Screening,isolation And Characterization Of Klebsiella Pneumoniae Strain GS7-1 Degrading Zearalenone

Mycotoxins are secondary metabolites produced by certain fungi such as Aspergillus,Penicillium,and Fusarium species which extensively pollute crops and agricultural products such as corn,wheat,peanuts,etc.,reducing the yield of crops and the quality of food and feed,causing huge economic losses worldwide.At the same time,it seriously threatens the health of humans and animals.Zearalenone（ZEN）is a mycotoxin produced by Fusarium species and is one of the main agricultural feed pollutants that cause reproductive disorders in livestock.Therefore,the urgent search to reduce zearalenone has become a current research hotspot.However,the physical and chemical detoxification methods before or after harvesting have different degrees of weakness in the elimination of mycotoxins.This makes the research of biodegradation and biotransformation methods aroused the attention of experts and scholars.In this study,soil samples were collected from corn and wheat farms in Hubei and Shandong provinces to screen microorganisms that can degrade ZEN.Among the isolated Klebsiella pneumoniae strain GS7-1,HPLC was used to evaluate the ability to detoxify ZEN from the Luria-Bertani（LB）broth medium.The results showed that 2 ppm ZEN was completely removed by K.pneumoniae GS7-1 within 96 hours.The removal of ZEN by K.pneumoniae strain GS7-1 is a slow and continuous process,and approximately 94.6%of the biodegradation is significant within 72 hours（p<0.05）.At the same time,I also studied the effects of degradation kinetics,p H and temperature factors on the degradation ability of ZEN.The optimum temperature for degradation of ZEN by K.pneumoniae strain GS7-1 is between 28°C and 60°C.The degradation rate of ZEN increased with the increase in incubation time.At 96 hours,ZEN（2ppm）was completely degraded.The strain can degrade at acidic and alkaline p H.At p H 5,p H 6 and p H 7,the removal rate of ZEN was approximately 61.29%,62.65%and 94.71%respectively.At an alkaline p H of 8 to 10,the degradation rate reaches 100%.Therefore,the optimal p H ranges from p H 7 to p H 10.This study also evaluated the degradation ability of K.pneumoniae strain GS7-1 cell components to the degradation of ZEN.By separating the extracellular supernatant of K.pneumoniae 7-1,washing the cell pellet,and the cell pellet suspension,the potential of the three components to remove ZEN was studied.The results showed that compared with other components,the culture supernatant degraded ZEN significantly（p<0.05）.No significant difference was observed in the first wash,which showed 30%degradation,while the second wash showed 25%degradation,and after the third wash,the cells settled in suspension in PBS No degradation was observed.Based on the above,the biodegradation of ZEN is a very feasible method for food and feed purification,because using physical and chemical agents may also eliminate mycotoxins,nutrients,and sensory-related compounds.The detoxification and biotransformation of ZEN were studied by the activity of the culture supernatant and the expected metabolites.Here,TLC,reversed-phase HPLC,ion source ESIMS and high-resolution Fourier transform mass spectrometry（HR-FTMS）analysis showed that after 72 hours of incubation,the ZEN concentration（20 ppm）exposed to the fermentation culture supernatant was greatly reduced,while C₂₁H₄₂O₉,C₃₅H₅₁O₅and C₂₂H₁₇O₃detected at m/z 461.27,552.38 and 330.12 respectively are ZEN metabolites,which can be regarded as degradation products produced by the activity of the fermentation culture supernatant.The possibility of reducing ZEN levels by K.pneumoniae GS7-1 isolated from agricultural soil is of great value for controlling ZEN contamination of food and feed.This is a discovery because it can be used in industry for potential food and feed.In fact,K.pneumoniae strain GS7-1 may be the source of effective enzymes for ZEN biotransformation.In conclusion,further studies focusing on ZEN metabolites’structure,toxicity and degradation mechanism are expected to further promote the development of new and genetically improved microbial ZEN detoxification strategies.Although K.pneumoniae strain GS7-1 is pathogenic,it is still considered to be a promising bacterium that degrades ZEN.The enzymes that cause this enzyme degradation should be further explored because detoxification enzymes can be used in the food and feed industries.