【作者】 郑卫江；

【导师】 姚文；

【作者基本信息】 南京农业大学 ， 动物营养与饲料科学， 2013， 博士

【摘要】 雌马酚是大豆苷原的微生物代谢产物,其生物活性(主要为类雌激素作用和抗氧化作用)是已报道的大豆异黄酮类物质代谢产物中最强的,因此被认为是大豆异黄酮类物质发挥生理作用的关键,但仅有30%-50%的人群能够产生雌马酚。研究表明个体肠道菌群结构的差异可能是导致个体产雌马酚能力差异的原因,微生物的种间氢转移与竞争可能在雌马酚生成过程中起着非常重要的作用。动物上特别是经常以富含大豆异黄酮类的大豆、豆粕等作为饲料来源的家畜,如猪个体间产雌马酚的能力如何?是否也存在个体差异?可否利用日粮,通过改变肠道菌群结构及其种间氢转移,而提高猪的雌马酚产生能力?从而提高大豆异黄酮对猪营养与健康的积极作用,这是本论文的研究目的与任务。本研究以猪为对象,研究了大白母猪产雌马酚能力的差异及与肠道微生物的相关性；结合体外和体内实验分析了乳果糖促进个体产雌马酚能力并提高大豆苷原抗氧化能力的可能性。1.大白母猪产雌马酚能力的个体差异比较本研究探讨了大白母猪个体间产雌马酚能力的差异及与肠道菌群结构之间的关系。结果显示,大白母猪粪便中雌马酚水平的个体差异系数为61.22%,尿液中的雌马酚水平的个体差异系数为48.33%,尿液中log1o(雌马酚／大豆苷原)均大于-1.0。聚类分析反映菌群结构的DGGE图谱显示大白母猪粪便中雌马酚浓度相近个体的菌群结构更相似。将粪便中总细菌、拟杆菌、硬壁菌、甲烷菌和硫酸还原菌的定量PCR结果与粪便和尿液中大豆苷原、雌马酚和log1o(雌马酚／大豆苷原)做相关性分析表明,粪便中雌马酚水平与总细菌、硬壁菌门、拟杆菌门和甲烷菌数量呈显著负相关,而粪便中log1o(雌马酚与大豆苷原)与总细菌、硬壁菌门、拟杆菌门、甲烷菌和硫酸还原菌均呈显著负相关；尿液样品中雌马酚和大豆苷原水平则与甲烷菌数量呈显著正相关。上述结果表明母猪肠道菌群产雌马酚能力高于人类,但仍存在个体差异,同时本研究首次在体比较了猪雌马酚生成与肠道氢气利用菌之间的关系。2.乳果糖对母猪粪样菌群体外代谢大豆苷原特性的影响以上一研究中高产雌马酚个体(粪便中雌马酚水平>15.0μg/g)的大白母猪粪便为接种物,利用体外培养技术探讨了乳果糖对母猪粪样菌群代谢大豆苷原产雌马酚特性的影响。结果显示,乳果糖显著促进了细菌的生长,累计产气量显著上升,改变了体外发酵的特性,显著提高了最大产气速率,并推迟了达到最大产气速率的时间。乳果糖改变了体外发酵体系中微生物的组成,增加了雌马酚的产量(大豆苷原组雌马酚水平为8.66±0.68μg/ml,而乳果糖+大豆苷原组雌马酚水平为19.26±0.24μg/ml)(P<0.05).此外,本研究首次发现在体外封闭发酵体系中,雌马酚可能通过二氢大豆苷原途径重新合成大豆苷原。本研究结果显示,乳果糖或许能够通过改变菌群结构及其氢转移而提高菌群产雌马酚的能力,为日粮途径提高个体产雌马酚能力提供新途径。3.日粮添加乳果糖及大豆苷原对去势雄性仔猪产雌马酚能力的影响试验选用40日龄去势雄性仔猪36头,根据体重随机分为三组：对照组、大豆苷原组和大豆苷原+乳果糖组。日粮中乳果糖的添加量为1%,大豆苷原添加量为50mg/kg,以观察饲喂21天后不同组别仔猪菌群结构和产雌马酚能力的差异。结果显示,乳果糖+大豆苷原组结肠食糜各短链脂肪酸水平与大豆苷原组相比,异丁酸(1.803mM比1.248mM)、异戊酸(2.660mM比1.866mM)、支链脂肪酸(BCFA)(4.463mM比3.115mM)和总挥发性脂肪酸(TVFA)(59.77mM比52.66mM)水平均显著升高(P<0.05)。此外,乳果糖+大豆苷原组粪便和尿液中雌马酚水平(分别为12.00±2.68μg/g和3.13±0.93μg/g)显著高于大豆苷原组(粪便和尿液中雌马酚水平分别为10.00±2.26μg/ml和2.11±0.82μg/ml);乳果糖+大豆苷原组粪便和尿液中雌马酚/大豆苷原比值分别为3.77±1.57和0.38±0.10,而大豆苷原组粪便和尿液中雌马酚/大豆苷原比值则显著低于乳果糖+大豆苷原组(分别为2.90±1.40和0.21±0.06)。PCR-DGGE对去势雄性仔猪粪便、结肠食糜和粘膜中总菌、甲烷菌和硫酸还原菌区系分析显示,乳果糖+大豆苷原组的图谱均归于同一簇；与大豆苷原组相比,添加乳果糖后粪便、结肠食糜和粘膜总细菌图谱中条带数显著降低；粪便中,甲烷菌的条带数显著增加,硫酸还原菌的条带数减少；结肠粘膜中,硫酸还原菌的条带数增加。Real-time PCR结果显示,相对于大豆苷原组,乳果糖+大豆苷原组粪便中拟杆菌数目、硬壁菌和拟杆菌占总菌的比例显著增多；而硫酸还原菌数目降低；大豆苷原组和乳果糖+大豆苷原组结肠粘膜硫酸还原菌数目较对照组显著降低。以上数据表明,在大豆苷原存在的条件下,乳果糖具有提高结肠食糜细菌代谢活力,改变粪便、结肠食糜和粘膜中细菌、甲烷菌和硫酸还原菌区系和数目的功能。最为重要的是,乳果糖+大豆苷原组肠道微生物产雌马酚的能力显著提高,因此我们认为乳果糖在体具有促仔猪肠道微生物产雌马酚的能力。4.日粮添加乳果糖及大豆苷原对去势雄性仔猪生长性能、免疫和肝脏抗氧化指标的影响本研究的动物选择、分组和饲养同上,以了解机体内雌马酚水平差异对试验动物生长性能、免疫和肝脏抗氧化指标的影响。结果表明,饲喂21天后,乳果糖+大豆苷原显著降低了仔猪腹泻率。但提高动物转化大豆苷原为雌马酚的能力对采食量、体重、日增重、各器官重量和指数均无显著影响(P>0.05)。大豆苷原组和乳果糖+大豆苷原组肝脏中总羟基和8-羟基脱氧鸟苷水平均显著低于对照组(P<0.05)；对照组肝脏中一氧化氮(NO)和蛋白质羰基水平最高,显著高于大豆苷原组(P<0.05),但与乳果糖+大豆苷原组无显著差异(P>0.05)。乳果糖+大豆苷原组肝脏中T-SOD、CuZn-SOD显著高于对照组和大豆苷原组(P<0.05)；但肝脏中过氧化氢酶、丙二醛、谷胱甘肽过氧化物酶、谷胱甘肽还原酶、谷胱甘肽、氧化型谷胱甘肽和谷胱甘肽／氧化型谷胱甘肽比值在三组动物间均无显著差异(P<0.05)。本章结果显示,大豆苷原能减少肝脏中氧化标记物的水平,增加抗氧化物酶的活性；而乳果糖的添加能提高肝脏的抗氧化活性。5.免疫应激条件下,日粮添加乳果糖及大豆苷原对去势雄性仔猪机体免疫和肝脏抗氧化性能的影响第四章研究结果发现日粮中添加乳果糖能够促进仔猪后肠发酵能力、显著提高动物的产雌马酚能力。本研究的目的是评价在LPS诱导的免疫应激状态下,通过乳果糖提高去势雄性仔猪转化大豆苷原为雌马酚的能力后对其机体免疫和肝脏抗氧化性能的影响。通过将第五章剩余6头去势雄性仔猪继续饲养,于第23天对所有动物进行LPS攻毒(25μg/kg BW).24小时后采集动物前腔静脉血液提取淋巴细胞,48小时将各组动物屠宰取样。结果表明,在免疫应激条件下,饲喂大豆苷原能显著增加外周血T和B淋巴细胞转化率,而乳果糖+大豆苷原组的T和B淋巴细胞转化率又显著高于大豆苷原组(P<0.05)；血液中白细胞介素和激素水平结果显示,对照组的白细胞介素6(IL-6)水平显著高于大豆苷原组和乳果糖+大豆苷原组(P<0.05)；大豆苷原组和乳果糖+大豆苷原组血液中皮质醇水平显著高于对照组(P<0.05)。大豆苷原能显著降低肝脏中总羟基、NO、8-羟基脱氧鸟苷和丙二醛水平(P<0.05),但乳果糖+大豆苷原没能促进对以上氧化标记物的抑制作用；此外大豆苷原显著降低了肝脏中T-SOD、CuZn-SOD、Mn-SOD及谷胱甘肽还原酶(GR)的活性,而乳果糖+大豆苷原T-SOD、Mn-SOD和谷胱甘肽还原酶(GR)活性显著高于大豆苷原组。以上数据说明,在LPS诱导的免疫应激条件下日粮大豆苷原能显著提高去势雄性仔猪体液免疫力,降低肝脏中氧化标记物水平和肝脏的抗氧化酶活性；乳果糖的添加能显著增加大豆苷原对T和B淋巴细胞转化的影响,提高肝脏抗氧化酶活性；但对细胞因子和激素水平,肝脏氧化标记物水平无促进作用。更多还原

【Abstract】 Equol, a gut microbial metabolite of daidzein, has been implicated to play a major role for potential beneficial effects of isoflavones, but it is produced by only30-50%of population. The gastrointestinal microbiota plays an important role in the metabolism of isoflavones, especially the formation of equol. Once formed, equol is in general metabolically stable. Equol exhibits a wide range of biological activies; including anti-oxidant and estrogenic properties. However, the factors for equol synthesis are unknown. It was found that the structure of intestinal microbiota and inter-species transference of hydrogen is very important for the production of equol. The motivations of this study are to character the equol-producing ability and its individual variation in pigs, and to develop the nutritional strategy to enhance pig equol-producing ability by modulating the structure of intestinal microbiota and inter-species transference of hydrogen. Therefore we first compared the equol-producing ability in large white sows, and then do further analyzed wether lactulose can enhance equol-producing capacity in vitro and in vivo.1. Individual differences of equol-producing capacity and the correlation with intestinal microbiota in sowsIn this study, the individual differences of equol-producing capacity and its correlation with intestinal microbiota in large white sows were assessed. Results showed that large white sows had a61.22%coefficient of variation inter-individual difference in faecal equol production. And had a48.33%coefficient of variation individual variation in urinary equol levels. The urinary log10(equol/daidzein) values were all above-1.0. Cluster analysis of denaturing gradient gel electrophoresis (DGGE) patterns from sows’ faeces showed that faecal samples had similar equol concentrations microbial composition. The population of total bacteria, firmicutes, bacteroidetes, and methanogen-producing bacteria correlated negatively with faecal equol production. The population of total bacteria, firmicutes, bacteroidetes, methanogen-producing and sulphate-reducing bacteria correlated negatively with faecal log10(equol/daidzein). A significant positive correlation between urinary equol and daidzein production populated with methanogen was also found. This study provide the first evidence that there is a relationship between equol production and the gut interspecies H2transfer in vivo.2. Lactulose promotes equol production and changes the microbial community during in vitro fermentation of daidzein by fecal inocula of sowsIn this study, the effect of lactulose on transformation of daidzein into equol by sows’fecal microbiota was investigated. Results showed that lactulose treatment improved bacteria growth parameters and modulated the kinetics of fermentation in vitro. Lactulose significantly increased total gas production, T1/2, Tmax, and Rmax. Furthermore, lactulose altered the microflora composition; increased equol production, reduced in the population of methanogen, and increased the sulfate-reducing bacteria population during24hours of incubation. Here, we report for the first time that in a certain condition (sealing or high pressure), equol might be able to re-transformed to daidzein via a dihydrodaidzein (DHD) pathway by further metabolism using lactulose as a substrate. This study proposes that "hydrogen-producing prebiotic" might be a novel approach to promote equol production in vivo or in vitro.3. Effects of dietary daidzein and lactulose on the production of equoOl in of barrowsThe present study was performed to investigate the effects of dietary supplemention of daidzein and lactulose on the equol-producing ability of barrows.36barrows aged40days were randomly allotted to one of three treatment groups. The control (C) was fed a soybean-free diet, divided Daidzein (D) and daidzein+lactulose (D+L) were the control diet containing50mg/kg daidzein and50mg/kg daidzein with1%lactulose, respectively. After21days of treatment, samples were taken for microflora and equol-producing ability analysis. The results showed that the isobutyric acid, isovaleric acid, branched chain fatty acid (BCFA) and total volatile fatty acid (TVFA) levels in the D+L group were higher than those in the D group.PCR-DGGE analysis showed the profiles of bacteria, methanogen-producing bacteria and sulfate-reducing bacteria in the colon digesta and mucosa had also changed by D+L. In addition, the bands number of bacteria, MPB and SRB in the feces, colon digesta and mucosa was manipulated by lactulose and daidzein. The number of firmicutes, bacteroidetes, and the percentage of bacteroidetes to bacteria were higher in the D+L group than D group. However, the SRB population in the D+L group was lower than that in the D group. Furthermore, methanogen-producing bacteria and sulfate-reducing bacteria in colon digesta were reduced by daidzein or daidzien with lactulose. Moreover, equol concentrations and the ratio of equol/daidzein were significantly increased by the addition of lactulose in barrows. Taken together; our data indicated that in the presence of daizein, dietary supplementation with lactulose can improve the microflora fermentation activity of colon digesta, shift the bacteria, MPB and SRB population and profile in the feces, colon digesta and mucosa. Most importantly, lactulose significantly increased the production of equol from faidzein in the intestine.4. Effects of dietary daidzein and lactulose on the growth performance, humoral immunity and liver antioxidant status in barrowsThe purpose of this study was to explore the effects of dietary daizein and lactulose on the growth performance, humoral immunity and liver antioxidant status in barrows. Results showed that no significant difference in the feed intake, average daily weight gain, body weight, organs weight and index among three groups. However, the D+L fed pigs had lower incidence of diarrhea as compared D and C groups. Blood T and B lymphocyte proliferation was no influenced by daidzien and lactulose. However, the liver oxidative biomarkers and antioxidant enzyme activities were affected. The total carbonyl,8-OH-dG levels in the D and D+L groups were higher than those in the C group. The NO and protein carbonyl concentrations were significantly lower in D group compared to those in the C group. The T-SOD and CuZn-SOD enzyme activities in D+L group were significantly higher as compared with the D and C groups. There were no differences in the levels of CAT, MDA, GSH-pX, GR, GSG, GSSG and the ratio of GSH/GSSG among three groups. Overall, daidzein can suppress the liver oxidative biomarkers, and lactulose had no addition effect on these parameters, but lactulose was found would improve the antioxidant enzymes of activities stimulated by daidzein in the liver of barrows.5. Effect of dietary daidzein and lactulose on humoral immunity and liver antioxidant status in barrows after LPS challengeIn this chapter, the effects of dietary daidzein and lactulose on humoral immunity and liver antioxidant status were studied.36barrows at40days old were randomly divided to one of three treatment groups. The control (C) barrows were fed a soybean-free control diet. Daidzein (D) and daidzein+lactulose (D+L) groups were fed the control diet containing with50mg/kg daidzein and50mg/kg daidzein with1%lactulose, respectively. After23days of treatment, half of pigs were intrapenearally injected with LPS (25μg/kg BW). The blood samples were then taken for lymphocyte proliferation after24hours of LPS induction.48hours later, animal were killed and the blood and liver samples were taken for analysis. Results showed that after LPS induction, daidzein increased T and B lymphocyte proliferation, and lactulose further enhanced cell proliferation. Plasma IL-6levels in the C group were significantly higher than the D and D+L groups. The plasma cortisol levels in the D and D+L groups was higher than those in the C group. Total carbonyl, NO,8-OH-dG, and MDA levels in the liver were reduced by daidzein treatment, but addition of fed animals further effected on these parameters. Moreover, D group had lower T-SOD, CuZn-SOD, Mn-SOD and GR enzyme activities as compared to the C group, but the lactulose+daidzein had higher T-SOD, Mn-SOD and GR activities than those in D group. These results suggest that dietary daidzein significantly modulated the humoral immunity and reduced oxidant biomarkers and anti-oxidant enzymes activity; and supplementation of lactulose can improve the humoral immunity and augment daidezin-induced antioxidant enzymes activity.更多还原