【作者】 孟祥晨；

【导师】 霍贵成；

【作者基本信息】 东北农业大学 ， 食品科学， 2002， 博士

【摘要】 双歧杆菌(Bifidobacterium)是人和动物肠道内重要的生理性细菌，它的存在直接影响消化道的健康状况，虽然此菌重要，但其作用机理尚需深入研究。所以本课题通过体内及体外实验方法，共计七个实验，包括所研究菌的分离与鉴定；生长特性的研究；影响其存活因素的研究；对病原菌的生物拮抗：对肠粘膜的粘附与定植；对试验动物的免疫调解机理以及含双歧杆菌产品的开发，系统研究了该菌的生理功能与作用机理及其应用。 应用TPY液体培养基，比较了两株商品菌株(B.adolescentis,Ba;B.infantis,Bi)、两株由产品分离菌株(B.longum,Bl;B.breve,Bbr)以及两株分离自婴儿粪便菌株(B.bifidum,Bb;B.longum-l,Bl-1)的生长与产酸能力，发现：Bi生长速度比Ba快，产酸能力却弱，前者无明显的生长迟滞期，后者却相反。剧和Bbr均无明显的生长迟滞期，培养48hr，菌液吸光值分别为2.348和2.162。培养Bb和Bl-1达48hr，其吸光值分别为1.400和1.447。商业生产株生长及产酸性状优于来自婴儿粪便的新分离株。 应用模拟胃液pH、胃液、肠液、肠胆盐环境，研究Ba、Bi、Bl和Bbr的抗消化道逆环境特性。在pH1.5酸性条件下作用1.5hr，存活率分别为0.14％、17.86％、49.09％和9.53％。在加有胃蛋白酶的模拟胃液中作用1.5hr，存活率分别为：45.39％、97.27％、84.53％和85.51％。在含胰蛋白酶的模拟肠液中作用12hr，存活率分别为：29.17％、47.97％、87.70％和77.27％。Ba、Bl和Bbr的活菌数随胆盐浓度升高而下降，2％胆盐浓度条件下作用12hr，三者的存活率分别为5.92％、13.96％和61.24％。而Bi在各胆盐浓度条件下，均有生长趋势，这种趋势随胆盐浓度升高而下降。消化道逆环境对此菌的存活有不同程度的影响，以Bi和Bl的耐性最好。 Ba、Bl、Bbr和Bi分别与沙门氏菌共同培养20小时后，前三者使沙门氏菌的菌数低于10~2cfu/mL，而Bi中却为1.3×10~8cfu／mL。将pH值接近4的四株菌的上清液和0.1M、pH4的乙酸-乳酸(3：2)混合酸液以一定比例加入培养基中，培养沙门氏菌，发现酸液对沙门氏菌的抑制作用最强：Ba、Bl和Bbr上清液的抑制强度相近，但不及酸液；Bi上清液的抑制作用最差。将酸液和上清液的pH调至7后，对沙门氏菌的生长趋势无影响，但各阶段菌数均低于对照组。 通过扫描电镜观察细菌在仔猪肠粘膜的粘附状况，发现肠道菌群与粘膜表面的粘液层有密切的联系。采用放射性代谢标记方法研究双歧杆菌与仔猪肠粘膜粘液糖蛋白的粘附，发现Ba的粘附能力最强，为44.61％；其次是Bbr，为33.20％；Bi和Bl分别是18.40％和11.78％。相同的实验方法观察到：所实验的糖、初乳粉以及Bi、Bl、大肠杆菌和沙门氏菌上清均能导致Ba粘附下降，而Ba和Bbr上清却有促进其粘附的作用。博土学位论文 双歧杆菌生理功gs＃性及其应用的研究 东北农业大学 应用动物实验方法，研究Bi和 BI对正常小白鼠免疫功能的调节作用以及对免疫力低下模型动物的预防与治疗作用。发现：两株菌对正常小白鼠体重、脾／体重和肾／体重比无显著性影响；对肝／体重比有增加趋势；能显著增加血液中IgA、lgG、C3、C4、IL－2。几－6和1卜8的含量（尸＜0．仍），扭效果优于扭；历可极显著增加lgM的含量（p＜0．01）。两株菌均可显著增加双歧杆菌（尸。0 05）和乳杆菌的数量，显著降低大肠杆菌的数量 （尸＜0．们）。结果表明：两株菌都能增强正常＊AL＊亿小白鼠的免疫功能，并具有良好调节肠道菌群能力，Bi效果优于m。 同时对＊＊m汇小白鼠施以环磷酞胺和双歧秆菌，发现；环磷酞胺导致体重、胸腺／体重、lgA、lgG、lgM、C。、IL－2和IL8显著下降中＜0．OJ），两株菌能显著预防IL2。IL－6和IL－8的下降（p＜0．OJ），Bi效果优于BI，对其他几个指标有轻微恢复作用，效果不显著。两株菌都可增加双歧杆菌与乳杆菌的数量，降低大肠杆菌数量（厂＜0．“）。 对免疫力低下的昆明小白鼠模型施以双歧杆菌后发现：两株菌对胸腺／体重、IL．二和几－8 的下降有轻微缓解作用，效果不显著。能显著增加乳杆菌和双歧杆菌的数量 （p、005），降低大肠杆菌的数量（厂＜00））。 通过优化Bi和 Ba增菌培养基，怪 Bi吸光值提高48％，使Ba生长迟滞期持续时间缩短约4个小时。采用优化条件制成的菌粉在4oC条件下贮存两个月后，菌的存活率在90％以上。 通过研究Bi和 BI在脱脂乳中的生长特性、影响其发酵的因素以及发酵乳的方式优化双歧杆菌酸奶的生产工艺。发现：选用m制作酸奶时，在乳中添加了0．3％酵母粉，低聚糖取代10％的蔗糖，接菌量为10％；选用Bi制作酸奶时，在乳中添加了0石％的酵母粉，低聚糖取代 20％的蔗糖，接菌量为 15％。39C条件下发酵，然后再按 1：l的比例与普通酸奶混合。所制作的酸奶在贮存期间，活菌数在 10’。filflllL以上。 基于上述实验结果，本课题的结论是：Bi和切具有良好的生产和抗逆性条件性状，有极大的开发与应用潜力。双歧杆菌的生物桔抗作用是营养竞争和代谢产物共同作用的结果，代谢产物中除乙酸和乳酸外，还有其他机制在起作用。该菌的粘更多还原

【Abstract】 Bifidobacterium is important inhabitant in intestinal tracts of people and animals. It has beneficial effects on intestinal health. But its functional mechanisms are not clear. Physiological function, functional mechanisms of Bifidobacterium and its applications in foods were investigated systematically by seven experiments in vitro and in vivo. They were composed of isolation and determination of Bifidobacterium, comparison of growth characteristics, factors influencing survivability, bioantaganism to pathogen, adherence and colonization to mucus membrane, immunomodulation mechanisms on trial animals and development of products containing Bifidobacterium.Growth and acidic ability of two commercial strains (B.adolescentis, Ba; B.infantis, Bi) , two strains isolated from commercial products (B.Iongum, Bl; B. breve, Bbr) and two strains isolated from infant faeces (B.b ifidum, Bb; B.Iongum- 1, Bl-1 ) were compared in TPY medium. The results showed that Bi grown faster than Ba. Acidic ability of Bi was lower than that of Ba. There was not obvious delaying stage in growth curve of Bi. Ba was reverse. There were also not obvious delaying stage in growth curve of BI and Bbr. Absorbance at 600nm of BI and Bbr were 2.348 and 2.162 respectively after 48 hours. Absorbance at 600nm of Bb and Bl-1 were 1.400 and 1.447 respectively after 48 hours. Commercial product strains were better than strains isolated from infant faeces in growth and acidic ability.Characteristics on gastrointestinal (GIT) condition tolerance of Ba, Bi, BI and Bbr were investigated in simulative pH of gastric acid, gastric juice, intestinal juice and intestinal bile salt. Survivability of Ba, Bi, BI and Bbr were 0.14%, 17.86%, 49.09% and 9.53% respectively after 1.5 hours in pH1.5 condition, and were 45.39%, 97.27%, 84.53% and 85.51% respectively after 1.5 hours in simulative gastric juice containing pepsin and were 29.17%, 47.97%, 87.70% and 77.27% respectively after 12 hours in simulative intestinal juice containing pancreatin. The amounts of viable Ba, BI, and Bbr decreased with the concentration of bile salt increased. The survivability were 5.92%, 1 3.96%. and 61.24% respectively after 12 hours in simulative intestinal juice containing 2% bile salt. The growth speed decreased with concentration of bile salt increased. The GIT conditions tolerance ability of Bifidobacterium strains was different. The tolerance of Bi and BI were best among the tested strains.The amounts of Salmonella were below 102cfu/mL after 20 hours in co-culturing with Ba, BI, and Bbr. The amounts of Salmonella were 1.3 X 108cfu/mL after 20 hours in co-culturing with Bi. When four strains supernatants and acetic acid-lactic acid (3:2) of pH4 were added to broth media to culture Salmonella, it was found that the inhibitory ability of acetic acid-lacticacid was the highest, then was that of Ba, Bl and Bbr supernatants, and the inhibitory ability of Bi supernatant was the lowest. When the pH of acid liquid and supernatants was adjusted to 7, there was no influence on growth trend of Salmonella. But the amounts of Salmonella in different growth stage were lower than the corresponding stage of the controlled group.The scan electron micrographs showed that the relationship of intestinal microflora with mucus was close. It was found that the adhesive percent of Ba was 44.6%, that of Bbr was 33.20%, that of Bi and Bl were 18.40% and 11.78% respectively by comparison of the adhesion of radioactively labeled Bifidobacterium strains to the mucus. The adhesive percent of Ba decreased when the tested sugars, colostrums powder and the supernatants of Bi, Bl, Salmonella and E.co//.were added, while the adhesive percent of Ba increased when the supernatants of Ba and Bbr were added.The immunomodulation of Bi and Bl on the specific and non-specific immunity of normal mouse, the prevention and the treatment of Bi and Bl on the mouse model with low immunity were investigated. It was found that two strains had no significant effects on weight, spleen/weight and kidney/weight. Ther更多还原