Analysis Of Fermentation Metabolites Of Mixed Bacteria Of Bacillus BL-21 And HNDF2

Bacillus sp.can produce a variety of antimicrobial substances which is widely used in bio-control of plant diseases and insect pests.This study mainly used Brevibacillus laterosporus BL-21 and Bacillus subtilis HNDF2 to do mixed-fermentation.On the basis of co-culture,the inhibitory effection,cell density and metabolites concentration was compared and analized between the single-fermentation and mixed-fermentation.We used CM-Sepharose Fast Flow ion exchange chromatography and Sephadex G75 gel filtration chromatography to obtain a substance which has antifungal acti-vity against Fusarium oxysporum.We also studied on the effect of HNDF2 to BL-21 in mixed-fermentation,and the antifungal results of co-culture fermentation of some actinomycetes mixed with BL-21 or HNDF2 was preliminary tested and analized.The results of the antibacterial between single-fermentation and mixed-fermentation showed that the metabolites of mixed-fermentation against Fusarium oxysporum inhibition zone diameter was 21.222±0.57 mm,metabolites of BL-21 single-fermentation inhibition zone diameter was 17.89 ±0.68 mm,metabolites of HNDF2 single-fermentation inhibition zone diameter was 9.11±0.57 mm.Significant analysis indicated that the inhibitory activity of mixed fermentation is higher than that of the two single-fermentation.There were four peaks after CM-Sepharose Fast Flow ion-exchange chromatography eluted of mixed-fermentation.A protein band.about 12kDa was detected in peak 3 by SDS-Page electrophoresis.After Sephadex G75 gel filtration chromatography,11 peaks were detected and collected.The results of HPLC-MS showed that there are two composition in peak 2,their m/z.are 508.0 and 530.9,seperatly.Tthere are some different between single-and mixed-fermentation.Only two peaks were observed in BL-21 single-fermentation on the process of ion-exchange chromatography with CM-Sepharose Fast Flow,while there was a protein band about 14kDa in peak 1 and a protein band about 12kDa in peak 2 with SDS-Page electrophoresis detecting,.Comparaing and analyzing the metabolites difference of mixed-fermentation from BL-21 single-fermentation,we can find that they are obviously different in the time of the peaks appearing and peak number.The impact of culture time,bacterial cell number and metabolites concentration of the seed liquid of HNDF2 to BL-21 in mixed fermentation were experimented,we drew conlusions as follows:The inhibitory effect of mixed fermentation is the best when HNDF2 was cultured 48h,the inhibition zone diameter was 24 ± 0.55mm;In the same way,when the cell concentration of HNDF2 was added to 9.84×107CFU/mL,the inhibition zone diameter of mixed fermentation was the largest,reaching 19.56=0.33mm;total seed liquid of HNDF2 stimulates BL-21 to produce stronger antifungal activity than that of only using thallus or matobolite.Some actinomycetes mixed-fermentate with BL-21 or HNDF2,the results showed that HD087,HD103 and HD006 can stimulate BL-21 to produce stronger antibacterial activity,the antibacterial activity of metabolites of mixed-fermentation is stronger than that of BL-21 single-fermentation.HD087 can stimulate HNDF2 to produce stronger antibacterial activity,too.But other actinomycete strains have not this role.