CN108676722A - Improve the store method of nitragin survival rate - Google Patents
Improve the store method of nitragin survival rate Download PDFInfo
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- CN108676722A CN108676722A CN201810771619.0A CN201810771619A CN108676722A CN 108676722 A CN108676722 A CN 108676722A CN 201810771619 A CN201810771619 A CN 201810771619A CN 108676722 A CN108676722 A CN 108676722A
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- rhizobium
- drawing protein
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
Abstract
The present invention relates to biotechnologies.To improve the survival rate of rhizobium, the invention discloses a kind of store methods improving nitragin survival rate, include the following steps：Selection and breeding rhizobium strain：Rhizobium strain is obtained into the rhizobium strain of selection and breeding through culture and selection and breeding；The rhizobium strain of the selection and breeding is fermented；Adsorbent is used as after soybean wire-drawing protein is sterilized；Rhizobium strain after fermentation is added in the adsorbent, cool place preserves.The method of the present invention substantially increases the survival rate of rhizobium.
Description
Technical field
The present invention relates to biotechnologies, and in particular to a kind of store method improving nitragin survival rate.
Background technology
The present invention belongs to the relevant technologies related to the present invention for the description of background technology, be only used for explanation and just
In understanding present disclosure, 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.
Legume, in occupation of very important effect, has greatly economic and social value in China plant library.
As a kind of gram-negative brevibacterium, thalline can be formed rhizobium after entering the root system of legume with legume symbiosis
Root nodule.Legume has the advantages that nitrogen fixing capacity is strong, amount of nitrogen fixation is big, resistance is strong with rhizobium symbiosis, is 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, and the fertilizer application amount in production can be reduced after, is improved the yield and quality, it is right
Nuisance free crops produce and reduce peasant and throw people, and environmental protection 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.
Invention content
The purpose of the embodiment of the present invention is a kind of store method improving nitragin survival rate, and 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 achieved by the following technical solution：
An embodiment of the present invention provides a kind of store methods improving nitragin survival rate, include the following steps：
Selection and breeding rhizobium strain：Rhizobium strain is obtained into the rhizobium strain of selection and breeding through culture and selection and breeding；
The rhizobium strain of the selection and breeding is fermented；
It will be used as adsorbent after the purification of soybean wire-drawing protein, dry sterilization；
Rhizobium strain after fermentation is added in the adsorbent, cool place preserves.
Further, the culture includes the following steps：
It after rhizobium are activated, is inoculated in rhizobium YMA fluid nutrient mediums, is cultivated at 28 DEG C with shaking culture
Under the conditions of after culture to zymotic fluid cell density is 8,000,000,000/ml or more in 180rpm shaking tables, be placed in Cord blood in 4 DEG C of refrigerator
It is spare.
Further, the parameter that the fermentation uses for：
Above-mentioned activated spawn is inoculated into seed fermentation tank and carries out three grade fermemtation, the parameter setting of fermentation tank is rotating speed
220rpm, it is 28 DEG C that tank, which presses 0.06rpm, cultivation temperature, and in 7.0-7.4, fermentation is completed for the pH controls 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 is impregnated with pure water, is gone
Except impurity, grease and water-soluble substance, then soybean wire-drawing protein is obtained into the high soybean of purity by centrifugal separation method and is drawn
Silk-fibroin..
Further, the drying uses low temperature drying seasoning：By the soybean wire-drawing protein after the purification, throw
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 and is inoculated with 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 rotating speed 60rpm, spray flow 0.06m3/h.
Further, the preservation uses aseptic packaging, room temperature to preserve, and its step are as follows：It will be big after the inoculation
Beans wire-drawing protein rhizobium are transferred to aseptic packaging workshop, are vacuum-packed using automatic packaging machine, and room temperature preserves at the drying of cool place.
Further, the soybean wire-drawing protein forms honeycomb structure before it is dried into eleven punch 11 processing.
According to the above aspect of the present invention, the control device and method of display device of the present invention at least has the advantages that：
The present invention, as adsorbent, substantially increases the survival during rhizobium preserve by using soybean wire-drawing protein
Rate.
Description of the drawings
Fig. 1 is wire-drawing protein schematic diagram in the store method embodiment of the invention for improving nitragin survival rate；
Fig. 2 is carrier adsorption microbial inoculum ability column in the store method embodiment of the invention for improving nitragin survival rate
Figure；
Fig. 3 is the microbial inoculum survival rate on absorption carrier in the store method embodiment of the invention for improving nitragin survival rate
Block diagram；
Fig. 4 is bacterium number block diagram of surviving after being adsorbed in the store method embodiment of the invention for improving nitragin survival rate.
Specific implementation mode
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 and to cannot function as the limit of the scope of the present invention in order to which those skilled in the art are easier to understand technical scheme 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 present invention, can replace or merge between different embodiments
Combination, therefore the present invention is it is also contemplated that include all possible combinations of recorded identical and/or different embodiment.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 embodiment of the every other possible combination of one or more of B, C, D, although the embodiment may be in the following contents
In have specific literature record.
An embodiment of the present invention provides a kind of store methods improving nitragin survival rate, include the following steps：
Selection and breeding rhizobium strain：Rhizobium strain is obtained into the rhizobium strain of selection and breeding through culture and selection and breeding；
The rhizobium strain of the selection and breeding is fermented；
It will be used as adsorbent after the purification of soybean wire-drawing protein, dry sterilization；
Rhizobium strain after fermentation is added in the adsorbent, cool place preserves.
The embodiment of the present invention uses soybean wire-drawing protein for adsorbent, substantially increases the survival during rhizobium preserve
Rate, said program 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 zymotic fluid cell density is 8,000,000,000/ml or more in 180rpm shaking tables, it is spare to be placed in Cord blood 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, zymotic fluid cell density can be improved.
The present invention does not limit the specific method of culture, as long as can complete to rihizobium japonicum Spawn incubation.
In other embodiments of the present invention, the fermentation of rhizobium carries out three grade fermemtation using fermentation tank, fermentation tank
Parameter setting is rotating speed 220rpm, and it is 28 DEG C that tank, which presses 0.06rpm, cultivation temperature, and the pH of culture medium, which is controlled, in fermentation process exists
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 zymotic fluid
Bacteria containing amount, the growth of 28 DEG C of most suitable rhizobium.
In other embodiments of the present invention, the purification of soybean wire-drawing protein uses physical purification method：It 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 high soybean wire-drawing protein of purity.
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 does not limit the specific method of purification, as long as the purification to soybean wire-drawing protein can be completed.
In other embodiments of the present invention, the drying of soybean wire-drawing protein uses low temperature drying seasoning：It 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：Low temperature drying drying pair, 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 does not limit dry specific method, as long as the drying to soybean wire-drawing protein can be completed.
In other embodiments of the present 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 is thorough, is conveniently operated, at low cost.
The present invention does not limit the specific method of sterilizing, as long as the sterilizing to soybean wire-drawing protein can be completed.
The soybean wire-drawing protein into eleven punch 11 processing, forms honeycomb structure before it is dried, increases specific surface area.
To note here is that：Rhizobium activation can also use following special culture medium in addition to using YMA medium
It is activated：
The high-concentration culturing base of superelevation thalline quantity is prepared by formula as below：Beef extract 2g；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, is obtained after culture medium activation used in the embodiment of the present invention
Rhizobium are conducive to and the combination of soybean wire-drawing protein, using 28 DEG C of CMC models to a concentration of 8,000,000,000/ml in the method for the present invention
After preserve, 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 shows through numerous studies compared with the rhizobium of low concentration, the rhizobium of larger concentration help to improve
Survival rate when preservation, especially after preserving the long period, effect becomes apparent from.
The detailed process that each parameter determines in this programme is given below：
The selection and 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
Fat 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, item is cultivated at 28 DEG C with shaking culture
After culture to zymotic fluid cell density is 8,000,000,000/ml or more in 180rpm shaking tables under part, it is standby to be placed in Cord blood in 4 DEG C of refrigerator
With.
Living bacteria count assay 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 is counted | 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 bacterium colony strain：It crosses on above-mentioned YMA medium solid plate respectively, it is solid to prepare rhizobium strain
Body plate single bacterium colony strain.
2) seed activation:Eugonic single bacterium colony is respectively connected to above-mentioned Y MA liquid training on picking solid plate tablet
It supports in the base culture solution of 250ml (in 500ml triangular flasks fill), it is spare after shake culture in 28 DEG C, the shaking table of 180rmp.
3) strain fermentation:Each strain seed liquor of 48h will be cultivated, being respectively connected to above-mentioned YMA by 10% inoculum concentration cultivates
In the fermentation medium culture solution of based formulas, 28 DEG C, shake culture in 180rpm shaking tables.
4) measurement of rhizobial growth situation：By the strain being inoculated in the shaking flask of culture medium prescription 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 thalline under each time conditions in zymotic fluid and becomes
Change situation.
Best fermentation manufacturing technique parameter determines
Method：
1) YMA root nodule bacterium culture mediums are prepared, are 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 by 10% inoculum concentration in culture medium, carries out full-automatic fermentation training
It supports.
3) setting of technological parameter：The parameter setting of fermentation tank is rotating speed 200rpm, and 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 thalline in thalline fermentation process in zymotic fluid for incubation time just
It is a variable, and is a particularly important parameter, directly affects the growth and development of thalline in zymotic fluid.What is fermented is first
Phase, the cell density in zymotic fluid is smaller, and the consumption of the oxygen in fermentation tank is little, at this moment if being passed through a large amount of air,
Although dissolved oxygen improves, but thalline does not need to so high dissolved oxygen, wastes the energy and machinery equipment.With culture
The increase of time, thalli growth breeding progress into exponential phase, and the speed of growth of thalline is constantly accelerated, oxygen demand
It is continuously increased, at this moment if dissolved oxygen content is low, the growth of thalline will be influenced, influence product quality.We pass through in thalline
When growing into exponential phase, increase filtrated air intake into tank body to ventilating the 40% of total amount, and from pair of thalline
In number growth period, this level of ventilation is maintained always, until fermentation terminates, so that the content of dissolved oxygen maintains in zymotic fluid
In a higher level, meets the needs of thalli growth is to oxygen.
Fermentation results are analyzed：
Carbon source and nitrogen source are to constitute the material base of phage structure, are the important elements of constitutive protein matter and nucleic acid, simultaneously
Also main energy sources are provided for microorganism growth.Suitable nitrogen source and carbon source are selected, the growth and development and breeding of microorganism are conducive to.
Different nitrogen sources and carbon source influences the biological characters such as zymotic fluid cell density and thalli morphology very big.This patent passes through not
The quantity variation of rhizobium thalline, 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 addition of best wheat bran and beancake powder has reached and has promoted rhizobial growth
Purpose, to increase thalline quantity and metabolite in zymotic fluid.
The principal element for influencing micropopulation growth and breeding has the height of dissolved oxygen concentration in the ingredient of culture medium, zymotic fluid
In low, zymotic fluid value, fermentation process in the temperature of culture solution, culture medium solvable nutriment concentration etc..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 and influences 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 thalline fermentation process cultivates
Time is a variable, is fermented initial stage, although thalline is in the vigorous metabolism growth phase, because its total number is few, therefore oxygen demand
Also few.With the passage of incubation time, thalline breeding enters exponential phase, and bacterial number is increased with geometry step velocity at this time,
Thalline 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 zymotic fluid
Amount, to meet the needs of rhizobial growth development.We when studying zymotechnique, thalline culture initial stage by dissolved oxygen control
System is 20%, when thalli growth is to exponential phase, by increase into the filtrated air intake of tank body, is risen to
30%, then it is increased to 40%, and since the exponential phase of thalline, 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 is compared with processing
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 preserve the long period.The addition of preservative with microbial metabolic products product as main component on being influenced
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
The principle of convenient material drawing, 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 best 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：It takes above-mentioned adsorbent in polypropylene plastics pocket respectively, i.e., installs poly- the third of carrier by 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, rises to about 70 DEG C or so and gradually fall after rise again extremely
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, to allow the beneficial microbe in rihizobium japonicum fertilizer 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 thalline 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 bacterium period samples, and with Microscope examination combination viable plate counts, detects in different time periods under each sterilising conditions
Viable bacteria survival condition determines best 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 addition is 5ml, with
The bacterium solution amount that 100g carriers can be added is carrier adsorption amount.Survival rate of the rhizobium in different absorption carriers measures will absorption
The carrier placement of microbial inoculum preserves in the cool, and 10g samples are taken out after 48h and are 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 samples 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 soybean wire-drawing protein (Fig. 1) and the load microbial inoculum of current common carrier.We
Detection finds that the absorption microbial inoculum ability of shown 4 kinds of absorption carriers is followed successively by:Vermiculite>Soybean wire-drawing protein>Turf>Perlite (figure
2).Survival rate of the rhizobium thalline on 4 kinds of absorption carriers as shown in Figure 3 is followed successively by:Soybean wire-drawing protein>Vermiculite>Turf>It is precious
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 the fertilizer efficiency of bigger.
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 be 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 prepared by microbial inoculum
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 solid fungicide prepared by primary sorbent, after being placed in room temperature preservation 6 months, in microbial inoculum viable count still greater than 1 ×
109CFU/g。
Soybean wire-drawing protein is a kind of excellent agricultural resource, and material is abundant, of low 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, also higher stability can make rhizobium keep activity in the even longer time ranges of 180d, to
It ensure that the fertilizer efficiency and product quality of rhizobium fertilizer.
The production and absorption and detection of 4 zymotic fluids and packaging
Rhizobium fertilizer is to use wire-drawing protein as carrier, biological production made of being stirred 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% thalline 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 in product and mass propagation, 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 thalline：By rhizobium strain be linked into Fresh after high temperature and pressure moist heat sterilization it is cold
But in 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
Zymotic fluid 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 zymotic fluids of rhizobium adsorbed in 20% ratio, a certain amount of pH adjusting agent stirs evenly in mixing agitator, 30
It is dried to the range of moisture qualification under the conditions of DEG C.
4) the detection above method of living bacteria count carries out the production repeated 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 to be placed at shady and cool drying and is preserved, avoid direct sunlight and temperature excessively high.
Interpretation of result
We have carried out the production of rhizobium fertilizer according to above-mentioned production technology, sample and have been 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 experiment 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 the living bacteria count of product this index, rhizobium survival number 1,500,000,000/g of average out to, and
It can keep stablizing relatively for fertilizer humidity.
Microbial inoculum prepared by the method for the present invention is adsorbed using soybean wire-drawing protein, can be improved the survival rate of rhizobium and be deposited
Live time, and be controlled in the concentration of rhizobium, it can further coordinate 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 air-dried, the microbial inoculum survival period after air-drying is more
It is long.
In other embodiments of the present invention, place is crushed to soybean wire-drawing protein in nanometer level before absorption
Reason, makes it have big surface area, is conducive to improve adsorption rate, ensures dry effect, extend the survival rate of rhizobium.
In other embodiments of the present invention, before absorption in nanometer level to soybean wire-drawing protein at eleven punch 11
Reason, makes it have honeycomb structure, is conducive to inner ventilation, ensures 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, any made by 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 improving nitragin survival rate, which is characterized in that include the following steps：
Selection and breeding rhizobium strain：Rhizobium strain is obtained into the rhizobium strain of selection and breeding through culture and selection and breeding；
The rhizobium strain of the selection and breeding is fermented；
It will be used as adsorbent after the purification of soybean wire-drawing protein, dry sterilization；
Rhizobium strain after fermentation is added in the adsorbent, cool place preserves.
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 mediums, with shaking culture in 28 DEG C of condition of culture
After culture to zymotic fluid cell density is 8,000,000,000/ml or more in lower 180rpm shaking tables, it is standby to be placed in Cord blood 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 for：
Above-mentioned activated spawn is inoculated into seed fermentation tank and carries out three grade fermemtation, the parameter setting of fermentation tank is rotating speed 220rpm,
It is 28 DEG C that tank, which presses 0.06rpm, cultivation temperature, and the pH controls of culture medium are 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 carried using physics
Pure method：The soybean wire-drawing protein is impregnated, removal impurity, grease and the substance for being dissolved in water with pure water, then soybean is drawn
Silk-fibroin obtains the high soybean wire-drawing protein of purity by centrifugal separation method.
5. store method according to claim 1, which is characterized in that the drying uses low temperature drying seasoning：It will
Soybean wire-drawing protein after the purification puts into dryer, 60 DEG C of low temperature dryings, water content 2% after drying.Powder again after drying
It is broken into the molecule that fineness is 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 the absorption is adsorbed using sterile spray, step
It is rapid as follows：
Soybean wire-drawing protein after the sterilizing-drying is transferred to sterile spray and is inoculated with tank, the root nodule fermented liquid is led to
It crosses sterile pipes to link with inoculation tank, be inoculated into soybean wire-drawing protein with spraying agitating mode, inoculum concentration 20%, blender turns
Fast 60rpm, spray flow 0.06m3/h。
8. store method according to claim 1, which is characterized in that the preservation uses aseptic packaging, room temperature to preserve,
Its step are as follows：Soybean wire-drawing protein rhizobium after the inoculation are transferred to aseptic packaging workshop, using automatic packaging machine
It is vacuum-packed, room temperature preserves 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 is handled, and forms honeycomb structure.
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| CN111960883A (en) * | 2020-07-28 | 2020-11-20 | 沧州市农林科学院 | Special bacterial fertilizer for soybean varieties and preparation method thereof |
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