Applied Microbiology and Biotechnology

, Volume 103, Issue 19, pp 7983–7995 | Cite as

Effect of Lactobacillus plantarum expressing multifunctional glycoside hydrolases on the characteristics of alfalfa silage

  • Jingui Guo
  • Yixiao Xie
  • Zhu Yu
  • Geng Meng
  • Zhe WuEmail author
Biotechnological products and process engineering


For the first time, Lactobacillus plantarum strains carrying heterologous genes encoding multifunctional glycoside hydrolases were constructed and used as additives for alfalfa silage. The chemical characteristics, nonstructural carbohydrate composition, and fermentation quality of alfalfa silage were examined. The supernatant of L. plantarum expressing CbXyn10C and Bgxg1 (LP11AG) showed activities on xylan, Avicel, and carboxymethylcellulose (CMC), while the supernatant of the wild-type L. plantarum showed no activity. When LP11AG was used as silage additive, the water-soluble carbohydrate content of alfalfa silage increased by 72%, 55%, and 155% compared with control when the silage was stored at 20 °C, 30 °C, and 40 °C, respectively. With LP11AG being used as an additive for the alfalfa silage stored at 20 °C, the hemicellulose, cellulose, and acid detergent ligninin (ADL) contents decreased by 17%, 6%, and 14% compared with the control (p < 0.05), respectively. Compared with the corresponding original contents, the contents of glucose, arabinose, galactose, and fructose detected in silage treated with LP11AG after 45 days of ensiling increased by 55%, 1494%, 68%, and 5% , respectively, when stored at 40 °C. Raffinose and stachyose, originally present in alfalfa, disappeared after ensiling. In conclusion, our results suggest that LP11AG provides a substantial benefit as a silage additive.


Alfalfa silage Lactobacillus plantarum Glycoside hydrolases Nonstructural carbohydrate composition Structural carbohydrate 


Funding information

This work was supported by the Modern Agro-industry Technology Research System (CARS-34), the National Natural Science Foundation of China (31702181), the National Key Research and Development Program of China (2017YFD0502100), and Fundamental Research Funds for the Central Universities (2018QC048).

Compliance with ethical standards

This article does not contain any studies with human participants or animal experiments.

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

253_2019_10097_MOESM1_ESM.pdf (980 kb)
ESM 1 (PDF 979 kb)


  1. Armentano LE, Pastore SC, Hoffman PC (1988) Particle size reduction of alfalfa silage did not alter nutritional quality of high forage diets for dairy cattle. J Dairy Sci 71(2):409–413. CrossRefGoogle Scholar
  2. Arshad M, Feyissa BA, Amyot L, Aung B, Hannoufa A (2017) MicroRNA156 improves drought stress tolerance in alfalfa (Medicago sativa) by silencing SPL13. Plant Sci 258:122–136. CrossRefGoogle Scholar
  3. Borrero J, Jimenez JJ, Gutiez L, Herranz C, Cintas LM, Hernandez PE (2011) Protein expression vector and secretion signal peptide optimization to drive the production, secretion, and functional expression of the bacteriocin enterocin A in lactic acid bacteria. J Biotechnol 156(1):76–86. CrossRefGoogle Scholar
  4. Brunecky R, Alahuhta M, Xu Q, Donohoe BS, Crowley MF, Kataeva IA, Yang SJ, Resch MG, Adams MW, Lunin VV, Himmel ME, Bomble YJ (2013) Revealing nature’s cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA. Science 342:1513–1516. CrossRefGoogle Scholar
  5. Coblentz WK, Muck RE, Borchardt MA, Spencer SK, Jokela WE, Bertram MG, Coffey KP (2014) Effects of dairy slurry on silage fermentation characteristics and nutritive value of alfalfa. J Dairy Sci 97(11):7197–7211. CrossRefGoogle Scholar
  6. Dean DB, Adesogan AT, Krueger N, Littell RC (2005) Effect of fibrolytic enzymes on the fermentation characteristics, aerobic stability, and digestibility of bermudagrass silage. J Dairy Sci 88(3):994–1003. CrossRefGoogle Scholar
  7. Denek N, Can A, Avci M, Aksu T, Durmaz H (2011) The effect of molasses-based pre-fermented juice on the fermentation quality of first-cut lucerne silage. Grass Forage Sc 66 (2):243-250. https://10.1111/j.1365-2494.2011.00783.xGoogle Scholar
  8. Desta ST, Yuan X, Li J, Shao T (2016) Ensiling characteristics, structural and nonstructural carbohydrate composition and enzymatic digestibility of Napier grass ensiled with additives. Bioresour Technol 221:447–454. CrossRefGoogle Scholar
  9. Ding W, Guo X, Ataku K (2014) Characterization of peptides in ensiled alfalfa treated with different chemical additives. Anim Sci J 84(12):774–781. CrossRefGoogle Scholar
  10. Japan Grassland Farming Forage Seed Association (1994) Guide book for quality evaluation of forage, Tokyo, pp 82–87Google Scholar
  11. Ke WC, Ding WR, Xu DM, Ding LM, Zhang P, Li FD, Guo XS (2017) Effects of addition of malic or citric acids on fermentation quality and chemical characteristics of alfalfa silage. J Dairy Sci 100(11):8958–8966. CrossRefGoogle Scholar
  12. Kim SK, Chung D, Himmel ME, Bomble YJ, Westpheling J (2017) Engineering the N-terminal end of CelA results in improved performance and growth of Caldicellulosiruptor bescii on crystalline cellulose. Biotechnol Bioeng 114(1):945–950. CrossRefGoogle Scholar
  13. Li J, Yuan X, Desta ST, Dong Z, Mugabe W, Shao T (2018a) Characterization of Enterococcus faecalis JF85 and Enterococcus faecium Y83 isolated from Tibetan yak (Bos grunniens) for ensiling Pennisetum sinese. Bioresour Technol 257:76–83. CrossRefGoogle Scholar
  14. Li J, Yuan X, Dong Z, Mugabe W, Shao T (2018b) The effects of fibrolytic enzymes, cellulolytic fungi and bacteria on the fermentation characteristics, structural carbohydrates degradation, and enzymatic conversion yields of Pennisetum sinese silage. Bioresour Technol 264:123–130. CrossRefGoogle Scholar
  15. Liu JR, Yu B, Zhao X, Cheng KJ (2007) Coexpression of rumen microbial beta-glucanase and xylanase genes in Lactobacillus reuteri. Appl Microbiol Biotechnol 77(1):117–124. CrossRefGoogle Scholar
  16. Ma X, Wang G, Li D, Hao Y (2016) Microcin v production in Lactobacillus plantarum LB-B1 using heterologous leader peptide from pediocin pa-1. Curr Microbiol 72(3):357–362.
  17. Mathiesen G, Sveen A, Brurberg MB, Fredriksen L, Axelsson L, Eijsink VG (2009) Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1. BMC Genomics 10(1):425. CrossRefGoogle Scholar
  18. Mierau I, Kleerebezem M (2005) 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol 68(6):705–717. CrossRefGoogle Scholar
  19. Morais S, Shterzer N, Lamed R, Bayer EA, Mizrahi I (2014) A combined cell-consortium approach for lignocellulose degradation by specialized Lactobacillus plantarum cells. Biotechnol Biofuels 7(1):112. CrossRefGoogle Scholar
  20. Morrison JM, Elshahed MS, Youssef N (2016) A multifunctional GH39 glycoside hydrolase from the anaerobic gut fungus Orpinomyces sp. strain C1A. Peer J 4(3):e2289. CrossRefGoogle Scholar
  21. Muck RE, Nadeau EMG, McAllister TA, Contreras-Govea FE, Santos MC, Kung L Jr (2018) Silage review: recent advances and future uses of silage additives. J Dairy Sci 101(5):3980-4000.
  22. Nadeau EM, Buxton DR, Russell JR, Allison MJ, Young JW (2000) Enzyme, bacterial inoculant, and formic acid effects on silage composition of orchardgrass and alfalfa. J Dairy Sci 83(7):1487–1502. CrossRefGoogle Scholar
  23. Nguyen STC, Freund HL, Kasanjian J, Berlemont R (2018) Function, distribution, and annotation of characterized cellulases, xylanases, and chitinases from CAZy. Appl Microbiol Biotechnol 102(4):1629–1637. CrossRefGoogle Scholar
  24. Ni K, Zhao J, Zhu B, Su R, Pan Y, Ma J, Zhou G, Tao Y, Liu X, Zhong J (2018) Assessing the fermentation quality and microbial community of the mixed silage of forage soybean with crop corn or sorghum. Bioresour Technol 265:563–567. CrossRefGoogle Scholar
  25. Nsereko VL, Rooke JA (2000) Characterisation of peptides in silages made from perennial ryegrass with different silage additives. J Sci Food Agric 80(6):725-731.<725::AID-JSFA602>3.0.CO;2-8
  26. Ozkose E, Akyol I, Kar B, Comlekcioglu U, Ekinci MS (2009) Expression of fungal cellulase gene in Lactococcus lactis to construct novel recombinant silage inoculants. Folia Microbiol 54(4):335–342. CrossRefGoogle Scholar
  27. Peng X, Su H, Mi S, Han Y (2016) A multifunctional thermophilic glycoside hydrolase from Caldicellulosiruptor owensensis with potential applications in production of biofuels and biochemicals. Biotechnol Biofuels 9(1):98. CrossRefGoogle Scholar
  28. Rossi F, Rudella A, Marzotto M, Dellaglio F (2001) Vector-free cloning of a bacterial endo-1,4-β-glucanase in lactobacillus plantarum and its effect on the acidifying activity in silage: use of recombinant cellulolytic lactobacillus plantarum as silage inoculant. Antonie Van Leeuwenhoek 80(2):139-147.
  29. Rud I, Jensen PR, Naterstad K, Axelsson L (2006) A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum. Microbiology 152(Pt 4):1011-1019.
  30. Shao Q, Chundawat SP, Krishnan C, Bals B, Sousa Lda C, Thelen KD, Dale BE, Balan V (2010) Enzymatic digestibility and ethanol fermentability of AFEX-treated starch-rich lignocellulosics such as corn silage and whole corn plant. Biotechnol Biofuels 3(1):12. CrossRefGoogle Scholar
  31. Sheperd AC, Maslanka M, Quinn D, Kung L Jr (1995) Additives containing bacteria and enzymes for alfalfa silage. J Dairy Sci 78(3):565–572. CrossRefGoogle Scholar
  32. Smith LH (1962) Theoretical carbohydrates requirement for alfalfa silage production1. Agron J 54 (4):291. https://10.2134/agronj1962.00021962005400040003xGoogle Scholar
  33. Talamantes D, Biabini N, Dang H, Abdoun K, Berlemont R (2016) Natural diversity of cellulases, xylanases, and chitinases in bacteria. Biotechnol Biofuels 9:133. CrossRefGoogle Scholar
  34. Tengerdy RP, Weinberg ZG, Szakacs G, Wu M, Linden JC, Henk LL, Johnson DE (2010) Ensiling alfalfa with additives of lactic acid bacteria and enzymes. J Sci Food Agric 55(2):215-228.
  35. Tian J, Li Z, Yu Z, Zhang Q, Li X (2017) Interactive effect of inoculant and dried jujube powder on the fermentation quality and nitrogen fraction of alfalfa silage. Anim Sci J 88(4):633-642.
  36. Tisma M, Planinic M, Bucic-Kojic A, Panjicko M, Zupancic GD, Zelic B (2018) Corn silage fungal-based solid-state pretreatment for enhanced biogas production in anaerobic co-digestion with cow manure. Bioresour Technol 253:220–226. CrossRefGoogle Scholar
  37. Xue X, Wang R, Tu T, Shi P, Ma R, Luo H, Yao B, Su X (2015) The N-terminal gh10 domain of a multimodular protein from Caldicellulosiruptor bescii is a versatile xylanase/β-glucanase that can degrade crystalline cellulose. Appl Environ Microbiol 81:3823–3833. CrossRefGoogle Scholar
  38. Yuan XJ, Wen AY, Wang J, Desta ST, Dong ZH, Shao T (2018) Effects of four short-chain fatty acids or salts on fermentation characteristics and aerobic stability of alfalfa (Medicago sativa L.) silage. J Sci Food Agric 98(1):328–335. CrossRefGoogle Scholar
  39. Zhang Q, Yu Z (2017) Characterization, identification and application of lactic acid bacteria isolated from Leymus chinensis silage. Grassl Sci 63(2):111–117. CrossRefGoogle Scholar
  40. Zhang Q, Yu Z, Wang X, Tian J (2018) Effects of inoculants and environmental temperature on fermentation quality and bacterial diversity of alfalfa silage. Anim Sci J 89(8):1085–1092. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Jingui Guo
    • 1
  • Yixiao Xie
    • 1
  • Zhu Yu
    • 1
  • Geng Meng
    • 2
  • Zhe Wu
    • 1
    Email author
  1. 1.College of Grassland Science and TechnologyChina Agricultural UniversityBeijingChina
  2. 2.College of Veterinary MedicineChina Agricultural UniversityBeijingChina

Personalised recommendations