瘤胃甲烷减排中草药筛选及目标中草药的作用机制研究

发布时间：2018-05-18 21:26

本文选题：紫苏子 + 甲烷产量　；参考：《浙江大学》2016年博士论文

【摘要】：减少反刍动物甲烷的排放对缓解温室效应和提高饲料利用率具有双重意义。中草药含有丰富的活性物质,是一种潜在的瘤胃甲烷抑制剂,可通过调控瘤胃微生物菌群提高饲料利用率并抑制甲烷产生。本研究首先利用体外模拟瘤胃发酵试验技术对百味中草药进行了筛选和验证；然后讨论了对抑制甲烷最有效的中草药,紫苏子(Perilla Frutescens Seed, PFS)的剂型和剂量效应,并通过实时定量PCR、 16SrDNA测序,探究了体外添加紫苏子提取物(Perilla Frutescens Seed Extracts, PFSE)对瘤胃微生物数量和种群的影响；最后以湖羊为模式动物,研究了在日粮中添加PFSE对湖羊消化功能、甲烷生成和瘤胃发酵的影响。1抑制甲烷生成的中草药筛选和验证1.1初步筛选利用体外模拟瘤胃发酵试验技术,发酵体系为10 ml,底物为0.1 g精粗比是30：70(DM)的玉米和羊草混合物。单因素试验设计,设置处理组100个,同时分别设置一个对照组和一个空白组,每个处理有3个重复。其中100个处理组分别为百味中草药的70%乙醇提取物,添加剂量为底物的4%(DM),以40 mg/ml的二甲基亚砜(DMSO)溶液的形式添加。对照组添加0.1 ml DMSO,空白组无底物且不添加中草药提取物及DMSO,以扣除人工瘤胃液的自身产气量。体外发酵24 h后测定产气量和甲烷产量。研究发现,有31味中草药降低甲烷产量达10%以上,其中降低甲烷产量最多的十味中草药依次是山竹皮(74.9%)、厚朴(60.1%)、乳香(45.4%)、苏木(44.8%)、五加皮(42.6%)、姜黄(42.6%)、蛇床子(38.8%)、远志(33.3%)、紫苏子(30.1%)和辛夷(29.0%)。1.2发酵验证对初筛中的31味中草药的降甲烷效果进行验证。同样利用体外模拟瘤胃发酵试验技术,发酵体系扩大为50 ml,底物为0.5 g的玉米和羊草混合物,精粗比为30：70(DM)。采用单因素实验设计,设置31个处理组,添加剂量仍然为底物的4%(DM),对照组添加0.5 ml DMSO,空白组和标准羊草组同筛选试验,每个处理有4个重复。体外发酵24 h后测定产气量、甲烷产量和其他瘤胃参数。在31味中草药提取物中,有10味产气量显著减少(P0.05),6味产气量显著增加(P0.05)；12味显著降低甲烷的产量(P0.05),只有桃仁提取物显著增加了甲烷的产量(P0.05)。除远志提取物之外,筛选试验中降低甲烷产量最多的10味中草药提取物在验证试验中被证实能够显著抑制甲烷生成(P0.05)。12味中草药提取物中,只有厚朴和酸枣仁在显著抑制甲烷生成的同时又没有减少产气量(P0.05)。在31味中草药提取物中,只有一味中草药提取物(辛夷)显著降低TVFA的浓度(P0.05),9味显著降低乙丙比(P0.05),其中有7味来自12味显著降低甲烷产量的中草药提取物。紫苏子在验证试验中降低甲烷产量的效果最好(63.6%),因此作为目标提取物做进一步研究。2紫苏子对瘤胃发酵和甲烷生成的影响及机制不同剂型紫苏子,即提取物、普通粉(80目)和超微粉(600目),主要成分均为长链脂肪酸,其中PFSE中十八碳不饱和脂肪酸含量最高(675 μg/mg),约是普通粉(295 μg/mg)的2倍,超微粉(118 μg/mg)的3倍。体外模拟瘤胃发酵试验结果表明,与高剂量PFSE (2%,0.2 mg/ml, DM)、普通粉和超微粉相比,添加低剂量的PFSE (1%,0.1 mg/ml, DM)显著降低甲烷产量(P0.05)并显著增加有效产气量(P0.05),对其他发酵参数没有显著影响(P0.05)。同样利用体外模拟瘤胃发酵试验技术,添加不同剂量PFSE,分别为底物干物质的0、1%、2%、3%和4%,体外发酵24h。结果表明,产气量、甲烷产量、TVFA、乙丙比以及干物质消化率均随PFSE剂量增加而线性降低,其中甲烷在添加量为1%时已下降显著(P0.05),而产气量和TVFA分别是在2%和3%时下降显著(P0.05)。实时定量PCR结果表明PFSE剂量不影响总细菌和总甲烷菌数量,但降低原虫数量(P0.05)。PFSE的添加对羧甲基纤维素酶、微晶纤维素酶和木聚糖酶的酶活性没有显著影响。利用MiSeq高通量测序平台对对照组(0%,0 mg/mL)、低剂量组(2%,0.2 mg/mL)和高剂量组(4%,0.4 mg/mL)样品微生物中细菌和古菌的16S rRNA基因进行扩增测序分析。(1) PFSE对细菌菌群的影响：不同处理组样品的细菌Chaol丰富度指数没有发生显著性变化(P0.05),Shannon多样性指数随PFSE添加量的增加显著降低。厚壁菌门、拟杆菌门和变形菌门占细菌总序列数的89.6%以上。其中,变形菌门相对丰度随PFSE浓度的增加而线性增加(P0.05),而拟杆菌门、纤维杆菌门、软壁菌门则线性降低(P0.05)。细菌OTUs数随PFSE剂量增加而降低。UniFrac分析表明添加0.4 mg/mL PFSE改变细菌群落,但0.2mg/mL添加浓度无影响。(2)PFSE对古菌菌群的影响：古菌Shannon指数随剂量增加而线性降低,但Chaol丰富度指数没有发生显著性变化。虽然qPCR结果显示古菌总数不受PFSE添加的影响,但测序数据显示Methanobrevibacter和Methanosphaera的相对丰度随PFSE剂量增加而增加,而Methanobacteriaceae的相对丰度随之降低。微生物与甲烷产量、瘤胃发酵参数存在相关性,根据相关系数将瘤胃微生物分为5组。组Ⅰ与甲烷产量和总VFA呈正相关(P0.05,r0.607),包括Fibrobacter、 Sphaerochaeta和其他八种未分类的细菌与未分类的Methanobacteriaceae。 Clostridium、Pedobacter、Anaeroplasma、Paludibacter、Ruminococcus、Zymomonas、 Luteimonas和其他七种未分类的细菌被划分为第2组,该组微生物与甲烷产量正相关,但与总VFA无关。与组Ⅰ相反,组Ⅲ中微生物与甲烷产量和总VFA都呈负相关(P0.05,r-0.598),包括 Roseomonas、Selenomonas、Shuttleworthia、 Pseudobutyrivibrio、Anaerovibrio、Ruminobacter、Succinivibrio、Methanosphaera和三种未分类的细菌。组Ⅳ微生物与甲烷产量呈负相关(P0.05,r-0.650),包括Methanobrevibacter和未分类的Sinobacteraceae。组Ⅴ由两种未分类的菌组成,其与甲烷产量和总VFA都无关。3紫苏子提取物对湖羊甲烷生成影响的在体评定采用反转实验设计,选取8只体况相近健康无病的成年公湖羊,设置对照组为基础日粮(300 g精料和700 g羊草),处理组为基础日粮上添加1%(DM)的PFSE。在湖羊日粮中添加1%(DM) PFSE,对湖羊的体重、干物质采食量、干物质消化率、粗蛋白消化率、中性洗涤纤维消化率和酸性洗涤纤维消化率均没有显著影响(P0.05),其中干物质消化率有降低的趋势(P=0.086)；以干物质采食量为单位,与对照组相比处理组有降低瘤胃甲烷生成的趋势(P=-0.075),而以可消化干物质和可消化纤维为单位,对照组和处理组瘤胃甲烷的生成量没有显著差异(P0.1)；添加PFSE对湖羊瘤胃的pH、NH3-N、乙酸、丙酸、丁酸、乙丙比和TVFA均没有显著影响(P0.1)。综上所述,中草药具有良好的抑制瘤胃甲烷生成的效果,可作为潜在的甲烷抑制剂做进一步开发；低剂量的PFSE能抑制甲烷生成而不对饲料发酵造成负面影响；本研究发现一些细菌、甲烷菌与甲烷产生存在相关关系,进一步对这些微生物的深入研究有助于开发甲烷减排的新策略。
[Abstract]:Reducing the emission of methane in ruminants has dual significance to mitigate the greenhouse effect and improve the feed utilization. The Chinese herbal medicine contains rich active substances, which is a potential rumen methane inhibitor, which can improve feed utilization and inhibit methane production by regulating the rumen microbial flora. The test technique was used to screen and verify the Chinese herbal medicine, and then the dosage and dosage effects of the most effective Chinese herbal medicine, Perilla Frutescens Seed (PFS), were discussed, and the extracts of the perilla seed (Perilla Frutescens Seed Extracts, PFSE) were investigated by real-time quantitative PCR and 16SrDNA sequencing. The effect of the number of microorganism and the population of the stomach; finally, taking Hu sheep as the model animal, the effects of adding PFSE on the digestive function of the sheep, methane production and rumen fermentation were screened and verified by the effect of PFSE on the inhibition of methane production by.1, and the preliminary screening and using in vitro simulated rumen fermentation test technology was used, the fermentation system was 10 ml, and the substrate was 0.1 g The precision ratio is 30:70 (DM) corn and Leymus chinensis mixture. A single factor trial was designed to set up 100 treatment groups, and a control group and a blank group were set up respectively. Each treatment had 3 repetitions. 100 of the treatment groups were 70% ethanol extracts of Chinese herbal medicine, 4% (DM), and two methylsub of 40 mg/ml. The addition of DMSO solution was added. The control group added 0.1 ml DMSO, the blank group had no substrate and no Chinese herbal extracts and DMSO, to deduct the gas production capacity of the artificial tumor gastric juice. After 24 h in vitro fermentation, the gas production and methane production were measured. The study found that 31 Chinese herbs reduced the methane production by more than 10%, which reduced the methane production most. The ten Chinese herbal medicines were in order of mangosteen (74.9%), Magnolia (60.1%), frankincense (45.4%), Su wood (44.8%), five Acanthopanax (42.6%), Curcuma (42.6%), Fructus Cnidium (38.8%), Polygala (33.3%), purple soda (30.1%) and Magnolia (29%).1.2 to verify the methane reduction effect of 31 Chinese herbal medicine in the initial screening. Technology, the fermentation system was expanded to 50 ml, the substrate was 0.5 g Corn and Leymus chinensis mixture, with the ratio of 30:70 (DM). By single factor experiment design, 31 treatment groups were set up, the dosage was still 4% (DM), the control group was 0.5 ml DMSO, the blank group and the standard weeds group were screened with 4 repetitions. 24 h in vitro fermentation. In the 31 flavors of Chinese herbal extracts, 10 flavors were significantly reduced (P0.05), 6 flavor production increased significantly (P0.05), and 12 flavor significantly reduced methane production (P0.05), only peach kernel extract significantly increased the production of methanes (P0.05). The 10 Chinese herbal extracts with the highest yield of low methane were proved to be able to significantly inhibit the Chinese herbal extracts of methane generation (P0.05).12 flavor in the test. Only Magnolia and Ziziphus Ziziphi had no reduction in methane production while reducing methane production (P0.05). In the 31 flavor herb extract, only a taste of Chinese herb extract (Xin Yi) The concentration of TVFA was significantly reduced (P0.05), and the 9 flavor significantly reduced the ethylene propylene ratio (P0.05), of which 7 flavors came from the Chinese herbal extracts of 12 flavors which significantly reduced the methane production. The effect of the perilla seed on the reduction of methane production was best (63.6%). As a target extract, the effect of.2 perilla seed on rumen fermentation and methane production was studied. The main components of the different dosage forms, the extract, the ordinary powder (80 orders) and the superfine powder (600 orders), are all long chain fatty acids, in which the content of eighteen carbon unsaturated fatty acids in PFSE is the highest (675 mu g/mg), about 2 times of the ordinary powder (295 mu g/mg) and 3 times of the ultrafine powder (118 micron g/mg). In vitro simulated rumen fermentation test results showed that the high dose was in high dose. PFSE (2%, 0.2 mg/ml, DM), the addition of low dose PFSE (1%, 0.1 mg/ml, DM) significantly reduced methane production (P0.05) and significantly increased the effective gas yield (P0.05), and had no significant effect on other fermentation parameters (P0.05). The same use of vitro simulated rumen fermentation technology, adding different doses of PFSE, was the substrate dry. 0,1%, 2%, 3% and 4% of the substance, the results of in vitro fermentation for 24h. showed that gas production, methane production, TVFA, EPDM and dry matter digestibility were linearly decreased with the increase of PFSE dose, and methane decreased significantly at 1% (P0.05), while gas production and TVFA decreased significantly at 2% and 3% respectively (P0.05). Real-time quantitative PCR results showed PFS The dose of E did not affect the number of total bacteria and total methanogens, but the addition of P0.05.PFSE had no significant influence on the enzyme activity of carboxymethyl cellulase, microcrystalline cellulase and xylanase. The low dose group (0%, 0 mg/mL), low dose group (2%, 0.2 mg/mL) and high dose group (4%, 0.4 mg/mL) samples were reduced by the MiSeq high throughput sequencing platform. The 16S rRNA gene of bacteria and archaea in the organism was amplified and sequenced. (1) the effect of PFSE on bacterial flora: there was no significant change in the Chaol richness index of the samples of different treatment groups (P0.05), and the Shannon diversity index decreased significantly with the increase of PFSE addition. The phylum Psalm gate, the bacteriobacteria and the deformable bacteria accounted for the total bacteria total. The relative abundance of deformable bacteria increased linearly with the increase of PFSE concentration (P0.05), while bacteriobacteria, bacilli, and soft wall bacteria were linearly decreased (P0.05). The OTUs number of bacteria decreased with the increase of PFSE dose and.UniFrac analysis showed that adding 0.4 mg/mL PFSE changed the bacterial community, but the concentration of 0.2mg/mL added did not affect the bacterial community. (2) the effect of PFSE on the Archaea group: the Shannon index of the Archaea decreased linearly with the increase of dose, but the Chaol richness index did not change significantly. Although the qPCR results showed that the total number of palaetobacteria was not affected by the addition of PFSE, the relative abundance of Methanobrevibacter and Methanosphaera increased with the increase of PFSE dose, while the sequence data showed that the relative abundance of Methanobrevibacter and Methanosphaera increased with the increase of PFSE dose, The relative abundance of Methanobacteriaceae decreased. There was a correlation between microbial and methane production and rumen fermentation parameters. Rumen microbes were divided into 5 groups according to the correlation coefficient. Group I was positively correlated with methane production and total VFA (P0.05, r0.607), including Fibrobacter, Sphaerochaeta, and other eight unclassified bacteria and unclassified Methanoba Cteriaceae. Clostridium, Pedobacter, Anaeroplasma, Paludibacter, Ruminococcus, Zymomonas, Luteimonas and other seven kinds of unclassified bacteria were divided into second groups, which were positively related to methane production, but were not related to total VFA. In contrast to group I, microbiology in group III was negatively correlated with methane production and total VFA (P0.05, r-0.598). Including Roseomonas, Selenomonas, Shuttleworthia, Pseudobutyrivibrio, Anaerovibrio, Ruminobacter, Succinivibrio, Methanosphaera and three kinds of unclassified bacteria. Group IV microbes are negatively correlated with methane production (P0.05, r-0.650), including Methanobrevibacter and unclassified Sinobacteraceae. group V consists of two unclassified bacteria. Compared with the methane production and total VFA, the effect of.3 purple soda extract on the formation of methane production in Hu sheep was evaluated by reverse experimental design, and 8 adult male sheep with similar health conditions were selected. The control group was set up as basal diet (300 g concentrate and 700 g Leymus chinensis), and the treatment group added 1% (DM) to the diet of lake sheep. Plus 1% (DM) PFSE, there was no significant effect on the weight of the sheep, the dry matter intake, the dry matter digestibility, the crude protein digestibility, the digestibility of neutral detergent fiber and the acid washing fiber digestibility (P0.05), and the dry matter digestibility was reduced (P=0.086), and the dry matter intake was reduced to the control group. The trend of methane production in the rumen (P=-0.075), but with digestible dry matter and digestible fiber as a unit, there was no significant difference in the production of methane in the control group and the treatment group (P0.1), and the addition of PFSE had no significant influence on the rumen of the sheep in the rumen of lake sheep, such as pH, NH3-N, acetic acid, propionic acid, butyric acid, ethylene propylene ratio and TVFA (P0.1). The effect of inhibiting the formation of rumen methane can be further developed as a potential methane inhibitor; low doses of PFSE can inhibit methane production without negative effects on feed fermentation; this study found that some bacteria, methane bacteria and methane production are related, and further research on these microbes is helpful to the development of these microorganisms. A new strategy for methane emission reduction.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S853.7

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