| Sweeteners are widely used in the food industry and can be classified into two categories:artificial sweeteners and natural sweeteners.Their main characteristic is a sweetness that is several hundred times higher than that of sucrose,and they also have low calorie properties that meet the demand for healthier foods among consumers.Research has shown that sweeteners can affect the growth of food microorganisms and the generation of metabolic products,thereby influencing the formation and stability of biofilms.Biofilms are an important survival mode that allows microorganisms to persist during food processing and storage.To investigate the impact of mainstream sweeteners on the formation of foodborne microbial biofilms,this paper selected three commercially available sweeteners(sucralose,acesulfame K,and stevia leaf extract)and applied them to the growth of five common foodborne microorganisms(Aeromonas hydrophila 、 Vibrio parahaemolyticus 、 Listeria monocytogenes、Escherichia coli、Pseudomonas fluorescens).The effects of sweeteners were determined by measuring the growth curves from 0-36 h and staining the biofilms with crystal violet.The results showed that most microorganisms in the experimental group had no significant differences compared to the control group,indicating that their growth and biofilm formation were not affected by the sweeteners mentioned above.However,we found that the stimulation of steviol glycosides had a specific physiological effect on V.parahaemolyticus(VP),and we confirmed that Rebaudioside A,as the main component of steviol glycosides,inhibited the growth of VP strains but significantly promoted its biofilm formation.In the preliminary studies,we identified a specific physiological response of VP strain to RA stimulation,which prompted us to investigate further the effects of RA on VP biofilm in this paper.By conducting CFU counting experiments and observing the morphology of VP biofilm using laser confocal microscopy,it was found that RA could increase the number of dead cells in VP biofilm and consequently increase its thickness.To explore the underlying mechanism,q PCR was used to detect the expression of VP-related genes.It was found that the expression of mam-7 gene in VP strain was significantly up-regulated compared to the control group after short-term stimulation with 5 m M RA.Mam-7 encodes an outer membrane protein,which mediates the adhesion of VP cells and enhances type III-mediated cell death.After using the phenol-sulfuric acid method and BCA method to detect the content of extracellular polymeric substances(EPS)components(extracellular polysaccharides and proteins)in VP biofilm,it was found that 1 m M and 5 m M RA significantly increased the content of extracellular proteins to 126.633±2.712 μg/m L and 189.867±3.521 μg/m L,respectively.Subsequently,we performed homology analysis on the mam7 protein of VP and selected Vibrio alginolyticus 、 Vibrio vulnificus 、 Vibrio cholerae and Salmonella typhimurium as research objects based on the degree of amino acid sequence matching.The results of the 0-36 h growth curve and crystal violet staining showed that V.alginolyticus,which had the highest degree of matching,exhibited similar physiological phenomena to VP under RA stimulation,namely growth inhibition and promoted biofilm formation.At the same time,q PCR detected a significant upregulation of the "mam7-like protein" gene expression in V.alginolyticus.Therefore,this study elucidated the mechanism by which RA affects the biofilm formation of the VP strain: RA stimulation mainly promoted the expression of VP’s mam7 gene,increased the protein content in EPS,enhanced biofilm adhesion,and increased the number of dead cells,resulting in an increase in biofilm formation due to enhanced cell aggregation and increased total EPS content.Furthermore,through homologous protein analysis and related experiments,it was confirmed that RA has a special response to the mam-7 gene,which is not limited to the VP strain,and other vibrios(such as V.alginolyticus)with highly similar amino acid sequences in the Vibrio genus also exhibit similar physiological phenomena under RA stimulation.This paper conducted a screening experiment using commercially available sweeteners to stimulate the formation of microbial biofilms on food.It was found that the VP strain exhibited a unique form of biofilm response to RA stimulation.To investigate this phenomenon,we systematically studied the effect of RA on VP biofilm formation and explained the mechanism of RA’s effect on VP biofilm formation at the gene transcription level.In addition,we also conducted a general exploration and found that RA is not the only sweetener that has a special effect on VP,which contributes to our understanding of the impact of natural sweeteners on the growth and pathogenic characteristics of foodborne microorganisms.This research is not only of great guiding significance for aquaculture,food safety,and health,but also has certain inspirations for the application research of natural sweeteners. |
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