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
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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。
Millet is the staple food in many developing countries of Asia and Africa. This study wasaimed to evaluate, the potential of millet dietary fibre fractions as a main carbon source of probioticfermentation and effect on growth, SCFA production and adhesion mechanism of probiotic bacteria. Infirst phase of study, Dietary fbres were extracted from four commercially available millet varietiesPearl millet (pennisetum glaucum), Foxtail millet (Setaria italica), Proso millet (Panicum miliacum),Finger Millet (Eleusine coracana) and separated into individual fractions of total dietary fbre (TDF),insoluble dietary fbre (IDF) and soluble dietary fbre (SDF). The current study focused on the “wholegrain”. The dietary fibres of millet varied between0.55-13.70g per100g.TDF varied significantlyamong tested millet varieties. Pear millet (PM) variety gave a highest yield of TDF while the fingermillet (FM) produced the least. The relative proportions of IDF in the different millet varieties were thesame as for TDF (Pearl millet (PM)SDF>IDF irrespective of millet variety, indicating that TDF is the best possible dietary fibre forSCFA production. Lactobacillus and Bifdobacterium spp. digested60–80%and75–85%of the milletfbre fractions from both millet samples respectively. The quantity of different SCFAs produced was inthe order acetate>propionate>butyrate. The fermentation of millet dietary fibre using nine co-culturesof four probiotics was also investigated. Interestingly, co-cultures of different genus showed more cellcount than combinations from same genus. However, maximum co-cultures showed more preferencefor glucose than fibre as a substrate. Co-cultures prepared of more than one genus reached a lower pHthan those made up of bacteria from the same genus, providing more evidence for the synergisticassociation in these co-cultures. Co-culture BB+BL+LR showed growth on all substrates tested (TDF,SDF, IDF and Glucose). Millet dietary fibre fractions fermentation with most of the co-culturessignificantly increased (p<0.05) the production of SCFA when compared to pure cultures results in ourprevious study. During all co-culture fermentation with fibre fractions, acetate was found the mostpredominant SCFA and contributed to more than90%of the total SCFA formation at each hour. Purebifidobacteria and lactobacilli adhered to dietary fibre fractions within20to40minutes. A higherpercentage of Bifidobacteria almost40-55%adhered to millet fibre while lactobacilli percentage wasless than40%. Adhesion was not influenced by NaCl or Tween80but, was decreased bypolysaccharides. The optimum temperature for adhesion was found37°C and live cells found essentialfor adhesion. Adhesion was significantly inhibited by protease and by low pH (pH3-4.15). Whileadhesion of probiotic combinations to fibre was influenced by the temperature, pH, and proteindigestive enzymes, but not affected by bile and NaCl. The probiotic co-cultures appear to have similarmechanisms of adhesion as the individual probiotics. Colonization of millet fibre by bacterial cells wasinfluenced by the temperature, with adhesion observed higher at37°C. This study observed enhancedadhesion for BB+BL and LA+LR when these combinations were together as BB+BL+LA+LR. Thisstudy involved low oxygen and highly reducing experimental conditions which were similar to humangut distal colon. Millet fibre was found a suitable substrate for probiotic microflora.
Millet is the staple food in many developing countries of Asia and Africa. This study wasaimed to evaluate, the potential of millet dietary fibre fractions as a main carbon source of probioticfermentation and effect on growth, SCFA production and adhesion mechanism of probiotic bacteria. Infirst phase of study, Dietary fbres were extracted from four commercially available millet varietiesPearl millet (pennisetum glaucum), Foxtail millet (Setaria italica), Proso millet (Panicum miliacum),Finger Millet (Eleusine coracana) and separated into individual fractions of total dietary fbre (TDF),insoluble dietary fbre (IDF) and soluble dietary fbre (SDF). The current study focused on the “wholegrain”. The dietary fibres of millet varied between0.55-13.70g per100g.TDF varied significantlyamong tested millet varieties. Pear millet (PM) variety gave a highest yield of TDF while the fingermillet (FM) produced the least. The relative proportions of IDF in the different millet varieties were thesame as for TDF (Pearl millet (PM)
引文
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