Antibacterial And Antibiofilm Of Garlic Ethanol Extract Against Bacillus Spp.Responsible For Spoilage

Bacillus spp.are the dominant bacteria responsible for spoilage of pre-rehydrated Sichuan yuba.These spoilage bacteria has strong capacity of producing heat-resistant spores and biofilm formation.The proliferation of spoilage strains can bring about negative changes in sensory and nutritional quality of products,and reduce the commercial value and shelf-life of Sichuan yuba.Organic sulfide compounds in Garlic have broad-spectrum antimicrobial activity including antibacterial and bactericidal effects.Therefore,using Organic sulfide compounds for control of food spoilage agents have recently attracted great interest.In this master's thesis,the inhibitory effect of garlic ethanol extract on bacterial cell proliferation and biofilm formation of four spoilage Bacillus including two Bacillus subtilis strains and two Bacillus amyloliquefaciens were investigated.In addition,the biofilm formation ability and growth conditions of four strains were measured.Furthermore,the inhibitory mechanism of garlic ethanol extract against cell proliferation and biofilm formation of spoilage Bacillus strains were clarified.The primary outcome are summarized as follows.1.The inhibition and mechanism of garlic ethanol extract(GEE)on the growth of Bacillus planktonic cell.The main components of GEE extracted by n-hexane were analyzed by GC-MS.Results showed that the relative content of disulfide was 70.08%in GEE.Inhibition zone diameters of GEE against strains DY1a,DY1b,DY2a and DY3were 31.7,25.7,30.3 and 30.0 mm,respectively.The MIC of GEE against four strains were 30 mg/m L.The MBC were 240,480,120 and 60 mg/m L respectively.Addition with 30 mg/m L GEE inhibited bacterial proliferation within the first 50 hours.The inhibition rate of 30 mg/m L GEE on the extracellular protease activity of strains DY1a and DY1b was 47.3%and 15.6%,while the effect on the strains DY2a and DY3 was not obvious.The planktonic bacteria treated by 30 mg/m L GEE still had metabolic activity.In addition,the alkaline phosphatase activity,conductivity and?-galactosidase in the extracellular medium of the GEE treated group were significantly higher than those of the untreated group(P<0.05),indicating that structure of the planktonic cell membrane was vigorously damaged.The SEM results showed that GEE addition resulted in shrinkage,pore collapse and rupture of planktonic cell surface.2.The biofilm forming ability and favorite conditions of B.amyloliquefaciens DY1a and DY1b have strong capacity of forming pellicles on the air-liquid interface of TSB medium,while B.subtilis DY2a and DY3 were weak.The pellicles growth of B.amyloliquefaciens DY1a and DY1b achieve to the maximum biomass after 48 h static culture.The biomass of pellicles formed in TSBS medium was significantly higher than that in other medium(P<0.05).The optimal cultural condition for pellicles formation of B.amyloliquefaciens were p H 7.0,temperature 37?,and initial inoculation 5-7 lg CFU/m L.3.The antibiofilm formation and pellicles removal effect of GEE on Bacillus.Various concentrations of GEE were added to TSBS medium to determine their anti-pellicles ability.GEE significantly inhibited pellicles formation of B.amyloliquefaciens DY1a and DY1b(P<0.05).The inhibition percent of 15 mg/m L GEE against bacteria within in the biofilm were 62.0%and 43.6%,respectively.GEE,at a concentration of15 mg/m L,could inhibit cell motility of strains.The inhibitory percent of GEE against swimming ability of DY1a and DY1b were 100%and 33.27%,respectively.The inhibitory percent responding to swarming ability were 100%and 92.0%,twitching ability were 78.6%and 71.6%.The addition of GEE showed no biofilm eradicating effect on biofilm of B.amyloliquefaciens at 48^th hours.On the contrary,the addition of GEE facilitated the biofilm reconstruction of B.amyloliquefaciens.The introduce of300 mg/m L GEE also enhanced the metabolic activity in pellicles by approximately1.6-folds,and increased protein content 2.5-folds,and protein/polysaccharide 2.6-folds,respectively.