Bacterial isolation and identification of natural Calculus
bovis and study of in vitro cultured Calculus bovis and
natural Calculus bovis on biochemical characteristics
本研究目的為篩選與鑑定天然牛黃的菌株、分析體外培養牛黃與天然牛黃之主要成分及其抗氧化能力。並進一步以二乙基亞硝胺誘發大鼠的肝臟損傷，探討上述兩種牛黃對肝臟的保護效果。
運用不同的方法鑑定分離得到的菌株，例如微生物的型態特性、鑑定細菌脂肪酸使用微生物鑑定系統（Microbial Identification System）、判斷微生物碳源利用生化特性（Biolog Identification System）與16S rDNA 的基因序列分析。本試驗分離得到一株短桿狀格蘭氏陰性菌。使用微生物鑑定系統鑑定細菌脂肪酸與判斷微
生物碳源利用生化特性的鑑定方法得到的結果，在資料庫中已知的微生物，並無此相符合的微生物資料，因此有可能為一株新型微生物，所以更進一步進行基因序列分析。從16S rDNA 基因序列分析得到的結果，在基因庫中亦無法找到相同基因序列的微生物，因此可確認為一株新型的桿菌。
天然牛黃中含有高量膽紅素、膽綠素與必需胺基酸，而且必需胺基酸占全部胺基酸的比值亦較高。體外培養牛黃與天然牛黃的脂肪酸組成皆以飽和脂肪酸為主。天然牛黃中微量元素鋅、鐵與錳的含量亦顯著高於體外培養牛黃。此二種牛黃的主要膽汁成分為膽酸、去氫膽汁酸與鵝去氧膽汁酸。將分離得到的微生物添加入豬膽汁中培養，培養期間樣品pH 值有稍微下降的趨勢。培養一日後微生物迅速增值。研究顯示，體外培養牛黃與天然牛黃皆具有良好的抗氧化能力。天然牛黃的還原力顯著高於體外培養牛黃。兩種牛黃皆具有高的DPPH 自由基清除與螯合亞鐵離子能力。兩種的牛黃皆有強抗氧化能力的抗
氧化劑。因為天然牛黃長期供不應求且價格昂貴，體外培養牛黃具有作為天然牛黃替代品的潛力。
在動物試驗中，大鼠經二乙基亞硝胺處理以誘發肝臟損傷，分析其血清中天門冬胺酸鹽轉胺酶、胺基丙酸轉胺酶、伽瑪麩胺基酸轉胺酶、鹼性磷酶與乳酸去氫酶。大鼠經二乙基亞硝胺處理後，血清中的天門冬胺酸鹽轉胺酶、胺基丙酸轉胺酶、伽瑪麩胺基酸轉胺酶、鹼性磷酶、與乳酸去氫酶皆顯著提高。體外培養牛黃與天然牛黃處理皆具有預防二乙基亞硝胺誘發肝損傷與氧化壓力之作用；顯示試驗動物經服用體外培養牛黃與天然牛黃均具有保護肝臟抵抗二乙基亞硝胺造成
的肝臟損傷的作用。The objectives of the study were to isolate and identify of bacteria of natural Calculus bovis, to analyze the major components of in vitro cultured Calculus bovis and
natural Calculus bovis, the antioxidative capacities of both kinds of Calculus bovis and effects of Calculus bovis on diethylnirosamine induced hepatic injury in rats.
Morphological features, bacterial fatty acid analysis of bacterium using Microbial Identification System, carbon source utilization profiles using Biolog system and 16S
rDNA sequencing were employed to identify isolated bacterium. This isolated bacterium was observed to be Gram-negative, rod-shaped and short chain. The results of
bacterial fatty acid analysis and physiological characteristics were not matched to the database. Based on 16S rDNA sequence analysis, this bacterium was classified as a novel Bacillus species.
The product of natural Calculus bovis was rich in bilirubin and biliverdin. The natural Calculus bovis had higher content of essential amino acids and a higher ratio of
essential amino acids to total amino acids. The dominant fatty acid composition of in vitro cultured Calculus bovis and natural Calculus bovis was saturated fatty acids. The
mineral contents of zinc, iron and manganese of natural Calculus bovis were significantly higher than those of in vitro cultured Calculus bovis. The major bile acids
of both kinds of Calculus bovis were cholic acid, dehydrocholic acid and chenodeoxycholic acid, respectively.
The results showed that the changes of pH values were slightly decreased throughout inoculation with starter. The initial microbial counts increased rapidly after
inoculation at d 1. The in vitro cultured Calculus bovis groups and the natural Calculus bovis group had good antioxidant activities. The reducing ability of natural Calculus bovis was significantly higher than that of in vitro cultured Calculus bovis. The in vitro cultured Calculus bovis and natural Calculus bovis showed high activities of the scavenging activities of DPPH radical and chelating abilities of ferrous ion. The in vitro cultured Calculus bovis and natural Calculus bovis were natural antioxidants with potent antioxidative activities. The in vitro cultured Calculus bovis might therefore be an alternative of natural Calculus bovis because of expensive and deficient natural Calculus bovis.
Aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, alkaline phosphatase and lactate dehydrogenase were determined in serum of rats. The
previous enzymes were estimated as markers of hepatoprotective studies. All the experimental animals administered with diethylnitrosamine were obviously by elevated serum aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase,
alkaline phosphatase and lactate dehydrogenase. The treatment of the in vitro cultured Calculus bovis and natural Calculus bovis prevented the diethylnitrosamine induced hepatic damage and oxidative stress. In conclusion, the results were shown that both of the experimental rats administered with the in vitro cultured Calculus bovis and natural Calculus bovis protected the liver against diethylnitrosamine induced hepatic injury.1 Chapter One -Abstract 1
1.1 Chinese abstract 1
1.2 English abstract 3
2. Chapter Two -Introduction 5
2.1 Introduction 7
2.2 Identification method of bacteria 8
2.2.1 Morphological properties 8
2.2.2 Bacterial fatty acid analysis 8
2.2.3 Determination of physiological characteristics 10
2.2.4 Isolation of 16S rDNA and sequencing 11
2.3 Role of major components in in vitro cultured Calculus bovis and natural Calculus bovis 14
2.3.1 Importance of Calculus bovis as Chinese medical material 14
2.3.2 Utilization of byproduct 15
2.3.3 Formation of natural Calculus bovis 16
2.3.4 Major components 17
2.3.4.1 Bile pigment 17
2.3.4.2 Zinc as an antioxidant 18
2.4 Antioxidative capacity of in vitro cultured Calculus bovis and natural Calculus bovis 21
2.4.1 Damage of reactive oxygen species 21
2.4.2 Antioxidant capacity 22
2.4.3 Natural antioxidant substances 23
2.4.4 Functional foods 24
2.5 Perspectives of chemoprevention of hepatoprotective properties 26
2.5.1 Chemoprevention 26
2.5.2 Hepatoinjury 30
2.5.3 Liver injury induced by diethylnitrosamine 31
2.5.4 Hepatoprotection 31
2.6 Objectives of this thesis research 34
2.7 References 35
Chapter Three -Identification of a novel Bacillus species isolated from Calculus bovis 42
3.1 Abstract 43
3.2 Introduction 44
3.3 Materials and methods 46
3.4 Results and discussion 52
3.4.1 Morphological properties 52
3.4.2 Bacterial fatty acid analysis 55
3.4.3 Determination of physiological characteristics 57
3.4.4 Isolation of 16S rDNA and sequencing 60
3.5 Conclusion 64
3.6 References 65
Chapter Four -Study of in vitro cultured Calculus bovis and natural Calculus bovis on major components 68
4.1 Abstract 69
4.2 Introduction 70
4.3 Materials and methods 72
4.4 Results and discussion 79
4.4.1 Yield percentage of in vitro cultured Calculus bovis 79
4.4.2 Bilirubin and biliverdin determination 82
4.4.3 Amino acid determination of Calculus bovis 84
4.4.4 Ratio of essential amino acids to total amino acids of Calculus bovis 87
4.4.5 Fatty acid analysis of Calculus bovis 89
4.4.6 Mineral content of Calculus bovis 92
4.4.7 Bile acid content of Calculus bovis 94
4.5 Conclusion 96
4.6 References 97
Chapter Five -Antioxidative capacity of in vitro cultured Calculus bovis and natural Calculus bovis 101
5.1 Abstract 101
5.2 Introduction 103
5.3 Materials and methods 105
5.4 Results and discussion 110
5.4.1 Determination of pH values 110
5.4.2 Microbiological determination 110
5.4.3 Measurement of antioxidant activity 114
5.4.4 Determination of reducing capacity 117
5.4.5 Analysis of DPPH radical scavenging activity 118
5.4.6 Chelating ability of ferrous ion 122
5.5 Conclusion 125
5.6 References 126
Chapter Six -Study of in vitro cultured Calculus bovis and natural Calculus bovis on diethylnitrosamine induced hepatic injury in rats 130
6.1 Abstract 131
6.2 Introduction 132
6.3 Materials and methods 134
6.4 Results and discussion 138
6.4.1 Aspartate aminotransferase 138
6.4.2 Alanine aminotransferase 142
6.4.3 Gamma glutamyl transferase 146
6.4.4 Alkaline phosphatase 149
6.4.5 Lactate dehydrogenase 152
6.5 Conclusion 155
6.6 References 156
Chapter Seven -Conclusion 15
