Studies On The Cultivation And Kinetic Models Of Fermentation By Clostridium Butyricum And Modulating Effect On Gastrointestinal Microflora In Antibiotic-associated Diarrheic Mice

Firstly, the strain of Clostridium butyricum used in this research was identified by Bergey's Manual of Systematic Bacteriology and the method of sequencing PCR-amplified 16S ribosomal DNA. And then the cultivation process and technology of this strain were fully discussed. The kinetic models of batch process and fed-batch process were also determined. Furthermore, this research studied the tolerance against low pH and bile concentrations of this strain comparing with some other probiotic strains in vitro. Finally, this study was designed to evaluate the safety of mixture of C. butyricum, Bifldobacterium adolescentis and Enterococcus faecalis by using acute toxicity test and Ames test, and evaluated the modulation effect on gastrointestinal microflora of the mixture in antibiotic-associated diarrheic mice. The primary results of this research were as follows:This strain of C. butyricum was identified by Bergey's Manual of Systematic Bacteriology and the method of sequencing PCR-amplified 16S ribosomal DNA. According to comparing the sequence in National Center Biotechnology Information (NCBI) using the programme of BLASTN, this research established a fast and precise method of molecular identification to identify the species of C. butyricum.The present study described the tolerance of acid and bile concentrations of C. butyricum, Lactobacillus, Bifldobacterium and Enterococcus strains. Comparison of data in this research showed C. butyricum not only can survive lower pH, but also higher bile condition than other probiotics.. In order to exert health-promoting probiotic effects, it is important for the bacterial strain to survive the inhospitable environment of the human gastrointestinal tract, so this property of C butyricum may provide an advantage in vivo over other probiotics.The cultivation conditions and medium compositions were optimized with response surface methodology (RSM). These experimental results showed that the optimum seed medium for incubating C. butyricum was composed of 2.44% (w/v) glucose, 2.08% yeast extract (w/v), 1% tryptone (w/v), 0.1% (NH₄)₂SO₄(w/v), 0.1% NaHCO₃(w/v), 0.02% MnSO₄ ? H₂O (w/v), 0.02% MgSO₄ ? 7H₂O (w/v), 0.002% CaCl₂ (w/v) and 2% agar (w/v) (if necessary) at pH 8.55, after incubation for 24 hrs in the optimum seed medium, the populations of the viable organisms could reach 1.03×10⁹CFU/mL. And the optimum fermentation medium for incubating C. butyricum was composed of 2% glucose (w/v), 3.98% soybean cake extract, 0.1% (NH₄)₂SO₄ (w/v), 0.124% NaHCO₃(w/v), 0.37% corn steep flour (w/v), 0.02% MnSO₄ ? H₂O (w/v), 0.02% MgSO₄ ? 7H₂O (w/v) and 0.002% CaCl₂(w/v) at pH 7.5, after incubating 24 hrs in the optimum fermentation medium, the populations of the viable organisms could reach 7.1 ×10⁸CFU/mL.This research discussed the microbial growth, product formation and substrate consumption kinetic models of C. butyricum in batch cultivation too. A simple model was proposed using Logistic equation for growth, the Luedeking-Piret equation for butyric acid production and Luedeking-Piret-like equation for glucose consumption. These models could suitably predict the batch fermentation progress,especially between 0 hr and 20 hr. The models were as follows:dx xMicrobial growth model: ^T = 0.1812(1 — )xat 1.64Glucose consumption model: ^T " 9-0603 — + 0.00967*at atProduct formation model: ^<sup>7 = 3.17 —— + 0.00175x .at atThis research also discussed the microbial growth, product formation and substrate consumption kinetic models of C. butyricum in fed-batch cultivation. These models could also suitably predict the fed-batch fermentation progress. The models were as follows:AYMicrobial growth model: ^T = (M 0.0083)XatGlucose consumption model: — = 0.0083(17.94 - S) - -dt 0.2701Product formation model: ^T —