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

doi:10.11686/cyxb2017374

摘要/Abstract

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60 d,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 plantarum subsp. 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。

关键词: 小花棘豆, 混贮, 微生物特性, 苦马豆素脱除

Abstract: To establish a method to use Oxytropis glabra as a silage crop, the influence of different ratios of O. glabra and 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. glabra and 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 JD6E, JD4D, JD10D, JD2E, and JD1F. After pretreatment of silage with boiling water, the swainsonine-removing rates of strains JD10D and JD2E were 100% and 97.76% respectively, but those of other strains decreased by 31.76%-100%. The swainsonine-removing rates of strains JD6D and JD1E dropped 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 JD10D and JD2E had the best ability to bind swainsonine, and strains JD6D and JD1E showed the best swainsonine-degradation ability. These four strains can be used as biological detoxification agents to remove swainsonine from O. glabra silage.

Key words: Oxytropis glabra, mixed silage, microbial characteristics, swainsonine-removing

陶雅, 李峰, 孙启忠, 柳茜, 高润, 徐春城. 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[J]. 草业学报, 2018, 27(8): 118-129.

TAO Ya, LI Feng, SUN Qi-zhong, LIU Qian, GAO Run, XU Chun-cheng. Microbial characteristics of Oxytropis glabra and corn mixed silage and screening for swainsonine-removing lactic acid bacteria[J]. Acta Prataculturae Sinica, 2018, 27(8): 118-129.

参考文献

[1] Zeng J F. Flora of China. Beijing: Science Press, 1998.
曾建飞. 中国植物志. 北京: 科学出版社, 1998.
[2] Ma Y Q. Flora of Inner Mongolia. Hohhot: Inner Mongolia People’s Publishing House, 1989.
马毓泉. 内蒙古植物志. 呼和浩特: 内蒙古人民出版社, 1989.
[3] Zhao Y Z, Liu L. Characteristics of Oxytropis flora ecological- geographic distribution. Journal of Inner Mongolia University, 1996, 27(1): 72-82.
赵一之, 刘丽. 内蒙古棘豆属 Oxytropis 植物区系生态地理分布特征. 内蒙古大学学报(自然科学版), 1996, 27(1): 72-82.
[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.
[5] Wang Z X. Chemical principals and nutritional evaluation of Oxytropis glabra and dynamic change of swainsonine in locoweed. Yangling: Northwest Agriculture and Forestry University, 2010.
王占新. 小花棘豆化学成分、营养成分评价及疯草苦马豆素动态变化规律. 杨凌: 西北农林科技大学, 2010.
[6] Liu Q, Sun Q Z, Hao H, et al . Study on alfalfa and corn mixed silage. China Dairy Cattle, 2017, (1): 59-62.
柳茜, 孙启忠, 郝虎, 等. 紫花苜蓿与全株玉米混合青贮研究. 中国奶牛, 2017, (1): 59-62.
[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 ochrocephala silage 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.
[11] Da N T, Zhao B Y, Dong F, et al . Study on utilization of Oxytropis glabra silage. Journal of Animal Toxicology, 2006, 21(1): 36-40.
达能太, 赵宝玉, 东风, 等. 小花棘豆的青贮利用研究. 动物毒物学, 2006, 21(1): 36-40.
[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, 16S ribosomal RNA and recA gene analysis. Systematic and Applied Microbiology, 2011, 34(3): 235-241.
[16] Ávila 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.
[18] Jia Q Z, Tao D Y, Deng L, et al . Isolation and identification of swainsonie degrading bactria. Acta Agriculturae Boreali-occidentalis Sinica, 2016, 25(10): 1554-1560.
贾琦珍, 陶大勇, 邓利, 等. 一株苦马豆素降解菌的分离与鉴定. 西北农业学报, 2016, 25(10): 1554-1560.
[19] Wang Y, Hu Y C, Yu Y T, et al . Characteristics of swainsonine-degrading bacteria YLZZ-2 and optimization of conditions for degradation. Acta Agrestia Sinica, 2010, 18(1): 89-92.
王妍, 胡延春, 余永涛, 等. 苦马豆素降解菌YLZZ-2的降解特性与条件优化. 草地学报, 2010, 18(1): 89-92.
[20] Hu Y C, Liu P, Zhao C R, et al . Screening and identification of endophytic bacteria of Oxytropis kansuensis Bunge for swainsonine degradation. Journal of Zhejiang University (Agriculture and Life Science), 2011, 37(5): 521-526.
胡延春, 刘鹏, 赵春蕊, 等. 甘肃棘豆中降解苦马豆素内生细菌的筛选与鉴定. 浙江大学学报(农业与生命科学版), 2011, 37(5): 521-526.
[21] Du P. The experimental techniques in dairy microbiology. Beijing: China Light Industry Press, 2008.
杜鹏. 乳品微生物学实验技术. 北京: 中国轻工业出版社, 2008.
[22] Ling D W, Dong X Z. The experimental techniques in identification of lactic acid bacteria. Beijing: China Light Industry Press, 1999.
凌代文, 东秀珠. 乳酸细菌分类鉴定及实验方法. 北京: 中国轻工业出版社, 1999.
[23] Pang H L, Zhang M, Qin G Y, et al . Identification of lactic acid bacteria isolated from corn stovers. Animal Science Journal, 2011, 82: 642-653.
[24] Mcdonald P, Henderson A R, Heron S J E. The biochemistry of silage. John Wiley & Sons, Ltd., 1991: 9-137.
[25] Muck R E. Initial bacterial numbers on lucerne prior to ensiling. Grass Forage Science, 1989, 44: 19-25.
[26] Cai Y. Identification and characterization of Enterococcus species isolated from forage crops and their influence on silage fermentation. Journal of Dairy Science, 1999, 82(11): 2466-2471.
[27] Hellings P, Bertin G, Vanbelle M. Effect of lactic acid bacteria on silage fermentation//Proceedings of the 15th international grassland congress. Kyoto, Japan: Kyotosyppan, 1985: 932-933.
[28] Pahlow G. Microbiology of inoculants, crops and silages//Small scale silage experiments. Sweden: Grovfoder, 1990.
[29] Rooke J A. The numbers of epiphytic bacteria on grass at ensilage on commercial farms. Journal of the Science of Food and Agriculture, 1990, 51(4): 525-533.
[30] Pahlow G, Muck R E, Driehuis F, et al . Microbiology of ensiling. Agronomy Monograph, Silage Science and Technology, 42: 31-93.
[31] Lin C, Bolsen K K, Brent B E, et al . Epiphytic lactic acid bacteria succession during the pre-ensiling and ensiling periods of alfalfa and maize. Journal of Applied Bacteriology, 1992, 73(5): 375-387.
[32] Ni K, Wang Y, Li D, et al . Characterization, identification and application of lactic acid bacteria isolated from forage paddy rice silage. PloS One, 2015, 10(3): e0121967.
[33] Muck R E. Silage microbiology and its control through additives. Revista Brasileira de Zootecnia, 2010, 39: 183-191.
[34] Tao Y, Li F, Gao F Q, et al . A study on fermentation quality of Amaranthus hypochondriacus and corn mixed silage and microbial characteristics. Acta Prataculturae Sinica, 2016, (12): 119-129.
陶雅, 李峰, 高凤芹, 等. 籽粒苋与青贮玉米混贮品质及微生物特性研究. 草业学报, 2016, (12): 119-129.
[35] Ruser B. Erfassung und Identifizierung des epiphytischen Milchsäurebakterienbesatzes auf Gras und Mais in Abhängigkeit von Standort, Sorte, Entwicklungsstadium sowie Ernte-und Klimaeinflüssen. Bundesforschungsanst. f. Landwirtschaft, 1989.
[36] Ennahar S, Cai Y, Fujita Y. Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis. Applied and Environmental Microbiology, 2003, 69(1): 444-451.
[37] Holzer M, Mayrhuber E, Danner H, et al . The role of Lactobacillus buchneri in forage preservation. Trends in Biotechnology, 2003, 21(6): 282-287.
[38] Qi Y Q, Zhang J T, Pan X H, et al . Binding of benzo (a) pyrene by Lactobacilli strains. Acta Microbiologica Sinica, 2011, 51(7): 956-964.
漆叶琼, 张佳涛, 潘向辉, 等. 乳杆菌吸附苯并芘的特性. 微生物学报, 2011, 51(7): 956-964.
[39] Ai L Z, Wu Y, Zhang H, et al . Effect of probiotic bacteria on aflatoxin. Food Science and Technology, 2004, (9): 12-15.
艾连中, 吴艳, 张灏, 等. 益生菌对黄曲霉毒素的作用. 食品科技, 2004, (9): 12-15.
[40] Park K Y, Jung K O, Rhee S H, et al . Antimutagenic effects of doenjang (Korean fermented soypaste) and its active compounds. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2003, 523: 43-53.
[41] Varga J, Kevei É, Tóth B, et al . Molecular analysis of variability within the toxigenic Aspergillus ochraceus species. Canadian Journal of Microbiology, 2000, 46(7): 593-599.
[42] Line J E, Brackett R E. Factors affecting aflatoxin B 1 removal by Flavobacterium aurantiacum . Journal of Food Protection, 1995, 58(1): 91-94.
[43] Haskard C A, El-Nezami H S, Kankaanpää P E, et al . Surface binding of aflatoxin B 1 by lactic acid bacteria. Applied and Environmental Microbiology, 2001, 67(7): 3086-3091.
[44] Han P F, He Z F, Li H J, et al . Research progress in microbial cellwall structure and its binding with mycotoxin. Food Science, 2012, 33(11): 294-298.
韩鹏飞, 贺稚非, 李洪军, 等. 微生物细胞壁结构及结合真菌毒素的研究进展. 食品科学, 2012, 33(11): 294-298.
[45] Takahashi-Ando N, Ohsato S, Shibata T, et al . Metabolism of zearalenone by genetically modified organisms expressing the detoxification gene from Clonostachys rosea . Applied and Environmental Microbiology, 2004, 70(6): 3239-3245.