Surface characteristics,adhesion and exopolymer of B. cereus as well as the formation and removal of its biofilm

• Main Authors: Peng Jui-Sen, 彭瑞森
• Other Authors: Chou, Cheng-Chun
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/07482410964332124143

博士 === 國立臺灣大學 === 食品科技研究所 === 87 === Bacillus is one of the major biofilm formation microorganisms in food industry. Biofilm developed when attached bacteria proliferates and produce exopolymer on the surface. In this study, the surface characteristics, exopolymer production and the adhesion of B. cereus cells to solid surface as well as the formation and removal of B. cereus biofilm were investigated.In diluted tryptic soy broth (DTSB) adhesion of Bacillus cereus to stainless steel was noted only after 18hr incubation when corresponding to its stationary phase. Cell surface hydrophobicity increased with the increase of incubation time. It was found that the cells had net negative charge at all growth phase measured. The adhesion of B. cereus vegetative cell to stainless steel was positively correlated with the cell surface hydrophobicity (R=0.995). However the adhesion of B. cereus vegetative cell to stainless steel was negatively correlated to cell surface charge.The surface hydrophobicity of B. cereus spores prepared in tryptone glucose extract agar (TGEA) was 10.54, Which was higher than it’s vegetative cells at log phase but lower than vegetative cells at stationary phase. The adhesion of B. cereus spore to stainless steel was about 100 times that of its vegetative cells. The adhesion of vegetative cells to stainless steel was not affected by the cell suspension menstrua. But the adhesion of spores to stainless steel varied with the spore suspension menstrua.The surface characteristics, exopolymer production and the adhesion of B. cereus to stainless steel surface grown in 5 different media were compared. Results showed that cells grown in diluted tryptic soy broth (DTSB) supplemented with 1 % lactose had the highest surface hydrophobicity of 18.72. In general, B. cereus cell had net negative surface charge, regardless of the cultativation medium. The soluble exopolymer production was higher when B. cereus was grown in SLB. While production of insoluble exopolymer by B. cereus was higher in DTSB supplemented with 1% of maltose, sucrose or fructose than in other medium. The protein content of the insoluble exopolymer was negatively linear correlated with B. cereus adhesion to stainless steel or with the cell surface hydrophobicity. The macromolecules adsorption on stainless steel was higher in SLB than in DTSB. The adsorbed macromolecular did not affected the adhesion of B. cereus to stainless steel.Grown in SLB, no significant difference (＞0.05) in the production of exopolymer by B. cereus was noted at the various incubation temperatures tested. However, the adhesion of B. cereus to stainless steel was highest at 25℃ among the cultativation temperatures tested. Besides, production of exopolymer by B. cereus increased as incubation time increased.Difference in the development of B. cereus biofilm on stainless surface was noted in the various culture media tested. When grown in diluted tryptic soy broth(DTSB), only separated single cell of B. cereus was found adherent on the surface of stainless steel with no formation of biofilm. On the other hand in slime broth, biofilm with single or multilayers of B. cereus cell developed on stainless steel surface. B. cereus formed biofilm more quickly on stainless grid or stainless steel surface presoiled with milk or slime broth than on clean stainless steel surface.In DTSB, P. diminuta CCRC 14888 also developed biofilm with multilayer of cells on stainless steel surface. Although presence of P. diminuta did not affect the adhesion and biofilm formation of B. cereus, the development of P. diminuta biofilm was hindered by the presence of B. cereus.Exposure of planktonic cells to sodium hypochlorite ( 25 and 50 ppm )，or Spartec ,a quaternary ammonium compound (100 and 200 ppm) for 30 sec resulted in population reduction of ca. 5.5～6.3 log CFU/ml whereas, exposure of the 4h and 8d attached cells to similar treatments resulted in a population reduction of only 4.2～4.8 and 1.3～2.3 log CFU/chip, respectively. Removal of the attached biofilm cells on milk presoiled stainless steel were less efficiently than from clean stainless steel by the sanitizers tested. The clean-in-place (CIP) procedure performed with Spec-Tak 1000, Dilac and Diverform Plus did not remove biofilm cells effectively from the surface. However, a more than 6.0 log CFU/chip reduction of biofilm cells was observed, when the time of alkaline treatment with Spec-Tak 1000 in the CIP was prolonged to 10 min and treatment temperature was elevated to 70℃.It was also observed that trypsin could remove more B. cereus biofilm than other enzymes tested. On the other hand, a slight reduction in the population of 0.66 log CFU/chip of the B. cereus in biofilm was noted after exposure to phosphate buffer solution for 60 min.