小花棘豆与玉米混贮微生物特性及脱除苦马豆素乳酸菌的筛选
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摘要
旨在探讨小花棘豆与玉米按不同比例混贮对微生物特性及乳酸菌多样性影响,挖掘具有脱除苦马豆素活性的乳酸菌,为小花棘豆青贮饲料的开发利用提供理论参考。以小花棘豆和全株玉米为原料,按不同比例10∶0(T1)、9∶1(T2)、8∶2(T3)7∶3(T4)、6∶4(T5)、5∶5(T6)、4∶6(T7)、0∶10(T8)混合青贮,室温发酵60d,检测青贮前、后微生物种类、数量变化,鉴定分离出的乳酸菌,并测定其对苦马豆素的脱除率。试验结果:1)原料中乳酸菌数量随着玉米混入量的增加逐渐升高,青贮饲料各处理间乳酸菌数量无显著差异;原料中的肠细菌各处理间无显著差异,经过青贮发酵后肠细菌均未检测到;好氧细菌数量在原料和青贮饲料中均随着玉米比例的增加逐渐减少;酵母菌数量在各处理间均无显著差异;原料中的霉菌数量随着玉米混入比例增加逐渐增加,而青贮饲料中的霉菌数量均在检出线以下。2)从小花棘豆与玉米混贮的原料中共分离得到乳酸菌菌株4株,经鉴定属于Lactococcus lactis subsp.hordniae、Lactococcus lactis subsp.lactis和Lactococcus taiwanensis 3种;从青贮饲料中分离出乳酸菌菌株9株,经鉴定属于Lactobacillus plantarumsubsp.plantarum、Lactobacillus brevis、Lactobacillus pentosus和Lactobacillus amylovorus 4种,当玉米混入比例升高时,原料中乳酸菌多样性有所增加,而青贮饲料中乳酸菌多样性有所下降。3)不同乳酸菌菌株对苦马豆素的脱除率均高于85%,其中菌株JD6E、JD4D、JD10D、JD2E和JD1F对苦马豆素的脱除率高达100%。经过热处理后,菌株JD10D和JD2E对苦马豆素的脱除率分别为100%和97.76%,而其他菌株对苦马豆素的脱除率下降31.76%~100%,其中菌株JD6D和JD1E经过热处理后对苦马豆素的脱除率分别下降到2.17%和0。结果表明,小花棘豆与玉米混贮可以增加青贮原料中乳酸菌的数量及多样性,降低青贮饲料和原料中好氧细菌的数量,有助于提高青贮成功率;乳酸菌对苦马豆素具有良好的脱除效果,菌株JD10D和JD2E对苦马豆素的吸附脱除作用最强,而菌株JD6D和JD1E对苦马豆素的降解作用最强,均可用作小花棘豆青贮脱除苦马豆素的发酵菌株。
To establish a method to use Oxytropis glabra as a silage crop,the influence of different ratios of O.glabraand corn(Zea mays)on microbial characteristics and the diversity of lactic acid bacteria in silage were investigated.We also screened for swainsonine-removing lactic acid bacterial strains.The proportions of O.glabra∶corn in the eight treatments were as follows:10∶0(T1),9∶1(T2),8∶2(T3),7∶3(T4),6∶4(T5),5∶5(T6),4∶6(T7)and 0∶10(T8).The microbial community structure and quantities were analyzed after fermentation of silage for 60 days.Lactic acid bacterial strains were isolated from fresh material and silage,and their removal rates of swainsonine were determined.The main results were as follows:1)The numbers of lactic acid bacteria in fresh material increased with increasing proportions of corn,but did not differ significantly among the silage materials.The mixing ratio did not affect the numbers of coliform bacteria in the fresh material,and coliform bacteria could not be detected after fermentation.The numbers of aerobic bacteria in fresh and silage material decreased with increasing proportions of corn.The amount of yeasts was not affected by the ratio of O.glabraand corn.When more corn was added,the number of molds increased,but molds were beneath the limits of detection in the silage materials.2)Four lactic acid bacterial strains were isolated from the fresh material:Lactococcus lactis subsp.Hordniae,Lactococcus lactis subsp.Lactis,and Lactococcus taiwanensis.Nine lactic acid bacterial strains were isolated from silage material,including Lactobacillus plantarum subsp.Plantarum,Lactobacillus brevis,Lactobacillus pentosus,and Lactobacillus amylovorus.As the proportion of corn increased,the diversity of lactic acid bacteria increased in fresh material but decreased in silage.3)The swainsonine-removing rates of all lactic acid bacterial strains were higher than 85%,and 100%for strains JD6 E,JD4 D,JD10 D,JD2 E,and JD1 F.After pretreatment of silage with boiling water,the swainsonine-removing rates of strains JD10 Dand JD2 Ewere 100% and 97.76% respectively,but those of other strains decreased by 31.76%-100%.The swainsonine-removing rates of strains JD6 Dand JD1 Edropped to 2.17%and 0,respectively,after the heat treatment.These results showed that with increasing proportions of corn in the mixture,the quantities and diversity of lactic acid bacteria in fresh material increased and the populations of aerobic bacteria both in raw material and silage decreased,which increased the success rate of ensilage.Some lactic acid bacteria were able to remove swainsonine.Strains JD10 Dand JD2 E had the best ability to bind swainsonine,and strains JD6 Dand JD1 Eshowed the best swainsonine-degradation ability.These four strains can be used as biological detoxification agents to remove swainsonine fromO.glabrasilage.
To establish a method to use Oxytropis glabra as a silage crop,the influence of different ratios of O.glabraand corn(Zea mays)on microbial characteristics and the diversity of lactic acid bacteria in silage were investigated.We also screened for swainsonine-removing lactic acid bacterial strains.The proportions of O.glabra∶corn in the eight treatments were as follows:10∶0(T1),9∶1(T2),8∶2(T3),7∶3(T4),6∶4(T5),5∶5(T6),4∶6(T7)and 0∶10(T8).The microbial community structure and quantities were analyzed after fermentation of silage for 60 days.Lactic acid bacterial strains were isolated from fresh material and silage,and their removal rates of swainsonine were determined.The main results were as follows:1)The numbers of lactic acid bacteria in fresh material increased with increasing proportions of corn,but did not differ significantly among the silage materials.The mixing ratio did not affect the numbers of coliform bacteria in the fresh material,and coliform bacteria could not be detected after fermentation.The numbers of aerobic bacteria in fresh and silage material decreased with increasing proportions of corn.The amount of yeasts was not affected by the ratio of O.glabraand corn.When more corn was added,the number of molds increased,but molds were beneath the limits of detection in the silage materials.2)Four lactic acid bacterial strains were isolated from the fresh material:Lactococcus lactis subsp.Hordniae,Lactococcus lactis subsp.Lactis,and Lactococcus taiwanensis.Nine lactic acid bacterial strains were isolated from silage material,including Lactobacillus plantarum subsp.Plantarum,Lactobacillus brevis,Lactobacillus pentosus,and Lactobacillus amylovorus.As the proportion of corn increased,the diversity of lactic acid bacteria increased in fresh material but decreased in silage.3)The swainsonine-removing rates of all lactic acid bacterial strains were higher than 85%,and 100%for strains JD6 E,JD4 D,JD10 D,JD2 E,and JD1 F.After pretreatment of silage with boiling water,the swainsonine-removing rates of strains JD10 Dand JD2 Ewere 100% and 97.76% respectively,but those of other strains decreased by 31.76%-100%.The swainsonine-removing rates of strains JD6 Dand JD1 Edropped to 2.17%and 0,respectively,after the heat treatment.These results showed that with increasing proportions of corn in the mixture,the quantities and diversity of lactic acid bacteria in fresh material increased and the populations of aerobic bacteria both in raw material and silage decreased,which increased the success rate of ensilage.Some lactic acid bacteria were able to remove swainsonine.Strains JD10 Dand JD2 E had the best ability to bind swainsonine,and strains JD6 Dand JD1 Eshowed the best swainsonine-degradation ability.These four strains can be used as biological detoxification agents to remove swainsonine fromO.glabrasilage.
引文
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[4]Li J K.The actuality and perspective commentary of development and utilization of China Oxytropis.China Herbivore Science,1997,4:3-5.李建科.中国棘豆属植物开发利用研究现状及前景评述.草与畜杂志,1997,4:3-5.
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[7]Wang L,Sun Q Z,Zhang H J.A study on quality of mixed silage of alfalfa and corn.Acta Prataculturae Sinica,2011,20(4):202-209.王林,孙启忠,张慧杰.苜蓿与玉米混贮质量研究.草业学报,2011,20(4):202-209.
[8]Huang X H,Li S C,Li D H,et al.Fermentation quality and content of poisonous substances in Sophora alopecuroides and corn straw mixed silage.Pratacultural Science,2013,30(10):1633-1639.黄晓辉,李树成,李东华,等.苦豆子和玉米秸秆的混合青贮.草业科学,2013,30(10):1633-1639.
[9]Ngwa T A,Nsahlai I V,Iji P A.Ensilage as a means of reducing the concentration of cyanogenic glycosides in the pods of Acacia sieberiana and the effect of additives on silage quality.Journal of the Science of Food and Agriculture,2004,84:521-529.
[10]Mo C H,Shen M H,Ma S Q.The toxicity test of Oxytropis ochrocephalasilage to sheep.Animal Husbandry&Veterinary Medicine,2013,45(8):92-94.莫重辉,沈明华,马双清.青贮黄花棘豆对羊的毒性试验.畜牧与兽医,2013,45(8):92-94.
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[12]Si B W.The dynamic changes of microbial flora in three shrubs silage and its fermentation characteristic.Beijing:Chinese Academy of Agriculture Sciences,2012.司丙文.三种灌木饲用植物青贮微生物种群动态变化与发酵特性.北京:中国农业科学院,2012.
[13]Borreani G,Tabacco E.The relationship of silage temperature with the microbiological status of the face of corn silage bunkers.Journal of Dairy Science,2010,93(6):2620-2629.
[14]McEniry J,O’kiely P,Clipson N J W,et al.The microbiological and chemical composition of silage over the course of fermentation in round bales relative to that of silage made from unchopped and precision-chopped herbage in laboratory silos.Grass and Forage Science,2008,63(3):407-420.
[15]Pang H,Qin G,Tan Z,et al.Natural populations of lactic acid bacteria associated with silage fermentation as determined by phenotype,16Sribosomal RNA and recA gene analysis.Systematic and Applied Microbiology,2011,34(3):235-241.
[16]vila C L S,Bravo Martins C E C,Schwan R F.Identification and characterization of yeasts in sugarcane silages.Journal of Applied Microbiology,2010,109(5):1677-1686.
[17]Parvin S,Nishino N.Bacterial community associated with ensilage process of wilted guinea grass.Journal of Applied Microbiology,2009,107(6):2029-2036.
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