Perfil fermentativo de silagem de milho inoculada com Lactobacillus buchneri.

Biological silage additives can assist in making silages by promoting a rapid reduction in silage pH and preventing aerobic deterioration. The current Lactobacillus buchnerion the market produces acetic acid slowly and identifying strains that would improve aerobic stability earlier in the ensiling process would be helpful. This study aimed to investigate the changes in microbial population, dry matter (DM) recovery and fermentation profile of maize silage with or without inoculation with L. buchneri after 45 days of ensiling. The wild L. buchneri strains were isolated from tropical maize silage in a previous study. Four strains of L. buchneri (56.22, 56.27, 56.28 and 56.29) were used as inoculants. Data from the silo openings were analyzed as a completely randomized design, with four replicates per treatment (inoculants). Selected strains did not affect the DM content, yeast and mould population, DM recovery, water-soluble carbohydrates (WSC), lactic acid and butyric acid of maize silage after 45 days of ensiling (p> 0.05). The pH, lactic acid bacteria (LAB) population and concentrations of acetic and propionic acids and ethanol were affected by inoculants (p 0,050). O pH, a população de bactérias do ácido láctico (BAL) e as concentrações de ácidos acético e propiônico e etanol foram afetados pelos inoculantes (p < 0,050). As linhagens 56.22, 56.27 e 56.28 apresentaram menor pH e menor concentração de ácido acético que a testemunha não tratada

Uso de novas estirpes de bactérias láticas como inoculantes para silagens de alfafa e milho e microbioma de silagens de grãos de milho e sorgo reidratados

This study was divided into five chapters Chapter 1 - The experiment was carried out under a completely randomized design with three replicates based on a 6 × 6 factorial arrangement, with 6 inoculants: T1- control (CTRL), T2- commercial inoculant containing Lactobacillus plantarum + Pediococcus pentosaceus (CI), T3- Lactobacillus pentosus 14.7SE (LPE), T4- Lactobacillus plantarum 3.7E (LP), T5- Pediococcus pentosaceus 14.15SE (PP), T6- Lactobacillus plantarum 3.7E + Pediococcus pentosaceus 14.15SE (LP+PP); and six fermentation periods: 1, 3, 7, 14, 28 and 56 days. Alfalfa was wilted for 6 h and increased the dry matter (DM) content to 368 g/kg as fed. The crude protein (CP) and yeast population decreased during the fermentation process. Highest pH decline rates in the first week of fermentation were observed for inoculated silages. Among inoculants, the PP strain resulted in lowest pH values from 14 d of fermentation and lowest acetic acid concentration in the last day of fermentation. Enterobacteria and molds populations were more efficiently controlled by new strains at day 56 and 28, respectively. The in vitro dry matter digestibility was higher in PP than LP silages (64.45 vs. 61.18% DM). Adding of P. pentosaceus alone resulted in positive influence on all evaluated parameters, thus providing better silage quality. Chapter 2 – We evaluated the effects of wild strains of Lactobacillus buchneri on chemical composition, fermentative profile and aerobic stability of corn silages after 90 days of fermentation. The experiment was carried out under a completely randomized design with three replicates and 13 treatments consisted in 1- water (CRTL), 2- commercial L. buchneri strain (CI), and 11 wild strains of L. buchneri: 3- strain 56.1, 4- strain 56.2, 5- strain 56.4, 6- strain 56.7, 7- strain 56.8, 8- strain 56.9, 9- strain 56.21, 10- strain 56.22, 11- strain 56.25, 12- strain 56.26, and 13- strain 56.27. A treatment effect (P 0.05) by treatment. Silage treated with 56.1 strain had higher aerobic stability than non-inoculated silages (68.25 vs. 36 h). The L. buchneri strain 56.1 has the potential to be used as microbial inoculant in corn silage. Chapters 3 and 4 - We explored the succession of bacterial and fungal populations, and evaluated the impacts caused by Lactobacillus plantarum + Propionibacterium acidipropionici and Lactobacillus buchneri inoculants on those communities of rehydrated corn and sorghum grains and their silages by next-generation sequencing after 0, 3, 7, 21, 90 and 360 days of fermentation. Proteobacteria was predominantly in both grains at the beginning of the fermentation and Firmicutes phylum throughout the fermentation periods. Species of Lactobacillus and Weissella were the main bacteria involved in the fermentation of rehydrated corn and sorghum grain silages. Aspergillus spp. molds were predominant in corn grain fermentation while the yeast Wickerhamomyces anomalus was the major fungal in sorghum grain silages. The inoculant containing L. plantarum and P. acidipropionici was more efficient in promoting a sharply growth of Lactobacillus spp. and maintaining greater stability of the bacterial community during longer periods of storage in both grains silages. The addition of inoculant did not have an influencial effect on fungal population of rehydrated sorghum grain silages. Chapter 5 - It was evaluated the effect of ensiling on the fermentation profile, corn silage processing score (CSPS) and long-chain fatty acids (LCFA) profile of whole-plant corn. Eleven corn hybrids were obtained at harvest. Each of the 11 samples was homogenized manually and allocated into 4 samples of approximately 600 g each. Each of the 4 samples was randomly assigned to 1 of 2 treatments (0 or 120 d of ensiling) and vacuum-sealed in nylon-polyethylene standard barrier vacuum pouches. Concentration of DM was unaffected (P > 0.10) by ensiling and averaged 36.2% as fed. The effects on pH is likely attributed to 7.7%-, 1.0%- and 1.2%-units greater (P 0.10) by ensiling and averaged 31.2% of DM and 28.8%, respectively. No effects of ensiling were observed on LCFA profile of major FA including C16:0, C18:0, C18:1, C18:2, and C18:3 FA (P > 0.10). Further research is warranted to elucidate under which conditions ensiling time enhances the CSPS.Este estudo foi dividido em cinco capítulos Capítulo 1 - O experimento foi conduzido em um delineamento inteiramente casualizado com três repetições, baseado em um arranjo fatorial 6 × 6, com 6 inoculantes: T1-Controle (CTRL), T2- Inoculante comercial contendo Lactobacillus plantarum + Pediococcus pentosaceus - (CI), T3- Lactobacillus pentosus 14.7SE (LPE), T4- Lactobacillus plantarum 3.7E (LP), T5- Pediococcus pentosaceus 14.15SE (PP), T6- Lactobacillus plantarum 3.7E + Pediococcus pentosaceus 14.15SE (LP + PP); e seis períodos de fermentação: 1, 3, 7, 14, 28 e 56 dias. A alfafa foi emurchecida por 6 h e aumentou o teor de matéria seca (MS) para 368 g/kg. A população de levedura e o teor de proteína bruta (PB) reduziram durante o processo de fermentação. Maiores taxas de declínio de pH na primeira semana de fermentação foram observadas para as silagens inoculadas. Entre os inoculantes, a estirpe PP resultou em menores valores de pH a partir de 14 d de fermentação e menor concentração de ácido acético no último dia de fermentação. As populações de enterobactérias e mofos foram mais eficientemente controladas pelas novas estirpes nos dias 56 e 28, respectivamente. A digestibilidade in vitro da matéria seca foi maior nas silagens inoculadas com PP do que LP (64.45 vs. 61.18% MS). A adição de P. pentosaceus influenciou positivamente todos os parâmetros avaliados, resultando em silagem de melhor qualidade. Capítulo 2 – Avaliamos os efeitos de estirpes selvagens de Lactobacillus buchneri sobre a composição química, perfil fermentativo e estabilidade aeróbia de silagens de milho após 90 dias de fermentação. O experimento foi conduzido em um delineamento inteiramente casualizado, com três repetições e 13 tratamentos constituídos por 1-água (CRTL), 2 - estirpe comercial de L. buchneri (CI) e 11 estirpes selvagens de L. buchneri: 3- estirpe 56.1, 4- estirpe 56.2, 5- estirpe 56.4, 6- estirpe 56.7, 7- estirpe 56.8, 8- estirpe 56.9, 9- estirpe 56.21, 10- estirpe 56.22, 11- estirpe 56.25, 12- estirpe 56.26 e 13- estirpe 56.27. Observou-se efeito de tratamento (P 0.05) pelos tratamentos. A silagem inoculada com 56.1 apresentou maior estabilidade aeróbia do que a silagem não inoculada (68.25 vs. 36 h). A cepa de L. buchneri 56.1 tem potencial para ser utilizada como inoculante microbiano em silagem de milho. Capítulos 3 e 4 - Exploramos as sucessões das populações bacterianas e fúngicas e avaliamos os impactos causados por inoculantes contendo Lactobacillus plantarum + Propionibacterium acidipropionici e Lactobacillus buchneri nessas populações em grãos de milho e sorgo reidratados e suas silagens por sequenciamento de nova geração após 0, 3, 7, 21, 90 e 360 dias de fermentação. Proteobacteria e Firmicutes foram predominantemente encontrados no início e durante os períodos intermediários de fermentação, respectivamente, em ambos os grãos. Espécies de Lactobacillus e Weissella foram as principais bactérias envolvidas na fermentação de silagens de grãos de milho e sorgo reidratados. Aspergillus spp. predominaram na fermentação de grãos de milho, enquanto a levedura Wickerhamomyces anomalus foi o principal fungo encontrado nas silagens de grãos de sorgo. O inoculante contendo L. plantarum e P. acidipropionici foi mais eficiente em promover crescimento de Lactobacillus spp. e manter maior estabilidade da comunidade bacteriana durante períodos mais longos de armazenamento em ambas as silagens, já a adição de inoculante não resultou em grandes alterações na população fúngica de silagens de grãos de sorgo reidratado. Capítulo 5 - Avaliou-se o efeito da ensilagem no perfil fermentativo, no corn silage processing score (CSPS) e no perfil de ácidos graxos de cadeia longa (AGCL) em planta inteira de milho. Onze híbridos de milho foram colhidos e cada uma das 11 amostras foi homogeneizada manualmente e alocada em 4 amostras de aproximadamente 600 g cada. Cada uma das 4 amostras foi aleatoriamente designada para 1 de 2 tratamentos (0 ou 120 d de ensilagem) e seladas a vácuo em bags. A concentração de MS não foi afetada (P > 0.10) pela ensilagem e teve média de 36.2% as fed. Os efeitos sobre o pH são provavelmente atribuídos a 7.7% -, 1.0% - e 1.2% - maiores concentrações de ácido lático, acético e isobutírico, respectivamente, para 120 d comparado com 0 d. As concentrações de N-NH3 aumentaram (P = 0.001) com a ensilagem, como esperado. As concentrações de amido e CSPS não foram afetadas (P > 0.10) pela ensilagem e tiveram média de 31.2% MS e 28.8%, respectivamente. Não foram observados efeitos de ensilagem no perfil de AGCL na maioria dos ácidos graxos incluindo C16:0, C18:0, C18:1, C18:2 e C18:3 (P > 0.10). Mais pesquisas são necessárias para elucidar sob quais condições o tempo de ensilagem aumenta o CSPS

Effect of wilting and bacterial inoculant on the microbial population and fermentation profile of alfalfa silage in tropical conditions

Avaliou-se o perfil fermentativo, a composição química e as populações microbianas de silagens de alfafa emurchecida ou não, tratadas com inoculante bacteriano em diferentes períodos de fermentação; bem como o isolamento, a identificação e a caracterização das bactérias do ácido lático (BAL) das silagens não inoculadas. Foi utilizado um esquema fatorial 2 × 2 × 6, ausência ou presença de emurchecimento (E), com e sem inoculante bacteriano (I) e seis períodos de fermentação (P) (1, 3, 7, 14, 28 e 56 dias), no delineamento inteiramente casualizado, com três repetições. A alfafa foi colhida aos 50 dias de rebrotação e picada em partículas de 1,5 cm. A alfafa foi emurchecida por 6h, no campo, no material submetido a esse tratamento. O inoculante usado foi o Sil-All® 4x4 W.S. (Alltech, Brasil), aplicado na taxa de 10 5 UFC/g (5 g/t de forragem). Após a aplicação dos tratamentos, a forragem foi ensilada em sacos plásticos de 25 × 35 cm e selados a vácuo. O emurchecimento da alfafa reduziu a capacidade tampão e as concentrações de carboidratos solúveis em água e aumentou o teor de MS da forragem de 133,9 g/kg MS para 233,4 g/kg MS, antes da ensilagem. Foi observado efeito (P < 0,05) de E sobre os teores de MS, PB, NIDA e FDN das silagens aos 56 dias de fermentação. As populações de BAL e fungos não foram afetadas por nenhum dos fatores estudados, enquanto as populações de enterobactérias e leveduras foram afetadas (P < 0,05) pelo P. O pH e a concentração de nitrogênio amoniacal foram afetados (P < 0,05) pela interação E × P, registrando-se menores valores destas variáveis nas silagens emurchecidas. Também foi observado efeito (P < 0,05) da interação E × P sobre o conteúdo dos ácidos lático, acético e propiônico, sendo registrados menores valores de ácido acético nas silagens emurchecidas em todos os períodos de fermentação. Menores concentrações de ácido propiônico também foram encontradas nas silagens emurchecidas. Excetuando o 1o dia de fermentação, foram observadas maiores concentrações de ácido lático nas silagens emurchecidas, no entanto, a partir do 7o dia, a concentração deste ácido decresceu em ambas as silagens emurchecidas ou não. A adição de inoculante bacteriano na presença ou não do emurchecimento, não promove melhorias no x processo fermentativo de silagem de alfafa com elevada umidade. A população de BAL na planta e nas silagens de alfafa submetidas ou não ao emurchecimento é diversa em nível de espécies, o Lactobacillus plantarum é a espécie predominante e apresenta comportamento distinto ao longo dos períodos de fermentação nas silagens não emurchecidas e emurchecidas.We aim with this study to evaluate the fermentation characteristics, chemical composition and microbial populations of wilted or unwilted alfalfa silage treated with bacterial inoculant in different periods of fermentation as well as the isolation, identification and characterization of lactic acid bacteria (LAB) from non-inoculated silage. We analyzed the data as a 2 × 2 × 6 factorial arrangement with the effects of wilting (W) (absence or presence), bacterial inoculant (I) (with and without) and period (P) (1, 3, 7, 14, 28 and 56 days), and their interaction in a completely randomized design with three replications. We harvested the alfalfa at 50 days of regrowth, chopped into particles of 1.5 cm and wilted for 6 hours in the field before ensiling. The commercial inoculant used was Sil-All® 4x4 W.S. (Alltech, Brazil), applied at the rate of 10 5 CFU/g of fresh forage. After the treatments forage was packed in plastic bags of 25 × 35 cm and sealed by using vacuum sealer. Wilting of alfalfa reduced buffering capacity and water soluble carbohydrate concentrations and increased the dry matter (DM) content of the forage 133.9 g/kg DM to 233.4 g/kg DM before ensiling. There was effect (P < 0.05) of W on the DM, crude protein, neutral detergent fiber and acid detergent insoluble nitrogen in the silages at 56 d. Populations of LAB and molds were not affected by any of the effects studied, however the populations of enterobacteria and yeasts were affected (P < 0.05) by P. The pH and ammonia nitrogen concentration were affected by the interaction W × P (P < 0.05), registering lower values of these variables on wilted silages. There was effect (P < 0.05) of the interaction W × P on the concentrations of lactic, acetic and propionic acid, resulting in lower values of acetic acid in the wilted silages in all fermentation periods. Lower concentrations of propionic acid were found in wilted silages. Except for the 1st day of fermentation, we observed higher concentrations of lactic acid in the wilted silages, however in both wilted and unwilted silages the values decreased from the 7th d of ensiling. The addition of inoculant in presence or absence of wilting, does not improve the fermentation process of alfalfa silage with high moisture content. The populations of LAB in the wilted or unwilted fresh-alfalfa and in the silages are diverse in species levels and the xii Lactobacillus plantarum is the predominant specie and presents different dynamic appearance over the fermentation period between unwilted and wilted silages.Conselho Nacional de Desenvolvimento Científico e Tecnológic

The chemical composition, fermentation profile, and microbial populations in tropical grass silages

The objective of this study was to evaluate the fermentation profile, chemical composition and microbial population and losses in the silages of signalgrass and Mombasa grass fertilized with the following levels of nitrogen (N): 0, 30, 60 and 90 kg/ha. The grasses were harvested at 70 days of regrowth, chopped and then ensiled in laboratory silos that had 20 kg of capacity and a snap-top cover and were fitted with Bunsen valves. Before ensiling, samples of the plants were used for the isolation and identification of lactic acid bacteria (LAB) in epiphytic microbiota. The design adopted was a 4 × 2 factorial arrangement, with four doses of N and two forage species, in a completely randomized design, with four replicates. The predominant species of LAB was Lactobacillus fermentum. The interaction between the N dose and forage species affected the dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF) and water soluble carbohydrates (WSC) of the silages. The pH values and gas losses were influenced only by the forage species, with higher values for the Mombasa grass. For the ammonia (NH3-N) levels and effluent losses, there was an effect of the interaction between the forage species and N doses, and the highest values of NH3-N and effluent losses were found in the Mombasa grass silage fertilized with 60 kg N/ha. Nitrogen fertilization reduces the levels of DM and WSC in the silages and also increases the levels of CP, NH3-N and effluent losses

The chemical composition, fermentation profile, and microbial populations in tropical grass silages

The objective of this study was to evaluate the fermentation profile, chemical composition and microbial population and losses in the silages of signalgrass and Mombasa grass fertilized with the following levels of nitrogen (N): 0, 30, 60 and 90 kg/ha. The grasses were harvested at 70 days of regrowth, chopped and then ensiled in laboratory silos that had 20 kg of capacity and a snap-top cover and were fitted with Bunsen valves. Before ensiling, samples of the plants were used for the isolation and identification of lactic acid bacteria (LAB) in epiphytic microbiota. The design adopted was a 4 × 2 factorial arrangement, with four doses of N and two forage species, in a completely randomized design, with four replicates. The predominant species of LAB was Lactobacillus fermentum. The interaction between the N dose and forage species affected the dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF) and water soluble carbohydrates (WSC) of the silages. The pH values and gas losses were influenced only by the forage species, with higher values for the Mombasa grass. For the ammonia (NH3-N) levels and effluent losses, there was an effect of the interaction between the forage species and N doses, and the highest values of NH3-N and effluent losses were found in the Mombasa grass silage fertilized with 60 kg N/ha. Nitrogen fertilization reduces the levels of DM and WSC in the silages and also increases the levels of CP, NH3-N and effluent losses

Effects of exogenous protease addition on fermentation and nutritive value of rehydrated corn and sorghum grains silages

Abstract The study objective was to evaluate the effects of the addition of exogenous protease on the fermentation and nutritive value of rehydrated corn and sorghum grain silages during various storage periods. Treatments were applied using a 2 × 6 × 3 factorial combination, with 2 types of rehydrated grains (corn and sorghum), 6 doses of the enzyme (0, 0.3, 0.6, 0.9, 1.2, and 1.5%, based on natural matter) and 3 fermentation periods (0, 60, and 90 days) in a completely randomized design, with 4 replications. The protease aspergilopepsin I, of fungal origin, produced by Aspergillus niger, was used. The lactic acid concentration increased linearly as the enzyme dose increased in corn (CG) and sorghum (SG) grain silages, at 60 and 90 days of fermentation. There was an increase in the concentrations of ammonia nitrogen and soluble protein, as well as the in situ starch digestibility in rehydrated CG and SG silages, compared to the treatment without the addition of protease. The addition of 0.3% exogenous protease at the moment of CG ensiling and 0.5% in rehydrated SG increased the proteolytic activity during fermentation, providing an increase in in situ starch digestibility in a shorter storage time

Effect of Various Strains of <em>Lactobacillus buchneri</em> on the Fermentation Quality and Aerobic Stability of Corn Silage

The aerobic deterioration of silage nutrients is inevitable in tropical countries, causing negative consequences in animal production systems. Aiming to minimize the losses, the effects of Lactobacillus buchneri strains on fermentation profile and aerobic stability of corn silages were evaluated. The experiment was conducted under a completely randomized design with 13 treatments and three replicates. The treatments were noninoculated, commercial L. buchneri (CI), and 11 wild strains of L. buchneri: LB-56.1, LB-56.2, LB-56.4, LB-56.7, LB-56.8, LB-56.9, LB-56.21, LB-56.22, LB-56.25, LB-56.26, and LB-56.27. The treatments could be divided into three different groups according to silage pH and acetic acid concentration. Silages inoculated with LB-56.1, LB-56.4, and LB-56.9 presented higher pH, whereas intermediate values were observed for LB-56.2, LB-56.7, and LB-56.8. The highest acetic acid production was observed for LB-56.1 and LB-56.7. On the other hand, lowest concentrations were found for CI, LB-56.22, LB-56.25, LB-56.26, and LB-56.27. Higher amounts of NH3–N were observed for LB-56.8, LB-56.21, LB-56.22, and LB-56.27 silages than others. Silage inoculation with CI, LB-56.1, LB-56.2, LB-56.4, LB-56.8, LB-56.9, and LB-56.25 strains had higher aerobic stability than others (59.7 vs. 41.2 h). The L. buchneri strains LB-56.1, LB-56.2, LB-56.4, LB-56.8, LB-56.9, and LB-56.25 provided potential features to improve the aerobic stability of corn silage