饲粮中添加发酵豆粕对断奶仔猪粪便菌群多样性的影响
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摘要
本试验采用高通量测序技术,研究饲粮中添加发酵豆粕对断奶仔猪粪便菌群多样性的影响。选取36日龄"杜×长×大"仔猪80头,随机分为A、B、C、D组,D组为对照组,饲喂基础饲粮; A、B、C组为试验组,分别在基础饲粮中添加5%、10%、15%的发酵豆粕。试验结束采集4组仔猪的粪便,采用Illumina Mi Seq高通量测序技术对其中细菌的16S rRNA基因的V3～V4区序列进行测序。结果表明:1)菌群的平均有效序列数为47 218条;在97%的相似水平下共产生了3 581个操作分类单元(OTUs);共检测到12个门、21个纲、33个目、64个科、111个属和137个种。2)α多样性指数在各组间均无显著性差异(P>0.05),但B组的Shannon指数最高,菌群最丰富。β多样性分析表明,4组菌群在统计学上存在显著差异(P<0.05),分组效果较好。3)门水平的优势菌为厚壁菌门,科与属水平的优势菌分别为乳酸菌科和乳酸菌属。B组的厚壁菌门相对丰度显著高于D组(P<0.05),乳酸菌属相对丰度显著高于A、D组(P<0.05),但与C组间差异不显著(P>0.05)。4)菌群主要参与的代谢通路为糖类代谢、氨基酸代谢、能量代谢、核酸代谢、辅酶和维生素代谢。除核酸代谢外,其余4种代谢通路中,B组菌群的相对丰度最高并依次为C、A、D组。综上所述,在仔猪饲粮中添加10%的发酵豆粕,能增加肠道菌群的多样性,显著增加肠道中厚壁菌门、乳酸菌科和乳酸菌属等有益菌的相对丰度,维持肠道的健康,促进营养物质的代谢。
This study was conducted to study the effects of dietary fermented soybean meal( FSM) on the fecal microbiota diversity of weaned piglets using Illumina high-throughput sequencing technology. A total of 80 piglets were randomly divided into A,B,C and D groups. As the control,the piglets of group D were fed a basal diet. The piglets of groups A,B and C were fed the basal diet added 5%,10% and 15% FSM,respectively.Feces of four groups were collected after the experiment. The V3 to V4 region of the 16 S rRNA gene of bacteria in faeces was sequenced using Illumina high-throughput sequencing technology. The results showed as follows: 1) in all of the samples,the average effective sequence number of bacteria was 47 218 and 3 581 operational taxonomic unit( OTUs) were produced at a similar level of 97% and 12 phylum,21 classes,33 orders,64 families,111 genera and 137 species were detected. 2) The alpha diversity indexes of fecal microbiota showed that there was no significant difference among groups( P > 0. 05),but the Shannon index of group B was the highest,indicating that the diversity of this group was the best. The beta analysis of diversity showed that the grouping effect was better,and there were significant differences between groups( P<0.05). 3) At the phylum level,the dominant bacteria in feces was Firmicutes. The relative abundance of Firmicutes of group B was significantly higher than that of group D( P<0.05). Both the family and genus levels,the dominant bacteria was Lactobacillus and Lactobacillus,respectively. The relative abundance of Lactobacillus of group B was significantly higher than that of groups A and D( P<0.05),but no significant differences between group B and group C( P>0.05). 4) It was predicted that the bacteria were mainly involved in metabolic pathways of carbohydrate,amino acid,energy,nucleic acid,and cofactors and vitamins. In addition to the nucleic acid metabolism,the relative abundance of bacteria of group B was the greatest and followed by groups C,A and D. In conclusion,adding 10% FSM to the piglet diets can increase the diversity of the intestinal bacteria,significantly increase the relative abundance of beneficial bacteria such as Firmicutes,Lactobacillus and Lactobacillus,maintain the healthy development and promote the metabolism of nutrients.[Chinese Journal of Animal Nutrition,2019,31( 6) : 2560-2571]
This study was conducted to study the effects of dietary fermented soybean meal( FSM) on the fecal microbiota diversity of weaned piglets using Illumina high-throughput sequencing technology. A total of 80 piglets were randomly divided into A,B,C and D groups. As the control,the piglets of group D were fed a basal diet. The piglets of groups A,B and C were fed the basal diet added 5%,10% and 15% FSM,respectively.Feces of four groups were collected after the experiment. The V3 to V4 region of the 16 S rRNA gene of bacteria in faeces was sequenced using Illumina high-throughput sequencing technology. The results showed as follows: 1) in all of the samples,the average effective sequence number of bacteria was 47 218 and 3 581 operational taxonomic unit( OTUs) were produced at a similar level of 97% and 12 phylum,21 classes,33 orders,64 families,111 genera and 137 species were detected. 2) The alpha diversity indexes of fecal microbiota showed that there was no significant difference among groups( P > 0. 05),but the Shannon index of group B was the highest,indicating that the diversity of this group was the best. The beta analysis of diversity showed that the grouping effect was better,and there were significant differences between groups( P<0.05). 3) At the phylum level,the dominant bacteria in feces was Firmicutes. The relative abundance of Firmicutes of group B was significantly higher than that of group D( P<0.05). Both the family and genus levels,the dominant bacteria was Lactobacillus and Lactobacillus,respectively. The relative abundance of Lactobacillus of group B was significantly higher than that of groups A and D( P<0.05),but no significant differences between group B and group C( P>0.05). 4) It was predicted that the bacteria were mainly involved in metabolic pathways of carbohydrate,amino acid,energy,nucleic acid,and cofactors and vitamins. In addition to the nucleic acid metabolism,the relative abundance of bacteria of group B was the greatest and followed by groups C,A and D. In conclusion,adding 10% FSM to the piglet diets can increase the diversity of the intestinal bacteria,significantly increase the relative abundance of beneficial bacteria such as Firmicutes,Lactobacillus and Lactobacillus,maintain the healthy development and promote the metabolism of nutrients.[Chinese Journal of Animal Nutrition,2019,31( 6) : 2560-2571]
引文
[1]陈斌.微生物发酵对豆粕中抗营养因子及营养价值的影响[D].硕士学位论文.杭州:浙江大学,2005.
[2]SOTAK-PEPER K M,GONZALEZ-VEGA J C,STEIN H H.Concentrations of digestible,metabolizable,and net energy in soybean meal produced in different areas of the United States and fed to pigs[J].Journal of Animal Science,2015,93(12):5694-5701.
[3]王格,董胜奇,张涛,等.基于高通量测序分析豆粕发酵过程中细菌群落结构及多样性[J].华中农业大学学报,2018,37(5):89-94.
[4]SOLOMON S G,OKOMODA V T,OGUCHE O.Nutritional value of raw Canavalia ensiformis and its utilization as partial replacement for soybean meal in the diet of Clarias gariepinus(Burchell,1822)fingerlings[J].Food Science&Nutrition,2018,6(1):207-213.
[5]徐倩.外加酶提高发酵豆粕水解度的研究[D].硕士学位论文.武汉:华中农业大学,2009.
[6]NG’ONG’OLA-MANANI T A,STLIE H M,M WANGWELA A M,et al.M etabolite changes during natural and lactic acid bacteria fermentations in pastes of soybeans and soybean-maize blends[J].Food Science&Nutrition,2014,2(6):768-785.
[7]章世元,全丽萍,徐健超,等.发酵豆粕对断奶仔猪生长性能、养分消化率和胃肠道发育的影响[J].中国饲料,2008(16):8-11.
[8]徐述亮,刘晓东,王丹丹,等.不同梯度发酵豆粕对仔猪生长性能和经济效益的影响[J].饲料工业,2013,34(24):48-51.
[9]单达聪,刘苹苹,王四新,等.发酵豆粕对仔猪生产性能及血液指标的研究[J].饲料研究,2011(6):1-4.
[10]王园.豆粕固态发酵条件及其对断奶仔猪饲用效果的研究[D].博士学位论文.北京:中国农业大学,2014:5.
[11]苏恺,蔡仁贤,熊罗英,等.发酵豆粕的营养价值及其在断奶仔猪日粮中的应用[J].西北农业学报,2014,23(4):117-123.
[12]VAN WINSEN R L,KEUZENKAMP D,URLINGSB A P,et al.Effect of fermented feed on shedding of Enterobacteriaceae by fattening pigs[J].Veterinary M icrobiology,2002,87(3):267-276.
[13]胡远亮.利用分子生物技术研究益生菌对断奶仔猪生长及粪便菌群的影响[D].博士学位论文.武汉:华中农业大学,2014:25-87.
[14]孙笑非,孙冬岩,王文娟,等.猪肠道微生物定植规律和生理功能[J].饲料研究,2011(9):70-72.
[15]POWER S E,O’TOOLE P W,STANTON C,et al.Intestinal microbiota,diet and health[J].British Journal of Nutrition,2014,111(3):387-402.
[16]刘虹,王琪,刘作华,等.猪肠道微生物区系的形成及营养调控[J].动物营养学报,2018,30(7):2480-2487.
[17]LIU H Y,IVARSSON E,DICKSVED J,et al.Inclusion of chicory(Cichorium intybus L.)in pigs’diets affects the intestinal microenvironment and the gut microbiota[J].Applied and Environmental M icrobiology,2012,78(12):4102-4109.
[18]李东萍,郭明璋,许文涛.16S rRNA测序技术在肠道微生物中的应用研究进展[J].生物技术通报,2015,31(2):71-77.
[19]叶雷,闫亚丽,陈庆森,等.高通量测序技术在肠道微生物宏基因组学研究中的应用[J].中国食品学报,2016,16(7):216-223.
[20]PAJARILLO E A B,CHAE J P,BALOLONG M P,et al.Assessment of fecal bacterial diversity among healthy piglets during the w eaning transition[J].The Journal of General and Applied M icrobiology,2014,60(4):140-146.
[21]杨巧丽.腹泻仔猪粪便微生物结构和功能及SLA-DQA基因与粪便菌群的相关性[D].博士学位论文.兰州:甘肃农业大学,2017:6.
[22]张煜.新型枯草芽孢杆菌的研发及其发酵饲料在仔猪上的应用研究[D].博士学位论文.杭州:浙江大学,2017:12.
[23]PANDA S,EL KHADER I,CASELLAS F,et al.Short-term effect of antibiotics on human gut microbiota[J].PLoS One,2014,9(4):e95476.
[24]KIM H B,BOREWICZ K,WHITE B A,et al.Longitudinal investigation of the age-related bacterial diversity in the feces of commercial pigs[J].Veterinary M icrobiology,2011,153(1/2):124-133.
[25]王一冰,张小平,黄怡,等.运用454焦磷酸测序技术对断奶前后仔猪肠道菌群的分析[J].动物营养学报,2013,25(10):2440-2446.
[26]SLIFIERZ M J,FRIENDSHIP R M,WEESE J S.Longitudinal study of the early-life fecal and nasal microbiotas of the domestic pig[J].BM C M icrobiology,2015,15(1):184.
[27]EVANS N J,BROWN J M,MURRAY R D,et al.Characterization of novel bovine gastrointestinal tract Treponema isolates and comparison w ith bovine digital dermatitis treponemes[J].Applied and Environmental M icrobiology,2011,77(1):138-147.
[28]YUAN L,CHANG J,YIN Q Q,等.发酵豆粕改善仔猪生长性能、营养物质消化率和肠道微生物菌群[J].王志林译.广东饲料,2017,26(1):51.
[29]AZCARATE-PERIL M A,FOSTER D M,CADENASM B,et al.Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosaassociated bacteria[J].Gut M icrobes,2011,2(4):234-243.
[30]O’HARA A M,SHANAHAN F.The gut flora as a forgotten organ[J].EM BO Reports,2006,7(7):688-693.
[31]SONNENBURG J L,XU J,LEIP D D,et al.Glycan foraging in vivo by an intestine-adapted bacterial symbiont[J].Science,2005,307(5717):1955-1959.
[32]YATSUNENKO T,REY F E,MANARY M J,et al.Human gut microbiome view ed across age and geography[J].Nature,2012,486(7402):222-227.
[33]CHOW J,LEE S M,SHEN Y,et al.Host-bacterial symbiosis in health and disease[J].Advances in Immunology,2010,107:243-274.
[34]O’TOOLE P W,CLAESSON M J.Gut microbiota:changes throughout the lifespan from infancy to elderly[J].International Dairy Journal,2010,20(4):281-291.
[35]WALKER A W,INCE J,DUNCAN S H,et al.Dominant and diet-responsive groups of bacteria w ithin the human colonic microbiota[J].The ISM E Journal,2011,5(2):220-230.
[36]EL KAOUTARI A,ARMOUGOM F,GORDON J I,et al.The abundance and variety of carbohydrate-active enzymes in the human gut microbiota[J].Nature Review s M icrobiology,2013,11(7):497-504.
[37]MACFARLANE G T,CUMMINGS J H,ALLISONC.Protein degradation by human intestinal bacteria[J].Microbiology,1986,132(6):1647-1656.
[38]黄沧海.仔猪复合益生乳酸杆菌制剂及其作用机理的研究[D].博士学位论文.北京:中国农业大学,2003.
[39]LOUIS P,FLINT H J.Diversity,metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine[J].FEM S M icrobiology Letters,2010,294(1):1-8.
[40]FLINT H J,BAYER E A,RINCON M T,et al.Polysaccharide utilization by gut bacteria:potential for new insights from genomic analysis[J].Nature Review s M icrobiology,2008,6(2):121-131.
[41]吴先华.发酵豆粕在断奶仔猪及生长育肥猪日粮中的应用研究[D].硕士学位论文.南宁:广西大学,2014:6.
[42]俞晓辉.发酵豆粕对断奶仔猪生长性能和肠道微生物的影响[D].硕士学位论文.南京:南京农业大学,2008:6.
[43]KIM Y G,LOHAKARE J D,YUN J H,et al.Effect of feeding levels of microbial fermented soy protein on the grow th performance,nutrient digestibility and intestinal morphology in w eaned piglets[J].Asian-Australasian Journal of Animal Sciences,2007,20(3):399-404.
[2]SOTAK-PEPER K M,GONZALEZ-VEGA J C,STEIN H H.Concentrations of digestible,metabolizable,and net energy in soybean meal produced in different areas of the United States and fed to pigs[J].Journal of Animal Science,2015,93(12):5694-5701.
[3]王格,董胜奇,张涛,等.基于高通量测序分析豆粕发酵过程中细菌群落结构及多样性[J].华中农业大学学报,2018,37(5):89-94.
[4]SOLOMON S G,OKOMODA V T,OGUCHE O.Nutritional value of raw Canavalia ensiformis and its utilization as partial replacement for soybean meal in the diet of Clarias gariepinus(Burchell,1822)fingerlings[J].Food Science&Nutrition,2018,6(1):207-213.
[5]徐倩.外加酶提高发酵豆粕水解度的研究[D].硕士学位论文.武汉:华中农业大学,2009.
[6]NG’ONG’OLA-MANANI T A,STLIE H M,M WANGWELA A M,et al.M etabolite changes during natural and lactic acid bacteria fermentations in pastes of soybeans and soybean-maize blends[J].Food Science&Nutrition,2014,2(6):768-785.
[7]章世元,全丽萍,徐健超,等.发酵豆粕对断奶仔猪生长性能、养分消化率和胃肠道发育的影响[J].中国饲料,2008(16):8-11.
[8]徐述亮,刘晓东,王丹丹,等.不同梯度发酵豆粕对仔猪生长性能和经济效益的影响[J].饲料工业,2013,34(24):48-51.
[9]单达聪,刘苹苹,王四新,等.发酵豆粕对仔猪生产性能及血液指标的研究[J].饲料研究,2011(6):1-4.
[10]王园.豆粕固态发酵条件及其对断奶仔猪饲用效果的研究[D].博士学位论文.北京:中国农业大学,2014:5.
[11]苏恺,蔡仁贤,熊罗英,等.发酵豆粕的营养价值及其在断奶仔猪日粮中的应用[J].西北农业学报,2014,23(4):117-123.
[12]VAN WINSEN R L,KEUZENKAMP D,URLINGSB A P,et al.Effect of fermented feed on shedding of Enterobacteriaceae by fattening pigs[J].Veterinary M icrobiology,2002,87(3):267-276.
[13]胡远亮.利用分子生物技术研究益生菌对断奶仔猪生长及粪便菌群的影响[D].博士学位论文.武汉:华中农业大学,2014:25-87.
[14]孙笑非,孙冬岩,王文娟,等.猪肠道微生物定植规律和生理功能[J].饲料研究,2011(9):70-72.
[15]POWER S E,O’TOOLE P W,STANTON C,et al.Intestinal microbiota,diet and health[J].British Journal of Nutrition,2014,111(3):387-402.
[16]刘虹,王琪,刘作华,等.猪肠道微生物区系的形成及营养调控[J].动物营养学报,2018,30(7):2480-2487.
[17]LIU H Y,IVARSSON E,DICKSVED J,et al.Inclusion of chicory(Cichorium intybus L.)in pigs’diets affects the intestinal microenvironment and the gut microbiota[J].Applied and Environmental M icrobiology,2012,78(12):4102-4109.
[18]李东萍,郭明璋,许文涛.16S rRNA测序技术在肠道微生物中的应用研究进展[J].生物技术通报,2015,31(2):71-77.
[19]叶雷,闫亚丽,陈庆森,等.高通量测序技术在肠道微生物宏基因组学研究中的应用[J].中国食品学报,2016,16(7):216-223.
[20]PAJARILLO E A B,CHAE J P,BALOLONG M P,et al.Assessment of fecal bacterial diversity among healthy piglets during the w eaning transition[J].The Journal of General and Applied M icrobiology,2014,60(4):140-146.
[21]杨巧丽.腹泻仔猪粪便微生物结构和功能及SLA-DQA基因与粪便菌群的相关性[D].博士学位论文.兰州:甘肃农业大学,2017:6.
[22]张煜.新型枯草芽孢杆菌的研发及其发酵饲料在仔猪上的应用研究[D].博士学位论文.杭州:浙江大学,2017:12.
[23]PANDA S,EL KHADER I,CASELLAS F,et al.Short-term effect of antibiotics on human gut microbiota[J].PLoS One,2014,9(4):e95476.
[24]KIM H B,BOREWICZ K,WHITE B A,et al.Longitudinal investigation of the age-related bacterial diversity in the feces of commercial pigs[J].Veterinary M icrobiology,2011,153(1/2):124-133.
[25]王一冰,张小平,黄怡,等.运用454焦磷酸测序技术对断奶前后仔猪肠道菌群的分析[J].动物营养学报,2013,25(10):2440-2446.
[26]SLIFIERZ M J,FRIENDSHIP R M,WEESE J S.Longitudinal study of the early-life fecal and nasal microbiotas of the domestic pig[J].BM C M icrobiology,2015,15(1):184.
[27]EVANS N J,BROWN J M,MURRAY R D,et al.Characterization of novel bovine gastrointestinal tract Treponema isolates and comparison w ith bovine digital dermatitis treponemes[J].Applied and Environmental M icrobiology,2011,77(1):138-147.
[28]YUAN L,CHANG J,YIN Q Q,等.发酵豆粕改善仔猪生长性能、营养物质消化率和肠道微生物菌群[J].王志林译.广东饲料,2017,26(1):51.
[29]AZCARATE-PERIL M A,FOSTER D M,CADENASM B,et al.Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosaassociated bacteria[J].Gut M icrobes,2011,2(4):234-243.
[30]O’HARA A M,SHANAHAN F.The gut flora as a forgotten organ[J].EM BO Reports,2006,7(7):688-693.
[31]SONNENBURG J L,XU J,LEIP D D,et al.Glycan foraging in vivo by an intestine-adapted bacterial symbiont[J].Science,2005,307(5717):1955-1959.
[32]YATSUNENKO T,REY F E,MANARY M J,et al.Human gut microbiome view ed across age and geography[J].Nature,2012,486(7402):222-227.
[33]CHOW J,LEE S M,SHEN Y,et al.Host-bacterial symbiosis in health and disease[J].Advances in Immunology,2010,107:243-274.
[34]O’TOOLE P W,CLAESSON M J.Gut microbiota:changes throughout the lifespan from infancy to elderly[J].International Dairy Journal,2010,20(4):281-291.
[35]WALKER A W,INCE J,DUNCAN S H,et al.Dominant and diet-responsive groups of bacteria w ithin the human colonic microbiota[J].The ISM E Journal,2011,5(2):220-230.
[36]EL KAOUTARI A,ARMOUGOM F,GORDON J I,et al.The abundance and variety of carbohydrate-active enzymes in the human gut microbiota[J].Nature Review s M icrobiology,2013,11(7):497-504.
[37]MACFARLANE G T,CUMMINGS J H,ALLISONC.Protein degradation by human intestinal bacteria[J].Microbiology,1986,132(6):1647-1656.
[38]黄沧海.仔猪复合益生乳酸杆菌制剂及其作用机理的研究[D].博士学位论文.北京:中国农业大学,2003.
[39]LOUIS P,FLINT H J.Diversity,metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine[J].FEM S M icrobiology Letters,2010,294(1):1-8.
[40]FLINT H J,BAYER E A,RINCON M T,et al.Polysaccharide utilization by gut bacteria:potential for new insights from genomic analysis[J].Nature Review s M icrobiology,2008,6(2):121-131.
[41]吴先华.发酵豆粕在断奶仔猪及生长育肥猪日粮中的应用研究[D].硕士学位论文.南宁:广西大学,2014:6.
[42]俞晓辉.发酵豆粕对断奶仔猪生长性能和肠道微生物的影响[D].硕士学位论文.南京:南京农业大学,2008:6.
[43]KIM Y G,LOHAKARE J D,YUN J H,et al.Effect of feeding levels of microbial fermented soy protein on the grow th performance,nutrient digestibility and intestinal morphology in w eaned piglets[J].Asian-Australasian Journal of Animal Sciences,2007,20(3):399-404.