本文選題：益生菌　切入點：乳酸菌　出處：《江南大學》2017年博士論文

【摘要】：鋁(Al,aluminum)不是人體必需的金屬元素并且沒有任何生理功能,但它卻廣泛存在于自然界中(地殼中含量最多的金屬元素),并在日常生活中被廣泛應(yīng)用。鋁離子可以蓄積在哺乳動物的大多數(shù)組織并造成損傷和病變,其中對神經(jīng)系統(tǒng)的損傷被廣泛研究。傳統(tǒng)螯合劑療法雖然可以促進鋁離子的排出,但具有不可避免的副作用,安全性存在缺陷。乳酸菌被公認為安全的食品級微生物,因已有報道表明乳酸菌可以吸附多種毒性金屬,維持腸道微生態(tài)平衡,并通過腸道菌群-腦腸軸影響神經(jīng)系統(tǒng)和行為表現(xiàn),表明其具備緩解鋁離子毒性的潛力,而且目前國內(nèi)外還沒有利用乳酸菌緩解鋁離子毒性的相關(guān)報道。因此本研究探索乳酸菌在緩解鋁離子毒性效應(yīng)中的效果及相關(guān)機制,用以深層次開發(fā)乳酸菌的新益生功能,以期挖掘更具應(yīng)用價值的益生功能乳酸菌,開發(fā)出新的解決鋁毒性問題的膳食策略和應(yīng)用。主要研究結(jié)果如下:建立了體外緩解鋁離子毒性潛力菌株的篩選模型,具體特性如下:高效的鋁離子吸附能力;優(yōu)良的鋁離子耐受能力和胃腸道的耐受能力;優(yōu)秀的抗氧化能力,可緩解鋁離子暴露帶來的氧化損傷。依據(jù)以上評價指標對30株乳酸菌菌株進行篩選,發(fā)現(xiàn)植物乳桿菌CCFM639在初始鋁離子濃度為5mg/L和50mg/L的條件下,其均體現(xiàn)出最強的吸附能力,分別為64.54%和26.83%;而且該菌株的鋁離子耐受能力特別突出,可以達到甚至超過2480 mg/L的最小抑菌濃度,即MIC,顯著高于該試驗的其他菌株測試能力。該菌株在模擬胃液和腸液中的存活率分別為90.69%和91.86%,說明其可以在腸道內(nèi)保持了較高的活性;同時,對抗氧化四個方面(羥自由基清除率、DPPH清除率、抗脂質(zhì)過氧化能力和還原能力)的能力進行了測試,結(jié)合主成分分析進一步綜合評價得出CCFM639具有較好的抗氧化能力。綜上,選擇植物乳桿菌CCFM639進行后續(xù)研究。利用傅里葉紅外光譜、超薄切片透射電鏡、掃描電鏡聯(lián)用能量色散X射線光譜和全基因組序列解析,確定了CCFM639菌體表面的多種功能基團參與了金屬鋁離子的吸附過程,多個功能基因參與緩解鋁離子毒性作用。通過急性和慢性鋁離子暴露動物模型的綜合效果評價,證明植物乳桿菌CCFM639菌株在鋁離子暴露動物模型中具有緩解鋁離子毒性作用。在急性實驗中,該菌株明顯增加存活率,促進糞便鋁離子的排出、降低鋁離子在血液、組織和器官中的蓄積、緩解氧化應(yīng)激狀態(tài),進而減輕肝臟的病理病變和降低異常肝細胞的數(shù)量。在慢性實驗中,該菌株降低鋁離子在血液、組織和器官中的蓄積和損傷、調(diào)節(jié)組織器官中微量元素的水平,減輕氧化應(yīng)激和生理生化指標、緩解組織病理損傷,以及完全避免了鋁離子暴露導(dǎo)致的羅非魚死亡。腸道是植物乳酸桿菌能夠直接發(fā)揮作用的器官,同時也是鋁離子的主要吸收部位,因此進一步研究了該菌株對腸道屏障及腸道功能的調(diào)控作用,以解析其緩解鋁毒性的具體機制。在體外腸道模型(Batch Culture Fermentation Model)中,鋁離子暴露顯著降低了雙歧桿菌和乳桿菌的數(shù)量,促進了擬桿菌、梭狀芽孢桿菌和腸桿菌的數(shù)量。而植物乳桿菌CCFM639對部分有益菌群有顯著提升作用,其中對雙歧桿菌和乳桿菌的作用最明顯。通過對體外腸道模型代謝產(chǎn)物水平的分析結(jié)果顯示,CCFM639可顯著提高代謝產(chǎn)物中丁酸鹽和乳酸鹽水平。在HT-29細胞模型和慢性鋁離子暴露小鼠模型中,鋁離子暴露破壞腸道屏障,增加了腸道通透性,損傷過程涉及氧化應(yīng)激,促炎反應(yīng)和緊密連接蛋白(TJs)的損傷。而灌胃CCFM639顯著緩解氧化應(yīng)激和促炎因子水平,對TJs的損傷也有一定恢復(fù)作用,顯著增加血清內(nèi)毒素水平,增加腸道通透性。在慢性鋁離子暴露羅非魚模型中,在門水平,鋁離子暴露顯著增加Bacteroidetes的豐度,而減少Firmicutes的豐度。在屬水平,鋁離子暴露明顯降低Plesiomonas和Deefgea的豐度,大大增加Flavobacterium,Enterovibrio,Porphyromonadaceae和Comamonadaceae的豐度。飼料添加CCFM639可顯著增加羅非魚糞便中的Lactobacilli含量,提高Deefgea的豐度,而降低Comamonadaceae的豐度。以上結(jié)果表明,植物乳桿菌CCFM639可調(diào)節(jié)羅非魚腸道菌群的結(jié)構(gòu)和組成,明顯提高腸道菌群中的有益菌群含量,同時有效降低有害菌群含量。通過慢性鋁離子暴露小鼠模型,分析了植物乳桿菌CCFM639與鋁離子暴露導(dǎo)致的行為異常之間的關(guān)聯(lián)性。慢性鋁離子暴露小鼠行為學(水迷宮、新物體識別和開放曠場)測試的結(jié)果表明鋁離子暴露可導(dǎo)致小鼠的學習記憶能力下降、對物體辨識度發(fā)生障礙以及憂慮行為,而CCFM639能夠顯著緩解和改善這些不利影響。此外,慢性鋁離子暴露組小鼠腦GSH水平以及SOD、GPx和CAT活性大大降低,促炎細胞因子水平和腦淀粉樣蛋白Aβ1-40和Aβ1-42含量顯著升高,小鼠腦組織中微量元素水平被影響以及腦緊密連接蛋白被破壞。灌胃植物乳桿菌CCFM639可顯著恢復(fù)這些指標,說明該菌能夠緩解慢性鋁離子暴露造成的腦損傷和行為異常,其可能原因是植物乳桿菌CCFM639可調(diào)節(jié)腸道屏障、腸道菌群及其代謝產(chǎn)物水平,進而通過腦腸軸而最終達到保護效果。
[Abstract]:Aluminum (Al, aluminum) is not essential metal elements without any physiological function, but it is widespread in nature (the content of metal elements in the crust, and the largest) are widely used in daily life. The aluminum ion can accumulate in most tissues of mammals and cause damage to the nervous system and the lesions. The injury has been extensively studied. Although traditional chelation therapy can promote the excretion of aluminum ions, but the side effect is inevitable, the defects of security. Lactic acid bacteria are recognized as food grade microorganism safety, as has been reported that lactic acid bacteria can absorb a variety of toxic metals, maintain the intestinal micro ecological balance, and through the intestinal bacteria group of brain gut axis affects the nervous system and behavior, show that it has the potential to ease aluminum ion toxicity, and there is no remission of aluminium toxicity by lactic acid bacteria Reported. The present study explored the lactic acid bacteria in alleviating the toxic effects of aluminum ion effect and related mechanism for deep development of new probiotic lactic acid bacteria function, in order to excavate the probiotic lactic acid bacteria has higher application value, the development and application of new dietary strategies to solve the problems. The main research of aluminum toxicity the results are as follows: to establish the screening model in vitro to alleviate Al toxicity of potential strains, the specific characteristics are as follows: aluminum ion efficient adsorption capacity; excellent aluminum tolerance ability and gastrointestinal tolerance; excellent antioxidation ability, can alleviate the oxidative damage caused by aluminum ion exposure. Based on the above evaluation index of 30 strains of lactic acid strain screening, discovery of Lactobacillus plantarum CCFM639 in the initial concentration of aluminium ions for the 5mg/L and 50mg/L conditions, which are reflected in the strongest adsorption capacity, respectively 64.54% and 26.83%; and the Aluminum ion tolerance ability was particularly prominent, minimum inhibitory concentration, even more than 2480 mg/L or MIC, significantly higher than that of other strains tested ability of the test. The survival rate of the strains in simulated gastric and intestinal fluids were 90.69% and 91.86%, which can keep high activity in the intestine; at the same time. Four aspects of anti oxidative (hydroxyl radical scavenging rate and DPPH clearance rate, anti lipid peroxidation and reduction ability) were tested for their ability, combined with principal component analysis to comprehensive evaluation results show that CCFM639 has good anti oxidation ability. Therefore, selection of Lactobacillus plantarum CCFM639 for further research. By Fourier transform infrared spectroscopy, ultra thin microscope, scanning electron microscopy combined with energy dispersive X ray spectra and genome sequence analysis, to determine the various functional groups on CCFM639 cell surface in aluminum ion The adsorption process, multiple functional genes involved in alleviating aluminum ion toxicity. Comprehensive evaluation of the animal model of acute and chronic exposure by aluminum ion, that Lactobacillus plantarum strain CCFM639 exposed to aluminum ion in the animal model with ease of aluminum ion toxicity. In acute experiments, the strains significantly increased the survival rate, promote discharge waste aluminum ions, reducing the accumulation of aluminum ions in the blood, tissues and organs, alleviate oxidative stress and reduce the pathological lesions in the liver and reduce the number of abnormal liver cells. In chronic experiment, the strain reducing aluminum ions in the blood, tissues and organs in the accumulation and damage regulation of trace elements the organ level, reduce stress and physiological and biochemical indexes of oxidation, relieve pathological damage, and completely avoid the aluminum ion exposure leads to death. The intestinal Lactobacillus is tilapia rod Bacteria can directly play the role of the organ, but also the main absorption site of aluminum ion, so further study of the role of the strain on the intestinal barrier and intestinal function, to analyze the mechanism of aluminum toxicity in vitro. Alleviate intestine model (Batch Culture Fermentation Model), aluminum ion exposure significantly decreased the number of Bifidobacterium and Lactobacillus, promoted the number of Bacteroides, Clostridium and Enterobacter. And Lactobacillus plantarum CCFM639 of probiotics has significantly improved the effect of Bifidobacterium and Lactobacillus bacteria plays the most important role. Through the analysis of the model in vitro intestinal metabolite levels showed that CCFM639 significantly increase in butyrate and lactate levels of metabolites. In HT-29 cell model and chronic aluminum ion exposure in mice, aluminum ion exposure increases the damage of intestinal barrier, intestinal pass Permeability damage process involving oxidative stress, inflammatory reaction and tight junction protein (TJs) and gastric perfusion injury. CCFM639 significantly alleviated oxidative stress and pro-inflammatory cytokines, damage of TJs has a certain recovery effect, significantly increased the serum endotoxin level and increased intestinal permeability. In chronic aluminum ion exposure model of tilapia in at the gate level, aluminum ion exposure significantly increased the abundance of Bacteroidetes, and reduce the abundance of Firmicutes. At the genus level, the aluminum ion exposure significantly decreased abundance of Plesiomonas and Deefgea, Enterovibrio, Flavobacterium increased significantly, the abundance of Porphyromonadaceae and Comamonadaceae. Feed additive CCFM639 can significantly increase the Lactobacilli content of tilapia in the stool, improve Deefgea abundance, decreased the abundance of Comamonadaceae. The above results showed that Lactobacillus plantarum CCFM639 can regulate the structure of intestinal microflora of tilapia and Composition, increase the content of beneficial bacteria in the intestinal microbiota, and effectively reduce the harmful bacteria content. A mouse model of chronic aluminum exposure by ion, analyses the relationship between Lactobacillus plantarum CCFM639 and aluminum ion exposure lead to abnormal behavior between chronic exposure of aluminum ions. Mice behavior (water maze, object recognition and new open field test) results show that aluminium ion exposure can lead to decreased learning and memory ability of mice, the identification of obstacles and concerns of object behavior, while CCFM639 can significantly alleviate and improve these adverse effects. In addition, chronic aluminum exposed mice brain GSH levels and SOD, GPx and CAT activity significantly reduced, proinflammatory the levels of cytokines and cerebral amyloid A beta 1-40 and beta 1-42 A content increased significantly, the levels of trace elements in mouse brain and brain is affected by the tight junction protein was damaged. Intragastric administration of Lactobacillus Coli CCFM639 can significantly restore these indexes, indicating that the bacteria can alleviate the chronic exposure of aluminum ions and brain damage caused by abnormal behavior, the possible reason is Lactobacillus plantarum CCFM639 can regulate the intestinal barrier, intestinal microflora and its metabolite levels, and then through the brain gut axis and the end to achieve the protective effect.

【學位授予單位】：江南大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：TS201.3

【參考文獻】

相關(guān)期刊論文 前7條

1 Qixiao Zhai;Tianqi Li;Leilei Yu;Yue Xiao;Saisai Feng;Jiangping Wu;Jianxin Zhao;Hao Zhang;Wei Chen;;Effects of subchronic oral toxic metal exposure on the intestinal microbiota of mice[J];Science Bulletin;2017年12期

2 Mi-Kyung Sung;Mi-Young Park;;Nutritional modulators of ulcerative colitis:Clinical efficacies and mechanistic view[J];World Journal of Gastroenterology;2013年07期

3 孫衛(wèi)文;黃越玲;張維雯;劉國彬;沈巖松;李敏雄;戴麗軍;陳盛強;;三十日齡Fmr1基因敲除小鼠的水迷宮實驗觀察[J];解剖學研究;2011年01期

4 張?zhí)觳?寧喜斌;;乳酸菌對自由基清除能力的研究[J];中國乳品工業(yè);2007年04期

5 徐海濱,嚴衛(wèi)星;淡水湖泊微囊藻毒素的污染和毒理學研究[J];衛(wèi)生研究;2002年06期

6 王墨林,崔行,張群業(yè);Alzheimer病模型大鼠腦生長抑素表達與學習能力改變[J];中國臨床神經(jīng)科學;2001年03期

7 劉潔生,謝麗玲,洪岸,陳式j(luò);神經(jīng)元煙堿乙酰膽堿受體受阻滯所致早老型癡呆動物模型的建立[J];衛(wèi)生研究;1999年04期

相關(guān)博士學位論文 前1條

1 李寶坤;乳酸桿菌冷凍干燥生理損傷機制及保護策略的研究[D];江南大學;2011年

相關(guān)碩士學位論文 前3條

1 李肖肖;植物乳桿菌對小鼠銅暴露毒性緩解作用的研究[D];江南大學;2014年

2 賴凱昭;飼料中添加復(fù)合益生菌對羅非魚生長性能、腸道蛋白酶活性和飼料消化率的影響[D];廣西大學;2012年

3 張永靜;飼料中鉛含量對建鯉的毒性效應(yīng)及3種非金屬礦吸附劑對其毒性緩解作用研究[D];南京農(nóng)業(yè)大學;2012年

，

本文編號：1711610