小米膳食纤维作为主要碳源对益生菌生长和发酵过程中短链脂肪酸产量的影响研究

论文摘要

小米是许多亚、非洲发展中国家的主食。本研究旨在评价小米膳食纤维分级物作为益生菌发酵主要碳源的可能性及其对生长、短链脂肪酸生产和附着机制的影响。在第一阶段研究中,分别从珍珠小米、狐尾小米、黍、龙爪小米这四种商业小米中提取了膳食纤维,并将其分级为总膳食纤维、不可溶膳食纤维、可溶膳食纤维。目前的研究重点主要集中在全谷物方向。小米膳食纤维含量在0.55-13.70g每100g范围内。总膳食纤维含量随着小米种类的不同而出现显著性差异。针对总膳食纤维含量,珍珠小米最高,而龙爪小米最低。不可溶膳食纤维的相对含量的趋势与总膳食纤维相似,（珍珠小米<狐尾小米<黍<龙爪小米）。然而,可溶性膳食纤维相对含量表现出不同,龙爪小米中可溶性膳食纤维相对含量最高,狐尾小米、珍珠小米、黍的可溶性膳食纤维含量分别为11.11%,9.57%和10.18%。在第二阶段研究中,四位志愿者摄入为期三个月的小米膳食后,分别采集了粪便中的微生物群样本。然而,在粪便微生物接种的小米膳食纤维分级物的体外发酵实验中,乳酸杆菌和双歧杆菌增长率分别达到9-12%和3-5%。在肠道微生物发酵后24小时,浓度为3%（v/v）的膳食纤维才足以促进其生长。相较于可溶性膳食纤维和不可溶膳食纤维,总膳食纤维的发酵对双歧杆菌和乳酸杆菌数量的增长更佳,而这一优势并未显示出显著性。在人类粪便接种发酵实验中,pH变化显著（p<0.05）,然而光密度和活菌数的变化并不显著。在志愿者摄入小米膳食前后,分别将其粪便微生物群接种于各部分膳食纤维中,接种物进行厌氧培养。通过汽液色谱对发酵生产的短链脂肪酸进行分析,将摄食三个月小米膳食的志愿者的粪便菌群接种于小米膳食纤维,短链脂肪酸产量显著高于摄食小米膳食前。以珍珠小米为碳源的发酵实验中短链脂肪酸产量最高,龙爪小米最低。在发酵6-24小时,短链脂肪酸产量最高。在三种膳食纤维中,以总膳食纤维为底物的短链脂肪酸发酵产量最高。实验结果表明,相对于其他种类小米,珍珠小米显示出作为膳食纤维来源的优势。根据第一阶段的研究结果,选择两种小米（珍珠小米和狐尾小米）以及四种益生菌（鼠李糖乳杆菌、嗜酸乳杆菌、长双歧杆菌、两歧双歧杆菌）,以小米膳食纤维为碳源进行体外发酵,生产短链脂肪酸。在发酵实验中,双歧杆菌显示出更长效缓慢而持续的发酵规律,这与乳酸杆菌截然不同。在培养基促进益生菌生长的实验中,乳酸菌在24小时发酵后显示出最高生长率,在不同的底物条件下均如此,然而双歧杆菌利用1.5%小米膳食纤维为碳源的条件下,在24-48小时的发酵后显示出最高生长率。在各种不同的膳食纤维底物中,短链脂肪酸产量为,总膳食纤维>可溶性膳食纤维>不可溶膳食纤维,不同小米种类的实验均表现这一结果。这表明,总膳食纤维最适合作为短链脂肪酸的生产碳源。乳酸菌和双歧杆菌分别消化60-80%和75-85%的小米膳食纤维分级物。短链脂肪酸产量由大到小乙酸>丙酸>丁酸。此外,还研究了采用四种益生菌不同组合的组合培养物对小米膳食纤维的发酵作用。值得注意的是,由不同属微生物构成的培养物较同一种微生物有更高的细胞数量。然而,相较于纤维,发酵效果最佳的组合培养物显示出对于葡萄糖的更高底物偏好性。而相对于单一属微生物,由多属微生物构成的组合培养物在发酵过程中到达更低的pH。这表明在发酵过程中组合培养物中存在协同作用。由鼠李糖乳杆菌、长双歧杆菌、两歧双歧杆菌组成的培养物可以在各种底物中生长（总膳食纤维、可溶性膳食纤维、不溶性膳食纤维和葡萄糖）。当使用菌种组合培养的情况下,小米膳食纤维发酵的短链脂肪酸产量显著高于此前研究中的单一菌种发酵。在组合发酵小米膳食纤维分级物的实验中,乙酸是最主要的产物,占每小时短链脂肪酸产量的90%。经纯化的双歧杆菌和乳酸杆菌在接种20-40分钟内即可附着于膳食纤维分级物。双歧杆菌对小米纤维的附着率达到40-55%,而乳酸杆菌仅为40%。氯化钠和吐温80对细菌细胞的附着影响不明显,但多聚糖显著降低吸附率。最适附着温度为37,且只有活细胞表现出附着现象。在蛋白酶和低pH（pH3-4.15）环境下,细胞定植受到抑制。不同益生菌组合对纤维的定植受温度,pH,蛋白消化酶的影响,而胆酸盐和氯化钠对其定植影响不大。益生菌共同培养物对纤维的附着机制与单一菌种附着相同。细菌细胞对小米纤维的定植受温度影响,在实验条件下,细菌细胞附着能力在37环境达到最大。当鼠李糖乳杆菌、嗜酸乳杆菌、长双歧杆菌、两歧双歧杆菌共同培养时,乳酸杆菌和双歧杆菌的附着能力有显著增强。本研究包括低氧以及高还原实验环境的模拟,这与人类肠道末端结肠的生理环境相似。本研究表明,小米纤维可以作为益生菌菌群的良好碳源。

论文目录

• ACKNOWLEDGEMENTS
• DEDICATION
• ABSTRACT
• 摘要
• LIST OF ABBREVIATIONS
• Table of Contents
• CHAPTER 1 GENRAL INTRODUCTION AND REVIEW OF LITERATURE
• 1.1 Introduction
• 1.2 Concept of Dietary fibre
• 1.2.1 Effects of dietary fibre on human health
• 1.2.2 Fibre determination
• 1.3 Millet
• 1.3.1 Millet varieties
• 1.3.1.1 Pearl Millet （pennisetum glaucum）
• 1.3.1.2 Foxtail Millet （Setaria italica）
• 1.3.1.3 Proso Millet （Panicum miliacum）
• 1.3.1.4 Finger Millet （Eleusine coracana）
• 1.3.2. Nutrition content of millet
• 1.4 Fermentation of dietary fibre
• 1.4.1 Mechanism of acetate, propionate and butyrate production by Bifidobacteria andLactobacillus species.
• 1.4.2 Factors affecting SCFA formation during fermentation
• 1.5 Bacterial adhesion
• 1.6 Statement of the problem and justification of research
• 1.7 Objectives of the research
• 1.7.1 Main Objective
• 1.7.2 Research Specific Objectives
• 1.8 References
• CHAPTER 2 Estimation of Dietary Fibre Content of Millet Varieties and Their Influence on the Growth of Human Faecal Microflora during InVitro Fermentation
• 2.1 Introduction
• 2.2 Materials and methods
• 2.2.1 Millet varieties
• 2.2.2 Sample preparation
• 2.2.3 Extraction and estimation of soluble, insoluble and total dietary fibre from millet varieties
• 2.2.4 Digestion of original sample with enzymes
• 2.2.5 Soluble/insoluble dietary fibre determination
• 2.2.6 Total dietary fibre determination
• 2.2.7 Filtration
• 2.2.8 Volunteers and their Dietary Schedule
• 2.2.9 Analysis of faecal samples
• 2.2.10 In vitro fermentation with human faecal inocula
• 2.2.11 Determination of pH, viable count and optical density
• 2.3 Statistical analysis
• 2.4 Results and discussion
• 2.4.1 Percentage of dietary fibre contents in millet varieties
• 2.4.2 Purity of extracted fibres
• 2.4.3 Faecal bacteria cell counts
• 2.4.4 Effect of millet varieties on the growth of faecal microorganisms
• 2.4.5 Determination of pH and optical density (OD
• 2.5. Conclusions
• 2.6. References
• CHAPTER 3 EFFECT OF MILLET FIBRE FRACTIONS ON IN VITRO FERMENTATION PRODUCTION OF SHORT CHAIN FATTY ACIDS USING HUMAN FAECAL MICROFLORA
• 3.1 Introduction
• 3.2 Materials and methods
• 3.2.1 Millet varieties
• 3.2.2 Fermentation substrate
• 3.2.3 Volunteers and their Dietary Schedule
• 3.2.4 Analysis of faecal samples and in vitro fermentation of extracted millet fbre fractions with faecal inocula
• 3.2.5 Determination of SCFA
• 3.2.6 Preparation of samples and standards for gas chromatography
• 3.2.7 Chemicals and reagents to prepare standards
• 3.2.8 GC experimental conditions
• 3.2.9 Stock standard solution preparation
• 3.2.10 Preparation of standard mix solution
• 3.2.11 Standard curve
• 3.2.12 Standards Retention time
• 3.2.13 Quantification of SCFA
• 3.3. Statistical analysis
• 3.4. Results and Discussion
• 3.4.1 SCFA formation before and after the consumption of diet
• 3.4.3 Variation in individual SCFA formation after fermentation of millet varieties with subject faecal microflora
• 3.5 Conclusion
• 3.6 References
• CHAPTER 4 INFLUENCE OF MILLET FIBRE FRACTIONS IN STIMULATING GROWTH OF PURE CULTURES OF PROBIOTICS AND ENHANCEMENT OF SHORT CHAIN FATTY ACID PRODUCTION UNDER IN VITRO FERMENTATION
• 4.1 Introduction
• 4.2 Materials and methods
• 4.2.1 Chemicals and Bacterial strains
• 4.2.2 Fermentation substrate
• 4.2.3 Preparation of cell suspension
• 4.2.4 In vitro fermentation of extracted millet fbre fractions using pure cultures of Lactobacillus and Bifidobacterium species
• 4.2.5 Determination of pH, viable count and optical density
• 4.2.6 Calculation of Specific growth rate
• 4.2.7 Determination of SCFA
• 4.2.8 Measurement of indigestible fibre percentage
• 4.2.8.1 Measurement of the indigestible soluble dietary fibre after fermentation of SDF
• 4.2.8.2 Measurement of the indigestible insoluble dietary fibre after fermentation of IDF
• 4.2.8.3 Measurement of indigestible total dietary fibre after fermentation of TDF
• 4.2.8.4 Calculation of % dry matter disappearance
• 4.3 Statistical analysis
• 4.4 Results and discussion
• 4.4.1 Probiotic growth during fermentation with millet fibre fractions
• 4.4.2 Optical density readings during fermentation
• 4.4.3 pH
• 4.4.4 Biomass yield
• 4.4.5 Specific growth rates
• 4.4.6. SCFA production
• 4.4.7 Indigestible material percentage
• 4.5 Conclusion
• 4.6 References
• CHAPTER 5 INFLUENCE OF MILLET FIBRE FRACTIONS IN STIMULATING GROWTH OF PURE CULTURES OF PROBIOTICS AND ENHANCEMENT OF SHORT CHAIN FATTY ACID PRODUCTION UNDER IN VITRO FERMENTATION
• 5.1 Introduction
• 5.2 Materials and Methods
• 5.2.1 Fermentation substrate
• 5.2.2 Chemicals and Bacterial strains/ Co-cultures
• 5.2.3 Cell suspension preparation
• 5.2.4 In vitro fermentation of extracted millet fbre fractions using co-cultures of Lactobacillus and bifidobacterium species
• 5.2.5 Determination of pH, viable count and optical density
• 5.2.6 Calculation of Specific growth rate
• 5.2.7 Determination of SCFA
• 5.2.8 Measurement of indigestible fibre percentage
• 5.3 Statistical analysis
• 5.4 Results and discussion
• 5.4.1 Probiotic co-cultures growth during fermentation with millet fibre fractions
• 5.4.2 Optical density
• 5.4.3 pH
• 5.4.4 Specific growth rates
• 5.4.5 SCFA production
• 5.4.6 Indigestible material percentage
• 5.5 Conclusion
• 5.6 References
• CHAPTER 6 STUDY of ADHESION MECHANISM OF PROBIOTICS TO INSOLUBLE, SOLUBLE AND TOTAL DIETARY FIBRE FRACTIONS OF MILLET
• 6.2 Materials and methods
• 6.2.1 Millet dietary fibre fractions
• 6.2.2 Bacterial culture media
• 6.2.3 Bacterial strains
• 6.2.4 Cell suspensions preparation
• 6.2.5 Bacterial adhesion to fibre
• 6.2.6 Effect of probiotic bacteria growth on adhesion
• 6.2.7 Study of probiotic adhesion mechanism
• 6.2.7.1 Fresh medium
• 6.2.7.2 Spent medium
• 6.2.7.3 Pepsin-treated spent medium
• 6.2.7.4 Pepsin-treated fibre
• 6.2.7.5 Pepsin-treated cells
• 6.2.7.6 Proteinase K-treated cells
• 6.2.7.7 Phosphate buffer
• 6.2.7.8 NaCl
• 6.2.7.9 Tween
• 6.2.7.10 Heat-treated cells, cells treated at room temperature （22-25°C） and human body temperature （37°C）
• 6.2.7.11 Effect of potential inhibitors
• 6.2.8 Bacterial adhesion under conditions of the human stomach and small intestine
• 6.2.8.1
• 6.2.8.2 Effect of acid and pepsin
• 6.2.8.3 Effect of bile
• 6.2.8.4 Effect of pancreatin
• 6.3 Statistical analysis
• 6.4 Results and discussion
• 6.4.1 Influence of time on adhesion
• 6.4.2 Influence of substrate concentration
• 6.4.4 Effect of growth
• 6.4.5 Effect of chemical
• 6.4.6 Effect of pH
• 6.4.7 Effect of pepsin and Proteinase
• 6.4.8 Effect of potential inhibitor
• 6.4.9 Effect of medium
• 6.4.10 Effect of simulated gastrointestinal conditions
• 6.5 Conclusion
• 6.6 References
• CHAPTER 7 EFFET OF ENVIRONMENTAL FACTORS ON THE ADHESION OF PROBIOTICS CO-CULTURES TO INSOLUBLE, SOLUBLE AND TOTAL DIETARY FIBRE FRACTIONS OF MILLET
• 7.1 Introduction
• 7.2 Materials and methods
• 7.2.1 Millet dietary fibre fractions
• 7.2.2 Bacterial culture media
• 7.2.3 Bacterial strains （Probiotic Co-cultures）
• 7.2.4 Cell suspensions preparation
• 7.2.5 Bacterial adhesion to fibre
• 7.2.6 Effect of probiotic bacteria growth on adhesion
• 7.2.7 Study of probiotic co-cultures adhesion mechanism
• 7.2.8 Probiotic co-cultures adhesion to fibre under modified conditions of the human stomach and small intestine
• 7.2.8.1 Effect of pH
• 7.2.8.2 Effect of acid and pepsin
• 7.2.8.3 Effect of bile
• 7.2.8.4 Effect of pancreatin
• 7.3 Statistical analysis
• 7.4 Results and discussion
• 7.4.1 Probiotic co-cultures adhesion to millet dietary fibre fractions
• 7.4.2 Effect of potential inhibitor
• 7.4.3 Effect of chemical
• 7.4.4 Effect of medium
• 7.4.5 Effect of enzyme
• 7.4.6 Effect of temperature
• 7.4.7 Effect of pH
• 7.4.8 Effect of substrate concentration
• 7.4.9 Effect of time and growth
• 7.4.10 Influence of simulating gastrointestinal conditions
• 7.5 Conclusion
• 7.6 Reference
• GENERAL CONCLUSIONS AND RECOMMENDATIONS
• 1. General conclusions
• 2. Key Innovation of thesis
• 3. Recommendations
• LIST OF PUBLICATIONS
• APPENDICES