【摘要】：農(nóng)業(yè)生物質資源是我國極其豐富的、可再生的生物燃料,化工原料和飼料來源。然而,由于農(nóng)作物秸稈細胞壁多糖成分的復雜性、合成酶系的多樣性和超分子結構的異質性導致其難以被畜禽有效利用,造成極大的浪費。厭氧真菌具有很強的降解植物組織的能力,是草食動物消化道內(nèi)一類重要的降解植物細胞壁的功能菌。本文以西農(nóng)薩能奶山羊瘤胃真菌為研究對象,利用亨蓋特厭氧滾管技術,分離獲得高植物細胞壁降解酶活性厭氧真菌,并研究其作為添加劑在全株玉米青貯中的應用效果,全面評價瘤胃厭氧真菌降解植物細胞壁功效及應用潛力,為今后瘤胃真菌資源的開發(fā)及在飼料工業(yè)的應用提供理論依據(jù)和技術支持。高植物細胞壁降解酶活性瘤胃真菌分離鑒定及酶學特性研究本研究采用亨蓋特厭氧滾管技術從西農(nóng)薩能奶山羊瘤胃中分離獲得12株具有細胞壁降解酶活性的厭氧真菌,通過形態(tài)學觀察、核糖體內(nèi)轉錄間隔區(qū)和28S rDNA D1/D2區(qū)基因序列分析確定其分類地位。測定12株真菌的植物細胞壁降解酶活性(木聚糖酶、羧甲基纖維素酶、微晶纖維素酶、乙酰酯酶和β-葡聚糖酶),并對活性最高菌株的木聚糖酶和乙酰酯酶進行酶學特性分析。結果表明,12株瘤胃厭氧真菌均為Piromyces屬,分別命名為Piromyces sp.CN1~Piromyces sp.CN12。其中,Piromyces sp.CN6的木聚糖酶、羧甲基纖維素酶和乙酰酯酶活性分別為1655.3、93.4和152.8 mU,顯著高于其他菌株(P0.05),Piromyces sp.CN3的微晶纖維素酶活性最高,但與Piromyces sp.CN6差異不顯著(P0.05),各菌株間β-葡聚糖酶活性差異不顯著(P0.05)。木聚糖酶與羧甲基纖維素酶、乙酰酯酶存在極顯著正相關(P0.01),與微晶纖維素酶呈顯著正相關(P0.05)。酶學特性表明,木聚糖酶最適反應溫度為50℃,最適pH為5.0,該酶在40℃和pH 5.0~8.0下較穩(wěn)定;K+、Co2+、Ca2+對其有激活作用,Zn2+、Cu2+、Mg2+、Fe2+、Mn2+抑制該酶活性。乙酰酯酶的最適反應溫度為50℃,最適pH為9.0,該酶在40℃和pH 5.0~10.0下較穩(wěn)定;Mg2+、K+、Ca2+對乙酰酯酶有激活作用,Zn2+、Fe2+、Co2+、Mn2+抑制該酶活性。本研究從西農(nóng)薩能奶山羊瘤胃內(nèi)容物分離獲得12株Piromyces屬厭氧真菌,Piromyces sp.CN6具有較高的植物細胞壁降解酶活性,其中木聚糖酶和乙酰酯酶在較寬的溫度和pH范圍內(nèi)均具有較高的活性,同時具有良好的熱穩(wěn)定性和酸堿穩(wěn)定性。Piromyces sp.CN6對全株玉米青貯發(fā)酵品質、營養(yǎng)成分及體外降解率的影響本研究旨在驗證試驗一所篩選的高植物細胞壁降解酶活性瘤胃真菌Piromyces sp.CN6作為青貯添加劑對全株玉米青貯發(fā)酵品質、營養(yǎng)成分及體外降解率的影響。試驗采用單因素完全隨機設計,每組設5個重復,以全株玉米為青貯原料,分別設為對照組、真菌組(105 TFU/g)和復合酶組(0.033 mg/g),聚乙烯袋真空包裝,室溫下存儲10 d、30 d、60 d后開封取樣。結果表明,真菌組和復合酶組青貯飼料達到1級優(yōu)良,對照組達到2級較好,瘤胃真菌Piromyces sp.CN6作為青貯接種劑能夠在青貯前期大量繁殖并附著在玉米秸稈莖葉表面。與對照組相比,真菌組和復合酶組均可顯著降低發(fā)酵30 d青貯飼料pH、乙酸含量和NH3-N/TN(P0.05),顯著降低發(fā)酵10 d、30 d、60 d青貯飼料NDF和ADF含量(P0.05),顯著提高發(fā)酵30 d青貯飼料可溶性碳水化合物含量、粗蛋白含量、乳酸含量和乳酸/乙酸比值(P0.05)。與對照組相比,真菌接種劑可顯著提高發(fā)酵30 d青貯飼料DM、NDF和ADF體外降解率(P0.05)。不同處理對青貯飼料干物質回收率無顯著影響(P0.05)。綜上,瘤胃真菌Piromyces sp.CN6能夠改善青貯飼料的發(fā)酵品質和營養(yǎng)成分,提高粗纖維的降解率。本研究從西農(nóng)薩能奶山羊瘤胃分離獲得12株Piromyces屬厭氧真菌,其中Piromyces sp.CN6具有較高的植物細胞壁降解酶活性,且各酶之間具有較高的相關性。酶學特性分析表明:Piromyces sp.CN6分泌的木聚糖酶和乙酰酯酶在較寬的溫度和pH范圍內(nèi)均具有較高的活性,同時具有良好的熱穩(wěn)定性和酸堿穩(wěn)定性。瘤胃厭氧真菌能夠改善青貯飼料的發(fā)酵品質和營養(yǎng)成分,提高粗纖維的降解率。
[Abstract]:The agricultural biomass resource is a rich and renewable bio-fuel, chemical raw material and feed source in China. However, due to the complexity of the polysaccharide component of the cell wall of the crop straw, the diversity of the synthetase system and the heterogeneity of the supramolecular structure cause it to be difficult to be effectively utilized by the livestock and poultry, resulting in great waste. The anaerobic fungus has strong ability to degrade plant tissue, and is an important functional bacterium for degrading plant cell wall in the digestive tract of herbivorous animal. In this paper, the rumen fungi of the nongaseng milk goat in the west of this article are the research objects, and the high plant cell wall degrading enzyme active anaerobic fungus is obtained by using the Henligate anaerobic rolling tube technology, and the application effect of the anaerobic fungus as an additive in the whole plant corn silage is studied. The effects of the rumen anaerobic fungi on the plant cell wall and the application potential are comprehensively evaluated, and the theoretical basis and technical support are provided for the development of the rumen fungus resources and the application of the feed industry in the future. In that present study,12 strain of anaerobic fungus with cell wall-degrading enzyme activity were obtained from the rumen of the western agricultural sacha milk goat by using the high-plant cell wall-degrading enzyme-active rumen fungus separation and identification and the enzyme-study property. The classification status of the ribosomal internal transcribed spacer region and the 28S rDNA D1/ D2 region gene sequence analysis was determined. The activity of plant cell wall-degrading enzymes (xylanase, methylcellulose, microcrystalline cellulose, and B-glucanase) of 12 strains of fungi was determined, and the xylanase and glyoxesterase of the highest active strain were analyzed. The results showed that the 12 strains of the rumen anaerobic fungi were Piromytsp. CN1 to Piromytsp. CN12, respectively. Among them, the activity of xylanase of Piromytsp. CN6 was 1655.3, 93.4 and 152.8mU, which was higher than that of other strains (P0.05). There was no significant difference in the activity of the E-glucanase between the strains (P0.05). There was a significant positive correlation between the xylanase and the methyl cellulose (P 0.01), and it was positively correlated with the microcrystalline cellulose (P <0.05). The optimum reaction temperature of xylanase is 50 鈩，

本文編號：2479409