【作者】 于卓腾；

【导师】 朱伟云；

【作者基本信息】 南京农业大学 ， 动物营养与饲料科学， 2007， 博士

【摘要】 大豆黄酮作为一种植物雌激素对人和动物的健康和生长有良好促进作用,因此开发利用黄酮类化合物正成为当前食品业与畜牧业的一个热点。许多报道表明,大豆黄酮的生物活性更多的是由其代谢终产物雌马酚来实现的,与大豆黄酮苷元相比,雌马酚与雌激素受体结合的亲和力、抗氧化活性、抗前列腺癌作用更强。而且雌马酚在体内相当稳定,不再发生降解,因此要想充分发挥大豆黄酮的生物学作用,必须使其尽可能转化成雌马酚,但雌马酚只能由肠道细菌特异性产生。许多学者认为,大豆黄酮对动物的生理效应在很大程度上决定于动物自身的肠道微生物菌群,但现有报道表明只有30-40%的人体内定植能够产生雌马酚的细菌,迄今为止,国内外有关大豆黄酮降解菌的研究多集中在人和小鼠上,而且分离获得的肠道内大豆黄酮降解菌只有少数几种,这其中的瓶颈在于大豆黄酮降解菌的分离条件(包括厌氧条件与分离培养基等)十分苛刻,如何突破这一瓶颈,大量分离肠道内具有降解大豆黄酮能力的微生物,从而全面了解大豆黄酮这一功能性物质在肠道内的降解过程,以及更深入地阐明大豆异黄酮在体内降解和发挥生理作用的形式,是该领域亟待解决的问题。本研究为阐明上述研究中的部分问题,采用动物试验、体外分离培养技术与分子生物学手段,研究了大豆黄酮对仔猪肠道微生物区系的影响、二花脸猪粪样菌群对大豆黄酮的代谢能力;并从低成本筛选的角度入手,获取了一种可大量富集大豆黄酮降解菌的培养基,在此基础上,从猪粪样中分离到数株降解大豆黄酮产生雌马酚的菌株,研究了这些降解大豆黄酮产雌马酚细菌的生理特性。试验结果阐明了大豆黄酮对仔猪肠道微生物区系的作用效应,揭示了仔猪肠道内产雌马酚微生物种类及其与宿主间的关系,同时也为肠道雌马酚产生菌的进一步研究拓宽了思路。1大豆黄酮对仔猪肠道微生物区系的影响选取江苏某猪场产期相近、胎次相似数目相当的杜/长/大三元杂交仔猪6窝,随机分为两组,每组三窝。一组为对照组,另一组为处理组,在7、9、11d对照组每头猪强饲脱脂乳1 mL、2 mL、3 mL,处理组每头猪强饲含大豆黄酮(10 mg/L)的脱脂乳1 mL、2 mL、3 mL。两组均于21d断奶。于14、21、24、35 d每窝各屠宰一头,无菌采集十二指肠、空肠、回肠、盲肠、结肠和直肠食糜。食糜细菌16S rRNA基因序列的V6-V8可变区经PCR扩增,扩增产物经DGGE电泳后再进行多样性分析,并对特殊条带进一步克隆测序。结果发现饲喂大豆黄酮后,处理组仔猪各肠段的DGGE条带数均高于对照组,盲肠、结肠和直肠中细菌的多样性指数增加;对照组中出现的3条优势条带在处理组中消失,经克隆测序后与GenBank登录序列比较,发现它们分别与Clostridium thermocellum,Lactobacillus pontis,Streptococcus sp.最相似。与对照组相比,在35d时处理组大肠中出现3条优势条带,经克隆测序后与GenBank登录序列比较,发现它们分别与Butyrate-producing bacterium SL7/1,Clostridium butyricum(NCIMB8082),Ruminococcus obeum clone 1-4最相似,相似性分别为95%,95%和96%。因此大豆黄酮可使仔猪大肠的微生物区系多样性增加,并且可选择性地促进某些菌群的富集。2体外培养研究二花脸猪粪样菌群对大豆黄酮的降解规律通过体外培养研究了不同日龄3窝(A、B、C)二花脸猪的粪样菌群对大豆黄酮的降解和雌马酚的产生规律。每窝各含1头母猪和3头仔猪。通过HPLC测定培养液中大豆黄酮和雌马酚的含量,并通过DGGE图谱分析每窝猪的粪样菌群的变化规律。结果表明12头二花脸猪中有8头猪的粪样菌群可将大豆黄酮降解成雌马酚。在3窝二花脸猪中,A窝母猪粪样菌群不能代谢大豆黄酮,但在其断奶后的两头仔猪粪样培养液中检测到雌马酚;B窝和C窝母猪粪样培养液中均检测到较高含量的雌马酚,在仔猪粪样培养液中发现B窝有1头仔猪,C窝有3头仔猪均检测到雌马酚。通过DGGE指纹图谱分析,每窝二花脸猪粪样培养液中可检测到雌马酚的猪的粪样细菌菌群之间的相似性均大于60%,高于它们与未检测到雌马酚的猪的粪样菌群的相似性。提示,二花脸猪粪样菌群中存在可降解大豆黄酮产生雌马酚的微生物。3产雌马酚微生物培养基的筛选及猪粪样中雌马酚产生菌的分离与鉴定为富集仔猪粪样中能够代谢大豆黄酮产雌马酚的微生物,本研究对三种不同培养基(M1、M2、M3)进行了筛选,并在获得其中一种可富集雌马酚产生菌的培养基的基础上,对仔猪粪样中的雌马酚产生菌进行了分离鉴定。结果发现,M1培养基中富集的微生物可以降解大豆黄酮产生大量的雌马酚。进一步对培养基M1中富集的微生物进行了分离鉴定,获得了3株(D1、D2、D3)能够代谢大豆黄酮产雌马酚的纯菌,通过革兰氏染色进行形态学观察和进一步作生理生化鉴定后发现D1和D2为Eubacterium属,D3为Enterococcus faecalis。系统进化树分析结果表明,D1与SNU-Julong732位于一个簇内,D2与SNU-NiuO16位于同一个簇内。通过体外厌氧培养进一步比较了雌马酚产生菌D1的代谢性质。结果发现补充葡萄糖、麦芽糖、果糖、菊粉、乙酸和丁酸的基础培养基均能促进菌株D1生长,其中果寡糖和菊粉比葡萄糖能更好促进菌株D1生长,并且添加寡糖组比单糖组能提高D1产雌马酚的浓度。短链脂肪酸乙酸和丁酸不能明显促进D1的生长,但能促进其产雌马酚的浓度;而乳酸对雌马酚的产生有抑制作用。以上结果说明通过该方法筛选的培养基能够有效的分离可将大豆黄酮转化成雌马酚的微生物,并且添加寡糖能促进雌马酚的产生。4体外继代培养研究仔猪粪样大豆黄酮降解菌群的变化规律采用体外继代培养法,结合PCR-DGGE技术,研究了二花脸仔猪粪样中大豆黄酮降解菌群的变化规律,并对富集培养液中的菌群进行了克隆测序。结果表明,二花脸仔猪粪样中微生物菌群在体外继代培养40代后,粪样菌群仍具有降解大豆黄酮产雌马酚的能力;PCR-DGGE的分析结果表明,随继代培养次数的增加,培养液中微生物区系的DGGE图谱上的条带数逐渐变少,一些条带消失,这些条带编号为A1,A2,A3,A4和A5,经克隆和切胶测序结果表明,A1与Megasphaera elsdenii strain YJ-4有91%的相似性,A2与Megasphaera elsdenii strain 5T有99%的相似性,A3与Clostridium sp.cTPY-17有87%的相似性,A4与Succinivibrio dextrinosolvens有96%的相似性,A5与Bacteroides sp.MANG有94%的相似性。而部分条带一直存在逐渐成为优势菌群。这些条带编号为QE3、QE4、QE7、QE8和QJ7。16SrRNA基因测序结果表明,QE3与Coporococcus comes strain ATCC 27758有92%的相似性;QE4与Clostridium bacterium JN18_V41_S具有93%的相似性;QE7与Eubacterium callanderi有99%的相似性;QE8与Clostridium glycyrrhizinilyticum有94%的相似性;QJ7与Bifidobacterium pseudolongum subsp.globosum strain 7#-3-7有94%的相似性。以上结果说明,混合粪样中存在一些难以分离到且在大豆黄酮降解中起重要作用的微生物,同时添加大豆黄酮可起到体外富集大豆黄酮降解菌作用。本文通过在体和体外试验研究了大豆黄酮与猪消化道微生物之间的相互作用,结果发现在在体试验中仔猪饲喂大豆黄酮后,其后肠微生物区系的多样性增加,且部分微生物明显得到富集;体外培养发现二花脸猪粪样菌群具有降解大豆黄酮产生雌马酚的能力;选择针对不同的菌群的培养基来筛选雌马酚产生菌,结果发现培养基M1中富集的细菌具有高产雌马酚能力;利用该培养基分离到3株可直接降解大豆黄酮产生雌马酚的细菌,通过体外发酵研究菌株D1对不同底物的代谢能力,结果发现寡糖可提高其产雌马酚的能力;将粪样菌群体外继代培养40代后发现其仍具有较高的产雌马酚能力,这提示在混合菌群中存在某些菌可能在大豆黄酮转化成雌马酚的过程中起关键作用。更多还原

【Abstract】 Daidzein is one of the major metabolites from isoflavones,and it can be further metabolized into equol with more estrogenic activity by intestinal microorganisms. However,little information is available on the daidzein effect on the intestinal microflora of animals,and only a few intestinal bacteria have so far been identified to metabolize daidzein.In the present work,a PCR/DGGE approach was used to investigate the effect of daidzein on the gastrointestinal bacterial community of piglets.For the first time this study investigated the possibility of conversion of daidzein to equol by pig fecal microflora. Three selective media used for enrichment of three large groups of bacteria were then compared for equol production and subsequently for the isolation of equol-producing bacteria from pig faecal materials.This dissertation was described in the following five sections.1 Effect of daidzein on the intestinal microflora of pigletsSix litters of piglets were randomly allocated into two groups:the control and the treatment.Each group had three litters.The piglets of the treatment group were fed with daidzein orally on Day 7,9 and 11.They were all weaned on Day 21.One piglet of each group was slaughtered on Day 14,21,24 and 35,respectively.The digesta of duodenum, jejunum,ileum,caecum,colon and rectum were collected and used for DNA extraction and followed by PCR/DGGE analysis.The diversity of intestinal microbial populations of the piglets was determined based on the shannon’s index.The 16S rRNA sequences of intestinal bacterial species were analysed.Piglets of the treatment group showed a higher bacterial diversity and more DGGE bands compared with that of the control on Days 24,35. Three DGGE bands existing in the control disappeared in the treatment group.The three clones corresponding to the three bands had their sequences most closely related to species of Clostridium thermocellum,Lactobacillus pontis and Streptococcus sp.,respectively.The other three DGGE bands were present only in the large intestine of piglets in the treatment group,and their corresponding clones had their 16S rRNA gene sequences most closely related to species of Butyrate-producing bacterium SL7/1,Clostridium butyricum, Ruminococcus obeum,respectively.The results suggested that daidzein could increase the diversity of the piglets intestinal bacterial community,and stimulate some specific bacteria to grow.2 In vitro fermentation reveals the conversion of daidzein to equol by fecal microflora of Erhualian pigsThe possibility of conversation of daidzein to equol by fecal microflora of Erhualian pigs was investigated by in vitro culture with fecal sample as inoculum.Faecal samples of three litters(A,B,C) of Erhualian pigs were collected.Each litter includes the sow and three piglets.Faecal samples were used to inoculate culture bottles with and without daidzein.After 48 h incubation,the cultures were taken to measure equol and daidzein concentrations by HPLC,and to analyze the microflora by PCR/DGGE.Results showed that microflora of eight pigs from the total 12 Erhualian pigs could transform daidzein into equol.For litter A,microflora of two piglets could metabolize daidzein to produce equol. For litter B,microflora of sow and one piglet could metabolize daidzein to produce equol. For litter C,microflora of sow and all the piglets could metabolize daidzein to produce equol.DGGE profiles revealed that in each litter,the pigs that could transform daidzein to equol showed higher similarities,while they had lower similarities to those that did not produce equol.The results showed that faecal microflora of Erhualian pigs had the ability to transform daidzein to equol.3 Medium selection for the isolation of equol-produciug bacterial strains from cultures of pig faecesThree selective media were compared for equol production using faeces from the Erhualian piglets which could transform daidzein into equol during in vitro fermentation as inoculum.Subsequently,three single bacterial strains capable of transforming daidzein into equol were isolated by using the selected medium.The bacterial strains were then identified using morphorlogical and biochemical parameters and by 16S rRNA gene sequence analysis.Results showed that among the three selective media,equol was detected only in the medium M1,which was selective medium for butyrate-producing bacteria,suggesting that butyrate-producing bacteria may transform daidzein to equol.Three single bacterial strains(D1,D2,D3) capable of transforming daidzein into equol in their pure cultures were then isolated using medium M1.At the genus level,D1 and D2 were both identified as Eubacterium sp.,while D3 as Enterococcus sp.based on morphological and physiological characteristics.The 16S rRNA gene sequence analysis showed that strains D1 and D2 were most closely related to previous daidzein-metabolisming bacteria isolated from humen and ruman faecal sample,respectively.Strain D3 was most closely related to Enterococcus faecium with sequence similarity as 97%.Strain D1 was further investigated in its characteristics of growth and equol production in vitro fermentation system.The results showed that the basal media supplemented with glucose,maltose,FOS,inulin,could stimulate the growth of strain D1,and media supplemented with FOS and inulin better supported it to grow.FOS and inulin could stimulate equol production.Media supplemented with acetate,butyrate and lactate failed to stimulate the growth of strain D1. But media supplemented with acetate and butyrate could stimulate equol production, whereas it was found that adding lactate was inhibitory for equol production.The results demonstrated that the present approach using a simple medium enabled to isolate bacterial strains capable of transforming daidzein into equol.The stains D1,D2 and D3 isolated could directly transforme daidzein into equol and oligosaccharide supplemented into the basal medium could stimulate the productiong of equol.4 The variation of faecal microorganisms of Erhualian pigs after being subcultured forty times in vitroMicroflora community change in the Erhualian pig facece and its equol production during subcultures was investigated.The results showed that the faecal bacteria were still able to transform daidzein into equol after being subcultured forty times.PCR/DGGE analysis showed that the number of DGGE bands decreased with the culture times increasing.Some DGGE bands in the culture disappeared,their 16S rRNA sequences most close to species of Megasphaera elsdenii strain YJ-4(91%),Megasphaera elsdenii strain 5T(99%),Clostridium sp.cTPY-17(87%),Succinivibrio dextrinosolvens(96%), Bacteroides sp.MANG(94%),respectively.Other bands enriched after 40 subcultures.The dominant bands remained in the culture after 40 subculturing had their 16S rRNA sequences most close to species of Coporococcus comes strain ATCC 27758(92%), Clostridium bacterium JN18_V41_S(93%),Eubacterium callanderi(99%), Bifidobacterium pseudolongum subsp,globosum strain 7#-3-7(94%),respectively.The results indicated that though some bacterial strains could not directly transform daidzein into equol,they play an important role during the time of daidzein being metabolized into equol.更多还原