilizing soybean
Wire-drawing protein is the solid fungicide of primary sorbent preparation, after being placed in room temperature preservation 6 months, in microbial inoculum viable count be still greater than 1 ×
109CFU/g。
Soybean wire-drawing protein is a kind of excellent agricultural resource, and material is abundant, low in cost, when selecting absorption carrier,
It is contemplated that selecting the absorption carrier as root nodule mushroom fertilizer.Rhizobium have in soybean wire-drawing protein preferable adsorption capacity and
Survival ability, there are also higher stability, rhizobium can be made to keep activity in 180d even longer time range, thus
It ensure that the fertilizer efficiency and product quality of rhizobium fertilizer.
The production and absorption and detection of 4 fermentation liquids and packaging
Rhizobium fertilizer is to use wire-drawing protein as carrier, biological production made of stirring after adsorbing Rhizobium Inoculant and being dry
Product.The biological characteristics of rhizobium are that heat-resisting quantity is poor, and general growth and survival temperature are 15-40 DEG C, when temperature reaches 60 DEG C
When above, 80% thallus will be dead, and temperature is higher, and death rate is faster, and The dead quantity is more.Therefore, rhizobium fertilizer
It produces, requires temperature in production process that must be strict controlled in 50 DEG C or less always in technique, be just effective to ensure that finished product fertilizers
Rhizobium living bacteria count in material is in higher level.In addition, to prevent the living contaminants and mass propagation in product, it should
It sterilizes to carrier, to kill the miscellaneous bacteria in carrier as much as possible.On the basis of this patent will be studied in front, further grind
Study carefully the production technology and its quality control standard of rhizobium fertilizer.
Method:
1) fermented and cultured of thallus: by rhizobium strain be linked into Fresh after high temperature and pressure moist heat sterilization it is cold
But into 30 DEG C of liquid YMA medium, fermented and cultured is carried out according to the microbial inoculum processing parameter formulated above, is turned out
Fermentation liquid is sub-packed in the microbial inoculum plastic barrel of 25L, spare.
2) the sterilization process condition that the sterilizing of carrier is studied and defined according to this patent, the plastics that soybean powder carrier is loaded on are compiled
It knits in bag, is transported into sterilizing chamber, sterilize, obtain meeting rhizobia fertilizer material manufacturing technique, suction by thoroughly sterilizing
Appendix body.
3) production method of rhizobium fertilizer takes adsorbent soybean wire-drawing protein, 8,000,000,000/ml or more of living bacteria count respectively
The fermentation liquids of rhizobium adsorbed in 20% ratio, a certain amount of pH adjusting agent stirs evenly in mixing agitator, 30
It dries under the conditions of DEG C to the range of moisture qualification.
4) the detection above method of living bacteria count carries out duplicate production three times in fertilizer, from the production of duplication of production three times
It is separately sampled in product, 1kg is taken every time, after mixing by three samples, weighs 1kg, by microorganism dilution plate counting method knot
Microscope examination routine operation is closed, living bacteria count is detected.
5) product of production is vacuumized postposition to save in a cool and dry place, avoids direct sunlight and temperature excessively high.
Interpretation of result
We have carried out the production of rhizobium fertilizer according to above-mentioned production technology, and sampling has carried out in fertilizer after production
The assay of living bacteria count.Analysis method is blood counting chamber combination viable plate count method, measures the root nodule in fertilizer
Bacterium living bacteria count amount and pH value are respectively 1,500,000,000/g and 6.8.
The rhizobium fermenting agent living bacteria count prepared in test is 8,000,000,000/g, in the ratio of manufacturing technique requirent 20%
Microbial inoculum is added, after being uniformly mixed with absorption carrier mixture, living bacteria count is 1,500,000,000/g, and process is given birth in triplicate respectively
Postpartum carries out product quality detection, in this index of the living bacteria count of product, rhizobium survival number 1,500,000,000/g of average out to, and
It is able to maintain the relatively stable of fertilizer humidity.
The microbial inoculum of the method for the present invention preparation, is adsorbed using soybean wire-drawing protein, and the survival rate of rhizobium can be improved and deposit
Live time, and be controlled in the concentration of rhizobium, it can further cooperate with soybean wire-drawing protein and improve survival rate, and
And in the microbial inoculum of preparation rhizobium Average Survival number it is very high, substantially increase the efficiency of microbial inoculum.
In some embodiments of the invention, the microbial inoculum of preparation can be carried out it is air-dried, air-dry after microbial inoculum survival period more
It is long.
In other embodiments of the invention, soybean wire-drawing protein is carried out at crushing in nanometer level before absorption
Reason, makes it have big surface area, is conducive to improve adsorption rate, guarantees dry effect, extend the survival rate of rhizobium.
In other embodiments of the invention, soybean wire-drawing protein is carried out at perforation in nanometer level before absorption
Reason, makes it have honeycomb structure, is conducive to inner ventilation, guarantees dry effect, extends the survival rate of rhizobium.
It is described above to be merely a preferred embodiment of the present invention, it is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of store method for improving nitragin survival rate, which comprises the steps of:
Breeding rhizobium strain: rhizobium strain is obtained into the rhizobium strain of breeding through culture and breeding；
The rhizobium strain of the breeding is fermented；
Adsorbent will be used as after the purification of soybean wire-drawing protein, dry sterilization；
Rhizobium strain after fermentation is added in the adsorbent with spraying agitating mode, cool place saves.
2. store method according to claim 1, which is characterized in that the culture includes the following steps:
It after rhizobium are activated, is inoculated in rhizobium YMA fluid nutrient medium, with shaking culture in 28 DEG C of condition of culture
After culture to fermentation liquid cell density is 8,000,000,000/ml or more in lower 180rpm shaking table, it is standby to be placed in cryo-conservation in 4 DEG C of refrigerator
With.
3. store method according to claim 1, which is characterized in that the parameter that the fermentation uses are as follows:
Above-mentioned activated spawn is inoculated into seed fermentation tank and carries out three grade fermemtation, the parameter setting of fermentor is revolving speed 220rpm,
Tank presses 0.06rpm, and cultivation temperature is 28 DEG C, and the pH control of culture medium is in 7.0-7.4 in fermentation process, every milliliter when fermentation is completed
Containing rhizobium 80-100 hundred million living.
4. store method according to claim 1, which is characterized in that the soybean wire-drawing protein purification is mentioned using physics
Pure method: the soybean wire-drawing protein is impregnated with pure water, removal impurity, grease and the substance for being dissolved in water, then soybean is drawn
Silk-fibroin obtains the soybean wire-drawing protein of purity is high by centrifugal separation method.
5. store method according to claim 1, which is characterized in that the drying uses low temperature drying seasoning: will
Soybean wire-drawing protein after purification, puts into dryer, 60 DEG C of low temperature dryings, and water content 2% after drying is ground into thin again after dry
Degree is the molecule of 70 mesh.
6. store method according to claim 1, which is characterized in that the sterilizing of the soybean wire-drawing protein is shone using cobalt source
Sterilization is penetrated, soybean wire-drawing protein is taken to be sub-packed in polypropylene plastics pocket, i.e., by the above-mentioned polypropylene for installing soybean wire-drawing protein
Polybag folding, which is rolled to play to seal well, prevents miscellaneous bacteria effect, is placed in radiation sterilization in cobalt source room.
7. store method according to claim 1, which is characterized in that absorption is adsorbed using sterile spray, and its step are as follows:
Soybean wire-drawing protein after sterilizing-drying is transferred to sterile spray inoculation tank, root nodule fermented liquid is passed through into sterile pipes
It links, is inoculated into soybean wire-drawing protein with spraying agitating mode, inoculum concentration 20%, agitator speed 60rpm with inoculation tank, sprayed
Drench flow 0.06m3/h。
8. store method according to claim 1, which is characterized in that the preservation uses aseptic packaging, is stored at room temperature,
Its step are as follows: the soybean wire-drawing protein rhizobium after inoculation being transferred to aseptic packaging workshop, using automatic packaging machine vacuum
It packs, is stored at room temperature at the drying of cool place.
9. store method according to claim 1, which is characterized in that the soybean wire-drawing protein is worn before it is dried
Hole processing, forms honeycomb structure.
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