体外法优化玉米——杂粕型饲粮的非淀粉多糖酶谱及其对育肥猪肠道微生物的影响

Non-Starch Polysaccharide Enzymes Cocktail of Corn-Miscellaneous Meal-Based Diet Optimization by In Vitro Method and Its Effects on Intestinal Microbiome in Finishing Pigs

【目的】利用体外模拟法优化玉米—杂粕型饲粮的非淀粉多糖(NSP)酶谱,并分析优化的非淀粉多糖酶谱对饲粮养分消化率和育肥猪肠道微生物组成和结构的影响,为饲粮高效利用和精准饲养提供数据支撑和理论参考。【方法】试验一在育肥猪玉米—杂粕型饲粮中分别添加不同水平的木聚糖酶、β-葡聚糖酶、纤维素酶、α-半乳糖苷酶、β-甘露聚糖酶和果胶酶6种NSP酶,采用胃—小肠体外模拟法测定体外回肠干物质消化率(IVIDMD);取IVIDMD达到最大值时各NSP酶的添加水平为该NSP酶0编码水平,按照六元二次回归正交旋转组合设计,进行体外消化试验,建立IVIDMD与NSP酶添加量的六元二次回归方程,筛选出玉米—杂粕型饲粮最优NSP酶谱(OEC);利用胃—小肠—大肠体外模拟消化法分析测定添加OEC前后饲粮的体外干物质消化率(IVDMD)、体外能量消化率(IVGED)和体外消化能(IVDE),以验证OEC的效果。试验二按照单因素完全随机设计,选用16头健康、体重相近去势公猪(117.8±1.66 kg),随机分为2个处理组,每个处理8头猪,对照组饲喂玉米—杂粕型基础饲粮,加酶组在基础饲粮中添加OEC;在试验开展第18天采用直肠擦拭法采集猪新鲜粪便,利用16S rRNA基因高通量测序对粪便微生物菌群的多样性及相对丰度进行分析,并进行功能预测。【结果】(1)在本试验条件下,玉米—杂粕型饲粮OEC为:纤维素酶1003 U·kg^(-1)、木聚糖酶18076 U·kg^(-1)、β-葡聚糖酶1377 U·kg^(-1)、β-甘露聚糖酶14765 U·kg^(-1)、α-半乳糖苷酶337 U·kg^(-1)和果胶酶138 U·kg^(-1);(2)在玉米—杂粕型饲粮中添加OEC使IVDMD由73.44%显著提高到76.26%(P<0.01),IVGED由74.03%显著提高到76.45%(P=0.01),IVDE由14.97 MJ·kg^(-1)显著提高到15.58 MJ·kg^(-1)(P<0.01);(3)在门水平上,共筛选出了12个相对丰度大于0.1%的菌门,其中Bacteroidota(拟杆菌门)、Firmicutes(厚壁菌门)和Spirochaetota(螺旋体门)为优势菌门,三者之和在组内占比达96%以上;(4)在属水平上,饲粮中添加OEC显著增加norank_f_F082,norank_f_Bacteroidales_RF16_group,Bacteroides(拟杆菌属)和Roseburia(氏菌属)的相对丰度(P<0.05),Eubacterium_ruminantium_group(P=0.083)有增加的趋势,而Oscillibacter(颤杆菌克)的相对丰度显著降低(P<0.05),Clostridium_sensu_stricto_1和norank_f__norank_o__WCHB1-41(P=0.083)有降低的趋势(P=0.052)。【结论】饲粮中添加体外法优化的NSP酶谱,显著提高了育肥猪玉米—杂粕型饲粮干物质和能量的体外消化率以及体外消化能,增加了纤维分解菌和产丁酸菌等有益菌在育肥猪肠道微生物中的占比,在一定程度上减少了有害菌的数量,优化了肠道微生态。

【Objective】The objective of this study was to optimize the non-starch polysaccharide(NSP)enzymes cocktail of the corn-miscellaneous meal-based diet for finishing pigs by using in vitro simulation method,and to analyze the effects of the optimal NSP enzymes cocktail(OEC)on dietary nutrient digestibility and intestinal microbial composition and structure of finishing pigs.Finally,it could provide data support and theoretical reference for efficient utilization of diets and precise feeding.【Method】In experiment 1,different levels of six NSP enzymes(xylanase,β-glucanase,cellulase,α-galactosidase,β-mannanase,and pectinase)were individually and respectively added to the corn-miscellaneous meal-based diet of finishing pigs.Then,in vitro ileal dry matter digestibility(IVIDMD)was determined by gastric-small intestinal simulation digestion method in vitro.When IVIDMD reached the maximum,the supplemental level of each NSP enzyme was the coding level of NSP enzyme 0.In vitro digestion experiments were carried out according to the six-element quadratic regression orthogonal rotation combination design.Meanwhile,the optimal NSP enzymes cocktail(OEC)of the corn-miscellaneous meal-based diet was selected by establishing the six-element quadratic regression equation between IVIDMD and the supplemental level of NSP enzymes.The in vitro dry matter digestibility(IVDMD),in vitro gross energy digestibility(IVGED)and in vitro digestible energy(IVDE)of diets before and after OEC addition were determined by gastric-small intestinal-large intestinal simulation digestion method in vitro to verify the effect of OEC.In experiment 2,16 healthy castrated barrows(117.8±1.66 kg)with similar body weight were randomly divided into two groups with eight pigs in each group.The pigs in the control group were fed the corn-miscellaneous meal-based diet,and the pigs in the enzyme-addition group were fed the basal diet supplemented with OEC.On the 18th day of the experiment,the fresh feces of pigs were collected by rectal wiping method,and the diversity and relative abundance of fecal microbiome were analyzed by high-throughput sequencing analysis of 16S rRNA gene,and the function was predicted.【Result】(1)Under the conditions of this experiment,the optimized NSP enzymes cocktail of corn-miscellaneous meal-based diet was as follows:cellulase 1003 U·kg^(-1),xylanase 18076 U·kg^(-1),β-glucanase 1377U·kg^(-1),β-mannanase 14765 U·kg^(-1),α-galactosidase 337 U·kg^(-1),and pectinase 138 U·kg^(-1).(2)Adding NSP enzymes cocktail optimized by in vitro method in corn-miscellaneous meal-based diet significantly increased the IVDMD from 73.44%to 76.26%(P<0.01),the IVGED from 74.03%to 76.45%(P=0.01),and the IVDE from 14.97 MJ·kg^(-1) to 15.58 MJ·kg^(-1)(P<0.01).(3)At the phylum level,a total of 12 phyla with relative abundances greater than 0.1%were selected,among which Bacteroidetes,Firmicutes,and Spirochetes were the dominant phyla,and the sum of these three phyla accounted for more than 96%in the group.(4)At the genus level,adding OEC in the diet significantly increased the relative abundance of Norank_F_F082,Norank_F_Bacteroidales_RF16_group,Bacteroides and Roseburia(P<0.05),and Eubacterium_ruminantium_group(P=0.083)had an increasing trend,while the relative abundance of Oscillibacter decreased significantly(P<0.05),and Clostridium_Sensu_Stricto_1 and Norank_F__Norank_O__WCHB1-41(P=0.083)showed a decreasing trend(P=0.052).【Conclusion】Dietary non-starch polysaccharide enzymes cocktail optimization by in vitro method increased in vitro digestibility of dry matter and energy and in vitro digestible energy of corn-miscellaneous meal-based diets in finishing pigs.It also increased the proportion of beneficial bacteria in intestinal microorganism,such as fiber decompose bacteria and butyric acid producing bacteria,and reduced the number of harmful bacteria to a certain extent,and optimized intestinal microecology.