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Fermentative profile and lactic acid bacterial dynamics in non-wilted and wilted alfalfa silage in tropical conditions

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Abstract

This study was conducted to evaluate the fermentative profile and microbial populations of wilted and non-wilted alfalfa silages ensiled with or without inoculant and the population dynamics of lactic acid bacteria (LAB) of wilted alfalfa plant and theirs silage. A 2 × 2 × 6 factorial arrangement was used, with the absence or presence of wilting (W), with and without bacterial inoculant (I) and six fermentation periods (P) (1, 3, 7, 14, 28 and 56 days), in a completely randomized design, with three replicates. The alfalfa was slightly wilted for 6 h and increased the dry matter content from 133.9 to 233.4 g/kg. It was performed the cultivation, followed by the isolation of LAB from samples of alfalfa forage before ensiling and its silage only in non-inoculated silages, after different fermentation periods. DNA was extracted from the isolated strains of LAB; the 16S rRNA gene sequences were amplified by PCR and the sequences were compared to those available from the GenBank database. Wilting provided silages with lower pH, ammonia nitrogen and acetic acid concentrations. The wilting process did not alter the amount of LAB; however, it affected the LAB diversity of the silages. The Lactobacillus plantarum was the predominant species in non-wilted and wilted silages.

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References

  1. Bao W, Mi Z, Xu H, Zheng Y, Kwok LY, Zhang H, Zhang W (2016) Assessing quality of Medicago sativa silage by monitoring bacterial composition with single molecule, real-time sequencing technology and various physiological parameters. Sci Rep 6:28358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Borreani G, Tabacco E, Schmidt RJ, Holmes BJ, Muck RE (2018) Silage review: Factors affecting dry matter and quality losses in silages. J Dairy Sci 101 (5)

  3. Bridge PD, Sneath PHA (1982) Streptococcus-gallinarum Sp-Nov and Streptococcus-oralis Sp-Nov. Intern J Systematic Bacteriol 32:410–415

    Article  Google Scholar 

  4. Cai Y, Benno Y, Ogawa M, Ohmomo S, Kumai S, Nakase T (1998) Influence of Lactobacillus spp. from an inoculant and of Weissella and Leuconostoc spp from forage crops on silage fermentation. Appl Environ Microbiol 64:2982–2987

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Chahrour W, Merzouk Y, Henni JE, Haddaji M, Kihal M (2013) Screening and identification of lactic acid bacteria isolated from sorghum silage processes in west Algeria. Afr J Biotechnol 12(14):1703–1709

    Google Scholar 

  6. Danner H, Holzer M, Mayrhuber E, Braun R (2003) Acetic acid increases stability of silage under aerobic conditions. Appl Environ Microbiol 69(1):562–567

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Deriaz RE (1961) Routine analysis of carbohydrate and lignin in herbage. J Sci Food Agric 12:150–160

    Article  Google Scholar 

  8. Fenton MP (1987) An investigation into the sources of lactic acid bacteria in grass silage. J Appl Bacteriol 62:181

    Article  Google Scholar 

  9. Fusco V, Quero GM, Cho GS, Kabisch J, Meske D, Neve H, Bockelmann W, Franz CMAP (2015) The genus Weissella: taxonomy, ecology and biotechnological potential. Front Microbiol 6:155

    Article  PubMed  PubMed Central  Google Scholar 

  10. Graf K, Ulrich A, Idler C, Klocke M (2016) Bacterial community dynamics during ensiling of perennial ryegrass at two compaction levels monitored by terminal restriction fragment length polymorphism. J Appl Microbiol 120

  11. Hartmann M, Windmer F (2006) Community structure analysis are more sensitive to differences in soil bacterial communities than anonymous diversity indices. Appl Environ Microbiol 72:7804–7812

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Heuer H, Krsek M, Baker P, Smalla K, Wellington EMH (1997) Analysis of Actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Appl Environ Microbiol 63:3233–3241

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Holt JG, Krieg NR, Sneath PHA (1994) Bergey’s manual of determinative bacteriology, 9th edn. Williams and Wilkins, Baltimore

    Google Scholar 

  14. Koeuth T, Versalovic J, Lupski JR (1995) Differential subsequence conservation of interspersed repetitive Streptococcus pneumoniae Box elements in diverse bacteria. Genome Res 5:408–418

    Article  CAS  PubMed  Google Scholar 

  15. Lin C, Bolsen KK, Brent BE, Hart RA, Dickerson JT (1992) Epiphytic microflora on alfalfa and whole-plant corn. J Dairy Sci 75:2484–2493

    Article  CAS  PubMed  Google Scholar 

  16. Liu Q, Chen M, Zhang J, Shi S, Cai Y (2012) Characteristics of isolated lactic acid bacteria and their effectiveness to improve stylo (Stylosanthes guianensis Sw.) silage quality at various temperatures. Anim Sci J 83:128–135

    Article  CAS  PubMed  Google Scholar 

  17. Mahanna B, Chase LE (2003) Practical application and solution to silage problems. In: silage science and technology, Proceedings. Madison: ASCSSA-SSSA, Agronomy 42:31–93

  18. Mahanna WC (1993) Silage fermentation and additive use in North America. In: Silage production from seed to animal, Proceedings, New York, NRAES, 85–95

  19. Mayra-Makinen A, Bigret M (1998) Industrial use and production of lactic acid bacteria. In: Lactic Acid Bacteria – Microbiology and Functional Aspects, 2nd edn. Marcel Dekker, New York, pp 73–102

  20. McDonald P, Henderson AR, Heron SJE (1991) Biochemistry of silage, 2.ed. Marlow Chalcombe Publications, New York, p 340

  21. Monteiro ALG, Costa C, Arrigoni MB (1998) Evaluation of Potential for Ensiling Alfalfa Cultivars (Medicago sativa L.) (In Portuguese). R Bras Zootec 27(5):1064–1068

    Google Scholar 

  22. Muck RE (1988) Factors influencing silage quality and their implications for management. J Dairy Sci 71:2992–3002

    Article  Google Scholar 

  23. Muck RE (1989) Initial bacterial numbers on lucerne prior to ensiling. Grass Forage Sci 44:19

    Article  Google Scholar 

  24. Nishino N, Li Y, Wang C, Parvin S (2012) Effects of wilting and molasses addition on fermentation and bacterial community in guinea grass silage. Lett Appl Microbiol 54:175–181

    Article  CAS  PubMed  Google Scholar 

  25. Ogunade IM, Jiang Y, Pech Cervantes AA, Kim DH, Oliveira AS, Vyas D, Weinberg ZG, Jeong KC, Adesogan AT (2018) Bacterial diversity and composition of alfalfa silage as analyzed by Illumina MiSeq sequencing: Effects of Escherichia coli O157:H7 and silage additives. J Dairy Sci 101:2048–2059

    Article  CAS  PubMed  Google Scholar 

  26. Ouwehand A, Salminen S, Isolauri E (2002) Probiotics: an overview of beneficial effects. Antonie Van Leeuwenhoek 82:279–289

    Article  CAS  PubMed  Google Scholar 

  27. Pahlow G, Muck RE, Driehuis F, Oude-Elferink SJWH, Spoelstra SF (2003) Microbiology of Ensiling. Silage Science Technology 42:31–93

    Google Scholar 

  28. Playne MJ, McDonald P (1966) The buffering constituents of herbage and of silage. J Sci Food Agric 17:264–268

    Article  CAS  Google Scholar 

  29. Rangrab LH, Mühlbach PRF, Berto JL (2000) Alfalfa silage harvested at the beginning of flowering and subjected to wilting and the action of biological additives (in Portuguese). R Bras de Zootec 29(2):349–356

    Article  Google Scholar 

  30. Ruser B (1989) Erfassung und Identifizierung des epiphytischen Micbsaure-bakterienbesatzes auf Gras und Mais in Abblingigkeit von Standort, Sorte, Entwicklungsstadium Sowieemte-und Klimaeinflussen. Dissertation Institute for Grassland and Forage Research

  31. Schachtsiek M, Hammes WP, Hertel C (2004) Characterization of Lactobacillus coryniformis DSM 20001T surface protein Cpf mediating coaggregation with and aggregation among pathogens. Appl Environ Microbiol 70:7078–7085

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Siegfried VR, Ruckemann H, Stumpf G (1984) Eine HPLC-Methode zur Bestimmung organischer Säuren in Silagen. Landwirtsch Forsch 37:298–304

    CAS  Google Scholar 

  33. Stevenson DM, Muck RE, Shinners KJ, Weimer PJ (2006) Use of real time PCR to determine population profiles of individual species of lactic acid bacteria in alfalfa silage and stored corn stover. Appl Mircrobiol Biotechnol 71:329–338

    Article  CAS  Google Scholar 

  34. Tagg JR, Dajani AS, Wannamaker LW (1976) Bacteriocins of gram-positive bacteria. Bacteriol Rev 40:722–756

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Tanaka O, Komatsu T, Oshibe A, Cai Y, Miyazaki S, Nakanishi K (2009) Production of 3-hydroxypropionaldehyde in silage inoculated with Lactobacillus coryniformis plus glycerol. Biosci Biotechnol Biochem 73:1494–1499

    Article  CAS  PubMed  Google Scholar 

  36. Tanaka O, Mori K, Ohmomo S (2000) Effect of inoculation with Lactobacillus curvatus on ensiling. Grassland Sci 46:148–152

    CAS  Google Scholar 

  37. Tanasupawat S, Shida O, Okada S, Komagata K (2000) Lactobacillus acidipiscis sp. nov. and Weissella thailandensis sp. nov., isolated from fermented fish in Thailand. Int J Syst Evol Microbiol 50 4:1479–1485

    Article  Google Scholar 

  38. Tohno M, Kobayashi H, Nomura M, Uegaki R, Cai Y (2012) Identification and characterization of lactic acid bacteria isolated from mixed pasture of timothy and orchardgrass, and its badly preserved silages. Anim Sci J 83:318–330

    Article  CAS  PubMed  Google Scholar 

  39. Tran TMT, Nguyen MT, Nguyen HV, Nishino N (2017) Effects of wilting and lactic acid bacteria inoculation on fermentation and microbial community of elephant grass silage produced in Vietnam. Grassland Sci 64(2):151–155

    Article  CAS  Google Scholar 

  40. Tyrolová Y, Výborná A (2011) The effects of wilting and biological and chemical additives on the fermentation process in field pea silage. Czech J Anim Sci 56:427–432

    Article  Google Scholar 

  41. Weatherburn MW (1967) Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 39:971–974

    Article  CAS  Google Scholar 

  42. Weinberg ZG, Ashbell G, Hen Y, Azrieli A (1993) The effect of applying lactic acid bacteria at ensiling on the aerobic stability of silages. J Appl Bacteriol 75:512–518

    Article  Google Scholar 

  43. Weise F (1969) The influence of initial plant microbial populations on the course of fermentation. In 3rd General Mtg Eur Grassl Fed, Proceedings. Braunschweig, 221

Download references

Acknowledgements

We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Instituto Nacional de Ciência e Tecnologia - Ciência Animal (INCT-CA, Brazil), and Fundação de Apoio a Pesquisa de Minas Gerais (FAPEMIG, Brazil) for their financial support.

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Authors and Affiliations

  1. Department of Animal Science, Federal University of Vicosa, Viçosa, MG, 36570-000, Brazil

    Mariele Cristina Nascimento Agarussi, Odilon Gomes Pereira, Vanessa Paula da Silva & Karina Guimarães Ribeiro

  2. University of Brasilia, Brasilia, DF, 70910-900, Brazil

    Eliana Santos Leandro

  3. Federal University of Bahia, Salvador, BA, 40110-909, Brazil

    Stefanie Alvarenga Santos

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  1. Mariele Cristina Nascimento Agarussi
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Correspondence to Mariele Cristina Nascimento Agarussi.

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Agarussi, M.C.N., Pereira, O.G., da Silva, V.P. et al. Fermentative profile and lactic acid bacterial dynamics in non-wilted and wilted alfalfa silage in tropical conditions. Mol Biol Rep 46, 451–460 (2019). https://doi.org/10.1007/s11033-018-4494-z

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