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研究生: 馬力可
Malick S. Bah
論文名稱: 大蒜提取物和海草酸鈣膠囊化在酸性條件下對Weisella confusa存活率的影響
Effects of garlic extract and calcium alginate encapsulation on the survival of Weisella confusa under simulated gastric juice treatment
指導教授: 郭嘉信
Jia-Hsin Guo
學位類別: 碩士
Master
系所名稱: 國際學院 - 熱帶農業暨國際合作系
Department of Tropical Agriculture and International Cooperation
畢業學年度: 106
語文別: 英文
論文頁數: 84
中文關鍵詞: 大蒜萃取抗菌微膠囊化海藻酸鈣益生菌
外文關鍵詞: garlic, extract, antimicrobial, encapsulation, calcium-alginate, probiotic
DOI URL: http://doi.org/10.6346/THE.NPUST.TAIC.015.2018.D04
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  • 大蒜是一種著名的藥用草本植物,具有廣泛的抗菌特性和癒合能力。該植物也被報導為益菌生,並可能改變腸道微生物群的組成,有利於乳酸菌生長。許多研究表明在益生菌為主的產品中益生菌在胃液模擬模型的酸性條件下存活率差。因此,提供益生菌活細胞物理保護是目前受到相當大的關注。海藻酸鈣中益生菌的微膠囊化是一種流行的技術,對酸性條件下的益生菌存活具有相當大的影響。因此,本研究的目的是研究大蒜提取物和海藻酸鈣包裹通過擠壓技術對酸性條件下Wesisella confusa細胞存活率的影響。該研究的結果表明,大蒜提取物對土麴黴(Aspergillus terreus)和金黃色葡萄球菌(Staphyloccocus aureus)具有一定劑量依賴性的抗菌活性。此外,發現了一種抵抗大蒜提取物抗菌活性的益生菌菌株。通過聚合酶鏈反應(PCR)擴增16S rRNA。基於核苷酸序列同源性和演化系統分析,將分離物鑑定為Weissella confusa(NCBI 基因庫登錄號:MG928528.1)。在NCBI-基因數據庫中進行比較時,分離的菌株揭示了序列與Weisella confusa原型的高度相似性(99%)。模擬胃部酸性條件顯示對Weisella confusa細菌的存活率具有99.9%的負面影響。然而,海藻酸鈣和大蒜提取物包裹Weisella confusa細菌細胞表明在酸性條件下存活率增加。由此推斷,海藻酸鈣和大蒜提取物
    膠囊化可作為在酸性條件下保護益生菌良好的替代選擇,並作為一種新型的食品應用。

    Garlic is a renowned medicinal herb with extensive antimicrobial properties and healing powers. This plant is also reported as a prebiotic and may change the composition of intestinal microbiota in favor of lactic acid bacteria. Numerous studies have reported the poor survival of probiotics in probiotics base products and in acidic conditions in a simulated model of gastric juice. Hence, providing probiotic living cells with a physical barrier is an approach currently receiving considerable interest. Microencapsulation of probiotics in calcium alginate is a modern technique that has considerable effects on probiotic survival in acidic condition. Thus, the aim of this study was to study the effects of garlic extract and calcium alginate encapsulation via extrusion technique on the survival rate of Weisella confusa under simulated gastric juice treatment. Results from this study shows that, garlic
    extract exhibited dose dependent antimicrobial activity against Aspergillus terreus and Staphyloccocus aureus. Moreover, a probiotic strain that resist the antimicrobial activity of garlic extract was discovered. The 16S rRNA was amplified by polymerase chain reaction (PCR). The isolate was identified as Weissella confusa (NCBI GenBank accession number: MG928528.1) based on nucleotide homology and phylogenetic analysis of the sequence. The isolated strain when compared in NCBI- GeneBank database reveals high similarity (99%) of the sequence Weisella confusa. The simulated gastric acidic condition shows to have a negative effect of 99.9% on the survival rate of the Weisella confusa bacteria. However, calcium alginate and garlic extract encapsulation of Weisella confusa bacteria cells indicated an increase in survival rate under acidic condition. It can be deduced that, calcium-alginate and garlic extract encapsulation of probiotic represents a good alternative to protect these bacteria under acidic condition and application as a novel food product.

    TABLE OF CONTENTS
    摘要………..…….………………………..………………….…………..……I
    ABSTRACT………………………………..……………....…........….….…III
    ACKNOWLEDGEMENTS.……………..………………………..…..……..V
    TABLE OF CONTENTS………………..…...….….......……..…………....VII
    LIST OF FIGURES..…..……………………..…...…...….….…..…..…...….X
    LIST OF TABLES……....……………………...…….…….………..…..….XI
    LIST OF ABBREVIATIONS…..……………..…........……………...….....XII

    1. INTRODUCTION………………………...…………….…………...….…1
    1.1 Background and motivation.…...…………………....…..……….…...1
    1.2 Importance of study…………..….…………….…,………………….3
    1.3 Research objectives…………………………….……….………….…4
    1.4 Experimental flow chart….….…………………………..……………5

    2. LIETRATURE REVIEWS…………………...….……………….……….6
    2.1 Garlic background information……………..……..…………….…….6
    2.1.1 Antimicrobial properties of garlic ……..…………………….....7
    2.2 Role of garlic for gastrointestinal health ………...……….…………...9
    2.3 Probiotics………………………………….……….………………...10
    2.3.1 Weisella confusa as probiotics………..…………….……..…...12
    2.3.2 Health functions of probiotics ………………....………..……..13
    2.3.3 Factors affecting Probiotics …………………....………….…..15
    2.3.4 Application of probiotics in the food industry…..……………..16
    2.4 Microencapsulation of probiotics ……….…………...……...……...18
    2.4.1 Microencapsulation in calcium alginate …..……...…...……....19

    3 MATERIALS AND METHODS…………...……….……………….…21
    3.1 Materials………...………..………………………...………….….…21
    3.1.0 Plant material………………………..……...…………..……..21
    3.1.1 Equipment…..…………………….…...……………….............21
    3.1.2 Reagents (source)…...…. ……………...………...……..……...21
    3.2 Methods………………………………………………………...……22
    3.2.1 Extraction……………………………….…...…………..……..22
    3.2.2 Growth media…………………….………………….….…......23
    3.2.3 Microorganisms..........................................................................24
    3.2.4 Design system to create anaerobic condition …..…...…………25
    3.2.5 Method of encapsulation…………………….……..…….....….26
    3.2.6 Antifungal activity of garlic against Aspergillus trreus….….....26
    3.2.7 Antibacterial activity of garlic against S. aureus…….…..….....27
    3.2.8 Garlic extract’s anti-probiotic screening test...…….……..…....27
    3.2.9 Isolate bacterium identification………………...…….………..28
    3.2.9.1 Isolate bacterium phenotypic characteristics…..….…….28
    3.2.9.2 Isolate bacterium genotypic identification……..…..…...28
    3.1.9.2.1 Genomic DNA extraction…….……………...…29
    3.1.9.2.2 Polymerase chain reaction (PCR)………;….…..30
    3.1.9.2.3 DNA purification…...……….………….…....…31
    3.1.9.2.4 Gel electrophoresis………………..…………....32
    3.1.9.2.5 16s RNA sequencing …..………….……;;;.…...32
    3.2.10 The effects of acidic condition on the survival rate of Weisella confusa confusa.............…………….………..……….……...33
    3.2.10.1 The effects of acidic condition on the survival rate of Weisella confusa free cells….................................……34
    3.2.10.2 The effect of acidic condition on the survival rate of encapsulated Weisella confusa cells…….............…….35
    3.2.11 Statistics………………...……….……...…….……….….......36

    3. RESULT AND DISCUSSSIONS……………...…………...….…...…..37
    4.1 Antifungal activity of garlic extract against Aspergillus terreus…..37
    4.2 Antibacterial activity of garlic against Staphylococcus aureus........39
    4.3 Garlic extract’s anti-probiotics test...…….…………..…...…....…..41
    4.4 Isolate bacterium morphological characteristics……...…....…..…..43
    4.5 The strain genomic identification ……...………………..…....……44
    4.6 The strain 16s RNA gene sequencing ……………….…...…….….44
    4.7 The effects of acidic condition on the survival rate of Weisella confusa free cells and mixture of Weisella confusa cells and garlic extract ……………………………………………………………....48
    4.8 The effect of acidic condition on the survival rate of encapsulated Weisella confusa cells and encapsulation of a mixture of Weisella confusa cells and garlic extract with calcium alginate…...................50
    4.9 Mean percentage (%) survival loss of Weisella confusa free cells and encapsulated cells under acid condition ................................……....52

    4. CONCLUSIONS…………...……………………...……......…..…….54
    REFERENCES………………..………………………….………………….57
    APPENDIX…………....………...……………………….…………….…....75
    BIOSKETCH OF AUTHOR………………………………………………...83

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