| Cronobacter sazakazii,a significant milk-borne contaminating bacteria with a death rate of 40% to 80%,cannot be detected in infant formula milk powder,and ‘the zero’ tolerance control standards were applied in international dairy import and export commerce.C.sakazakii mostly polluted the equipment and floor of milk powder processing workplace.Traditional sterilizing treatments were difficult to control the strain because of C.sakazakii’s high environmental tolerance.As a result,it is critical to do theoretical study on the factors influencing C.sakazakii resistance,and research in this field has only recently begun.Based on earlier research,this study investigated for the first time.The impact of a series of component alterations in C.sakazakii carotenoids on cell properties,particularly environmental resistance.Furthermore,in order to investigate its possible mechanism of action,lipidomics was used for the first time to analyze and compare different mutants of C.sakazakii,revealing the effects of different carotenoids components on bacterial lipids,laying a foundation for explaining the mechanism of C.sakazakii carotenoids affecting stress resistance.In this study,the original strain was Cronobacter sakazakii BAA-894.To begin,five essential genes involved in carotenoid production were knocked out using Red homologous recombination technique to generate Δcrt B,Δcrt I,Δcrt Y,Δcrt Z and Δcrt X strains,and the components of carotenoids were qualitatively or quantitatively examined.The stress resistance of several key cell characteristics(growth rate,outer membrane permeability,membrane fluidity,and so on)and five important environmental stresses(acid,alkali,high osmotic pressure,oxidation,and drying)were then systematically analyzed and compared to reveal the effects of carotenoid composition changes on C.sakazakii’s environmental stress resistance.Finally,using differential lipidomics,the alterations in lipidomicss in different mutant strains and BAA-894 were investigated,as well as the impact of carotenoid components on cell membrane lipids.The main findings of the present research were as follows:(1)Construction and phenotypic evaluation of five carotenoid genes deletion strains.Crt B,crt Y,crt Z,crt I,and crt X were knocked out in C.sakazakii,and five mutuants were produced,confirming that they could synthesize distinct carotenoids components: LC-MS/MS demonstrated that BAA-894 created disaccharide zeaxanthin,whereas HPLC revealed thatΔcrt B produced geranylgeranyl pyrophosphate(GGPP),Δcrt Y produced lycopene,Δcrt Z produced β-carotene,Δcrt I synthesized phytoene,and Δcrt X produced zeaxanthin.Changes in these carotenoid components had no effect on the growth of the strain.(2)The effects of carotenoid content on cell properties and tolerance to environmental stress in C.sakazakii.Environmental stress resistance is closely related to cell characteristics.Firstly,it was discovered that the extracellular membrane permeability,membrane fluidity,and biofilm formation ability of the five mutants were significantly increased compared to BAA-894,the membrane fluidity was decreased,and the biofilm formation ability was weakened,indicating that changes in carotenoid components of C.sakazakii affected the cell characteristics of the strains.A comparison of the five environmental resistances of different strains revealed that,when compared to the wild-type strain,C.sakazakii showed different changes in acid,alkali,high permeability,and drying after changing the carotenoid composition,and the tolerance of multiple strains decreased.It is worth noting that the β-carotene hydroxylase(Crt Z)deletion strain(accumulated β-carotene)significantly decreased resistance to five environmental stresses and was an important candidate target for antimicrobial agents.(3)Changes in carotenoid composition and their impact on the lipidomics of C.sakazakii.According to differential lipidomics,the unsaturated PE abundance of BAA-894 was 42.80%,but it increased to 50.70%-52.66% in the five mutants.The saturated PE abundance of BAA-894 was 3.16%,while that of the five mutants was 2.94%-3.79%,indicating that the change of carotenoid composition led to the increase of unsaturation of important lipid PE.Monounsaturated and diunsaturated PE were also the primary unsaturated PEs,and each strain’s percentage ranged from 99.40% to 99.70%.Monounsaturated and diunsaturated PE were also the primary unsaturated PEs,and each strain’s percentage ranged from 99.40% to 99.70%.Additionally,the primary forms of unsaturated PE abundance in the five carotenoid mutants included PE(16:0/16:1),PE(16:0/18:1),PE(17:1/16:0),PE(16:1/18:1),PE(17:1/18:1),and PE(18:1/18:1).Since the interaction of carotenoid-lipid molecules in the cell membrane is related to the change in environmental stress resistance brought on by the change in carotenoid components,the change in carotenoid components will consequently result in significant changes in the lipidomics,especially the significant increase of unsaturated PE. |
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