發(fā)布時間：2018-05-18 21:26

  本文選題：紫蘇子 + 甲烷產(chǎn)量��； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：減少反芻動物甲烷的排放對緩解溫室效應(yīng)和提高飼料利用率具有雙重意義。中草藥含有豐富的活性物質(zhì),是一種潛在的瘤胃甲烷抑制劑,可通過調(diào)控瘤胃微生物菌群提高飼料利用率并抑制甲烷產(chǎn)生。本研究首先利用體外模擬瘤胃發(fā)酵試驗技術(shù)對百味中草藥進行了篩選和驗證；然后討論了對抑制甲烷最有效的中草藥,紫蘇子(Perilla Frutescens Seed, PFS)的劑型和劑量效應(yīng),并通過實時定量PCR、 16SrDNA測序,探究了體外添加紫蘇子提取物(Perilla Frutescens Seed Extracts, PFSE)對瘤胃微生物數(shù)量和種群的影響；最后以湖羊為模式動物,研究了在日糧中添加PFSE對湖羊消化功能、甲烷生成和瘤胃發(fā)酵的影響。1抑制甲烷生成的中草藥篩選和驗證1.1初步篩選利用體外模擬瘤胃發(fā)酵試驗技術(shù),發(fā)酵體系為10 ml,底物為0.1 g精粗比是30：70(DM)的玉米和羊草混合物。單因素試驗設(shè)計,設(shè)置處理組100個,同時分別設(shè)置一個對照組和一個空白組,每個處理有3個重復(fù)。其中100個處理組分別為百味中草藥的70%乙醇提取物,添加劑量為底物的4%(DM),以40 mg/ml的二甲基亞砜(DMSO)溶液的形式添加。對照組添加0.1 ml DMSO,空白組無底物且不添加中草藥提取物及DMSO,以扣除人工瘤胃液的自身產(chǎn)氣量。體外發(fā)酵24 h后測定產(chǎn)氣量和甲烷產(chǎn)量。研究發(fā)現(xiàn),有31味中草藥降低甲烷產(chǎn)量達10%以上,其中降低甲烷產(chǎn)量最多的十味中草藥依次是山竹皮(74.9%)、厚樸(60.1%)、乳香(45.4%)、蘇木(44.8%)、五加皮(42.6%)、姜黃(42.6%)、蛇床子(38.8%)、遠志(33.3%)、紫蘇子(30.1%)和辛夷(29.0%)。1.2發(fā)酵驗證對初篩中的31味中草藥的降甲烷效果進行驗證。同樣利用體外模擬瘤胃發(fā)酵試驗技術(shù),發(fā)酵體系擴大為50 ml,底物為0.5 g的玉米和羊草混合物,精粗比為30：70(DM)。采用單因素實驗設(shè)計,設(shè)置31個處理組,添加劑量仍然為底物的4%(DM),對照組添加0.5 ml DMSO,空白組和標準羊草組同篩選試驗,每個處理有4個重復(fù)。體外發(fā)酵24 h后測定產(chǎn)氣量、甲烷產(chǎn)量和其他瘤胃參數(shù)。在31味中草藥提取物中,有10味產(chǎn)氣量顯著減少(P0.05),6味產(chǎn)氣量顯著增加(P0.05)；12味顯著降低甲烷的產(chǎn)量(P0.05),只有桃仁提取物顯著增加了甲烷的產(chǎn)量(P0.05)。除遠志提取物之外,篩選試驗中降低甲烷產(chǎn)量最多的10味中草藥提取物在驗證試驗中被證實能夠顯著抑制甲烷生成(P0.05)。12味中草藥提取物中,只有厚樸和酸棗仁在顯著抑制甲烷生成的同時又沒有減少產(chǎn)氣量(P0.05)。在31味中草藥提取物中,只有一味中草藥提取物(辛夷)顯著降低TVFA的濃度(P0.05),9味顯著降低乙丙比(P0.05),其中有7味來自12味顯著降低甲烷產(chǎn)量的中草藥提取物。紫蘇子在驗證試驗中降低甲烷產(chǎn)量的效果最好(63.6%),因此作為目標提取物做進一步研究。2紫蘇子對瘤胃發(fā)酵和甲烷生成的影響及機制不同劑型紫蘇子,即提取物、普通粉(80目)和超微粉(600目),主要成分均為長鏈脂肪酸,其中PFSE中十八碳不飽和脂肪酸含量最高(675 μg/mg),約是普通粉(295 μg/mg)的2倍,超微粉(118 μg/mg)的3倍。體外模擬瘤胃發(fā)酵試驗結(jié)果表明,與高劑量PFSE (2%,0.2 mg/ml, DM)、普通粉和超微粉相比,添加低劑量的PFSE (1%,0.1 mg/ml, DM)顯著降低甲烷產(chǎn)量(P0.05)并顯著增加有效產(chǎn)氣量(P0.05),對其他發(fā)酵參數(shù)沒有顯著影響(P0.05)。同樣利用體外模擬瘤胃發(fā)酵試驗技術(shù),添加不同劑量PFSE,分別為底物干物質(zhì)的0、1%、2%、3%和4%,體外發(fā)酵24h。結(jié)果表明,產(chǎn)氣量、甲烷產(chǎn)量、TVFA、乙丙比以及干物質(zhì)消化率均隨PFSE劑量增加而線性降低,其中甲烷在添加量為1%時已下降顯著(P0.05),而產(chǎn)氣量和TVFA分別是在2%和3%時下降顯著(P0.05)。實時定量PCR結(jié)果表明PFSE劑量不影響總細菌和總甲烷菌數(shù)量,但降低原蟲數(shù)量(P0.05)。PFSE的添加對羧甲基纖維素酶、微晶纖維素酶和木聚糖酶的酶活性沒有顯著影響。利用MiSeq高通量測序平臺對對照組(0%,0 mg/mL)、低劑量組(2%,0.2 mg/mL)和高劑量組(4%,0.4 mg/mL)樣品微生物中細菌和古菌的16S rRNA基因進行擴增測序分析。(1) PFSE對細菌菌群的影響：不同處理組樣品的細菌Chaol豐富度指數(shù)沒有發(fā)生顯著性變化(P0.05),Shannon多樣性指數(shù)隨PFSE添加量的增加顯著降低。厚壁菌門、擬桿菌門和變形菌門占細菌總序列數(shù)的89.6%以上。其中,變形菌門相對豐度隨PFSE濃度的增加而線性增加(P0.05),而擬桿菌門、纖維桿菌門、軟壁菌門則線性降低(P0.05)。細菌OTUs數(shù)隨PFSE劑量增加而降低。UniFrac分析表明添加0.4 mg/mL PFSE改變細菌群落,但0.2mg/mL添加濃度無影響。(2)PFSE對古菌菌群的影響：古菌Shannon指數(shù)隨劑量增加而線性降低,但Chaol豐富度指數(shù)沒有發(fā)生顯著性變化。雖然qPCR結(jié)果顯示古菌總數(shù)不受PFSE添加的影響,但測序數(shù)據(jù)顯示Methanobrevibacter和Methanosphaera的相對豐度隨PFSE劑量增加而增加,而Methanobacteriaceae的相對豐度隨之降低。微生物與甲烷產(chǎn)量、瘤胃發(fā)酵參數(shù)存在相關(guān)性,根據(jù)相關(guān)系數(shù)將瘤胃微生物分為5組。組Ⅰ與甲烷產(chǎn)量和總VFA呈正相關(guān)(P0.05,r0.607),包括Fibrobacter、 Sphaerochaeta和其他八種未分類的細菌與未分類的Methanobacteriaceae。 Clostridium、Pedobacter、Anaeroplasma、Paludibacter、Ruminococcus、Zymomonas、 Luteimonas和其他七種未分類的細菌被劃分為第2組,該組微生物與甲烷產(chǎn)量正相關(guān),但與總VFA無關(guān)。與組Ⅰ相反,組Ⅲ中微生物與甲烷產(chǎn)量和總VFA都呈負相關(guān)(P0.05,r-0.598),包括 Roseomonas、Selenomonas、Shuttleworthia、 Pseudobutyrivibrio、Anaerovibrio、Ruminobacter、Succinivibrio、Methanosphaera和三種未分類的細菌。組Ⅳ微生物與甲烷產(chǎn)量呈負相關(guān)(P0.05,r-0.650),包括Methanobrevibacter和未分類的Sinobacteraceae。組Ⅴ由兩種未分類的菌組成,其與甲烷產(chǎn)量和總VFA都無關(guān)。3紫蘇子提取物對湖羊甲烷生成影響的在體評定采用反轉(zhuǎn)實驗設(shè)計,選取8只體況相近健康無病的成年公湖羊,設(shè)置對照組為基礎(chǔ)日糧(300 g精料和700 g羊草),處理組為基礎(chǔ)日糧上添加1%(DM)的PFSE。在湖羊日糧中添加1%(DM) PFSE,對湖羊的體重、干物質(zhì)采食量、干物質(zhì)消化率、粗蛋白消化率、中性洗滌纖維消化率和酸性洗滌纖維消化率均沒有顯著影響(P0.05),其中干物質(zhì)消化率有降低的趨勢(P=0.086)；以干物質(zhì)采食量為單位,與對照組相比處理組有降低瘤胃甲烷生成的趨勢(P=-0.075),而以可消化干物質(zhì)和可消化纖維為單位,對照組和處理組瘤胃甲烷的生成量沒有顯著差異(P0.1)；添加PFSE對湖羊瘤胃的pH、NH3-N、乙酸、丙酸、丁酸、乙丙比和TVFA均沒有顯著影響(P0.1)。綜上所述,中草藥具有良好的抑制瘤胃甲烷生成的效果,可作為潛在的甲烷抑制劑做進一步開發(fā)；低劑量的PFSE能抑制甲烷生成而不對飼料發(fā)酵造成負面影響；本研究發(fā)現(xiàn)一些細菌、甲烷菌與甲烷產(chǎn)生存在相關(guān)關(guān)系,進一步對這些微生物的深入研究有助于開發(fā)甲烷減排的新策略。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S853.7

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2 孫鳳莉;墨鋒濤;李茜;鄭長山;魏忠華;左曉磊;劉榮昌;;反芻動物甲烷減量排放調(diào)控措施[A];冀農(nóng)杯2008“綠色奧運”科技論文集[C];2008年

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