Acessibilidade / Reportar erro

Natamycin as a potential silage additive: A lab trial using sugarcane to assess greenhouse gas emissions

ABSTRACT

The objective of this study was to evaluate natamycin, Lactobacillus buchneri (LB), or their combination on the chemical composition, loss, fermentative profile, and aerobic stability as well as gas production and composition of sugarcane silages. The treatments were (wet basis): no additive (control), 10 g t−1 of natamycin (N10), 5 × 104 cfu g−1 of LB, and the combination of 4 g t−1 of natamycin and 2.5 × 104 cfu g−1 of LB (NLB). Sugarcane was chopped (10 mm), treated with the additives, and ensiled in experimental silos (four replicates). The silos remained stored for 51 days. The LB inoculation, alone or in combination with natamycin, increased the acetic acid content (by 105 and 78% respectively) and decreased ethanol content (by 83 and 71% respectively) when compared to N10 treatment and the control. A decrease in both dry matter and gas losses was observed in the LB (by 72 and 78%, respectively) and N10 (by 69 and 77%, respectively) silages compared with the control, but not the combination. The N10 treatment reduced greenhouse gas (GHG) emission by 86% compared with the control silage. Control and N10 silages deteriorated to the same extent with aerobic exposure, whereas LB and NLB presented higher aerobic stability. The use of natamycin alone is not recommended when ethanol and aerobic stability are concerns. However, natamycin may be considered for the composition of blend additives to decrease greenhouse gas emission and fermentative loss in silages. Further studies must be carried out to optimize doses of natamycin in blend additives.

Keywords:
ethanol; gas production; inoculant; methane; yeast

1. Introduction

Crop and livestock production systems are claimed as important sources of greenhouse gases (GHG), accounting for approximately 10 and 14.5% of total GHG emissions worldwide ( Gerber et al., 2013Gerber, P. J.; Hristov, A. N.; Henderson, B.; Makkar, H.; Oh, J.; Lee, C.; Meinen, R.; Montes, F.; Ott, T.; Firkins, J.; Rotz, A.; Dell, C.; Adesogan, A. T.; Yang, W. Z.; Tricarico, J. M.; Kebreab, E.; Waghorn, G; Dijkstra, J. and Oosting, S. 2013. Technical options for the mitigation of direct methane and nitrous oxide emissions from livestock: a review. Animal 7:220-234. https://doi.org/10.1017/S1751731113000876
https://doi.org/10.1017/S175173111300087...
; Tubiello et al., 2013Tubiello, F. N.; Salvatore, M.; Rossi, S.; Ferrara, A.; Fitton, N. and Smith, P. 2013. The FAOSTAT database of greenhouse gas emissions from agriculture. Environmental Research Letters 8:015009. https://doi.org/10.1088/1748-9326/8/1/015009
https://doi.org/10.1088/1748-9326/8/1/01...
; Tubiello et al., 2015Tubiello, F. N.; Salvatore, M.; Ferrara, A. F.; House, J.; Federici, S.; Rossi, S.; Biancalani, R.; Golec, R. D. C.; Jacobs, H.; Flammini, A.; Prosperi, P.; Cardenas-Galindo, P.; Schmidhuber, J.; Sanchez, M. J. S.; Srivastava, N. and Smith, P. 2015. The Contribution of agriculture, forestry and other land use activities to global warming, 1990–2012. Global Chance Biology 21:2655-2660. https://doi.org/10.1111/gcb.12865
https://doi.org/10.1111/gcb.12865...
). However, silage production has been scarcely studied as a possible source of GHG ( Grossi et al., 2019Grossi, G.; Goglio, P.; Vitali, A. and Williams, A. G. 2019. Livestock and climate change: impact of livestock on climate and mitigation strategies. Animal Frontiers 9:69-76. https://doi.org/10.1093/af/vfy034
https://doi.org/10.1093/af/vfy034...
).

Ensiling is one of the most common methods for long-term forage storage for ruminants, and its fermentation can lead to on-farm GHG emission. According to Henriksson et al. (2012)Henriksson, M.; Cederberg, C. and Swensoon, C. 2012. Impact of cultivation strategies and regional climate on greenhouse gas emissions from grass/clover silage. Acta Agriculturae Scandinavica, Section A - Animal Science 62:233-237. https://doi.org/10.1080/09064702.2013.797010
https://doi.org/10.1080/09064702.2013.79...
, higher dry matter (DM) losses during fermentation are linked to higher GHG emission from productive systems based on silage. However, fermentation gases were not assessed by those authors.

Several microorganisms are responsible for fermentation loss and gas emission from silage (e.g., heterofermentative lactic acid bacteria [LAB], clostridia, enterobacteria). Yeast activity in silages, for instance, increases gas emission (mostly CO2) as well as volatile organic compound synthesis (mainly ethanol), reducing air quality and greatly contributing to greenhouse effect ( McDonald et al., 1991McDonald, P.; Henderson, A. R. and Heron, S. 1991. The biochemistry of silage. 2nd ed. Chalcombe Publications, Marlow. ; Hafner et al., 2010Hafner, S. D.; Montes, F.; Rotz, C. A. and Mitloehner, F. 2010. Ethanol emission from loose corn silage and exposed silage particles. Atmospheric Environment 44:4172-4180. https://doi.org/10.1016/j.atmosenv.2010.07.029
https://doi.org/10.1016/j.atmosenv.2010....
; Borreani et al., 2018Borreani, G.; Tabacco, E.; Schmidt, R. J.; Holmes, B. J. and Muck, R. E. 2018. Silage review: Factors affecting dry matter and quality losses in silages. Journal of Dairy Science 101:3952-3979. https://doi.org/10.3168/jds.2017-13837
https://doi.org/10.3168/jds.2017-13837...
).

In Brazil, sugarcane ( Saccharum officinarum ) is one of the main crops studied for ensiling purposes. Since sugarcane is highly prone to ethanolic fermentation due to its high sugar availability and indigenous yeast population ( Pedroso et al., 2005Pedroso, A. F.; Nussio, L. G.; Paziani, S. F.; Loures, D. R. S.; Igarasi, M. S.; Coelho, R. M.; Packer, I. H.; Horii, J. and Gomes, L. H. 2005. Fermentation and epiphytic microflora dynamics in sugar cane silage. Scientia Agricola 62:427-432. https://doi.org/10.1590/S0103-90162005000500003
https://doi.org/10.1590/S0103-9016200500...
; Ávila et al., 2010aÁvila, C. L. S.; Valeriano, A. R.; Pinto, J. C.; Figueiredo, H. C. P.; Rezende, A. V. and Schwan, R. F. 2010a. Chemical and microbiological characteristics of sugar cane silages treated with microbial inoculants. Revista Brasileira de Zootecnia 39:25-32. https://doi.org/10.1590/S1516-35982010000100004
https://doi.org/10.1590/S1516-3598201000...
), many studies have been conducted to determine suitable additives to avoid yeast development and reduce gas production ( Pedroso et al., 2005Pedroso, A. F.; Nussio, L. G.; Paziani, S. F.; Loures, D. R. S.; Igarasi, M. S.; Coelho, R. M.; Packer, I. H.; Horii, J. and Gomes, L. H. 2005. Fermentation and epiphytic microflora dynamics in sugar cane silage. Scientia Agricola 62:427-432. https://doi.org/10.1590/S0103-90162005000500003
https://doi.org/10.1590/S0103-9016200500...
; Ávila et al., 2014Ávila, C. L. S.; Carvalho, B. F.; Pinto, J. C.; Duarte, W. F. and Schwan, R. F. 2014. The use of Lactobacillus species as starter cultures for enhancing the quality of sugar cane silage. Journal of Dairy Science 97:940-951. https://doi.org/10.3168/jds.2013-6987
https://doi.org/10.3168/jds.2013-6987...
). Thus, sugarcane silage can be considered a good model for assessing gas production and its control through additives.

Lactobacillus buchneri (LB) is an obligate heterofermentative bacterium that has been widely studied as a sugarcane additive to reduce yeast activity. It converts not only sugars but also lactic acid into acetic acid, which reduces yeast count, ethanol synthesis, and fermentative loss and increases aerobic stability compared with untreated silage ( Holzer et al., 2003Holzer, M.; Mayrhuber, E.; Danner, H. and Braun, R. 2003. The role of Lactobacillus buchneri in forage preservation. Trends in Biotechnology 21:282-287. https://doi.org/10.1016/S0167-7799(03)00106-9
https://doi.org/10.1016/S0167-7799(03)00...
; Pedroso et al., 2008Pedroso, A. F.; Nussio, L. G.; Loures, D. R. S.; Paziani, S. F.; Ribeiro, J. L.; Mari, L. J.; Zopollatto, M.; Schmidt, P.; Mattos, W. R. S. and Horii, J. 2008. Fermentation, losses, and aerobic stability of sugarcane silages treated with chemical or bacterial additives. Scientia Agricola 65:589-594. https://doi.org/10.1590/S0103-90162008000600004
https://doi.org/10.1590/S0103-9016200800...
; Ávila et al., 2010bÁvila, C. L. S.; Bravo Martins, C. E. C. and Schwan, R. F. 2010b. Identification and characterization of yeasts in sugarcane silages. Journal of Applied Microbiology 109:1677-1686. https://doi.org/10.1111/j.1365-2672.2010.04796.x
https://doi.org/10.1111/j.1365-2672.2010...
; Ávila et al., 2012Ávila, C. L. S.; Pinto, J. C.; Oliveira, D. P. and Schwan, R. F. 2012. Aerobic stability of sugar cane silages with a novel strain of Lactobacillus sp. Isolated from sugar cane. Revista Brasileira de Zootecnia 41:249-255. https://doi.org/10.1590/S1516-35982012000200003
https://doi.org/10.1590/S1516-3598201200...
). However, the results of LB inoculation in sugarcane silages are highly variable ( Schmidt, 2009Schmidt, P. 2009. Improved efficiency of sugarcane ensiling for ruminant supplementation. p.47-72. In: Proceedings of the 1st International Symposium on forage Quality and Conservation. FEALQ, Piracicaba. ).

The design of new additives might be helpful to better decrease DM loss as well as GHG emissions from ensiling ( Muck et al., 2018Muck, R. E.; Nadeau, E. M. G.; McAllister, T. A.; Contreras-Govea, F. E.; Santos, M. C. and Kung Jr, L. 2018. Silage review: Recent advances and future uses of silage additives. Journal of Dairy Science 101:3980-4000. https://doi.org/10.3168/jds.2017-13839
https://doi.org/10.3168/jds.2017-13839...
), and their combination with chemicals can improve the results of microbial additive inoculation.

Natamycin is a polyene macrolide (bacteriocin) produced by Streptomyces natalensis, Streptomyces chattanoogensis, or Streptomyces gilvosporeus and is commonly used as a food additive (e.g., in hard cheeses, sausages, wine, fermented olive) to avoid yeast spoilage ( Hondrodimou et al., 2011Hondrodimou, O.; Kourkoutas, Y. and Panagou, E. Z. 2011. Efficacy of natamicyn to control fungal growth in natural black olive fermentation. Food Microbiology 28:621-627. https://doi.org/10.1016/j.fm.2010.11.015
https://doi.org/10.1016/j.fm.2010.11.015...
; Dalhoff and Levy, 2015Dalhoff, A. A. H. and Levy, S. B. 2015. Does use of the polyene natamycin as a food preservative jeopardise the clinical efficacy of amphotericin B? A word of concern. International Journal of Antimicrobial Agents 45:564-567. https://doi.org/10.1016/j.ijantimicag.2015.02.011
https://doi.org/10.1016/j.ijantimicag.20...
; Wang et al., 2016Wang, M.; Wang, S.; Zong, G.; Hou, Z.; Liu, F.; Liao, D. J. and Zhu, X. 2016. Improvement of natamycin production by cholesterol oxidase overexpression in Streptomyces gilvosporeus . Journal of Microbiology and Biotechnology 26:241-247. https://doi.org/10.4014/jmb.1505.05033
https://doi.org/10.4014/jmb.1505.05033...
). Natamycin binds ergosterol (fatty acid present in the cell wall of molds and yeasts), thereby impairing the selective permeability of the cell ( Welscher et al., 2008Welscher, Y. M.; Napel, H. H.; Balangué, M. M.; Souza, C. M.; Riezman, H.; Kruiff, B. and Breukink, E. 2008. Natamycin blocks fungal growth by binding specialy to ergosterol without permeabilizing the membrane. The Journal of Biological Chemistry 283:6393-6401. https://doi.org/10.1074/jbc.m707821200
https://doi.org/10.1074/jbc.m707821200...
). It is poorly absorbed in the digestive system of mammals and totally excreted in the feces, making it safe for use in human food and feed (EAEMP, 1998). Recently, Shah et al. (2020)Shah, A. A.; Wu, J.; Qian, C.; Liu, Z.; Mobashar, M.; Tao, Z.; Zhang, X. and Zhong, X. 2020. Ensiling of whole plant hybrid Pennisetum with Natamycin and Lactobacillus plantarum impacts on fermentation characteristics and meta-genomic microbial community at low temperature. Journal of the Science of Food and Agriculture 100:3378-3385. https://doi.org/10.1002/jsfa.10371
https://doi.org/10.1002/jsfa.10371...
reported a low yeast count in elephant grass silage treated with natamycin. Pinto et al. (2020)Pinto, S.; Warth, J. F. G.; Novinski, C. O. and Schmidt, P. 2020. Effects of natamycin and Lactobacillus buchneri on the fermentative process and aerobic stability of maize silage. Journal of Animal and Feed Sciences 29:82-89. https://doi.org/10.22358/jafs/118179/2020
https://doi.org/10.22358/jafs/118179/202...
observed the synergistic effect of combining natamycin and LB in corn silages. However, information about the use of natamycin as an additive in sugarcane silage as well as its combination with LB has never been reported. Thus, we hypothesized that the use of natamycin, alone or combined with a commonly used additive (LB), would be an important tool for mitigating GHG emission from sugarcane silage. Our objective was to evaluate gas production, chemical composition, aerobic stability, and fermentative loss resulting from sugarcane silages dosed with natamycin, with or without L. buchneri .

2. Material and Methods

2.1. Ensiling

The research was performed in Curitiba (25°25'42" S and 49°16'24" W), located in the state of Paraná, Brazil. Sugarcane was grown and harvested in Paranavaí (23°4'26" S and 52°27'55" W).

The sugarcane variety RB 72-454 (12 months regrowth, second harvest) was manually harvested (no chopping) in July 2012 ( Table 1 ), and forage was carried to Curitiba the same day. The next day, the forage was processed in a stationary forage chopper adjusted to a theoretical length of 10 mm. Some moisture loss may have occurred between the harvest and processing (∼20 h) stages; however, it was not measured.

Table 1
Dry matter content, Brix grade, and pH in fresh sugarcane before ensiling

The processed forage was split into small piles (four piles per treatment), and additives were manually sprayed and mixed into the forage. Four treatments were tested: control with no additive (distilled water only); 10 g t−1 (wet basis) of natamycin (N10); 5 × 104 cfu g−1 of L. buchneri NCIMB 40788 (LB – Lalsil Cana – Lallemand Animal Nutrition); and a combination of 4 g t−1 of natamycin and 2.5 × 104 cfu g−1 of LB (NLB). The doses for treatments N10 and NLB were established to present similar costs to the LB treatment. All the additives were diluted with distilled water and applied at the rate of 2 L t−1. After spraying and mixing, a laboratory silo (20 L plastic bucket) was manually filled up with fresh forage from each pile (14.8 kg) and compacted (density of 220 kg DM m−3).

The gas volume (GV) produced was recorded daily using a 1-L low-density polypropylene cylinder, as described by Restellato et al. (2019)Restellato, R.; Novinski, C. O.; Pereira, L. M.; Silva, E. P. A.; Volpi, D.; Zopollatto, M.; Schmidt, P and Faciola, A. P. 2019. Chemical composition, fermentative losses, and microbial counts of total mixed ration silages inoculated with different Lactobacillus species. Journal of Animal Science 97:1634-1644. https://doi.org/10.1093/jas/skz030
https://doi.org/10.1093/jas/skz030...
( Figure 1 ). The silo lid was equipped with silicone hose that passed through a three-way stopcock for gas measurement and sampling. One cylinder (Ø-6.5 cm; 43 cm long) was connected to the stopcock of each silo by a silicone hose. In the bottom of the cylinder, a 2-mm hole was made and a hollow metal pin inserted to connect the hose. These cylinders were placed with the mouth facing down and immersed in water to avoid any gas leakage. The lid and all the connections were sealed airtight. Direct GV measurements were taken several times per day, and the gas was released after recording the volume. The total GV was the sum of all measurements of each silo during the fermentation period. To record the effluent production, the lab silos were previously filled in the bottom with dry sand (1.0 kg), covered with a thin plastic screen and two layers of cheesecloth. The silos remained stored for 51 days.

Figure 1
Schematic illustration of the gas collector device (GCD) and effluent collector device (ECD). Adapted from Souza (2015)Souza, C M. 2015. Impacto ambiental da produção de silagens: Revisão da literatura e avaliação experimental em silos laboratoriais. Dissertação (M. Sc.). Universidade Federal do Paraná, Curitiba. .

2.2. Laboratory analyses

Fresh samples were taken from each treatment before ensiling to determine the dry matter content (55 °C during 72 h) in a forced-air- oven as well as Brix grade by using a refractometer ( Table 1 ). From another sub-sample of each treatment, an aqueous extract ( Kung Jr. et al., 2000Kung Jr., L.; Robinson, J. R.; Ranjit, N. K.; Chen, J. H.; Golt, C. M. and Pesek, J. D. 2000. Microbial populations, fermentation end-products, and aerobic stability of corn silage treated with ammonia or a propionic acid-based preservative. Journal of Dairy Science 83:1479-1486. https://doi.org/10.3168/jds.S0022-0302(00)75020-X
https://doi.org/10.3168/jds.S0022-0302(0...
) was prepared, and its pH value was measured using a potentiometer (model WTW 330i).

Gas samples were taken from each silo over two consecutive days (14 and 15 days after closing the silos) using a 20 mL polypropylene disposable syringe equipped with a stopcock. The syringe was connected to the system, and 10 movements of suction and expulsion were performed to homogenize the gas content before collecting the gas sample. The syringes containing the samples were packed with ice and immediately sent to the laboratory. Concentrations of CO2, N2O, and CH4 were determined using a gas chromatograph (Shimadzu 14-A). The average gas composition of each silo was applied to the total gas produced by the same silo throughout the trial to calculate the total CO2 equivalent (GHG) produced per ton of ensiled DM, according to Houghton et al. (2001)Houghton, J. T.; Ding, Y.; Griggs, D. J.; Noguer, M.; van der Linden, P. J.; Dai, X.; Maskell, K. and Johnson, C. A. (eds.) 2001. Climate Change 2001: The Scientific Basis. Contribution of Working Group I to the Third Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, United Kingdom. . To reach those values, the gas composition (ppm for CO2 and CH4, and ppb for N2O) was transformed to mg/m3 using the molar mass of the molecules and the ideal gas equation. The result was applied to the total volume of gas produced per kg of forage to obtain the weight of the produced molecules (mg/kg of forage). The CO2-equivalent production was estimated by applying correction factor 25 to CH4 and 298 to N2O.

Before opening, the silos were weighed again, and the gravimetric gas losses and DM loss were estimated using the initial and final weight of each silo and the DM contents of the fresh sugarcane and silages ( Jobim et al., 2007Jobim, C. C.; Nussio, L. G.; Reis, R. A. and Schmidt, P. 2007. Avanços metodológicos na avaliação da qualidade da forragem conservada. Revista Brasileira de Zootecnia 36:101-119. https://doi.org/10.1590/S1516-35982007001000013
https://doi.org/10.1590/S1516-3598200700...
). Upon opening, the first 5 cm were discharged, and the remaining silage was homogenized (in plastic bags) and sampled for further analyses.

A sub-sample from each silo (500 g) was dried in a forced-air oven at 55 °C for 72 h and milled (1-mm sieve) for laboratory analyses. The absolute dry matter (105 °C), ash, and crude protein (CP) contents were determined ( AOAC, 2000AOAC - Association of Official Analytical Chemistry. 2000. Official methods of analysis. 17th ed. AOAC International, Gaithersburg, MD. ). The neutral detergent fiber with thermostable amylase and ash-inclusive (aNDF) and acid detergent fiber (ADF) concentrations were sequentially assessed in accordance with Mertens (2002)Mertens, D. R. 2002. Gravimetric determination of amylase-treated neutral detergent fiber in feeds with refluxing in beaker or crucibles: collaborative study. Journal of AOAC International 85:1217-1240. and Van Soest (1963)Van Soest, P. J. 1963. Use of detergents in the analysis of fibrous feeds. II. A rapid method for the determination of fiber and lignin. Journal of the Association of Official Agricultural Chemists 46:829-835. , respectively.

The fermentative profile was assessed for the silage extract obtained by hydraulic pressing the wet samples of each silo. The samples were prepared in accordance with Palmquist and Conrad (1971)Palmquist, D. L. and Conrad, H. R. 1971. Origin of plasma fatty acids in lactating cows fed high grain or high fat diets. Journal of Dairy Science 54:1025-1033. https://doi.org/10.3168/jds.s0022-0302(71)85966-0
https://doi.org/10.3168/jds.s0022-0302(7...
and frozen for further analyses. The concentration of volatile fatty acids, lactic acid, and ethanol was performed by gas chromatography ( Palmquist and Conrad, 1971Palmquist, D. L. and Conrad, H. R. 1971. Origin of plasma fatty acids in lactating cows fed high grain or high fat diets. Journal of Dairy Science 54:1025-1033. https://doi.org/10.3168/jds.s0022-0302(71)85966-0
https://doi.org/10.3168/jds.s0022-0302(7...
).

The DM content, fermentative profile, and chemical composition variables of the silages were corrected for volatiles ( Weissbach, 2011Weissbach, F. 2011. The future of forage conservation. p.319-363. In: Daniel, J. L. P.; Zopolatto, M. and Nussio, L. G., eds. Proceedings of the II International Symposium on Forage Quality and Conservation. São Pedro, Brazil. ).

2.3. Aerobic stability

For assessing the aerobic stability, silage from each replicate was unpacked and homogenized, and 3 kg of silage was transferred to 20-L plastic buckets (no pressing) and exposed to air in a temperature-controlled room at 25 °C for 120 h. A mercury-in-glass thermometer was positioned in the center of the forage mass in each bucket, and the temperature was recorded twice daily (at 08:00 and 15:00 h). A second set of buckets was used to take daily samples of the silage for pH measurement. Samples were taken from a central point of each bucket. Aerobic stability was defined as the time (h) taken for the silage temperature to rise 2 °C above room temperature ( Kung Jr. et al., 2000Kung Jr., L.; Robinson, J. R.; Ranjit, N. K.; Chen, J. H.; Golt, C. M. and Pesek, J. D. 2000. Microbial populations, fermentation end-products, and aerobic stability of corn silage treated with ammonia or a propionic acid-based preservative. Journal of Dairy Science 83:1479-1486. https://doi.org/10.3168/jds.S0022-0302(00)75020-X
https://doi.org/10.3168/jds.S0022-0302(0...
). The maximum temperature, time (h) to reach maximum temperature, and cumulative temperature were also assessed to evaluate aerobic deterioration intensity ( Novinski et al., 2012Novinski, C. O.; Junges, D.; Schmidt, P.; Rossi Junior, P.; Carvalho, J. P. G. and Teixeira, R. A. 2012. Methods of lab silos sealing and fermentation characteristics and aerobic stability of sugarcane silage treated with microbial additive. Revista Brasileira de Zootecnia 41:264-270. https://doi.org/10.1590/S1516-35982012000200005
https://doi.org/10.1590/S1516-3598201200...
).

2.4. Statistical analysis

Statistical analysis was performed using the MIXED procedure of SAS (Statistical Analysis System, version 9.0) as a completely randomized design with four treatments and four replicates, totalizing 16 experimental units (silos). The model included the fixed effect of additives. The results were analyzed with ANOVA, and the means were compared using the Tukey test at a 5% significance level.

Silage pH during aerobic exposure was analyzed using repeated measurements over time. The model included the fixed effects of additive, length of aerobic exposure, and their interactions. A covariance structure was chosen by considering the lowest Akaike Information Criterion ( Littel et al., 1998Littell, R. C.; Henry, P. R. and Ammerman, C. B. 1998. Statistical analysis of repeated measures data using SAS procedures. Journal of Animal Science 76:1216-1231. https://doi.org/10.2527/1998.7641216x
https://doi.org/10.2527/1998.7641216x...
). The structures of covariance tested included variance compounds (VC), compound symmetry (CS), first-order autoregressive (AR 1), and unstructured (UN).

3. Results

Additives did not change the pH, lactic acid, and butyric acid values of the silages ( Table 2 ). The control and N10 silages presented higher concentrations of propionic acid and ethanol but a lower concentration of acetic acid than the LB and NLB silages.

Table 2
Fermentative profile of sugarcane silages treated with natamycin, Lactobacillus buchneri , or their combination

The control presented higher GV and DM losses than the LB and N10 silages. However, the combination of additives was not capable of reducing the DM and gas losses ( Table 3 ). Effluent production was not affected by the treatments. All the treated silages presented lower GV production than the control. The control silage had the highest GHG value compared with the additive-treated silages. However, the additive combination led to higher GHG emissions than the N10 silage.

Table 3
Gas volume produced, fermentative losses, and greenhouse gases equivalent (GHG) in sugarcane silages treated with natamycin, Lactobacillus buchneri , or their combination

The untreated silage presented higher CO2 emissions than the treated silages ( Table 4 ). Of the inoculated silages, natamycin reduced CO2 emission compared with NLB treatment. The control silage presented higher contents of CH4 and N2O in its gas than the N10 treatment.

Table 4
Gas composition of sugarcane silages treated with natamycin, Lactobacillus buchneri , or their combination

Silage treated with LB presented the highest aerobic stability, followed by NLB, N10, and control ( Table 5 ). In addition, LB inoculation increased the time taken to reach the maximum temperature and reduced the accumulated temperature during aerobic exposure compared with the other treatments.

Table 5
Aerobic stability (AS) of sugarcane silages treated with natamycin, Lactobacillus buchneri , or their combination

The pH values were similar among the treatments up to 48 h of aerobic exposure ( Figure 2 ). However, in the N10 silages, pH increased after 72 h and was higher than in all other the treatments, remaining high until the end of aerobic exposure (120 h). The LB silage had the lowest pH until the end of the trial, followed by the NLB silage.

Figure 2
pH values during aerobic exposure of sugarcane silages treated with natamycin (N10), L. buchneri (LB), or their combination (NLB).

Regarding the chemical composition, the DM, ash, NDF, and ADF contents were similar for all the treatments; however, the N10 silages presented high CP content compared with the control and NLB silages, and similar to the LB-treated silages ( Table 6 ).

Table 6
Chemical composition (g kg−1 DM unless stated) of sugarcane silages treated with natamycin, Lactobacillus buchneri , or their combination

4. Discussion

In sugarcane silages, for each 1 g kg−1 DM of ethanol accumulated, an extra DM loss of 0.65 g kg−1 DM is expected ( Rabelo et al., 2019Rabelo, C. H. S.; Härter, C. J.; Ávila, C. L. S. and Reis, R. A. 2019. Meta‐analysis of the effects of Lactobacillus plantarum and Lactobacillus buchneri on fermentation, chemical composition and aerobic stability of sugarcane silage. Grassland Science 65:3-12. https://doi.org/10.1111/grs.12215
https://doi.org/10.1111/grs.12215...
). This effect was also detected in our trial. Ethanol is highly volatile and may worsen air conditions in silage-based livestock systems ( Hafner et al., 2013Hafner, S. D.; Howard, C.; Muck, R. E.; Franco, R. B.; Montes, F.; Green, P. G.; Mitloehner, F.; Trabue, S. L. and Rotz, C. A. 2013. Emission of volatile organic compounds from silage: compounds, sources, and implications. Atmospheric Environment 77:827-839. https://doi.org/10.1016/j.atmosenv.2013.04.076
https://doi.org/10.1016/j.atmosenv.2013....
). Moreover, high ethanol synthesis is also linked to CO2 synthesis ( McDonald et al., 1991McDonald, P.; Henderson, A. R. and Heron, S. 1991. The biochemistry of silage. 2nd ed. Chalcombe Publications, Marlow. ), which contributes to GHG emission.

As expected, LB inoculation, alone or in combination with natamycin, increased acetic acid content (by 105 and 78%, respectively) and decreased propionic acid (by 69 and 54%, respectively) and ethanol content (by 83 and 71%, respectively) in sugarcane silage due to a probable decrease in yeast activity ( Pedroso et al., 2008Pedroso, A. F.; Nussio, L. G.; Loures, D. R. S.; Paziani, S. F.; Ribeiro, J. L.; Mari, L. J.; Zopollatto, M.; Schmidt, P.; Mattos, W. R. S. and Horii, J. 2008. Fermentation, losses, and aerobic stability of sugarcane silages treated with chemical or bacterial additives. Scientia Agricola 65:589-594. https://doi.org/10.1590/S0103-90162008000600004
https://doi.org/10.1590/S0103-9016200800...
; Ávila et al., 2010bÁvila, C. L. S.; Bravo Martins, C. E. C. and Schwan, R. F. 2010b. Identification and characterization of yeasts in sugarcane silages. Journal of Applied Microbiology 109:1677-1686. https://doi.org/10.1111/j.1365-2672.2010.04796.x
https://doi.org/10.1111/j.1365-2672.2010...
; Ávila et al., 2012Ávila, C. L. S.; Pinto, J. C.; Oliveira, D. P. and Schwan, R. F. 2012. Aerobic stability of sugar cane silages with a novel strain of Lactobacillus sp. Isolated from sugar cane. Revista Brasileira de Zootecnia 41:249-255. https://doi.org/10.1590/S1516-35982012000200003
https://doi.org/10.1590/S1516-3598201200...
). In addition, LB use also led to lower DM and gas losses (72 and 78%, respectively). No effect was reported for the combination of additives, possibly because the NLB treatment was a result of a combination of smaller doses of both the additives. Our results contrast with those of Pinto et al. (2020)Pinto, S.; Warth, J. F. G.; Novinski, C. O. and Schmidt, P. 2020. Effects of natamycin and Lactobacillus buchneri on the fermentative process and aerobic stability of maize silage. Journal of Animal and Feed Sciences 29:82-89. https://doi.org/10.22358/jafs/118179/2020
https://doi.org/10.22358/jafs/118179/202...
, who reported low yeast counts and dry matter losses in maize silage treated with a combination of natamycin and LB compared with the control silage; however, no decrease in the yeast counts and dry matter losses was reported when additives were used alone. Those authors used higher doses in combination with the additives than we did. Thus, the effectiveness of natamycin combined with LB may be dependent on the target silage (e.g., chemical composition, initial microbial counts) as well as the dose.

Interestingly, we did not observe any differences in the lactic acid content and pH values among the silages. Besides soluble sugars, LB also uses lactic acid as a substrate, which may result in decreased lactic acid content ( Oude Elferink et al., 2001Oude Elferink, S. J. W. H.; Krooneman, J.; Gottschal, J. C.; Spoelstra, S. F.; Faber, F. and Driehuis, F. 2001. Anaerobic conversion of lactic acid to acetic acid and 1,2-propanediol by Lactobacillus buchneri . Applied and Environmental Microbiology 67:125-132. https://doi.org/10.1128/AEM.67.1.125-132.2001
https://doi.org/10.1128/AEM.67.1.125-132...
). Contrary to expectations, the ethanol concentration of the N10 silage was similar (P>0.05) to that of the control silage. Our hypothesis is that the natamycin additive had a certain amount of effectiveness against yeast during the initial stages of sugarcane fermentation (hours or days) but lost its efficacy over the course of fermentation when used alone or in combination with LB. Different from acetic acid, which needs to be synthetized in silage to start acting against fungi, natamycin is readily available to interact with ergosterol in yeast membranes and decrease its activity ( Welscher et al., 2008Welscher, Y. M.; Napel, H. H.; Balangué, M. M.; Souza, C. M.; Riezman, H.; Kruiff, B. and Breukink, E. 2008. Natamycin blocks fungal growth by binding specialy to ergosterol without permeabilizing the membrane. The Journal of Biological Chemistry 283:6393-6401. https://doi.org/10.1074/jbc.m707821200
https://doi.org/10.1074/jbc.m707821200...
). Indeed, a decrease in DM and gas losses was observed in N10 silages (69.15 and 76.83%, respectively) compared with the control. Gas production, the main source of DM losses in silages, achieves its peak in the first few days of fermentation ( Schmidt et al., 2012Schmidt, P.; Novinski, C. O.; Carneiro, E. W. and Bayer, C. 2012. Greenhouse gas emissions from fermentation of corn silage. p.448-449. In: Kuoppala, K.; Rinne, M. and Vanhatalo, A., eds. Proceedings of the XVI International Silage Conference, Hämeenlinna, Finland. ).

We supposed that natamycin activity might be curtailed by low pH in silage. Under acidic conditions, natamycin is broken down, producing mycosamine and other inactive degradation products (amphoteric aponatamycin, acidic di-natamycinolidediol, and nonionic di-decarboxy-anhydronatamycinolidediol) ( Brik, 1976Brik, H. 1976. New high-molecular decomposition products of natamycin (pimaricin) with intact lactone-ring. The Journal of Antibiotics 29:632-637. https://doi.org/10.7164/antibiotics.29.632
https://doi.org/10.7164/antibiotics.29.6...
; Brik, 1981Brik, H. 1981. Natamycin. p.513-561. In: Analytical profiles of drug substances. Florey, K., ed. Academic Press, New York, NY. ; Dalhoff and Levy, 2015Dalhoff, A. A. H. and Levy, S. B. 2015. Does use of the polyene natamycin as a food preservative jeopardise the clinical efficacy of amphotericin B? A word of concern. International Journal of Antimicrobial Agents 45:564-567. https://doi.org/10.1016/j.ijantimicag.2015.02.011
https://doi.org/10.1016/j.ijantimicag.20...
), which seems to reduce natamycin effectiveness in the advanced stages of fermentation and after opening the silo. On the other hand, acetic acid is not negatively affected by acidic conditions. In our trial, the average pH was 3.9, an unfavorable condition for natamycin action ( Stark and Tan, 2003Stark, J. and Tan, H. S. 2003. Natamycin. p.179-195. In: Food preservatives. 2nd ed. Russel, N. J. and Gould, G. W., eds. Springer, New York, NY. https://doi.org/10.1007/978-0-387-30042-9_9
https://doi.org/10.1007/978-0-387-30042-...
; Delves-Broughton et al., 2005Delves-Broughton, J.; Thomas, L. V.; Doan, C. H. and Davidson, P. M. 2005. Natamicyn. p.275-289. In: Antimicrobials in food. 3rd ed. Davidson, P. M.; Sofos, J. N. and Branen, A. L., eds. CRC Press, Boca Raton, FL. ; Hanušová et al., 2012Hanušová, K.; Dobiáš, J. and Voldřich, M. 2012. Assessment of functional properties and antimicrobial efficiency of polymer films with lacquer layer containing natamycin in cheese packaging. Journal of Food and Nutrition Research 51:145-155. ). In addition, sugarcane presents high lactic acid production and a fast pH drop ( Custódio et al., 2016Custódio, L.; Morais, G.; Daniel, J. L. P.; Pauly, T. and Nussio, L. G. 2016. Effects of chemical and microbial additives on clostridium development in sugarcane ( Saccharum officinarum L.) ensiled with lime. Grassland Science 62:135-143. https://doi.org/10.1111/grs.12124
https://doi.org/10.1111/grs.12124...
), which also result in lowered natamycin effectiveness during fermentation. Shah et al. (2020)Shah, A. A.; Wu, J.; Qian, C.; Liu, Z.; Mobashar, M.; Tao, Z.; Zhang, X. and Zhong, X. 2020. Ensiling of whole plant hybrid Pennisetum with Natamycin and Lactobacillus plantarum impacts on fermentation characteristics and meta-genomic microbial community at low temperature. Journal of the Science of Food and Agriculture 100:3378-3385. https://doi.org/10.1002/jsfa.10371
https://doi.org/10.1002/jsfa.10371...
reported lower yeast counts in Napier grass silage treated with natamycin for 60 days than in untreated silage. Additionally, the authors reported lower lactic acid levels than those of our silages. Indeed, Napier grass presents a low level of soluble carbohydrates to draw on slow pH drop rate ( Desta et al., 2016Desta, S. T.; Yuan, X.; Li, J. and Shao, T. 2016. Ensiling characteristics, structural and nonstructural carbohydrate composition and enzymatic digestibility of Napier grass ensiled with additives. Bioresource Technology 221:447-454. https://doi.org/10.1016/j.biortech.2016.09.068
https://doi.org/10.1016/j.biortech.2016....
), reinforcing the above hypothesis. Nonetheless, trials evaluating the effects of different natamycin doses on silage fermentation and microbial counts over time are required to draw conclusions about this topic in sugarcane silages.

Normally, untreated sugarcane silage presents a high predominance of yeast fermentation, leading to higher gas yield and, consequently, GHG emission (as observed in our study). The use of additives, in general, reduced GV as well GHG emission (g CO2 eq t−1 fresh forage) by 68% compared with untreated silage, probably because of the lowered CO2 emissions. Natamycin decreased total GV production by 61% compared with the control, whereas the GHG emission was reduced by 86% in natamycin-treated silages in comparison with the non-treated silage. Nitrous oxide, methane, and carbon dioxide were less representative in the gas composition of natamycin silage compared with the control. Those three gases are the main generators of global warming, and methane and nitrous oxide are, respectively, 23 and 298 times more harmful than carbon dioxide ( Houghton et al., 2001Houghton, J. T.; Ding, Y.; Griggs, D. J.; Noguer, M.; van der Linden, P. J.; Dai, X.; Maskell, K. and Johnson, C. A. (eds.) 2001. Climate Change 2001: The Scientific Basis. Contribution of Working Group I to the Third Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, United Kingdom. ). Despite the relatively high amount of GHG emission from untreated sugarcane silage (311 g CO2 eq t−1 fresh forage), those values are small when compared with the estimates of GHG emission from feedlot cattle (5.6 kg CO2 eq per kg of live weight gain) or dairy cattle (1.1 kg CO2 eq per kg of milk) ( Phetteplace et al., 2001Phetteplace, H. W.; Johnson, D. E. and Seidl, A. F. 2001. Greenhouse gas emissions from simulated beef and dairy livestock systems in the United States. Nutrient Cycling in Agroecosystems 60:99-102. https://doi.org/10.1023/A:1012657230589
https://doi.org/10.1023/A:1012657230589...
).

With the yeast population being the major cause of gas losses in sugarcane silages, other ensiled crops (e.g., corn or grass) present significantly lower ethanol synthesis and gas emission rates. Schmidt et al. (2012)Schmidt, P.; Novinski, C. O.; Carneiro, E. W. and Bayer, C. 2012. Greenhouse gas emissions from fermentation of corn silage. p.448-449. In: Kuoppala, K.; Rinne, M. and Vanhatalo, A., eds. Proceedings of the XVI International Silage Conference, Hämeenlinna, Finland. observed GV production in corn silages lower than the untreated sugarcane silage from our trial (424 vs 7282 L t−1 DM); this was also the case with GHG emission (14.5 vs 311 g CO2 eq t−1 fresh forage). Contrary to our data, Gomes et al. (2019)Gomes, A. L. M.; Jacovaci, F. A.; Bolson, D. C.; Nussio, L. G.; Jobim, C. C. and Daniel, J. L. P. 2019. Effects of light wilting and heterolactic inoculant on the formation of volatile organic compounds, fermentative losses and aerobic stability of oat silage. Animal Feed Science and Technology 247:194-198. https://doi.org/10.1016/j.anifeedsci.2018.11.016
https://doi.org/10.1016/j.anifeedsci.201...
reported higher GV (27640 vs 10880 L t−1 DM) as well as ethanol synthesis (5.60 vs 1.43 g kg−1 DM) in wilted oat silage treated with LB than in untreated silage. Those authors indirectly estimated GV, which may have led to much higher values than ours.

The large yeast population typically found in sugarcane silage ( Ávila et al., 2010bÁvila, C. L. S.; Bravo Martins, C. E. C. and Schwan, R. F. 2010b. Identification and characterization of yeasts in sugarcane silages. Journal of Applied Microbiology 109:1677-1686. https://doi.org/10.1111/j.1365-2672.2010.04796.x
https://doi.org/10.1111/j.1365-2672.2010...
; Santos et al., 2015Santos, W. C. C.; Nascimento, W. G.; Magalhães, A. L. R.; Silva, D. K. A.; Silva, W. J. C. S.; Santana, A. V. S. and Soares, G. S. C. 2015. Nutritive value, total losses of dry matter and aerobic stability of the silage from three varieties of sugarcane treated with commercial microbial additives. Animal Feed Science and Technology 204:1-8. https://doi.org/10.1016/j.anifeedsci.2015.03.004
https://doi.org/10.1016/j.anifeedsci.201...
) makes it prone to deterioration after exposure to air. The deterioration rate is also related to the amount of substrate (e.g., sucrose, lactic acid) and the presence, or lack, of protective substances (e.g., short-chain fatty acids) ( Ávila et al., 2012Ávila, C. L. S.; Pinto, J. C.; Oliveira, D. P. and Schwan, R. F. 2012. Aerobic stability of sugar cane silages with a novel strain of Lactobacillus sp. Isolated from sugar cane. Revista Brasileira de Zootecnia 41:249-255. https://doi.org/10.1590/S1516-35982012000200003
https://doi.org/10.1590/S1516-3598201200...
; Wilkinson and Davies, 2013Wilkinson, J. M. and Davies, D. R. 2013. The aerobic stability of silage: key findings and recent developments. Grass and Forage Science 68:1-19. https://doi.org/10.1111/j.1365-2494.2012.00891.x
https://doi.org/10.1111/j.1365-2494.2012...
). As observed in our trial, the acetic acid formed during LB development reduced spoilage after silo opening ( Table 5 ; Figure 2 ). The weakness of acetic acid (pka 4.76) contributes to its effectiveness against yeast under the typical acidic conditions found in silages ( Danner et al., 2003Danner, H.; Holzer, M.; Mayrhuber, E. and Braun, R. 2003. Acetic acid increases stability of silage under aerobic conditions. Applied and Environmental Microbiology 69:562-567. https://doi.org/10.1128/AEM.69.1.562-567.2003
https://doi.org/10.1128/AEM.69.1.562-567...
). Different from our data, a meta-analysis performed by Rabelo et al. (2019)Rabelo, C. H. S.; Härter, C. J.; Ávila, C. L. S. and Reis, R. A. 2019. Meta‐analysis of the effects of Lactobacillus plantarum and Lactobacillus buchneri on fermentation, chemical composition and aerobic stability of sugarcane silage. Grassland Science 65:3-12. https://doi.org/10.1111/grs.12215
https://doi.org/10.1111/grs.12215...
did not show positive effects related to aerobic stability of LB-treated sugarcane silage despite the higher acetic acid content in those silages.

Natamycin alone had no effect on aerobic stability, indicating a lack of action of this compound to inhibit yeast growth after opening the silos. This effect can be related to the short half-life of that compound after being applied to low pH media, such as silages ( Brik, 1976Brik, H. 1976. New high-molecular decomposition products of natamycin (pimaricin) with intact lactone-ring. The Journal of Antibiotics 29:632-637. https://doi.org/10.7164/antibiotics.29.632
https://doi.org/10.7164/antibiotics.29.6...
; Brik, 1981Brik, H. 1981. Natamycin. p.513-561. In: Analytical profiles of drug substances. Florey, K., ed. Academic Press, New York, NY. ; Stark and Tan, 2003Stark, J. and Tan, H. S. 2003. Natamycin. p.179-195. In: Food preservatives. 2nd ed. Russel, N. J. and Gould, G. W., eds. Springer, New York, NY. https://doi.org/10.1007/978-0-387-30042-9_9
https://doi.org/10.1007/978-0-387-30042-...
; Delves-Broughton et al., 2005Delves-Broughton, J.; Thomas, L. V.; Doan, C. H. and Davidson, P. M. 2005. Natamicyn. p.275-289. In: Antimicrobials in food. 3rd ed. Davidson, P. M.; Sofos, J. N. and Branen, A. L., eds. CRC Press, Boca Raton, FL. ; Hanušová et al., 2012Hanušová, K.; Dobiáš, J. and Voldřich, M. 2012. Assessment of functional properties and antimicrobial efficiency of polymer films with lacquer layer containing natamycin in cheese packaging. Journal of Food and Nutrition Research 51:145-155. ; Dalhoff and Levy, 2015Dalhoff, A. A. H. and Levy, S. B. 2015. Does use of the polyene natamycin as a food preservative jeopardise the clinical efficacy of amphotericin B? A word of concern. International Journal of Antimicrobial Agents 45:564-567. https://doi.org/10.1016/j.ijantimicag.2015.02.011
https://doi.org/10.1016/j.ijantimicag.20...
). In contrast, Pinto et al. (2020)Pinto, S.; Warth, J. F. G.; Novinski, C. O. and Schmidt, P. 2020. Effects of natamycin and Lactobacillus buchneri on the fermentative process and aerobic stability of maize silage. Journal of Animal and Feed Sciences 29:82-89. https://doi.org/10.22358/jafs/118179/2020
https://doi.org/10.22358/jafs/118179/202...
did not find positive effects of natamycin or LB alone, but their combination increased aerobic stability.

In general, the additives used in our trial did not cause any changes in the DM content or chemical composition of the silages. Unexpectedly, the silage treated with natamycin showed higher CP values than the control and NLB silages, while LB-treated silages presented intermediate CP values. According to Rosi et al. (1987)Rosi, I.; Costamagna, L. and Birtuccion, M. 1987. Screening for extracellular acid protease(s) production by wine yeasts. Journal of the Institute of Brewing 93:322-324. https://doi.org/10.1002/j.2050-0416.1987.tb04511.x
https://doi.org/10.1002/j.2050-0416.1987...
, yeasts are capable of producing extracellular proteases that consume part of the CP. In addition, yeasts are involved in the nitrogen cycle due to nitrous oxide (N2O) reductase production ( Shoun et al., 2012Shoun, H.; Fushinobu, S.; Jiang, L.; Kim, S. W. and Wakagi, T. 2012. Fungal denitrification and nitric oxide reductase cytochrome p450nor. Philosophical Transactions of the Royal Society B. Biological Sciences 367:1186-1194. https://doi.org/10.1098/rstb.2011.0335
https://doi.org/10.1098/rstb.2011.0335...
). As observed, silage treated with natamycin showed the lowest N2O production as well as the highest protein content. Shah et al. (2020)Shah, A. A.; Wu, J.; Qian, C.; Liu, Z.; Mobashar, M.; Tao, Z.; Zhang, X. and Zhong, X. 2020. Ensiling of whole plant hybrid Pennisetum with Natamycin and Lactobacillus plantarum impacts on fermentation characteristics and meta-genomic microbial community at low temperature. Journal of the Science of Food and Agriculture 100:3378-3385. https://doi.org/10.1002/jsfa.10371
https://doi.org/10.1002/jsfa.10371...
reported lower protein degradation in elephant grass silage treated with natamycin than in untreated silage. It is possible that the presence of natamycin and acetic acid impaired protein metabolization by yeasts.

As expected, LB was effective in controlling ethanol synthesis as well as reducing fermentation losses and improving aerobic stability in sugarcane silages. In contrast, natamycin was effective only with GV-related variables and did not present a synergistic effect when combined with LB.

5. Conclusions

Natamycin alone as well as its combination with Lactobacillus buchneri decrease the volume of gas and greenhouse gases emitted by sugarcane silages. Due to the lack of effectiveness in reducing ethanol content or improving the aerobic stability, natamycin seems to be effective only in the early stages of ensiling process. Further studies must be carried out for natamycin to be recommended for composing blend additives with microbial or other chemicals.

References

  • AOAC - Association of Official Analytical Chemistry. 2000. Official methods of analysis. 17th ed. AOAC International, Gaithersburg, MD.
  • Ávila, C. L. S.; Valeriano, A. R.; Pinto, J. C.; Figueiredo, H. C. P.; Rezende, A. V. and Schwan, R. F. 2010a. Chemical and microbiological characteristics of sugar cane silages treated with microbial inoculants. Revista Brasileira de Zootecnia 39:25-32. https://doi.org/10.1590/S1516-35982010000100004
    » https://doi.org/10.1590/S1516-35982010000100004
  • Ávila, C. L. S.; Bravo Martins, C. E. C. and Schwan, R. F. 2010b. Identification and characterization of yeasts in sugarcane silages. Journal of Applied Microbiology 109:1677-1686. https://doi.org/10.1111/j.1365-2672.2010.04796.x
    » https://doi.org/10.1111/j.1365-2672.2010.04796.x
  • Ávila, C. L. S.; Pinto, J. C.; Oliveira, D. P. and Schwan, R. F. 2012. Aerobic stability of sugar cane silages with a novel strain of Lactobacillus sp. Isolated from sugar cane. Revista Brasileira de Zootecnia 41:249-255. https://doi.org/10.1590/S1516-35982012000200003
    » https://doi.org/10.1590/S1516-35982012000200003
  • Ávila, C. L. S.; Carvalho, B. F.; Pinto, J. C.; Duarte, W. F. and Schwan, R. F. 2014. The use of Lactobacillus species as starter cultures for enhancing the quality of sugar cane silage. Journal of Dairy Science 97:940-951. https://doi.org/10.3168/jds.2013-6987
    » https://doi.org/10.3168/jds.2013-6987
  • Borreani, G.; Tabacco, E.; Schmidt, R. J.; Holmes, B. J. and Muck, R. E. 2018. Silage review: Factors affecting dry matter and quality losses in silages. Journal of Dairy Science 101:3952-3979. https://doi.org/10.3168/jds.2017-13837
    » https://doi.org/10.3168/jds.2017-13837
  • Brik, H. 1976. New high-molecular decomposition products of natamycin (pimaricin) with intact lactone-ring. The Journal of Antibiotics 29:632-637. https://doi.org/10.7164/antibiotics.29.632
    » https://doi.org/10.7164/antibiotics.29.632
  • Brik, H. 1981. Natamycin. p.513-561. In: Analytical profiles of drug substances. Florey, K., ed. Academic Press, New York, NY.
  • Custódio, L.; Morais, G.; Daniel, J. L. P.; Pauly, T. and Nussio, L. G. 2016. Effects of chemical and microbial additives on clostridium development in sugarcane ( Saccharum officinarum L.) ensiled with lime. Grassland Science 62:135-143. https://doi.org/10.1111/grs.12124
    » https://doi.org/10.1111/grs.12124
  • Dalhoff, A. A. H. and Levy, S. B. 2015. Does use of the polyene natamycin as a food preservative jeopardise the clinical efficacy of amphotericin B? A word of concern. International Journal of Antimicrobial Agents 45:564-567. https://doi.org/10.1016/j.ijantimicag.2015.02.011
    » https://doi.org/10.1016/j.ijantimicag.2015.02.011
  • Danner, H.; Holzer, M.; Mayrhuber, E. and Braun, R. 2003. Acetic acid increases stability of silage under aerobic conditions. Applied and Environmental Microbiology 69:562-567. https://doi.org/10.1128/AEM.69.1.562-567.2003
    » https://doi.org/10.1128/AEM.69.1.562-567.2003
  • Delves-Broughton, J.; Thomas, L. V.; Doan, C. H. and Davidson, P. M. 2005. Natamicyn. p.275-289. In: Antimicrobials in food. 3rd ed. Davidson, P. M.; Sofos, J. N. and Branen, A. L., eds. CRC Press, Boca Raton, FL.
  • Desta, S. T.; Yuan, X.; Li, J. and Shao, T. 2016. Ensiling characteristics, structural and nonstructural carbohydrate composition and enzymatic digestibility of Napier grass ensiled with additives. Bioresource Technology 221:447-454. https://doi.org/10.1016/j.biortech.2016.09.068
    » https://doi.org/10.1016/j.biortech.2016.09.068
  • EAEMP - European Agency for the Evaluation of Medicinal Products. 1998. Committee for Veterinary Medicinal Products: Natamycin Summary Report. EMEA/MRL/342/98-Final. Available at: <https://www.ema.europa.eu/en/documents/mrl-report/natamycin-summary-report-committee-veterinary-medicinal-products_en.pdf> Accessed on: Jan. 05, 2020.
    » https://www.ema.europa.eu/en/documents/mrl-report/natamycin-summary-report-committee-veterinary-medicinal-products_en.pdf
  • Gerber, P. J.; Hristov, A. N.; Henderson, B.; Makkar, H.; Oh, J.; Lee, C.; Meinen, R.; Montes, F.; Ott, T.; Firkins, J.; Rotz, A.; Dell, C.; Adesogan, A. T.; Yang, W. Z.; Tricarico, J. M.; Kebreab, E.; Waghorn, G; Dijkstra, J. and Oosting, S. 2013. Technical options for the mitigation of direct methane and nitrous oxide emissions from livestock: a review. Animal 7:220-234. https://doi.org/10.1017/S1751731113000876
    » https://doi.org/10.1017/S1751731113000876
  • Gomes, A. L. M.; Jacovaci, F. A.; Bolson, D. C.; Nussio, L. G.; Jobim, C. C. and Daniel, J. L. P. 2019. Effects of light wilting and heterolactic inoculant on the formation of volatile organic compounds, fermentative losses and aerobic stability of oat silage. Animal Feed Science and Technology 247:194-198. https://doi.org/10.1016/j.anifeedsci.2018.11.016
    » https://doi.org/10.1016/j.anifeedsci.2018.11.016
  • Grossi, G.; Goglio, P.; Vitali, A. and Williams, A. G. 2019. Livestock and climate change: impact of livestock on climate and mitigation strategies. Animal Frontiers 9:69-76. https://doi.org/10.1093/af/vfy034
    » https://doi.org/10.1093/af/vfy034
  • Hafner, S. D.; Montes, F.; Rotz, C. A. and Mitloehner, F. 2010. Ethanol emission from loose corn silage and exposed silage particles. Atmospheric Environment 44:4172-4180. https://doi.org/10.1016/j.atmosenv.2010.07.029
    » https://doi.org/10.1016/j.atmosenv.2010.07.029
  • Hafner, S. D.; Howard, C.; Muck, R. E.; Franco, R. B.; Montes, F.; Green, P. G.; Mitloehner, F.; Trabue, S. L. and Rotz, C. A. 2013. Emission of volatile organic compounds from silage: compounds, sources, and implications. Atmospheric Environment 77:827-839. https://doi.org/10.1016/j.atmosenv.2013.04.076
    » https://doi.org/10.1016/j.atmosenv.2013.04.076
  • Hanušová, K.; Dobiáš, J. and Voldřich, M. 2012. Assessment of functional properties and antimicrobial efficiency of polymer films with lacquer layer containing natamycin in cheese packaging. Journal of Food and Nutrition Research 51:145-155.
  • Henriksson, M.; Cederberg, C. and Swensoon, C. 2012. Impact of cultivation strategies and regional climate on greenhouse gas emissions from grass/clover silage. Acta Agriculturae Scandinavica, Section A - Animal Science 62:233-237. https://doi.org/10.1080/09064702.2013.797010
    » https://doi.org/10.1080/09064702.2013.797010
  • Holzer, M.; Mayrhuber, E.; Danner, H. and Braun, R. 2003. The role of Lactobacillus buchneri in forage preservation. Trends in Biotechnology 21:282-287. https://doi.org/10.1016/S0167-7799(03)00106-9
    » https://doi.org/10.1016/S0167-7799(03)00106-9
  • Hondrodimou, O.; Kourkoutas, Y. and Panagou, E. Z. 2011. Efficacy of natamicyn to control fungal growth in natural black olive fermentation. Food Microbiology 28:621-627. https://doi.org/10.1016/j.fm.2010.11.015
    » https://doi.org/10.1016/j.fm.2010.11.015
  • Houghton, J. T.; Ding, Y.; Griggs, D. J.; Noguer, M.; van der Linden, P. J.; Dai, X.; Maskell, K. and Johnson, C. A. (eds.) 2001. Climate Change 2001: The Scientific Basis. Contribution of Working Group I to the Third Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, United Kingdom.
  • Jobim, C. C.; Nussio, L. G.; Reis, R. A. and Schmidt, P. 2007. Avanços metodológicos na avaliação da qualidade da forragem conservada. Revista Brasileira de Zootecnia 36:101-119. https://doi.org/10.1590/S1516-35982007001000013
    » https://doi.org/10.1590/S1516-35982007001000013
  • Kung Jr., L.; Robinson, J. R.; Ranjit, N. K.; Chen, J. H.; Golt, C. M. and Pesek, J. D. 2000. Microbial populations, fermentation end-products, and aerobic stability of corn silage treated with ammonia or a propionic acid-based preservative. Journal of Dairy Science 83:1479-1486. https://doi.org/10.3168/jds.S0022-0302(00)75020-X
    » https://doi.org/10.3168/jds.S0022-0302(00)75020-X
  • Littell, R. C.; Henry, P. R. and Ammerman, C. B. 1998. Statistical analysis of repeated measures data using SAS procedures. Journal of Animal Science 76:1216-1231. https://doi.org/10.2527/1998.7641216x
    » https://doi.org/10.2527/1998.7641216x
  • McDonald, P.; Henderson, A. R. and Heron, S. 1991. The biochemistry of silage. 2nd ed. Chalcombe Publications, Marlow.
  • Mertens, D. R. 2002. Gravimetric determination of amylase-treated neutral detergent fiber in feeds with refluxing in beaker or crucibles: collaborative study. Journal of AOAC International 85:1217-1240.
  • Muck, R. E.; Nadeau, E. M. G.; McAllister, T. A.; Contreras-Govea, F. E.; Santos, M. C. and Kung Jr, L. 2018. Silage review: Recent advances and future uses of silage additives. Journal of Dairy Science 101:3980-4000. https://doi.org/10.3168/jds.2017-13839
    » https://doi.org/10.3168/jds.2017-13839
  • Novinski, C. O.; Junges, D.; Schmidt, P.; Rossi Junior, P.; Carvalho, J. P. G. and Teixeira, R. A. 2012. Methods of lab silos sealing and fermentation characteristics and aerobic stability of sugarcane silage treated with microbial additive. Revista Brasileira de Zootecnia 41:264-270. https://doi.org/10.1590/S1516-35982012000200005
    » https://doi.org/10.1590/S1516-35982012000200005
  • Oude Elferink, S. J. W. H.; Krooneman, J.; Gottschal, J. C.; Spoelstra, S. F.; Faber, F. and Driehuis, F. 2001. Anaerobic conversion of lactic acid to acetic acid and 1,2-propanediol by Lactobacillus buchneri . Applied and Environmental Microbiology 67:125-132. https://doi.org/10.1128/AEM.67.1.125-132.2001
    » https://doi.org/10.1128/AEM.67.1.125-132.2001
  • Palmquist, D. L. and Conrad, H. R. 1971. Origin of plasma fatty acids in lactating cows fed high grain or high fat diets. Journal of Dairy Science 54:1025-1033. https://doi.org/10.3168/jds.s0022-0302(71)85966-0
    » https://doi.org/10.3168/jds.s0022-0302(71)85966-0
  • Pedroso, A. F.; Nussio, L. G.; Paziani, S. F.; Loures, D. R. S.; Igarasi, M. S.; Coelho, R. M.; Packer, I. H.; Horii, J. and Gomes, L. H. 2005. Fermentation and epiphytic microflora dynamics in sugar cane silage. Scientia Agricola 62:427-432. https://doi.org/10.1590/S0103-90162005000500003
    » https://doi.org/10.1590/S0103-90162005000500003
  • Pedroso, A. F.; Nussio, L. G.; Loures, D. R. S.; Paziani, S. F.; Ribeiro, J. L.; Mari, L. J.; Zopollatto, M.; Schmidt, P.; Mattos, W. R. S. and Horii, J. 2008. Fermentation, losses, and aerobic stability of sugarcane silages treated with chemical or bacterial additives. Scientia Agricola 65:589-594. https://doi.org/10.1590/S0103-90162008000600004
    » https://doi.org/10.1590/S0103-90162008000600004
  • Pinto, S.; Warth, J. F. G.; Novinski, C. O. and Schmidt, P. 2020. Effects of natamycin and Lactobacillus buchneri on the fermentative process and aerobic stability of maize silage. Journal of Animal and Feed Sciences 29:82-89. https://doi.org/10.22358/jafs/118179/2020
    » https://doi.org/10.22358/jafs/118179/2020
  • Phetteplace, H. W.; Johnson, D. E. and Seidl, A. F. 2001. Greenhouse gas emissions from simulated beef and dairy livestock systems in the United States. Nutrient Cycling in Agroecosystems 60:99-102. https://doi.org/10.1023/A:1012657230589
    » https://doi.org/10.1023/A:1012657230589
  • Rabelo, C. H. S.; Härter, C. J.; Ávila, C. L. S. and Reis, R. A. 2019. Meta‐analysis of the effects of Lactobacillus plantarum and Lactobacillus buchneri on fermentation, chemical composition and aerobic stability of sugarcane silage. Grassland Science 65:3-12. https://doi.org/10.1111/grs.12215
    » https://doi.org/10.1111/grs.12215
  • Restellato, R.; Novinski, C. O.; Pereira, L. M.; Silva, E. P. A.; Volpi, D.; Zopollatto, M.; Schmidt, P and Faciola, A. P. 2019. Chemical composition, fermentative losses, and microbial counts of total mixed ration silages inoculated with different Lactobacillus species. Journal of Animal Science 97:1634-1644. https://doi.org/10.1093/jas/skz030
    » https://doi.org/10.1093/jas/skz030
  • Rosi, I.; Costamagna, L. and Birtuccion, M. 1987. Screening for extracellular acid protease(s) production by wine yeasts. Journal of the Institute of Brewing 93:322-324. https://doi.org/10.1002/j.2050-0416.1987.tb04511.x
    » https://doi.org/10.1002/j.2050-0416.1987.tb04511.x
  • Santos, W. C. C.; Nascimento, W. G.; Magalhães, A. L. R.; Silva, D. K. A.; Silva, W. J. C. S.; Santana, A. V. S. and Soares, G. S. C. 2015. Nutritive value, total losses of dry matter and aerobic stability of the silage from three varieties of sugarcane treated with commercial microbial additives. Animal Feed Science and Technology 204:1-8. https://doi.org/10.1016/j.anifeedsci.2015.03.004
    » https://doi.org/10.1016/j.anifeedsci.2015.03.004
  • Schmidt, P. 2009. Improved efficiency of sugarcane ensiling for ruminant supplementation. p.47-72. In: Proceedings of the 1st International Symposium on forage Quality and Conservation. FEALQ, Piracicaba.
  • Schmidt, P.; Novinski, C. O.; Carneiro, E. W. and Bayer, C. 2012. Greenhouse gas emissions from fermentation of corn silage. p.448-449. In: Kuoppala, K.; Rinne, M. and Vanhatalo, A., eds. Proceedings of the XVI International Silage Conference, Hämeenlinna, Finland.
  • Shah, A. A.; Wu, J.; Qian, C.; Liu, Z.; Mobashar, M.; Tao, Z.; Zhang, X. and Zhong, X. 2020. Ensiling of whole plant hybrid Pennisetum with Natamycin and Lactobacillus plantarum impacts on fermentation characteristics and meta-genomic microbial community at low temperature. Journal of the Science of Food and Agriculture 100:3378-3385. https://doi.org/10.1002/jsfa.10371
    » https://doi.org/10.1002/jsfa.10371
  • Shoun, H.; Fushinobu, S.; Jiang, L.; Kim, S. W. and Wakagi, T. 2012. Fungal denitrification and nitric oxide reductase cytochrome p450nor. Philosophical Transactions of the Royal Society B. Biological Sciences 367:1186-1194. https://doi.org/10.1098/rstb.2011.0335
    » https://doi.org/10.1098/rstb.2011.0335
  • Souza, C M. 2015. Impacto ambiental da produção de silagens: Revisão da literatura e avaliação experimental em silos laboratoriais. Dissertação (M. Sc.). Universidade Federal do Paraná, Curitiba.
  • Stark, J. and Tan, H. S. 2003. Natamycin. p.179-195. In: Food preservatives. 2nd ed. Russel, N. J. and Gould, G. W., eds. Springer, New York, NY. https://doi.org/10.1007/978-0-387-30042-9_9
    » https://doi.org/10.1007/978-0-387-30042-9_9
  • Tubiello, F. N.; Salvatore, M.; Rossi, S.; Ferrara, A.; Fitton, N. and Smith, P. 2013. The FAOSTAT database of greenhouse gas emissions from agriculture. Environmental Research Letters 8:015009. https://doi.org/10.1088/1748-9326/8/1/015009
    » https://doi.org/10.1088/1748-9326/8/1/015009
  • Tubiello, F. N.; Salvatore, M.; Ferrara, A. F.; House, J.; Federici, S.; Rossi, S.; Biancalani, R.; Golec, R. D. C.; Jacobs, H.; Flammini, A.; Prosperi, P.; Cardenas-Galindo, P.; Schmidhuber, J.; Sanchez, M. J. S.; Srivastava, N. and Smith, P. 2015. The Contribution of agriculture, forestry and other land use activities to global warming, 1990–2012. Global Chance Biology 21:2655-2660. https://doi.org/10.1111/gcb.12865
    » https://doi.org/10.1111/gcb.12865
  • Van Soest, P. J. 1963. Use of detergents in the analysis of fibrous feeds. II. A rapid method for the determination of fiber and lignin. Journal of the Association of Official Agricultural Chemists 46:829-835.
  • Wang, M.; Wang, S.; Zong, G.; Hou, Z.; Liu, F.; Liao, D. J. and Zhu, X. 2016. Improvement of natamycin production by cholesterol oxidase overexpression in Streptomyces gilvosporeus . Journal of Microbiology and Biotechnology 26:241-247. https://doi.org/10.4014/jmb.1505.05033
    » https://doi.org/10.4014/jmb.1505.05033
  • Weissbach, F. 2011. The future of forage conservation. p.319-363. In: Daniel, J. L. P.; Zopolatto, M. and Nussio, L. G., eds. Proceedings of the II International Symposium on Forage Quality and Conservation. São Pedro, Brazil.
  • Welscher, Y. M.; Napel, H. H.; Balangué, M. M.; Souza, C. M.; Riezman, H.; Kruiff, B. and Breukink, E. 2008. Natamycin blocks fungal growth by binding specialy to ergosterol without permeabilizing the membrane. The Journal of Biological Chemistry 283:6393-6401. https://doi.org/10.1074/jbc.m707821200
    » https://doi.org/10.1074/jbc.m707821200
  • Wilkinson, J. M. and Davies, D. R. 2013. The aerobic stability of silage: key findings and recent developments. Grass and Forage Science 68:1-19. https://doi.org/10.1111/j.1365-2494.2012.00891.x
    » https://doi.org/10.1111/j.1365-2494.2012.00891.x

Publication Dates

  • Publication in this collection
    25 Nov 2020
  • Date of issue
    2020

History

  • Received
    29 Jan 2020
  • Accepted
    10 Sept 2020
Sociedade Brasileira de Zootecnia Universidade Federal de Viçosa / Departamento de Zootecnia, 36570-900 Viçosa MG Brazil, Tel.: +55 31 3612-4602, +55 31 3612-4612 - Viçosa - MG - Brazil
E-mail: rbz@sbz.org.br