Romero J J, Zarate M A, Arriola K G, Gonzalez C F, Silva-Sanchez C, Staples C R, Adesogan A T
Department of Animal Sciences, University of Florida, Gainesville 32608.
Department of Microbiology and Cell Science, University of Florida, Gainesville 32608.
J Dairy Sci. 2015 Apr;98(4):2555-67. doi: 10.3168/jds.2014-8059. Epub 2015 Feb 11.
Our objectives were to evaluate the effects of 12 exogenous fibrolytic enzyme products (EFE) on ruminal in vitro neutral detergent fiber digestibility (NDFD) and preingestive hydrolysis of a 4-wk regrowth of bermudagrass haylage (BH), to examine the accuracy of predicting NDFD with EFE activity measures, and to examine the protein composition of the most and least effective EFE at increasing NDFD. In experiment 1, effects of 12 EFE on NDFD of BH were tested. Enzymes were applied in quadruplicate to culture tubes containing ground BH. The suspension was incubated for 24 h at 25 °C before addition of rumen fluid media and further incubation for 24 h at 39 °C. The experiment was repeated twice. In addition, regression relationships between EFE activity measures and NDFD were examined. Compared with the values for the control, 9 EFE-treated substrates had greater NDFD (37.8 to 40.4 vs. 35.6%), 6 had greater total VFA concentration (59.1 to 61.2 vs. 55.4 mM), and 4 had lower acetate-to-propionate ratios (3.03 to 3.16 vs. 3.24). In experiment 2, EFE effects on preingestive fiber hydrolysis were evaluated by incubating enzyme-treated and untreated bermudagrass suspensions in quadruplicate for 24 h at 25 °C and examining fiber hydrolysis measures. Compared with values for the control, 3 EFE reduced neutral detergent fiber concentration (62.8 to 63.7 vs. 67.3%), 10 increased release of water-soluble carbohydrates (26.8 to 58.5 vs. 22.8 mg/g), and 8 increased release of ferulic acid (210 to 391 vs. 198 μg/g). Regression analyses revealed that enzyme activities accurately [coefficient of determination (R(2)) = 0.98] predicted preingestive hydrolysis measures (water-soluble carbohydrates, ferulic acid), moderately (R(2) = 0.47) predicted neutral detergent fiber hydrolysis, but poorly (R(2) ≤ 0.1) predicted dry matter and NDFD. In experiment 3, proteomic tools were used to examine the protein composition of the most and least effective EFE at improving NDFD. Relative to the least effective, the most effective EFE at increasing NDFD contained 10 times more endoglucanase III, 17 times more acetylxylan esterase with a cellulose-binding domain 1, 33 times more xylanase III, 25 times more β-xylosidase, and 7.7 times more polysaccharide monooxygenase with cellulose-binding domain 1 and 3 times more swollenin. The most effective EFE had a much greater quantity of fibrolytic enzymes and key proteins necessary for hemicellulose and lignocellulase deconstruction. This study identified several EFE that increased the NDFD and in vitro fermentation of 4-wk BH and revealed why some EFE are more effective than others.
我们的目标是评估12种外源纤维分解酶产品（EFE）对瘤胃体外中性洗涤纤维消化率（NDFD）以及4周龄百慕大青贮干草（BH）采食前水解的影响，检验用EFE活性指标预测NDFD的准确性，并分析在提高NDFD方面最有效和最无效的EFE的蛋白质组成。在实验1中，测试了12种EFE对BH的NDFD的影响。将酶一式四份应用于装有磨碎BH的培养管中。悬浮液在25℃下孵育24小时，然后加入瘤胃液培养基，并在39℃下进一步孵育24小时。该实验重复了两次。此外，还研究了EFE活性指标与NDFD之间的回归关系。与对照组相比，9种经EFE处理的底物具有更高的NDFD（37.8%至40.4%对35.6%），6种具有更高的总挥发性脂肪酸浓度（59.1至61.2 mM对55.4 mM），4种具有更低的乙酸与丙酸比率（3.03至3.16对3.24）。在实验2中，通过将酶处理和未处理的百慕大草悬浮液一式四份在25℃下孵育24小时并检测纤维水解指标，评估了EFE对采食前纤维水解的影响。与对照组相比，3种EFE降低了中性洗涤纤维浓度（62.8%至63.7%对67.3%），10种增加了水溶性碳水化合物的释放量（26.8至58.5 mg/g对22.8 mg/g），8种增加了阿魏酸的释放量（210至391 μg/g对198 μg/g）。回归分析表明，酶活性能够准确地[决定系数（R²）= 0.98]预测采食前水解指标（水溶性碳水化合物、阿魏酸），适度地（R² = 0.47）预测中性洗涤纤维水解，但对干物质和NDFD的预测效果较差（R²≤0.1）。在实验3中，使用蛋白质组学工具分析在提高NDFD方面最有效和最无效的EFE的蛋白质组成。相对于最无效的EFE，在增加NDFD方面最有效的EFE所含的内切葡聚糖酶III多10倍，含纤维素结合结构域1的乙酰木聚糖酯酶多17倍，木聚糖酶III多33倍，β-木糖苷酶多25倍，含纤维素结合结构域1的多糖单加氧酶多7.7倍，膨胀素多3倍。最有效的EFE具有更多数量的纤维分解酶以及半纤维素和木质纤维素解构所需的关键蛋白质。本研究鉴定出了几种能提高4周龄BH的NDFD和体外发酵能力的EFE，并揭示了为何有些EFE比其他的更有效。
