Analysis Of Growth Status And Metabolic Markers Of Staphylococcus Aureus In Meat Matrix

Staphylococcus aureus is widely spread in the nature and can be found in the air,water,dust,excretion of humans and animals.It is highly polluting food,seriously affecting food safety and threatening consumers' health.In recent years,food poisoning caused by S.aureus is still one of the major food safety issues that threaten the health of consumers.With the development of metabolomics research technology,searching for characteristic metabolites of foodborne pathogenic bacteria has become an important direction for the rapid detection and identification of pathogenic bacteria.Analysis of volatile metabolites of foodborne pathogens can be used as a new method for the identification of pathogenic bacteria in the field of clinic and food research.Previous reports and our research have found that S.aureus produces 3-methylbutanal and 3-methyl-butanoic acid,and the two metabolites are special to S.aureus.Although the characteristic volatile metabolites of S.aureus have been found,the factors affecting the release of the metabolic markers have not been completely investigated.The stability of metabolic markers is still unclear.China is the world's largest consumer of pork,and the total pork consumption of Chinese accounts for about half of the world.Raw pork and its products are highly susceptible to microbial contaminations during processing,transportation and sales.S.aureus is a serious pathogen contaminating in meat.In the present study,ground pork and pork broth were used as substrates.Firstly,the specificity of the volatile metabolic markers of S.aureus was analyzed,and the growth and metabolic markers of different S.aureus strains were explored.Then the effects of mixed cultures on the growth and metabolic markers of S.aureus were revealed.Finally,the effects of food preservatives on the growth and metabolic marker yield and metabolism key enzyme gene expressions were illustrated.The research results are as follows:(1)In nutrient broth,trypticase soy broth as well as brain heart infusion broth,the signals of 3-methylbutanal and 3-methylbutanoic acid were only detected in S.aureus,not detected in Escherichia coli O157:H7 and Salmonella enteritidis.On ground pork,the signals of 3-methylbutanal and 3-methylbutanoic acid were also only detected in S.aureus,not detected in E.coli O157:H7 and S.enteritidis.In pork broth,the signal of 3-methylbutanoic acid was only detected in S.aureus,not detected in E.coli O157:H7 and S.enteritidis.The specificity of metabolic markers to S.aureus was not affected by meat substrate.(2)On ground pork,both 3-methylbutanal and 3-methylbutanoic acid were detected in seven S.aureus strains.The signal of 3-methylbutanal was earlier than 3-methyl-butanoic acid.The signal intensity of 3-methylbutanal increased then decreased rapidly,while the signal intensity of 3-methylbutanoic acid kept increasing during the whole cultivation process.In pork broth,only S.aureus B83 and S.aureus 134 were detected 3-methylbutanal and the signal intensity of 3-methylbutanal increased then decreased rapidly.The 3-methylbutanoic acid was detected in seven S.aureus strains,and the signal intensity increased steadily.Compared to the transient 3-methylbutanal,3-methylbutanoic acid was a more reliable and stable marker.During the whole culture process from 0 to 24 h,the correlations between the production of 3-methylbutanoic acid and the growth of S.aureus were significant(p<0.01),and the linear regressions obtained by F detection were also significant(p<0.01).(3)On ground pork,both 3-methylbutanal and 3-methylbutanoic acid were not detected in mixed cultures of S.aureus and E.coli O157:H7(1:1,10:1),S.aureus and S.enteritidis(1:1),and three bacteria(1:1:1).These two metabolites were detected in the cultures of S.aureus mixed with E.coli O157:H7(100:1)and with S.enteritidis(10: 1,100:1).In pork broth,3-methylbutanoic acid were detected in mixed cultures of S.aureus and E.coli O157:H7(1:1),S.aureus and S.enteritidis(1:1),three bacteria(1:1:1).In comparison with the single culture,the maximum growth levels of S.aureus in mixed cultures were reduced,,while the signal intensities of 3-methylbutanoic acid from mixed cultures were not distinctly repressed.During the whole culture process from 0 to 24 h,the correlations between the production of 3-methylbutanoic acid and the growth of S.aureus were significant(p<0.01),and the linear regressions obtained by F detection were also significant(p<0.01).(4)At the maximum limit of national standard,the inhibition to S.aureus of four preservatives was: nisin(0.5 g/L)> chitosan(6.0 g/L)> sodium benzoate(2.0 g/ L)> sodium nitrite(0.15 g/L).In trypticase soy broth and pork broth,four preservatives(sodium nitrite 0.15 g/L,chitosan 3.0 g/L,sodium benzoate 2.0 g/L,Nisin 0.0625 g/L)had negative effects on the transcription level of ilvE gene encoding aminotransferase and aldH gene encoding aldehyde dehydrogenase of S.aureus,and the transcription level of kadh gene encoding ?-ketoacid dehydrogenase was not significantly affected.All four preservatives reduced the release of 3-methylbutanoic acid.The results revealed that the effects of different culture modes and the addition of food preservatives on the growth and the release of metabolic markers of S.aureus on pork matrix.The stability of metabolic markers of S.aureus was analyzed in detail.These would lay a foundation for the establishment of the detection technique of S.aureus in meat based on the targeted metabolism.