研究简报 Short communications

基于宏基因组学技术检测全株玉米青贮期间和暴露空气后的微生物多样性

展开
  • 内蒙古民族大学动物科学技术学院, 通辽 028000
胡宗福(1979-),男,内蒙古呼伦贝尔人,讲师,博士研究生,从事动物营养与饲料研究。E-mail:huzongfusohu@163.com

收稿日期: 2017-04-29

  网络出版日期: 2017-09-28

基金资助

国家自然科学基金项目(31160474);优质肉牛生产核心技术研究与示范创业人才团队资助项目

Microbial Diversity of Whole-Plant Maize during Ensilage and after Air Exposure Analyzed by Metagenomics Technology

Expand
  • College of Animal Science and Technology, Inner Mongolia University for the Nationalities, Tongliao 028000, China

Received date: 2017-04-29

  Online published: 2017-09-28

摘要

本试验旨在分析全株玉米青贮发酵期间和暴露空气后的发酵品质及微生物多样性,监测其微生物群落组成动态变化。试验分别在3个发酵时期取样,即青贮第5天(F5组)、青贮第40天(F40组)和青贮40 d开袋暴露空气后第3天(A3组),每次取3袋作为3个重复。采用宏基因组学技术,测定全株玉米青贮发酵期间和暴露空气后微生物的16S rDNA V3~V4区序列,比较3个发酵时期样品微生物群落的组成和丰度信息,通过Alpha多样性和主成分分析,考察和比较全株玉米青贮发酵期间和暴露空气后的微生物多样性。结果表明:全株玉米经过40 d的青贮发酵,可使pH及中性洗涤纤维(NDF)和酸性洗涤纤维(ADF)含量显著下降(P<0.05),乳酸含量显著升高(P<0.05),具有良好的发酵品质和营养价值,且发酵40 d后短时间开袋暴露空气对其发酵品质和营养价值无显著影响(P>0.05)。3组样本通过Illumina Miseq测序平台共获得122 371条高质量有效序列,聚类为239个操作分类单元,经分类学鉴定分属16个门,163个属。在门水平上,厚壁菌门(Firmicutes)始终占优势地位,其丰度在F5、F40和A3组中分别为57.57%、74.65%、78.82%,呈增长趋势。发酵前期(F5组)、发酵后期(F40组)和开袋期(A3组)优势菌属均为乳杆菌属(Lactobacillus),其丰度分别为49.78%、64.46%和45.34%,但在开袋期芽孢乳杆菌属(Sporolactobacillus)的比例上升明显,至28.46%。综上,全株玉米青贮自然发酵升高了产乳酸乳杆菌属的丰度,有利于提高全株玉米青贮的发酵品质,但开袋暴露空气3 d对全株玉米青贮的微生物多样性产生了影响,而通过Miseq高通量测序技术,能够全面了解全株玉米青贮期间和暴露空气后微生物群落组成及丰度上的变化。

本文引用格式

胡宗福, 常杰, 萨仁呼, 王思珍, 牛化欣 . 基于宏基因组学技术检测全株玉米青贮期间和暴露空气后的微生物多样性[J]. 动物营养学报, 2017 , 29(10) : 3750 -3760 . DOI: 10.3969/j.issn.1006-267x.2017.10.038

Abstract

The objective of this experiment was to analyze the fermentation quality and microbial diversity of whole-plant maize during ensilage and after air exposure, and to monitor the dynamic changes of microbial community composition. There were 3 sampling time points in this experiment including fermentation at the 5th day (F5 group), fermentation at the 40th day (F40 group) and exposed air at the 3rd day after ensilage for 40 days (A3 group), and each sampling time point took 3 bags as 3 replicates. Microbial 16S rDNA V3 to V4 region of whole-plant maize during ensilage and after air exposure were sequenced by metagenomics technology, and the microbial community composition and abundance of 3 groups were compared. Alpha diversity and principal component analysis were carried out to investigate the microbial diversity of whole-plant maize during ensilage and after air exposure. The results showed that the contents of pH, neutral detergent fiber (NDF) and acid detergent fiber (ADF) of the whole-plant maize after 40 days of silage fermentation were significantly decreased (P<0.05), and the lactic acid content was significantly increased (P<0.05), which had a good fermentation quality and nutritional value, and short-time exposed air after 40 days for silage had no significantly effects on fermentation quality and nutritional value (P>0.05). A total of 122 371 high-quality effective sequences and 239 operational taxonomic units (OTU) were obtained by Illumina Miseq sequencing platform, and totally 16 phyla, 163 genuses of bacteria were identified in samples of 3 groups. At the level of phylum, Firmicutes was dominant from beginning to end, and its abundance in F5, F40 and A3 groups was 57.57%, 74.65% and 78.82%, respectively, and showed a growth trend. Dominant species of the early stage for fermentation (F5 group), the late stage for fermentation (F40) and the aerobic period (A3 group) were Lactobacillus, which abundance was 49.78%, 64.46% and 45.34%, respectively, but the proportion of Sporolactobacillus in aerobic period was obvious increased by 28.46%. In conclusion, the natural fermentation of whole-plant maize silage significantly increases the abundance of Lactobacillus, which is beneficial to improve the fermentation quality of whole-plant maize silage, but exposed air at the 3rd day after ensilage has effect on the microbial diversity of whole-plant maize silage. By Miseq high-throughput sequencing technology, we can fully understand the changes of microbial community composition and abundance of whole-plant maize during ensilage and after air exposure.

参考文献

[1] BERTHIAUME R,MANDELL I,FAUCITANO L,et al.Comparison of alternative beef production systems based on forage finishing or grain-forage diets with and without promotants:1.Feedlot performance,carcass quality,and production costs[J].Journal of Animal Science,2006,84(8):2168-2177.  

[2] HATEW B,BANNINK A,VAN LAAR H,et al.Increasing harvest maturity of whole-plant corn silage reduces methane emission of lactating dairy cows[J].Journal of Dairy Science,2016,99(1):354-368.  

[3] ELLIS J L,BANNINK I K,HINDRICHSEN R D,et al.The effect of lactic acid bacteria included as a probiotic or silage inoculant on in vitro rumen digestibility,total gas and methane production[J].Animal Feed Science and Technology,2016,211:61-74.

[4] CARRILLO J A,HE Y H,LI Y K,et al.Integrated metabolomic and transcriptome analyses reveal finishing forage affects metabolic pathways related to beef quality and animal welfare[J].Scientific Reports,2016,6:25948,doi:10.1038/srep25948.

[5] LIN C J,BOLSEN K K,BRENT B E,et al.Epiphytic lactic acid bacteria succession during the pre-ensiling and ensiling periods of alfalfa and maize[J].Journal of Applied Microbiology,2010,73(5):375-387.

[6] MUYZER G,DE WAAL E C,UITTERLINDEN A G.Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified gene encoding for 16S rRNA[J].Applied and Environmental Microbiology,1993,59(3):695-700.

[7] 韩吉雨,侯先志,杨凯,等.PCR-DGGE方法分析内蒙古不同地区青贮玉米中乳酸菌群多样性[J].动物营养学报,2009,21(6):974-981.

[8] 王红梅,孙启忠,屠焰,等.呼伦贝尔草原野生牧草青贮中优良乳酸菌的分离及鉴定[J].草业学报,2016,25(8):189-196.

[9] CAPORASO J G,LAUBER C L,WALTERS W A,et al.Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms[J].The ISME Journal,2012,6(8):1621-1624.  

[10] 张和平,于洁.乳酸菌基因组学研究新进展[J].中国食品学报,2016,16(2):1-8.

[11] JIANG Y,OGUNADE I M,QI S,et al.Effects of the dose and viability of Saccharomyces cerevisiae.1.Diversity of ruminal microbes as analyzed by Illumina MiSeq sequencing and quantitative PCR[J].Journal of Dairy Science,2017,100(1):325-342.  

[12] LI L H,SUN Y M,YUAN Z H,et al.Effect of microalgae supplementation on the silage quality and anaerobic digestion performance of Manyflower silvergrass[J].Bioresource Technology,2015,189:334-340.

[13] 刘晶晶.生物添加剂对柳枝稷青贮的作用及机理研究[D].博士学位论文.北京:中国农业大学,2015.

[14] 陶莲,刁其玉.青贮发酵对玉米秸秆品质及菌群构成的影响[J].动物营养学报,2016,28(1):198-207.

[15] BAO W C,MI Z H,XU H Y,et al.Assessing quality of Medicago sativa silage by monitoring bacterial composition with single molecule,real-time sequencing technology and various physiological parameters[J].Scientific Reports,2016,6:28358,doi:10.1038/srep28358.

[16] 万楚筠,钮琰星,黄凤洪,等.对羟基联苯比色法测定乳酸显色反应条件的研究[J].食品工业科技,2013,34(7):322-324,353.

[17] 张丽英.饲料分析及饲料质量检测技术[M].2版.北京:中国农业大学出版社,2003:53-56,70-74.

[18] OWENS V N,ALBRECHT K A,MUCK R E,et al.Protein degradation and fermentation characteristics of red clover and alfalfa silage harvested with varying levels of total nonstructural carbohydrates[J].Crop Science,1999,39(6):1873-1880.  

[19] DENNIS K L,WANG Y W,BLATNER N R,et al.Adenomatous polyps are driven by microbe-instigated focal inflammation and are controlled by IL-10-producing T cells[J].Cancer Research,2013,73(19):5905-5913.  

[20] ZHOU Y,DROUIN P,LAFRENIÈRE C.Effect of temperature (5-25℃) on epiphytic lactic acid bacteria populations and fermentation of whole-plant corn silage[J].Journal of Applied Microbiology,2016,121(3):657-671.  

[21] 兴丽,韩鲁佳,刘贤,等.乳酸菌和纤维素酶对全株玉米青贮发酵品质和微生物菌落的影响[J].中国农业大学学报,2004,9(5):38-41.

[22] 许庆方,张翔,崔志文,等.不同添加剂对全株玉米青贮品质的影响[J].草地学报,2009,17(2):157-161.

[23] SANTOS A O,ÁVILA C L S,SCHWAN R F.Selection of tropical lactic acid bacteria for enhancing the quality of maize silage[J].Journal of Dairy Science,2013,96(12):7777-7789.  

[24] MUCK R E.Recent advances in silage microbiology[J].Agriculture Food Science,2013,22(1):3-15.

[25] RANJIT N K,TAYLOR C C,KUNG L,Jr.Effect of Lactobacillus buchneri 40788 on the fermentation,aerobic stability and nutritive value of maize silage[J].Grass Forage Science,2002,57(2):73-81.  

[26] DOLCI P,TABACCO E,COCOLIN L,et al.Microbial dynamics during aerobic exposure of corn silage stored under oxygen barrier or polyethylene films[J].Applied and Environmental Microbiology,2011,77(21):7499-7507.  

[27] TOHNO M,KOBAYASHI H,NOMURA M,et al.Identification and characterization of lactic acid bacteria isolated from mixed pasture of timothy and orchardgrass,and its badly preserved silage[J].Journal of Animal Science,2002,83(4):318-330.

[28] SMOKER A N.Screening of Sporolactobacillus and Bacillus Strains for Use in Silage Inoculation[D].Master Thesis.Corvallis:Oregon State University,1999.

[29] KHARAZIANA Z A,JOUZANIA G S,AGHDAS M,et al.Biocontrol potential of Lactobacillus strains isolated from corn silages against some plant pathogenic fungi[J].Biological Control,2017,110(1):33-43.

[30] 刘磊,朱立贤.芽孢乳杆菌对肉仔鸡生产性能、肠道发育和微生物菌群的影响[J].动物营养学报,2011,23(12):2136-2142.

[31] DRIEHUIS F,ELFERINK S J W H O.The impact of the quality of silage on animal health and food safety:a review[J].Veterinary Quarterly,2000,22(4):212-216.  

[32] WAMBACQ E,VANHOUTTE I,AUDENAERT K,et al.Occurrence,prevention and remediation of toxigenic fungi and mycotoxins in silage:a review[J].Journal of the Science of Food and Agriculture,2016,96(7):2284-2302.  

[33] VISSERS M M M,TE GIFFEL M C,DRIEHUIS F,et al.Minimizing the level of Bacillus cereus spores in farm tank milk[J].Journal of Dairy Science,2007,90(7):3286-3293.  

[34] MCDONALD P,HENDERSON A R,HERON S J E.The biochemistry of silage[M].2nd ed.Marlow:Chalcombe Publications, 1991:81-151.

[35] DUNIÈRE L,SINDOU J,CHAUCHEYRAS-DURAND F,et al.Silage processing and strategies to prevent persistence of undesirable microorganisms[J].Animal Feed Science and Technology,2013,182(1/2/3/4):1-15.
文章导航

/