CN104099278B - A kind of method cultivating high yield Paenibacillus polymyxa - Google Patents
A kind of method cultivating high yield Paenibacillus polymyxa Download PDFInfo
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Abstract
The invention discloses a kind of method cultivating high yield Paenibacillus polymyxa, comprise the following steps: (1) weighs following raw material blending by weight: rice husk 10-15, Semen Maydis powder 30-40, Rhizoma Solani tuber osi 10-20, tea grounds cake 5-10, water 10-15, dipotassium hydrogen phosphate 0.1-0.2, manganese sulfate 0.18-0.22；(2) by after being standing and soak for 3.5-4.5 hour after mixing, pack autoclaving 40-50 minute, accesses liquid seeds bacterium solution after temperature drops to 38-42 DEG C, inoculum concentration 9-11%, after stirring and evenly mixing, 36-38 DEG C after fermentation culture 70-74 hour, stopping fermentation, period timing sampling checks, to obtain final product.
Description
Technical field
The present invention relates to culture medium field, be exactly a kind of method cultivating high yield Paenibacillus polymyxa.
Background technology
Current Paenibacillus polymyxa many employings liquid submerged fermentation technology, produce then through spray drying, equipment requirements is high, complex manufacturing, and the raw material that solid fermentation adopts is usually cheap agricultural byproducts (such as grass meal, wheat bran etc.), also relatively liquid fermentation is simple for the equipment adopted, and production cost is significantly less than liquid fermentation.So each manufacturer is all trying to explore the solid-fermented technique of bacillus cereus in recent years, and minority manufacturer is had to start exploratory production.It is found by experiment that rice sheath blight disease, Oil Tea Anthracnose etc. are had good effect by Paenibacillus polymyxa, carry out Paenibacillus polymyxa different component culture medium solid fermentation based on this and produce the test of bacterium amount, in the hope of simplifying production technology, improve yield, reduce production cost.
Summary of the invention
It is an object of the invention to provide a kind of method cultivating high yield Paenibacillus polymyxa.
Above-mentioned purpose is realized by below scheme:
A kind of method cultivating high yield Paenibacillus polymyxa, it is characterised in that: comprise the following steps:
(1) following raw material blending is weighed by weight: rice husk 10-15, Semen Maydis powder 30-40, Rhizoma Solani tuber osi 10-20, tea grounds cake 5-10, water 10-15, dipotassium hydrogen phosphate 0.1-0.2, manganese sulfate 0.18-0.22；
(2) by after being standing and soak for 3.5-4.5 hour after mixing, pack autoclaving 40-50 minute, accesses liquid seeds bacterium solution after temperature drops to 38-42 DEG C, inoculum concentration 9-11%, after stirring and evenly mixing, 36-38 DEG C after fermentation culture 70-74 hour, stopping fermentation, period timing sampling checks, to obtain final product
A kind of described method cultivating high yield Paenibacillus polymyxa, it is characterised in that: after adopting spore Gram’s staining, basis of microscopic observation calculates spore rate.
The invention have the benefit that the present invention passes through to optimize the dispensing combination of Paenibacillus polymyxa solid fermentation culture medium, reach to improve the effect of yield, tea grounds cake can play in fermentation culture and promotes bacterium amount to increase and improve the effect of spore rate,
Detailed description of the invention
Embodiment 1, a kind of method cultivating high yield Paenibacillus polymyxa, comprise the following steps:
(1) by weight (kg) ratio weighs following raw material blending: rice husk 15, Semen Maydis powder 30, Rhizoma Solani tuber osi 20, tea grounds cake 7, water 12, dipotassium hydrogen phosphate 0.2, manganese sulfate 0.2；
(2) by after being standing and soak for 4 hours after mixing, pack autoclaving 50 minutes, after temperature drops to 40 DEG C, access liquid seeds bacterium solution, inoculum concentration 10%, after stirring and evenly mixing, 37 DEG C of fermentation culture, after 74 hours, stop fermentation, and period timing sampling checks, to obtain final product
Count plate: adopting mixing plate viable bacteria counting method, make variable concentrations diluent by 10 times of dilution methods, draw 0.1mL respectively and be placed in sterile petri dish from 3 suitable diluents, each dilution factor makees three plates.Pour about 12mL into by melting in advance and being cooled in each culture dish of solid culture basal orientation of about 50 DEG C, shake up, after to be solidified, put into 37 DEG C of incubators are inverted and cultivate 24h, carry out colony count.
After adopting spore Gram’s staining, basis of microscopic observation calculates spore rate.
26,300,000,000 g of viable count-1, spore rate reach 92%.
Embodiment 2,
A kind of method cultivating high yield Paenibacillus polymyxa, comprises the following steps:
(1) by weight (kg) ratio weighs following raw material blending: rice husk 15, Semen Maydis powder 30, Rhizoma Solani tuber osi 20, tea grounds cake 7, water 12, dipotassium hydrogen phosphate 0.2, manganese sulfate 0.2, citric acid 0.1, the bagasse 0.1 after drying, sodium chloride 0.2, Fumaric acid 0.1, tapioca starch 0.2；
(2) by after being standing and soak for 4 hours after mixing, pack autoclaving 50 minutes, after temperature drops to 40 DEG C, access liquid seeds bacterium solution, inoculum concentration 10%, after stirring and evenly mixing, 37 DEG C of fermentation culture, after 74 hours, stop fermentation, and period timing sampling checks, to obtain final product
Count plate: adopting mixing plate viable bacteria counting method, make variable concentrations diluent by 10 times of dilution methods, draw 0.1mL respectively and be placed in sterile petri dish from 3 suitable diluents, each dilution factor makees three plates.Pour about 12mL into by melting in advance and being cooled in each culture dish of solid culture basal orientation of about 50 DEG C, shake up, after to be solidified, put into 37 DEG C of incubators are inverted and cultivate 24h, carry out colony count.
After adopting spore Gram’s staining, basis of microscopic observation calculates spore rate.
27,300,000,000 g of viable count-1, spore rate reach 92%.The additive such as citric acid, tapioca starch can promote the growth of strain immediate stability more, and spore rate is high.
Comparative example:
One, materials and methods
1. material
Strain is that Paenibacillus polymyxa (Paenibacilluspolymyxa) is separated by this test chamber soil, and preserves.
Liquid seed culture medium: Rhizoma Solani tuber osi 20%, glucose 2%, NaCl0.5%, K2HPO41%.
Solid fermentation culture medium raw material and reagent: wheat bran, rice husk, Semen Maydis powder, Rhizoma Solani tuber osi, tea grounds cake (oil tea side-product), dipotassium hydrogen phosphate, manganese sulfate etc., concrete formula is in Table 1.
Capital equipment: steam sterilizer, superclean bench, temperature control shaking table, temperature control biochemical cultivation case, balance, microscope.
2. method
(1) treatment formulations is in Table one: often process 4 repetitions.
One 9 kinds of solid fermentation nutrient media componentses of table and content
(2) method
After processing and each component being added to the water immersion 4 hours, pack autoclaving 45 minutes, after temperature drops to about 40 DEG C, access liquid seeds bacterium solution, inoculum concentration 10%, after stirring and evenly mixing, fermentation culture.37 DEG C fermentation 72 hours after, stop fermentation, period timing sampling check.
Count plate: adopting mixing plate viable bacteria counting method, make variable concentrations diluent by 10 times of dilution methods, draw 0.1mL respectively and be placed in sterile petri dish from 3 suitable diluents, each dilution factor makees three plates.Pour about 12mL into by melting in advance and being cooled in each culture dish of solid culture basal orientation of about 50 DEG C, shake up, after to be solidified, put into 37 DEG C of incubators are inverted and cultivate 24h, carry out colony count.
Spore rate: after adopting spore Gram’s staining, basis of microscopic observation calculates.
Two, result and analysis
Table two different component solid fermentation culture medium viable count and spore rate statistical table
From result of the test: viable count and the impact of spore rate of Paenibacillus polymyxa are differed greatly by the solid fermentation culture medium of different component proportioning.Processing 1 best results, viable count, spore rate reach 26,300,000,000 g respectively-1, 92%；Processing 9 results worst, viable count, spore rate are respectively only up to 17,100,000,000 g-1, 71%；Differ greatly between process.Simultaneously it appeared that the lifting that the addition of tea grounds cake may advantageously facilitate the increase of bacterium amount and brood cell leads, this is highly profitable for the composition of culture medium.
Conclusion: rice husk (10%-15%), Semen Maydis powder (30%-40%), Rhizoma Solani tuber osi (10%-20%), tea grounds cake (5%-10%), water (10%-15%), dipotassium hydrogen phosphate (0.1%), manganese sulfate (0.2%) are the Paenibacillus polymyxa more excellent combinations of solid fermentation culture medium；Tea grounds cake can play promotion in fermentation culture or bacterium amount increases and improves the effect of spore rate, in current bibliographical information, there is not yet and added in Paenibacillus polymyxa nutrient media components.
Claims (2)
1. the method cultivating high yield Paenibacillus polymyxa, it is characterised in that: comprise the following steps: (1) weighs following raw material blending by weight: rice husk 10-15, Semen Maydis powder 30-40, Rhizoma Solani tuber osi 10-20, from the tea grounds cake 5-10 of oil tea side-product, water 10-15, dipotassium hydrogen phosphate 0.1-0.2, manganese sulfate 0.18-0.22；(2) by after being standing and soak for 3.5-4.5 hour after mixing, pack autoclaving 40-50 minute, accesses liquid seeds bacterium solution after temperature drops to 38-42 DEG C, inoculum concentration 9-11%, after stirring and evenly mixing, 36-38 DEG C after fermentation culture 70-74 hour, stopping fermentation, period timing sampling checks, to obtain final product.
2. a kind of method cultivating high yield Paenibacillus polymyxa according to claim 1, it is characterised in that: after adopting spore Gram’s staining, basis of microscopic observation calculates spore rate.
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