The Studying Of Solid-state Fermentation Of Streptomyces Avermitilis And Its Activity Against To Nematode

In recent years,with the increased focusing on environmental protection and food safety,pesticides are developed towards the direction of non-toxic and easily degradable formulation.As a highly efficient,low-toxic and easily degradable biological pesticide,avermectin has been extensively studied and widely used.Avermectins are a class of broad-spectrum insecticidal antibiotics produced by Streptomyces avermitilis.It contains8 components of which the B1 component is the most effective one against nematodes.At present,avermectins are mainly produced by liquid submerge fermentation.However,the liquid submerge fermentation has brought huge harm to the environment.For example,there are many waste residue and water generated by the production process.At the same time,a large amount of organic solvents difficult-to-be degraded are added to the process of avermectin production and formulation.The chemical ingredients in avermectin product have shown big damage to the plants and soil.Therefore,developing a new type of fermentation process,which has little waste liquid and residue,low risk of environmental pollution from the organic solvent,and good products with better resistance to insects,has become the current hot top of avermectin production and research.The solid-state fermentation?SSF?is ofte used for the antibiotics production.SSF has the advantages of less waste residue,less waste liquid,less organic solvents and better environmental compatibility.So,this study plans to develop a better production method of avermectins with environmental compatibility by using SSF.In this paper,the solid-state fermentation process conditions of S.avermitilis AVM5were studied.The viable cell count was taken as the optimization index during studying medium compositions and conditions for solid-state fermentation by S.avermitilis AVM5.The yields of avermectins were determined by HPLC method.The soild fermentation products were mixed into the soil samples containing nematodes to investigate its resiatant to nematodes.The main results were as follows:Firstly,one-factor test and orthogonal test were used to select solid fermentation medium components and conditions.The solid-state fermentation conditions by canned bottle culture were determined as follows,28°C of culture temperature,96 hours of seed liquid age,20 mL of inoculum volume,45%initial water content,initial pH 7.3,culture cycle for 10 days.The medium components of the solid state fermentation were also determined.The optimal carbon source was 11 g of rice flour mixed with 7 g of cassava residue,and the nitrogen source was 11 g of soybean meal mixed with 9 g of beef powder.The adding amounts of inorganic salt were 0.40%NaCl?0.50%K₂HPO₄?0.50%MgSO₄·₇H2O?0.10%CaCl₂?0.01%FeSO₄·7H₂O and 0.04%CoCl₂·6H₂O,respectively.Based on the optimization above,the growth stage of the viable cell was determined by testing the growth curve of S.avermitilis AVM5 in solid state fermentation process.The maximum viable cell count(1.41×10¹⁰ cfu/g per dry basis)was received after cultured,which was 29 times higher than the initial culture.Secendly,the HPLC conditions for detecting avermectins were determined as follows,Agilent ZORBAX Eclipse C18 column,245 nm for UV detection wavelength,mobile phase with methanol:water=95:5,1.0 mL/min of set flow rate,20?L of sample injection volume,40°C of column temperature.The sample was pretreated by 5 times volume methanol and soaking extraction for 2 hours,supplemented with ultrasonic treatment for 25 min,and shaking once every 30 minutes.The treated sample was filtered through the 0.45?m membrane,and then was analyzed by HPLC.Analysis of HPLC chromatograms of solid fermentation products revealed that avermectins content were positively correlated with the number of viable cells.However,the production of avermectins was often delayed behind that of the number of viable cells,that was,the cells were lysed and avermectin was then released from them,which led to the increasing of avermectins content.Avermectins production reached its maximum yields of at about0.62 mg/g per dry base at 40 d by prolonging the fermentation cycle.Thirdly,the solid fermentation product was mixed into the soil sample containing nematodes,and the number of nematode was significantly reduced.It was determined that the avermectin released from the product instead of viable cells had the insecticidal activity.The maximum insecticidal efficiency of solid state fermentation products could reach up to 37%,and the diluted products by 100 times could keep insecticidal activity even.Through the study mentioned above,the solid fermentation process of avermectin was preliminarily optimized,which provided a good guidance for replacing liquid submerge fermentation of avermectins.This study will be beneficial to the production and application of avermectin products with good environmental compatibility.