研究论文 RESEARCHPAPER

乳酸杆菌的分离鉴定及其对皇竹草青贮饲料发酵品质的影响

  • 谢婷霞 ,
  • 曹雪笛 ,
  • 谭玉兰 ,
  • 李果 ,
  • 晋蕾 ,
  • 刘书亮 ,
  • 敖晓琳 ,
  • 赵珂 ,
  • 邹立扣
展开
  • 1. 四川农业大学资源学院, 成都 611130;
    2. 四川农业大学食品学院, 雅安 615014
谢婷霞(1994—),女,四川巴中人,硕士研究生,从事资源利用与植物保护的研究。E-mail:xietingxia0615@163.com

收稿日期: 2021-12-07

  网络出版日期: 2022-06-14

基金资助

四川省科技计划项目(2021ZDZX0012);四川省科技厅项目(2020ZYD003)

Isolation and Identification of Lactobacillus and Their Effects on Fermentation Quality of Pennisetum sinense Silage

  • XIE Tingxia ,
  • CAO Xuedi ,
  • TAN Yulan ,
  • LI Guo ,
  • JIN Lei ,
  • LIU Shuliang ,
  • AO Xiaolin ,
  • ZHAO Ke ,
  • ZOU Likou
Expand
  • 1. College of Resources, Sichuan Agricultural University, Chengdu 611130, China;
    2. College of Food Science, Sichuan Agricultural University, Ya'an 615014, China

Received date: 2021-12-07

  Online published: 2022-06-14

摘要

本试验旨在探讨将从黑麦草青贮饲料中分离获得的乳酸杆菌添加到皇竹草青贮中对其发酵品质及微生物多样性的影响。采用传统可培养法分离乳酸杆菌,通过形态学鉴定和16S rRNA测序比对后保存备用。在切碎的皇竹草样品中分别添加分离的植物乳杆菌LAB158(Lactobacillus plantarum LAB158)、玉米乳杆菌MZN1(Lactobacillus zeae MZN1)和草本乳杆菌P-1-12(Lactobacillus herbaceous P-1-12)作为试验组,同时添加等量无菌水作为对照组,打包后经60 d青贮发酵,对青贮样品进行感官评定,并分析其营养成分含量和微生物群落结构。结果表明:与对照组相比,3个试验组青贮饲料的色泽评分和pH评分均显著提高(P<0.05),干物质、粗蛋白质、粗脂肪和乳酸等营养成分含量也显著提高(P<0.05),同时LAB158组甘氨酸和丙氨酸等鲜味氨基酸含量也显著提高(P<0.05)。基于高通量测序发现,各组青贮饲料中优势菌在门水平上均是厚壁菌门,且相对丰度以对照组最低;其次为变形菌门,且相对丰度以对照组最高。各组优势菌在属水平上均为乳杆菌属,另外还有鞘氨醇杆菌属和魏斯氏菌属等。由此可见,添加乳酸杆菌能够改善青贮饲料的发酵品质,提高其营养品质,Alpha多样性分析发现添加植物乳杆菌LAB158和玉米乳杆菌MZN1后,微生物丰富度和多样性均得到提高,综合判定添加植物乳杆菌LAB158菌株的青贮效果更好。

本文引用格式

谢婷霞 , 曹雪笛 , 谭玉兰 , 李果 , 晋蕾 , 刘书亮 , 敖晓琳 , 赵珂 , 邹立扣 . 乳酸杆菌的分离鉴定及其对皇竹草青贮饲料发酵品质的影响[J]. 动物营养学报, 2022 , 34(6) : 4060 -4070 . DOI: 10.3969/j.issn.1006-267x.2022.06.065

Abstract

This experiment was conducted to investigate the effects of Lactobacillus isolated from ryegrass silage on fermentation quality and microbial diversity of Pennisetum sinese silage. Lactobacillus was isolated by traditional culture method, and then preserved for use after morphological identification and 16S rRNA sequencing comparison. The isolated Lactobacillus plantarum LAB158, Lactobacillus zeae MZN1 and Lactobacillus herbaceous P-1-12 were added into the mince samples of Pennisetum sinese as experimental groups, respectively, and the control group was added with the same amount of sterile water. The silage samples were fermented for 60 days after packaging, and the sensory evaluation, nutrient contents and microbial community structure were measured. The results showed that compared with the control group, the color score and pH score of silage in 3 experimental groups were significantly increased (P<0.05), and the contents of dry matter, crude protein, ether extract and lactic acid were also significantly increased (P<0.05), while the contents of umami amino acids such as glycine and alanine in LAB158 group were also significantly increased (P<0.05). Based on high-throughput sequencing, it was found that the dominant bacteria in each group were Firmicutes at phylum level, and the relative abundance was the lowest in the control group; Proteobacteria followed, and the relative abundance was the highest in the control group. At the genus level, the dominant bacteria in each group were Lactobacillus, Sphingobacterium and Weissella, etc. Thus it can be seen that Lactobacillus can improve the fermentation quality and nutritional quality of silage. Alpha diversity analysis shows that adding Lactobacillus plantarum LAB158 and Lactobacillus zeae MZN1 can increase the richness and diversity of microbial community. It is comprehensively determined that Lactobacillus plantarum LAB158 strain has better silage effect.

参考文献

[1] 张涛, 崔宗均, 李建平, 等.不同发酵类型青贮菌制剂对青贮发酵的影响[J].草业学报, 2005, 14(3):67-71. ZHANG T, CUI Z J, LI J P, et al.The effect of different fermentation type of silage inoculants bacteria on the fermentation of silage[J].Acta Prataculturae Sinica, 2005, 14(3):67-71.(in Chinese)
[2] 孙蕊, 贾鹏禹, 武瑞, 等.高效液相色谱法快速测定青贮饲料中4种有机酸的含量[J].饲料研究, 2019, 42(4):77-80. SUN R, JIA P Y, WU R, et al.Fast determination of four kinds of organic acids in silage by high performance liquid chromatography[J].Feed Research, 2019, 42(4):77-80.(in Chinese)
[3] MUCK R E, NADEAU E M G, MCALLISTER T A, et al.Silage review:recent advances and future uses of silage additives[J].Journal of Dairy Science, 2018, 101(5):3980-4000.  
[4] SHAH A A, YUAN X, KHAN R U, et al.Effect of lactic acid bacteria-treated King grass silage on the performance traits and serum metabolites in New Zealand white rabbits (Oryctolagus cuniculus)[J].Journal of Animal Physiology and Animal Nutrition, 2018, 102(2):e902-e908.
[5] 王思伟, 李魁英, 张海娜, 等.花生秧、全株玉米不同混合比例及添加剂对青贮发酵品质和营养价值的影响[J].草业科学, 2019, 36(9):2413-2422. WANG S W, LI K Y, ZHANG H N, et al.Mixed ratios and additives affect the quality of peanut vines and whole-plant corn in mixed silages[J].Pratacultural Science, 2019, 36(9):2413-2422.(in Chinese)
[6] 孙志强, 吴哲, 王炳, 等.不同品种和乳酸菌添加剂对全株玉米青贮品质和有氧稳定性的影响研究[J].中国草地学报, 2019, 41(1):83-88. SUN Z Q, WU Z, WANG B, et al.The effect of different hybrids and lactic acid bacteria additives on the quality and aerobic stability of the whole plant corn silage[J].Chinese Journal of Grassland, 2019, 41(1):83-88.(in Chinese)
[7] 辛亚芬, 陈晨, 曾泰儒, 等.青贮添加剂对微生物多样性影响的研究进展[J].生物技术通报, 2021, 37(9):24-30. XIN Y F, CHEN C, ZENG T R, et al.Research progress in the effects of additives to silage on microbial diversity[J].Biotechnology Bulletin, 2021, 37(9):24-30.(in Chinese)
[8] WANG Y, HE L W, XING Y Q, et al.Dynamics of bacterial community and fermentation quality during ensiling of wilted and unwilted Moringa oleifera leaf silage with or without lactic acid bacterial inoculants[J].mSphere, 2019, 4(4):e00341-19.
[9] 李雁冰, 玉柱, 孙娟娟.不同乳酸菌添加剂对青贮黑麦草和青贮玉米微生物群集的影响[J].草地学报, 2015, 23(2):387-393. LI Y B, YU Z, SUN J J.Microbial communities of whole crop corn and wilted Italian ryegrass silage inoculated with and without different lactic acid bacteria inoculants[J].Acta Agrestia Sinica, 2015, 23(2):387-393.(in Chinese)
[10] 尹福泉, 吴征敏, 王志敬, 等.皇竹草和喷浆玉米皮混合对瘤胃发酵特性的影响[J].草业科学, 2018, 35(7):1797-1804. YIN F Q, WU Z M, WANG Z J, et al.Effect of the combination of Pennisetum sinese and sprouting corn bran on rumen fermentation characteristics[J].Pratacultural Science, 2018, 35(7):1797-1804.(in Chinese)
[11] LI J F, YUAN X J, DONG Z H, et al.The effects of fibrolytic enzymes, cellulolytic fungi and bacteria on the fermentation characteristics, structural carbohydrates degradation, and enzymatic conversion yields of Pennisetum sinese silage[J].Bioresource Technology, 2018, 264:123-130.
[12] 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. ZHANG L Y.Feed analysis and feed quality testing technology[M].2nd ed.Beijing:China Agricultural University Press, 2003.(in Chinese)
[13] 王杰, 张养东, 郑楠, 等.青贮饲料感官评定研究进展[J].中国奶牛, 2019(1):1-3. WANG J, ZHANG Y D, ZHENG N, et al.Research progress in sensory evaluation of silage[J].China Dairy Cattle, 2019(1):1-3.(in Chinese)
[14] 陆永祥, 赵嫚, 陈良寅, 等.布氏乳杆菌和甲酸对青藏高原不同物候期燕麦青贮饲料发酵品质和细菌群落的影响[J].草地学报, 2020, 28(6):1736-1743. LU Y X, ZHAO M, CHEN L Y, et al.Effects of Lactobacillus brucei and formic acid addition on fermentation quality and bacterial community of oat silage of different harvest stages in the Tibetan Plateau[J].Acta Agrestia Sinica, 2020, 28(6):1736-1743.(in Chinese)
[15] 陶莲, 刁其玉.利用MiSeq高通量测序技术分析风干处理及青贮发酵对玉米秸秆菌群构成及丰度的影响[J].中国农业科学, 2015, 48(增刊):94-103. TAO L, DIAO Q Y.Effects of ensiling and natural drying on composition and abundance of bacterium of corn stalk analyzed by MiSeq sequencing technology[J].Scientia Agricultura Sinica, 2015, 48(Suppl):94-103.(in Chinese)
[16] HESS M, SCZYRBA A, EGAN R, et al.Metagenomic discovery of biomass-degrading genes and genomes from cow rumen[J].Science, 2011, 331(6016):463-467.  
[17] LUO C W, TSEMENTZI D, KYRPIDES N, et al.Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample[J].PLoS One, 2012, 7(2):e30087.
[18] DEGNAN P H, OCHMAN H.Illumina-based analysis of microbial community diversity[J].The ISME Journal, 2012, 6(1):183-194.  
[19] 吴进东.添加剂对全株玉米青贮饲料品质的影响[J].中国草食动物, 2006, 26(6):49-51. WU J D.Effects of additives on the quality of whole corn silage[J].China Herbivores, 2006, 26(6):49-51.(in Chinese)
[20] CAI Y, BENNO Y, OGAWA M, et al.Influence of Lactobacillus spp. from an inoculant and of Weissella and Leuconostoc spp. from forage crops on silage fermentation[J].Applied and Environmental Microbiology, 1998, 64(8):2982-2987.  
[21] 黄媛, 代胜, 梁龙飞, 等.不同添加剂对构树青贮饲料发酵品质及微生物多样性的影响[J].动物营养学报, 2021, 33(3):1607-1617. HUANG Y, DAI S, LIANG L F, et al.Effects of different additives on fermentation quality and microbial diversity of paper mulberry silage[J].Chinese Journal of Animal Nutrition, 2021, 33(3):1607-1617.(in Chinese)
[22] OLIVEIRA M R, JOBIM C C, NEUMANN M, et al.Effects of inoculation with homolactic bacteria on the conservation of wheat silage stored in bunker-silos[J].Italian Journal of Animal Science, 2018, 17(1):81-86.  
[23] 寇江涛, 雷佳星, 张海波, 等.不同添加剂对皇竹草青贮品质的影响[J].中国畜牧杂志, 2021, 57(10):178-182. KOU J T, LEI J X, ZHANG H B, et al.Effects of different additives on silage quality of Pennisetum sinese[J].Chinese Journal of Animal Science, 2021, 57(10):178-182.(in Chinese)
[24] 塔娜, 魏日华, 德庆哈拉, 等.对禾草源同型发酵和/或异型发酵乳酸菌发酵无芒雀麦青贮有氧稳定性的评价[J].动物营养学报, 2017, 29(4):1301-1311. TA N, WEI R H, DE Q H L, et al.Evaluation of aerobic stability of Bromus inermis Leyss. silage fermented by homofermentative and/or heterofermentative lactic acid bacteria from grass[J].Chinese Journal of Animal Nutrition, 2017, 29(4):1301-1311.(in Chinese)
[25] HOU M L, GENTU G, LIU T Y, et al.Silage preparation and fermentation quality of natural grasses treated with lactic acid bacteria and cellulase in meadow steppe and typical steppe[J].Asian-Australasian Journal of Animal Sciences, 2017, 30(6):788-796.
[26] ZHOU Y, DROUIN P, LAFRENIōRE 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.  
[27] 杨文琦, 龙宣杞, 崔卫东.玉米青贮中细菌多样性分析[J].新疆农业科学, 2013, 50(8):1424-1433. YANG W Q, LONG X Q, CUI W D.Bacteria diversity analysis in corn silage[J].Xinjiang Agricultural Sciences, 2013, 50(8):1424-1433.(in Chinese)
[28] 张娟, 史文娇, 韩雪林, 等.苹果酸和嗜酸乳杆菌对圆叶决明青贮品质及微生物多样性的影响[J].动物营养学报, 2021, 33(12):6941-6952. ZHANG J, SHI W J, HAN X L, et al.Effects of malic acid and Lactobacillus acidophilus on quality and microbial diversity of Chamaecrista rotundifolia silage[J].Chinese Journal of Animal Nutrition, 2021, 33(12):6941-6952.(in Chinese)
[29] CAI Y M, SUYANANDANA P, SAMAN P, et al.Classification and characterization of lactic acid bacteria isolated from the intestines of common carp and freshwater prawns[J].The Journal of General and Applied Microbiology, 1999, 45(4):177-184.  
[30] 闫绍鹏, 杨瑞华, 冷淑娇, 等.高通量测序技术及其在农业科学研究中的应用[J].中国农学通报, 2012, 28(30):171-176. YAN S P, YANG R H, LENG S J, et al.High-fluxed DNA sequencing technology and its application in agricultural science research[J].Chinese Agricultural Science Bulletin, 2012, 28(30):171-176.(in Chinese)
[31] 张想峰, 艾尼瓦尔·艾山, 买热木尼沙·吾甫尔, 等.新疆小芦苇青贮中乳酸杆菌的分离鉴定[J].新疆农业科学, 2009, 46(6):1327-1331. ZHANG X F, AISHAN A N W E, WUFUER M R M N S, et al.Isolation and identification of lactobacillus from Xinjiang small reed silage[J].Xinjiang Agricultural Sciences, 2009, 46(6):1327-1331.(in Chinese)
[32] YANG L L, YUAN X J, LI J F, et al.Dynamics of microbial community and fermentation quality during ensiling of sterile and nonsterile alfalfa with or without Lactobacillus plantarum inoculant[J].Bioresource Technology, 2019, 275:280-287.
[33] 石娇, 王光西, 丁曼琳, 等.雷氏普罗威登斯菌耐药性及耐药基因的单中心研究[J].中国感染控制杂志, 2021, 20(7):597-601. SHI J, WANG G X, DING M L, et al.A single center study on antimicrobial resistance and antimicrobial resistance genes of Providencia rettgeri[J].Chinese Journal of Infection Control, 2021, 20(7):597-601.(in Chinese)
文章导航

/