| Staphylococcus aureus(S.aureus)is one of the most common foodborne pathogens related to foodborne disease outbreaks via contaminated food which is widely distributed.Particularly,larges of number of frozen and refrigerated food were contaminated by S.aureus annually.As a common storage condition for frozen food during transportation,distribution and storage,freezing doesn’t seem to be entirely safe due to the cold tolerance of S.aureus.Once the frozen food is thawed or out of cold chain temperature control,the pathogen may grow immediately,if present,which represents a potential hazard of S.aureus to well thrive even after storage under freezing temperature.Thus,it is necessary to elucidate the molecular mechanisms in response to freezing stress for controlling them.Biochanin A,an isoflavonoid found in chickpea(Cicer arietinum),in peanuts and in other legumes,has been reported to exhibit the bioactive potentials including anti-microbial.While it has not been reported whether it has anti-biofilm activity.This study aimed to monitor and predict the survival kinetics of S.aureus under cold chain dynamic temperature,explore the biofilm formation,adhesion ability and molecular mechanism in regulating the response exposed to freezing stress and evaluate the anti-biofilm activity of the biochanin A against S.aureus.Collectively,the current work provided potential targets for developing strategy to control S.aureus biofilm.The main research contents and results of this paper are as follows:(1)Construct survival kinetics model of Staphylococcus aureus under cold chain temperature conditionsThe survival curve of S.aureus in raw pork under dynamic temperature profile across freezing and thawing combined with the static curves were used for fitting analysis.The One-step analysis was used to determine the model parameters,which integrates the primary model(Logistical model)and secondary model(Huang square root model)into one tertiary model for fitting analysis.The results of kinetic analysis showed that Tmin(minimum growth temperature)was 6.85℃.S.aureus may die off at a rate of 3.63×10-3 Log CFU/mL per h per℃ below the Tmin.Subsequently,validation results indicated the integrated model can accurately predict the behavior of S.aureus regardless of isothermal or nonisothermal conditions with the root-meansquare errors(RMSE<0.44 Log CFU/g,73.9%of the errors of prediction falls within± 0.5 Log CFU/g),accuracy factors Af and bias factors Bf were 1.05 and 0.98,respectively,which were both close to 1.The results demonstrated that the one-step analysis method is an accurate and efficient method for directly constructing predictive models and estimating kinetic parameters of microbial growth and survival in food under cold chain conditions.(2)Proteomics signature of S.aureus in regulating the response exposed to freezing stressProteomics signature of S.aureus after freezing stress(-20℃,5 days)with 2hours resuscitation based on tandem mass tag(TMT)labeling and LC-MS/MS was analyzed.A total of 1993 proteins were identified,of which 1882 proteins were quantified.Result showed that 335 proteins were upregulated,and 225 proteins were downregulated(P<0.05).Gene Ontology,KEGG pathway and protein domain analysis revealed the ribosome function,metabolism,RNA repair,QS system,stress response and biofilm-related proteins are differentially regulated(P<0.05).Moreover,significant lysine acetylation and malonylation signals in S.aureus response to freezing stress were observed.Collectively,the current work provided research basis for comprehending the molecular mechanism of S.aureus in response to freezing stress and presented potential targets for developing strategy to control S.aureus.(3)Analysis of S.aureus biofilm formation and adhesion to host cells in response to freezing stressThe biofilm formation of S.aureus during different freezing storage(1,2,4,5,6,7 and 9 days)and resuscitated at 37℃ for different hours(0-7 hours,at 1-h intervals)were investigated.Result showed that the 5 days storage were the minimum duration time at which higher biofilm formation potential of the freezing group were initially showed.And biofilm formation significantly(P<0.05)increased with the extension of recovery time.In addition,PIA and eDNA in EPS were investigated further via dotblot assay and agarose gel electrophoresis method,respectively.The result indicated that biofilm formation ability in S.aureus was significantly increased via increasing EPS production in response to freezing stress.Furthermore,the effect of freezing stress on the adhesion to A549 cells was investigated which indicated the adhesion to host cell were increased and the release of inflammatory factors were also increased.(4)Activity of biochanin A against S.aureus biofilmMolecular docking demonstrated that biochanin A conducted strong interactions with pathogenicity-related proteins(Ica A,Sortase A and Enolase)mediating the increased biofilm formation in response to freezing stress.Then,the anti-biofilm activity of biochanin A against S.aureus were confirmed.The results indicated that biochanin A had anti-biofilm activity against S.aureus for both freezing treatment and non-treatment group.Result showed that biochanin A was effective in disrupting the preformed biofilm with 24.8%-52.1%reduction(P<0.05)in biofilm biomass at 128μg/mL,whereas,significantly(P<0.01)reduction by 46.2%-78.4%was observed at 256 μg/mL.In addition,result showed that biochanin A effectively eradicated established S.aureus biofilms on different food-contact materials.At 256 μg/mL,biochanin A reduced the viable counts by 3.08 Log/cm2 and 2.71 Log/cm2 on stainless steel and glass,respectively.In addition,biochanin A can effectively inhibited the biofilms formation by regulating the expression of biofilm-related genes(icaA,srtA,eno)mediating the suppression of the EPS release and intercellular adhesion aggregation.This study provides a theoretical basis for comprehending the molecular mechanism of S.aureus in response to freezing stress and presented potential targets for developing strategy to control S.aureus. |
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