【作者】 孔青；

【导师】 何国庆；

【作者基本信息】 浙江大学 ， 食品科学， 2006， 博士

【摘要】 本论文从丁酸梭菌的菌体形态、生理生化和16S rRNA序列分析菌种鉴定开始,在此基础上,系统研究了丁酸梭菌培养条件的优化,并建立了丁酸梭菌分批发酵和补料分批发酵的动力学模型;另外,在体外研究了丁酸梭菌的耐酸、耐胆汁特性,并在体内研究了丁酸梭菌和青春双歧杆菌、粪肠球菌混菌调节抗生素引起的腹泻小鼠肠道菌群平衡的作用。论文的主要研究结果如下: 利用伯杰氏细菌分类鉴定系统和16S rRNA序列分析的鉴定方法对实验室保存的丁酸梭菌进行鉴定,通过设计引物并将扩增出的16S rRNA序列在National Center Biotechnology Information（NCBI）上使用BLASTN程序进行比对,尝试了一种丁酸梭菌分子鉴定的快速、准确方法: 研究了丁酸梭菌的耐酸、耐胆汁特性,通过和其它几种常用益生菌体外实验的比较,证明丁酸梭菌比乳杆菌、肠球菌、双歧杆菌更耐酸、更耐胆汁浓度,这个结论表明了丁酸梭菌更能耐受肠道环境、能在肠道停留更长时间,具有很好的应用价值; 利用响应面实验设计的科学方法对丁酸梭菌的培养条件进行系统优化,最终确定培养丁酸梭菌的种子培养基为:2.44%葡萄糖（w/v）,2.08%酵母浸膏（w/v）,1%胰蛋白胨（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）和2%琼脂（w/v）（配固体培养基时用）,初始pH为8.55,丁酸梭菌在此培养基中厌氧培养24小时后活菌数可达1.03×10⁹ CFU/mL:培养丁酸梭菌的发酵培养基为:2%葡萄糖（w/v）,3.98%豆饼粉提取液,0.1%（NH₄）₂SO₄（w/v）,0.124%NaHCO₃（w/v）,0.37%玉米浆（w/v）,0.02%MnSO₄·H₂O（w/v）,0.02%MgSO₄·7H₂O（w/v）,0.002%CaCl₂（w/v）和2%琼脂（w/v）（配固体培养基时用）,初始pH为7.5,丁酸梭菌在此培养基中厌氧培养24小时后活菌数可达7.1×10⁸CFU/mL,筛选出合适的培养基配方,为下步的研究以及今后大规模的生产提供依据; 首次考察了丁酸梭菌在5 L发酵罐中自然分批发酵动力学过程,并建立了相关模型,实验中采用Logistic方程描述了菌体生长的动力学过程,采用Leudeking-Piret方程描述了产物合成的动力学过程,用Leudeking-Piret-Like方程描述了底物消耗的动力学过程,从检验结果可知,模型可以较好的（尤其在发酵的前20小时内）解释实际发酵中细胞生长、底物利用和产物合成的动力学规律。所得模型方程如下: 细胞生长模型:（dx）/（dt）=0.1812（1-x/1.64）x 底物消耗模型:-（dS）/（dt）=9.0603（dx）/（dt）+0.00967x 产物合成模型:（dP）/（dt）=3.17（dx）/（dt）+0.00175x;更多还原

【Abstract】 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 — <lp ’ X — 0.0083PatSpecific growth rate model: M = 0.15282 (1-----———4.4584Specific product formation rate model: %=3.3825 JI+0.03928.Finally, this study was designed to evaluate the safety of mixture of C. butyricum, Bifidobacterium 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 oral maximum tolerant dose of this mixture of probiotics was more than 10, 000 mg/kg body weight in mice;and no increase in the number of revertant colonies occurred in the four test strains at any concentrations of mixture of probiotics, either in the presence or absence of S9 mix, and similar results were obtained from the duplicate test, namely no obvious dose-response relationship had been found and therefore the Ames test result of mixture of probiotics was negative. Therefore, the mixture of C. butyricum, Bifidobacterium adolescentis and Enterococcus faecalis was no toxicity according to the criteria of acute toxic classifications （Ministry of Health PR China）.Consumption of mixture of C. butyricum, Bif. adolescentis and Ent. faecalis at the dose of 5X 105 CFU/kg body weight to 5x106 CFU/kg body weight per day was already beneficial to the ecosystem of the intestinal tract by increasing the populations of good bacteria and reducing the populations of unwanted bacteria, the quantity of consumed probiotics was much lower than the findings of other researchers using one probiotic strain alone. So this mixture of probiotics showed good potential using in food and feed industry.更多还原