New Papiliotrema laurentii UFV-1 strains with improved acetic acid tolerance selected by adaptive laboratory evolution.

The production of yeast oil from lignocellulosic biomasses is impaired by inhibitors formed during the pretreatment step, mainly acetic acid. Herein, we applied Adaptive Laboratory Evolution (ALE) to select three Acetic acid Tolerant Strains (ATS) of P. laurentii UFV-1. Different phenotypes emerged alongside evolution. The ATS II presented trade-offs in the absence of acetic acid, suggesting that it displays a specialized phenotype of tolerance to growth on organic acids. On the other hand, ATS I and ATS III presented phenotypes associated with the behavior of generalists. ATS I was considered the most promising evolved strain as it displayed the oleaginous phenotype in all conditions tested. Thus, we applied whole-genome sequencing to detect the mutations that emerged in this strain during the ALE. We found alterations in genes encoding proteins involved in different cellular functions, including multidrug resistance (MDR) transporters, energy metabolism, detoxification, coenzyme recycling, and cell envelope remodeling. To evaluate acetic acid stress responses, both parental and ATS I strains were cultivated in chemostat mode in the absence and presence of acetic acid. In contrast to ATS I, the parental strain presented alterations in the cell envelope and cell size under acetic acid stress conditions. Furthermore, the parental strain and the ATS I presented differences regarding acetic acid assimilation. Contrary to the parental strain, the ATS I displayed an increase in unsaturated fatty acid content irrespective of acetic acid stress, which might be related to improved tolerance to acetic acid. Altogether, these results provided insights into the mechanisms involved with the acetic acid tolerance displayed by ATS I and the responses of P. laurentii to this stressful condition.

PCR screening reveals abundance of bovicin-like bacteriocins among ruminal Streptococcus spp. isolated from beef and dairy cattle.

AIMS: To investigate the inhibitory activity and the distribution of biosynthetic genes encoding bovicin-like bacteriocins among ruminal Streptococcus isolated from beef and dairy cattle. METHODS AND RESULTS: Most isolates were classified as Streptococcus equinus and Streptococcus lutetiensis based on 16S rRNA sequencing. The antimicrobial activity of 150 ruminal streptococci isolated from beef and dairy cattle were tested by deferred inhibition assays and their genetic diversity was characterized by BOX-PCR. The frequency of biosynthetic genes associated with the biosynthesis of bovicin-like bacteriocins (bovicin HC5 and bovicin 255) was investigated by PCR screening. Approximately 33% of the ruminal streptococci isolated from Nellore heifers showed inhibitory activity in vitro with the majority harbouring genes for bacteriocin biosynthesis. In contrast, streptococci from Holstein cows showed limited inhibitory activity and a lower frequency of bacteriocin biosynthetic genes. CONCLUSIONS: Streptococcus from the rumen of beef and dairy cattle exhibit remarkable differences in inhibitory activity and distribution of genes associated with the biosynthesis of prototypical bovicins (bovicin HC5 and bovicin 255). SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings demonstrate that bovicin HC5 is distributed among ruminal streptococci from different breeds of cattle. The high degree of conservation of the bovicin HC5 structural gene among strains of ruminal streptococci suggests that random genetic drift is not a dominant force in the evolution of this bacteriocin.

Dietary nucleotide supplementation as an alternative to in-feed antibiotics in weaned piglets.

Nucleotides are important to cell growth and division and are crucial to the rapid proliferation of such cells as the intestinal mucosa and immune cells. Accordingly, the nucleotide requirements of animals are high during periods of rapid growth and periods of stress like post-weaning period. Thus, nucleotide supplementation may be a possible alternative to in-feed antibiotics as growth promoter in this phase. The study aimed to evaluate dietary nucleotide supplementation as an alternative to in-feed antibiotics on performance and gut health of weaned piglets. Ninety-six 21-day-old piglets, weighing 7.44 ±â€¯0.65 kg, were allocated into 1 of 3 treatments (8 pens per treatment; 4 pigs per pen) in a 14-day trial. Dietary treatments consisted of control: corn-soybean meal-based diet; nucleotides: control +2 g/kg of a nutritional additive with purified nucleotides; and antibiotic: control +0.8 g/kg of antibiotic growth promoter based on colistin and tylosin. Performance variables and fecal score were not affected (P > 0.05) by supplementing nucleotide or antibiotic. Nucleotides treatment had similar effect to antibiotic and superior to control (P < 0.05) on enhancing duodenum villus height, jejunum crypt depth, and reduction of Paneth cellular area. Duodenum and ileum of animals supplemented with nucleotides or antibiotics had higher (P < 0.05) number of proliferating cells than did those of control animals, whereas the jejunum of animals that received antibiotic diets presented more (P < 0.05) proliferating cells than either the nucleotides or control animals. Jejunum of nucleotide-treated piglets showed a greater number of apoptotic cells than those fed antibiotic or control diets (P < 0.05). Nucleotides and antibiotic treatments decreased the B lymphocyte counts in duodenum and ileum (P < 0.05) but increased in the jejunum (P < 0.05), when compared to the control treatment. Relative abundance of mitogen-activated protein kinases-6, haptoglobin, and tumor necrosis factor-α mRNA was not influenced (P > 0.05) by treatments. In the ileal, antibiotic supplementation reduced total bacteria quantification compared to nucleotide supplementation or the control (P < 0.05), whereas nucleotides supplementation increased enterobacteria proliferation compared to the antibiotic or control diets (P < 0.05). However, nucleotides and antibiotic reduced (P < 0.05) colon total bacteria quantification when compared to control. These results suggest that the nucleotides source used to weaned piglets improved gut health by modulating the local immune response and modulating intestinal mucosa development, and, therefore, nucleotides may be an alternative to antibiotics as growth promoters.

Identification and characterization of yeasts from bovine rumen for potential use as probiotics.

AIMS: The aim was to isolate, identify and characterize yeasts present in rumen fluid and to select strains showing potential as probiotics. METHODS AND RESULTS: Rumen fluid was sampled from 4 herds of dairy and beef cattle and 77 yeast isolates were identified. Initial screening was based on the capacity to maintain viability in a medium with different ruminal conditions. A second screening in fresh rumen fluid to assess the growth of inoculated yeasts and evaluate in vitro neutral detergent fibre digestibility (NDF-D), pH and acid accumulation was conducted. The yeast population ranged from 3·84 to 6·76 log10 CFU per ml. The main species of yeast found were Pichia kudriavzevii, Candida rugosa, C. pararugosa, C. ethanolica and Magnusiomyces capitatus. Strains CCMA 933 (C. rugosa) and CCMA 970 (C. pararugosa) showed greater ability to survive in ruminal fluid and stimulated the production of acids. Isolate CCMA 967 (C. ethanolica) survived and improved the NDF-D. CONCLUSION: Pichia kudriavzevii was the dominant yeast found in the cattle herds. Strains CCMA 933, CCMA 970 and CCMA 967 showed properties that could be useful as potential probiotics for cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to select yeasts from the rumen fluid, with the potential to be used as probiotic, based on the ruminal conditions.

The effects of increasing amounts of milk replacer powder added to whole milk on passage rate, nutrient digestibility, ruminal development, and body composition in dairy calves.

The aim of this study was to evaluate the effects on feed intake, calf performance, feed efficiency, fecal score, passage rate, apparent nutrient digestibility, development of rumen and other organs, and body composition of increasing the total solids content of liquid feed (whole milk) by adding increasing amounts of milk replacer powder during the preweaning period. Crossbred Holstein-Gyr calves (n=32) were assigned to 1 of 4 treatments (n=8 per group), which consisted of different total solids concentrations: 12.5, 15.0, 17.5, and 20.0% of liquid feed. Calves received 6 L of liquid per day, divided into 2 equal meals (0800 and 1600 h) and provided in buckets, from 5 to 55d of age. Starter and water were provided ad libitum during the entire experiment. At 56d of age, animals were killed. Laboratory analysis determined that the actual total solids contents of the liquid feed were 13.5, 16.1, 18.2, and 20.4%, for the proposed 12.5, 15.0, 17.5, and 20.0% total solids treatments, respectively. The osmolality of liquid feed treatments was 265 to 533 mOsm/L. Fecal score was similar among treatments, except for wk 2 and 7. Intake of liquid feed was similar among treatments from 6wk of age. During wk 4, 5, and 6, we detected a linear decrease in starter intake. After wk 7, we observed greater starter intake for calves fed approximately 16.1% total solids. Water intake, feed efficiency, and withers height were similar among treatments. Increasing concentrations of total solids in liquid feed quadratically affected average daily gain, final body weight, and empty body weight. We observed a greater average daily gain for calves fed approximately 20.4% total solids. Passage rate, nutrient digestibility, development of pre-stomachs and intestine, and body composition were similar among treatments. Increasing the concentration of total solids in liquid feed up to 20.4% reduced starter intake between 4 and 6wk of life, but increased average daily gain. It did not affect passage rate, nutrient digestibility, ruminal and organ development, or body composition in calves during the preweaning period, indicating that this strategy may be a viable alternative for feeding without increasing the total volume of liquid feed provided to dairy calves.

Effect of protein supplementation on ruminal parameters and microbial community fingerprint of Nellore steers fed tropical forages.

In tropical regions, protein supplementation is a common practice in dairy and beef farming. However, the effect of highly degradable protein in ruminal fermentation and microbial community composition has not yet been investigated in a systematic manner. In this work, we aimed to investigate the impact of casein supplementation on volatile fatty acids (VFA) production, specific activity of deamination (SAD), ammonia concentration and bacterial and archaeal community composition. The experimental design was a 4×4 Latin square balanced for residual effects, with four animals (average initial weight of 280±10 kg) and four experimental periods, each with duration of 29 days. The diet comprised Tifton 85 (Cynodon sp.) hay with an average CP content of 9.8%, on a dry matter basis. Animals received basal forage (control) or infusions of pure casein (230 g) administered direct into the rumen, abomasum or divided (50 : 50 ratio) in the rumen/abomasum. There was no differences (P>0.05) in ruminal pH and microbial protein concentration between supplemented v. non-supplemented animals. However, in steers receiving ruminal infusion of casein the SAD and ruminal ammonia concentration increased 33% and 76%, respectively, compared with the control. The total concentration of VFA increased (P0.05) in species richness and diversity of γ-proteobacteria, firmicutes and archaea between non-supplemented Nellore steers and steers receiving casein supplementation in the rumen. However, species richness and the Shannon-Wiener index were lower (P<0.05) for the phylum bacteroidetes in steers supplemented with casein in the rumen compared with non-supplemented animals. Venn diagrams indicated that the number of unique bands varied considerably among individual animals and was usually higher in number for non-supplemented steers compared with supplemented animals. These results add new knowledge about the effects of ruminal and postruminal protein supplementation on metabolic activities of rumen microbes and the composition of bacterial and archaeal communities in the rumen of steers.

Differences in the antibacterial activity of nisin and bovicin HC5 against Salmonella Typhimurium under different temperature and pH conditions.

AIMS: To compare the action of nisin and bovicin HC5 in combination with EDTA on Salmonella Typhimurium under different environmental conditions. METHODS AND RESULTS: Salmonella Typhimurium was treated in BHI broth containing EDTA (1·5 mmol l(-1)) and nisin or bovicin HC5 (200 AU ml(-1)) under different pH and temperature conditions, and according to a central composite design with two factors (temperature and pH). Cell viability was evaluated on plate count agar for 48 h. The combination of nisin or bovicin HC5 with EDTA was able to inhibit the growth of Salmonella, but the temperature and pH conditions promoting inhibition were distinct for each bacteriocin. Nisin was bactericidal over a broad range of temperature and pH, while bovicin HC5 was bacteriostatic in most conditions and bactericidal only in specific conditions (pH >6·0 and temperature >30°C). Salmonella Typhimurium did not show tolerance to bovicin HC5 or cross-tolerance between these lantibiotics. CONCLUSIONS: Nisin and bovicin HC5 both inhibited the growth of Salmonella, but the activity of each bacteriocin was differently influenced by environmental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Nisin and bovicin HC5 have the potential to inhibit the growth of Salmonella, but environmental conditions should be considered to establish optimal conditions for its application.

Accuracy of the estimates of ammonia concentration in rumen fluid using different analytical methods / Exatidão das estimativas de concentração de amônia em fluido ruminal utilizando diferentes métodos analíticos

The accuracy of two different methods in measuring the ammonia nitrogen (N-NH3) concentration in rumen fluid were evaluated: a catalyzed indophenol colorimetric reaction (CICR) and the Kjeldahl distillation (KD). Five buffered standard solutions containing volatile fatty acids, true protein, and known ammonia concentrations (0, 3, 6, 12, and 24 N-NH3 mg/dL) were used to simulate rumen fluid. Different ratios (10:1, 7.5:1, 5:1, 2.5:1, 1:1, 1:2.5, 1:5, 1:7.5, and 1:10) of a potassium hydroxide solution (KOH, 2 mol/L) to standard solutions were evaluated by the KD method. The accuracy of each method was evaluated by adjusting a simple linear regression model of the estimated N-NH3 concentrations on the N-NH3 concentrations in the standard solutions. When the KD method was used, N-NH3 was observed to be released from the deamination of true protein (P<0.05), and an incomplete recovery of N-NH3 was observed (P<0.05), except for 7.5:1 and 5:1 ratios of KOH solution to standard solutions (P>0.05). The estimates of the N-NH3 concentration obtained by the CICR method were found to be accurate (P>0.05). After the accuracy evaluation, ninety-three samples of rumen fluid were evaluated by the CICR and KD methods (using the 5:1 ratio of KOH solution to rumen fluid sample), assuming that the CICR estimates would be accurate. The N-NH3 concentrations obtained by the two methods were observed to be different (P<0.05) but strongly correlated (r = 0.9701). Thus, it was concluded that the estimates obtained by the Kjeldahl distillation using a 5:1 ratio of KOH solution to rumen fluid sample can be adjusted to avoid biases. Furthermore, a model to adjust the N-NH3 concentration is suggested.(AU)

Avaliou-se a exatidão de dois métodos para mensuração da concentração de nitrogênio amoniacal (N-NH3) em fluido ruminal: reação colorimétrica catalisada por indofenol (RCCI) e destilação de Kjeldahl (DK). Cinco soluções-tampão contendo ácidos graxos voláteis, proteína verdadeira e concentrações conhecidas de amônia (0, 3, 6, 12 e 24 N-NH3mg/dL) foram utilizadas para simular o fluido ruminal. Diferentes relações (10:1; 7,5:1; 5:1; 2,5:1; 1:1; 1:2,5; 1:5; 1:7,5 e 1:10) entre a solução de hidróxido de potássio (KOH, 2 mol/L) e as soluções-padrão foram avaliadas no método DK. A exatidão de cada método foi avaliada por intermédio do ajustamento de um modelo de regressão linear simples das concentrações estimadas de N-NH3sobre as concentrações reais de N-NH3nas soluções-padrão. Com a utilização da DK observou-se que N-NH3foi liberada a partir da deaminação de proteína verdadeira (P<0,05). Adicionalmente, a utilização da DK promoveu recuperação incompleta da N-NH3(P<0,05), com exceção das razões entre a solução de KOH e as soluções-padrão de de 7,5:1 e de 5:1 (P>0,05). As estimativas de concentração de amônia obtidas por intermédio do método de RCCI foram exatas (P>0,05). Após a avaliação da exatidão, noventa e três amostras de fluido ruminal foram avaliadas por intermédio dos métodos de RCCI e de DK (neste caso usando a razão entre solução de KOH e fluido ruminal de 5:1). Nesta avaliação assumiu-se que as estimativas obtidas com a RCCI seriam exatas. Observou-se que as concentrações de N-NH3obtidas pelos dois métodos foram diferentes (P<0,05), mas fortemente correlacionadas (r = 0,9701). Concluiu-se que as estimativas obtidas com a DK utilizando-se razão entre a solução de KOH e a amostra de fluido ruminal de 5:1 poderiam ser ajustadas para correções de vieses Um modelo para realização desse ajustamento foi sugerido.(AU)

Accuracy of the estimates of ammonia concentration in rumen fluid using different analytical methods / Exatidão das estimativas de concentração de amônia em fluido ruminal utilizando diferentes métodos analíticos

The accuracy of two different methods in measuring the ammonia nitrogen (N-NH3) concentration in rumen fluid were evaluated: a catalyzed indophenol colorimetric reaction (CICR) and the Kjeldahl distillation (KD). Five buffered standard solutions containing volatile fatty acids, true protein, and known ammonia concentrations (0, 3, 6, 12, and 24 N-NH3 mg/dL) were used to simulate rumen fluid. Different ratios (10:1, 7.5:1, 5:1, 2.5:1, 1:1, 1:2.5, 1:5, 1:7.5, and 1:10) of a potassium hydroxide solution (KOH, 2 mol/L) to standard solutions were evaluated by the KD method. The accuracy of each method was evaluated by adjusting a simple linear regression model of the estimated N-NH3 concentrations on the N-NH3 concentrations in the standard solutions. When the KD method was used, N-NH3 was observed to be released from the deamination of true protein (P<0.05), and an incomplete recovery of N-NH3 was observed (P<0.05), except for 7.5:1 and 5:1 ratios of KOH solution to standard solutions (P>0.05). The estimates of the N-NH3 concentration obtained by the CICR method were found to be accurate (P>0.05). After the accuracy evaluation, ninety-three samples of rumen fluid were evaluated by the CICR and KD methods (using the 5:1 ratio of KOH solution to rumen fluid sample), assuming that the CICR estimates would be accurate. The N-NH3 concentrations obtained by the two methods were observed to be different (P<0.05) but strongly correlated (r = 0.9701). Thus, it was concluded that the estimates obtained by the Kjeldahl distillation using a 5:1 ratio of KOH solution to rumen fluid sample can be adjusted to avoid biases. Furthermore, a model to adjust the N-NH3 concentration is suggested.

Avaliou-se a exatidão de dois métodos para mensuração da concentração de nitrogênio amoniacal (N-NH3) em fluido ruminal: reação colorimétrica catalisada por indofenol (RCCI) e destilação de Kjeldahl (DK). Cinco soluções-tampão contendo ácidos graxos voláteis, proteína verdadeira e concentrações conhecidas de amônia (0, 3, 6, 12 e 24 N-NH3mg/dL) foram utilizadas para simular o fluido ruminal. Diferentes relações (10:1; 7,5:1; 5:1; 2,5:1; 1:1; 1:2,5; 1:5; 1:7,5 e 1:10) entre a solução de hidróxido de potássio (KOH, 2 mol/L) e as soluções-padrão foram avaliadas no método DK. A exatidão de cada método foi avaliada por intermédio do ajustamento de um modelo de regressão linear simples das concentrações estimadas de N-NH3sobre as concentrações reais de N-NH3nas soluções-padrão. Com a utilização da DK observou-se que N-NH3foi liberada a partir da deaminação de proteína verdadeira (P<0,05). Adicionalmente, a utilização da DK promoveu recuperação incompleta da N-NH3(P<0,05), com exceção das razões entre a solução de KOH e as soluções-padrão de de 7,5:1 e de 5:1 (P>0,05). As estimativas de concentração de amônia obtidas por intermédio do método de RCCI foram exatas (P>0,05). Após a avaliação da exatidão, noventa e três amostras de fluido ruminal foram avaliadas por intermédio dos métodos de RCCI e de DK (neste caso usando a razão entre solução de KOH e fluido ruminal de 5:1). Nesta avaliação assumiu-se que as estimativas obtidas com a RCCI seriam exatas. Observou-se que as concentrações de N-NH3obtidas pelos dois métodos foram diferentes (P<0,05), mas fortemente correlacionadas (r = 0,9701). Concluiu-se que as estimativas obtidas com a DK utilizando-se razão entre a solução de KOH e a amostra de fluido ruminal de 5:1 poderiam ser ajustadas para correções de vieses Um modelo para realização desse ajustamento foi sugerido.

Use of phages against antibiotic-resistant Staphylococcus aureus isolated from bovine mastitis.

Bovine mastitis is the primary disease of dairy cattle worldwide and it causes large economic losses. Among several microorganisms that are the causative agents of this disease, Staphylococcus aureus is the most prevalent. Although antibiotic therapy is still the most widely used procedure for the treatment of bovine mastitis, alternative means of treatment are necessary due to the presence of antibiotic residues in milk, which is a growing concern because of its interference with the production of milk derivatives and the selection of resistant bacterial strains. The use of bacteriophages as a tool for the control of pathogens is an alternative treatment to antibiotic therapy. In this work, to obtain phages with the potential for use in phage therapy as a treatment for mastitis, we isolated and identified the bacteria from the milk of mastitis-positive cows. A total of 19% of the animals from small and medium farms of the Zona da Mata Mineira, Brazil, was positive for bovine mastitis, and bacteria of the genus Staphylococcus were the most prevalent pathogens. The majority of the S. aureus isolates tested was resistant to penicillin and ampicillin. In parallel, we isolated 10 bacteriophages able to infect some of these S. aureus isolates. We determined that these phages contained DNA genomes of approximately 175 kb in length, and the protein profiles indicated the presence of 4 major proteins. Electron microscopy revealed that the phages are caudate and belong to the Myoviridae family. The isolates exhibited interesting features for their use in phage therapy such as a high lytic potential, a wide range of hosts, and thermostability, all of which favor their use in the field.

Ingestão e digestibilidade aparente em ovinos alimentados com silagens de capim-elefante inoculadas com Streptococcus bovis / Intake and apparent digestibility in sheep fed elephant grass silage inoculated with Streptococcus bovis

Foram avaliadas a ingestão e a digestibilidade aparente de silagens de capim, utilizando-se 20 ovinos adultos, sem raça definida, castrados, distribuídos em delineamento inteiramente ao acaso, com quatro tratamentos: silagem de capim-elefante; silagem de capim-elefante inoculada com 10(6)UFC/g de()estirpes de Enterococcus faecium; silagem de capim-elefante inoculada com 10(6)UFC/g de estirpes de Streptococcus bovis JB1; e silagem de capim-elefante inoculada com 10(6)UFC/g de()estirpes de S. bovis HC5, e cinco repetições por tratamento. O período de avaliação foi de 21 dias, sendo 15 para adaptação e seis para as coletas. Observou-se diferença (P<0,05) entre o consumo de matéria seca, sendo a silagem-controle a que apresentou os valores mais baixos - 1,43%PV ou 42,79g/kg PV0,75/dia -, enquanto a silagem inoculada com S. bovis HC5 apresentou o melhor consumo de matéria seca, 2,3% PV ou 56,10g/kg PV0,75/dia. Menores valores de digestibilidade da matéria seca ocorreram na silagem-controle, 42,8% da MS, seguida pela silagem de capim-elefante inoculada com E. faecium e S. bovis JB1. Para a digestibilidade aparente da fibra em detergente neutro, houve efeito dos inoculantes (P<0,05) com valores acima de cinco pontos percentuais em relação à silagem inoculada, 54,7%.

The intake and digestibility were evaluated in grass silage, using 20 adult sheep, mixed breed, distributed in a completely randomized design with four treatments: elephant grass silage without inoculant, elephant grass silage inoculated with 10(6)FCU/g of strains of Enterococcus faecium, elephant grass silage inoculated with 10(6)FCU/g strains of Streptococcus bovis JB1, elephant grass silage inoculated with 10(6)FCU/g of strains of S. bovis HC5 and five replicates per treatment. The evaluation period lasted 21 days being 15 for diet adaptation and six for the collections. There was a statistical difference (P<0.05) between the intake of dry matter of the silages assessed, and the highest values were recorded in the silages inoculated with microbial inoculants. Silage without inoculation had the lowest values (P<0.05) of dry matter intake (1.43% BW or 42.79g/kg BW0,75/day) and silage inoculated with Streptococcus bovis HC5 showed higher dry matter intake (2.3% or 56.10BW g/kg BW0,75/day). The lowest digestibility of dry matter occurred in the control silage (42.8% DM) followed by elephant grass silage inoculated with E. faecium and S. bovis JB1, respectively. The apparent digestibility of neutral detergent fiber was affected by inoculation (P<0.05) with values higher than five percentage points compared to elephant grass silage without inoculant (54.7%).

Ingestão e digestibilidade aparente em ovinos alimentados com silagens de capim-elefante inoculadas com Streptococcus bovis / Intake and apparent digestibility in sheep fed elephant grass silage inoculated with Streptococcus bovis

Foram avaliadas a ingestão e a digestibilidade aparente de silagens de capim, utilizando-se 20 ovinos adultos, sem raça definida, castrados, distribuídos em delineamento inteiramente ao acaso, com quatro tratamentos: silagem de capim-elefante; silagem de capim-elefante inoculada com 10(6)UFC/g de()estirpes de Enterococcus faecium; silagem de capim-elefante inoculada com 10(6)UFC/g de estirpes de Streptococcus bovis JB1; e silagem de capim-elefante inoculada com 10(6)UFC/g de()estirpes de S. bovis HC5, e cinco repetições por tratamento. O período de avaliação foi de 21 dias, sendo 15 para adaptação e seis para as coletas. Observou-se diferença (P<0,05) entre o consumo de matéria seca, sendo a silagem-controle a que apresentou os valores mais baixos - 1,43%PV ou 42,79g/kg PV0,75/dia -, enquanto a silagem inoculada com S. bovis HC5 apresentou o melhor consumo de matéria seca, 2,3% PV ou 56,10g/kg PV0,75/dia. Menores valores de digestibilidade da matéria seca ocorreram na silagem-controle, 42,8% da MS, seguida pela silagem de capim-elefante inoculada com E. faecium e S. bovis JB1. Para a digestibilidade aparente da fibra em detergente neutro, houve efeito dos inoculantes (P<0,05) com valores acima de cinco pontos percentuais em relação à silagem inoculada, 54,7%.(AU)

The intake and digestibility were evaluated in grass silage, using 20 adult sheep, mixed breed, distributed in a completely randomized design with four treatments: elephant grass silage without inoculant, elephant grass silage inoculated with 10(6)FCU/g of strains of Enterococcus faecium, elephant grass silage inoculated with 10(6)FCU/g strains of Streptococcus bovis JB1, elephant grass silage inoculated with 10(6)FCU/g of strains of S. bovis HC5 and five replicates per treatment. The evaluation period lasted 21 days being 15 for diet adaptation and six for the collections. There was a statistical difference (P<0.05) between the intake of dry matter of the silages assessed, and the highest values were recorded in the silages inoculated with microbial inoculants. Silage without inoculation had the lowest values (P<0.05) of dry matter intake (1.43% BW or 42.79g/kg BW0,75/day) and silage inoculated with Streptococcus bovis HC5 showed higher dry matter intake (2.3% or 56.10BW g/kg BW0,75/day). The lowest digestibility of dry matter occurred in the control silage (42.8% DM) followed by elephant grass silage inoculated with E. faecium and S. bovis JB1, respectively. The apparent digestibility of neutral detergent fiber was affected by inoculation (P<0.05) with values higher than five percentage points compared to elephant grass silage without inoculant (54.7%).(AU)

The effect of carbon and nitrogen sources on bovicin HC5 production by Streptococcus bovis HC5.

AIMS: To investigate the effect of media composition and agroindustrial residues on bovicin HC5 production by Streptococcus bovis HC5. METHODS AND RESULTS: Batch cultures of S. bovis HC5 were grown in basal medium containing different carbon and nitrogen sources. The activity of cell-free and cell-associated bovicin HC5 was determined in culture supernatants and acidic extracts obtained from cell pellets, respectively. Streptococcus bovis HC5 produced bovicin using a variety of carbon and nitrogen sources. The highest specific activity was obtained in media containing 16 g l(-1) of glucose, after 16 h of incubation. The peak in cell-free and cell-associated bovicin HC5 activity was detected when S. bovis HC5 cultures reached stationary phase. The bovicin HC5 specific activity and bacterial cell mass increased approximately 3-fold when yeast extract and trypticase (0.5 and 1.0 g l(-1), respectively) were added together to the basal medium. Streptococcus bovis HC5 cultures produced bovicin HC5 in cheese whey and sugar cane juice and maximal volumetric productivity was obtained after 12 h of incubation. CONCLUSIONS: Streptococcus bovis HC5 is a versatile lactic acid bacterium that can utilize several carbon and nitrogen sources for bovicin HC5 production. This bacterium could be a useful model to study bacteriocin production in the rumen ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of agroindustrial residues as carbon sources could have an economical impact on bovicin HC5 production. To our knowledge, this is the first report to show the use of sugar cane juice for bacteriocin production by lactic acid bacteria.

Bovicin HC5 reduces thermal resistance of Alicyclobacillus acidoterrestris in acidic mango pulp.

AIMS: To test the effect of bovicin HC5 against vegetative cells and endospores of Alicyclobacillus acidoterrestris DSMZ 2498 in synthetic media and in acidic mango pulp. METHODS AND RESULTS: Alicyclobacillus acidoterrestris was grown in synthetic medium at 40 degrees C and pH 4.0. The effect on vegetative cells was assayed by adding bovicin HC5 to synthetic medium (40-160 AU ml(-1)) or to mango pulp (100 AU ml(-1)) at various pH values and determining the effect on growth (OD(600 nm)) and viable cell number, respectively. The effect of bovicin HC5 on spore germination and thermal sensitivity of A. acidoterrestris was tested in mango pulp (pH 4.0) containing 80 AU ml(-1) of bovicin HC5. Bovicin HC5 was bactericidal against vegetative cells of A. acidoterrestris at different pH values and showed sporicidal activity against endospores of this bacterium. When spores of A. acidoterrestris were heat treated in the presence of bovicin HC5, D-values decreased 77% to 95% compared to untreated controls at temperatures ranging from 80 to 95 degrees C. CONCLUSION: Bovicin HC5 was bactericidal and sporicidal against A. acidoterrestrsi DSMZ 2498. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicated that bovicin HC5 has potential to prevent spoilage of acidic fruit juices by thermocidophilic spore-forming bacteria.

Avaliação de ionóforos pela técnica da perda do potássio celular e produção de gases in vitro / Ionophores evaluation by intracellular potassium depletion and in vitro gas production

Dois estudos foram realizados com vacas lactantes utilizadas como unidade experimental e doadoras de líquido ruminal, sendo as populações de bactérias utilizadas para avaliar a ação de níveis crescentes de lasalocida e monensina na resistência à perda de potássio intracelular, e para produção de gases in vitro. A perda de potássio (Kmax) da lasalocida foi menor para a população de bactérias obtidas do líquido de rúmen de vacas submetidas a dietas com monensina, óleo de soja e monensina mais óleo de soja (19,4 a 25,4 por cento) quando comparada com a perda de potássio em vacas submetidas a dietas sem ionóforo e óleo de soja (30,1 por cento). O mesmo ocorreu para a perda de potássio da monensina, em que o menor valor foi de 6,5 por cento para monensina mais óleo e o maior, de 29,5 por cento, para o controle. Necessita-se de alta concentração de monensina (Kd= 2,3µM), porém baixa de lasalocida (Kd= 0,2µM) para causar a metade da perda máxima de potássio intracelular da população de bactérias do rúmen de vacas submetidas a dietas com monensina. As populações de bactérias de vacas submetidas às dietas com monensina foram sensíveis à lasalocida. As amostras incubadas com própolis produziram menor volume de gases (12,9ml/100g de MS)(AU)

Two studies were carried out with lactating cows as experimental units and ruminal fluid donors. The ruminal bacteria population was used to evaluate the action of increasing levels of lasalocid and monensin on resistance of intracellular potassium depletion and in vitro gas production intracellular depletion potassium (Kmax) of lasalocid was lower to ruminal bacteria population obtained from rumen of cows fed diets with monensin, soybean oil and monensin plus soybean oil (19.4 to 25.4 percent) when compared to cows fed with control diet (30.1 percent). The same occurred for intracellular depletion potassium (Kmax) of monensin, in which the lowest value was 6.5 percent to monensin plus soybean oil and the greatest was 29.5 percent to control. High monensin concentration (Kd= 2.3µM) and low lasalocid concentration (Kd= 0.2µM) were necessary to cause half of maximum potassium depletion in ruminal bacteria population from cows fed diet with monensin. The ruminal bacteria population from cows feed diet with monensin were sensible to lasalocid. In vitro gas production showed the lowest volume when diets were incubated with propolis (12,9ml/100g of DM)(AU)

Avaliação de ionóforos pela técnica da perda do potássio celular e produção de gases in vitro / Ionophores evaluation by intracellular potassium depletion and in vitro gas production

Dois estudos foram realizados com vacas lactantes utilizadas como unidade experimental e doadoras de líquido ruminal, sendo as populações de bactérias utilizadas para avaliar a ação de níveis crescentes de lasalocida e monensina na resistência à perda de potássio intracelular, e para produção de gases in vitro. A perda de potássio (Kmax) da lasalocida foi menor para a população de bactérias obtidas do líquido de rúmen de vacas submetidas a dietas com monensina, óleo de soja e monensina mais óleo de soja (19,4 a 25,4 por cento) quando comparada com a perda de potássio em vacas submetidas a dietas sem ionóforo e óleo de soja (30,1 por cento). O mesmo ocorreu para a perda de potássio da monensina, em que o menor valor foi de 6,5 por cento para monensina mais óleo e o maior, de 29,5 por cento, para o controle. Necessita-se de alta concentração de monensina (Kd= 2,3µM), porém baixa de lasalocida (Kd= 0,2µM) para causar a metade da perda máxima de potássio intracelular da população de bactérias do rúmen de vacas submetidas a dietas com monensina. As populações de bactérias de vacas submetidas às dietas com monensina foram sensíveis à lasalocida. As amostras incubadas com própolis produziram menor volume de gases (12,9ml/100g de MS)

Two studies were carried out with lactating cows as experimental units and ruminal fluid donors. The ruminal bacteria population was used to evaluate the action of increasing levels of lasalocid and monensin on resistance of intracellular potassium depletion and in vitro gas production intracellular depletion potassium (Kmax) of lasalocid was lower to ruminal bacteria population obtained from rumen of cows fed diets with monensin, soybean oil and monensin plus soybean oil (19.4 to 25.4 percent) when compared to cows fed with control diet (30.1 percent). The same occurred for intracellular depletion potassium (Kmax) of monensin, in which the lowest value was 6.5 percent to monensin plus soybean oil and the greatest was 29.5 percent to control. High monensin concentration (Kd= 2.3µM) and low lasalocid concentration (Kd= 0.2µM) were necessary to cause half of maximum potassium depletion in ruminal bacteria population from cows fed diet with monensin. The ruminal bacteria population from cows feed diet with monensin were sensible to lasalocid. In vitro gas production showed the lowest volume when diets were incubated with propolis (12,9ml/100g of DM)

Bactericidal effect of bovicin HC5 and nisin against Clostridium tyrobutyricum isolated from spoiled mango pulp.

AIMS: To test the effect of bovicin HC5--a bacteriocin from Streptococcus bovis HC5--against the strains of Clostridium tyrobutyricum isolated from canned spoiled mango pulp. METHODS AND RESULTS: Bovicin HC5 [40-160 arbitrary unit (AU) ml(-1)] reduced the specific growth rate and increased the lag phase duration of the bacterial isolates inoculated in brain heart infusion media at 30 degrees C. The inhibitory activity of bovicin HC5 (100 AU ml(-1)) in mango pulp was bactericidal and more pronounced at acidic conditions. When C. tyrobutyricum was inoculated into mango pulp with bovicin HC5, gas production was not observed. Cultures that were successively transferred in the presence of sublethal doses of bovicin HC5 did not become resistant. CONCLUSIONS: The addition of bovicin HC5 to mango pulp might be effective in preventing deterioration by spoilage bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Bovicin HC5 and nisin have the potential to increase the shelf life of canned fruit pulps.

Effect of bovicin HC5 on growth and spore germination of Bacillus cereus and Bacillus thuringiensis isolated from spoiled mango pulp.

AIMS: To use bovicin HC5 to inhibit predominant bacteria isolated from spoiled mango pulp. METHODS AND RESULTS: Bovicin HC5 and nisin were added to brain heart infusion (BHI) medium (40-160 AU ml(-1)) or mango pulp (100 AU ml(-1)) and the growth of Bacillus cereus and Bacillus thuringiensis was monitored. Cultures treated with bovicin HC5 or nisin showed longer lag phases and grew slower in BHI medium. Bovicin HC5 and nisin were bactericidal and showed higher activity in mango pulp at acidic pH values. To determine the effect on spore germination and D values, mango pulp containing bovicin HC5 was inoculated with 10(6) and 10(9) spores per ml(-1), respectively, from each strain tested. Bovicin HC5 reduced the outgrowth of spores from B. cereus and B. thuringiensis, but thermal sensitivity was not affected. CONCLUSIONS: Bovicin HC5 was bactericidal against B. cereus and B. thuringiensis isolated from spoiled mango pulp. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus cereus and B. thuringiensis had not been previously isolated from spoiled mango pulp and bovicin HC5 has the potential to inhibit such bacteria in fruit pulps.

Efeito do pH in vitro sobre a resistência de bactérias do rúmen à perda de potássio intracelular e efeito do pH e de ionóforos sobre a produção de amônia e proteína microbiana / In vitro effect of pH on resistance of ruminal bacteria to intracellular potassium depletion, and effect of pH and ionophores on ammonia and microbial protein production

Em dois estudos, o líquido ruminal de bovinos mantidos sob pastagem foi usado para incubação in vitro em diferentes meios artificiais com valores de pH 5,5 e 7,0, para avaliar a ação de níveis crescentes de monensina na resistência à perda de potássio de bactérias do rúmen e verificar o efeito de monensina e lasalocida na produção de amônia e de proteína microbiana em pH 5,5 e 7,0. O meio utilizado para determinar a perda de potássio interferiu nos valores absolutos de potássio. A concentração de monensina necessária para causar a metade da perda máxima de potássio foi de 2,77µM em pH 5,5 e 0,056µM em pH 7,0, evidenciando que as bactérias incubadas em meios com pH 5,5 foram mais resistentes à monensina que aquelas incubadas em meios com pH 7,0. Os ionóforos e a acidez do meio reduziram a produção de amônia, e não se observou interação entre eles. Os ionóforos, independente do pH, inibiram a produção de amônia em 56%. A acidez inibiu a produção de amônia em 50,5%, independente do ionóforo. Os efeitos dos ionóforos e da acidez foram aditivos quando a inibição máxima ocorreu pelo uso de ionóforos com pH baixo (75,2%). A produção de proteína microbiana foi menor quando a lasalocida estava presente no meio de cultura com baixo valor de pH.

Efeito do pH in vitro sobre a resistência de bactérias do rúmen à perda de potássio intracelular e efeito do pH e de ionóforos sobre a produção de amônia e proteína microbiana / In vitro effect of pH on resistance of ruminal bacteria to intracellular potassium depletion, and effect of pH and ionophores on ammonia and microbial protein production

Em dois estudos, o líquido ruminal de bovinos mantidos sob pastagem foi usado para incubação in vitro em diferentes meios artificiais com valores de pH 5,5 e 7,0, para avaliar a ação de níveis crescentes de monensina na resistência à perda de potássio de bactérias do rúmen e verificar o efeito de monensina e lasalocida na produção de amônia e de proteína microbiana em pH 5,5 e 7,0. O meio utilizado para determinar a perda de potássio interferiu nos valores absolutos de potássio. A concentração de monensina necessária para causar a metade da perda máxima de potássio foi de 2,77µM em pH 5,5 e 0,056µM em pH 7,0, evidenciando que as bactérias incubadas em meios com pH 5,5 foram mais resistentes à monensina que aquelas incubadas em meios com pH 7,0. Os ionóforos e a acidez do meio reduziram a produção de amônia, e não se observou interação entre eles. Os ionóforos, independente do pH, inibiram a produção de amônia em 56%. A acidez inibiu a produção de amônia em 50,5%, independente do ionóforo. Os efeitos dos ionóforos e da acidez foram aditivos quando a inibição máxima ocorreu pelo uso de ionóforos com pH baixo (75,2%). A produção de proteína microbiana foi menor quando a lasalocida estava presente no meio de cultura com baixo valor de pH.(AU)

Ruminal fluid from steers fed on pasture was incubated with artificial media at pH 5.5 and 7.0 in two experiments. In the first, the effect of monensin level on resistance of ruminal bacteria to potassium depletion was evaluated; in the second, effects of the ionophores monensin and lasalocid on ammonia and protein production were quantified. In experiment 1, culture media affected potassium level. The monensin concentration needed to cause half maximal potassium depletion was 2.77µM at pH 5.5 but was 0.056µM at pH 7.0, showing that bacteria incubated at pH 5.5 were more tolerant to monensin than those incubated at pH 7.0. Both ionophores as well as increased acidity caused decreased ammonia production. Both ionophores inhibited ammonia production by 56%, independently of pH. In cultures incubated at pH 5.5 compared to pH 7.0, ammonia production was decreased by 50.5%, independently of the ionophores. Therefore, effects of ionophores and acidity were additive, and the maximum inhibition occurred in the presence of an ionophore at low pH (75.2%). Microbial protein production was lowest when lasalocid was present in a low pH culture medium, causing inhibition of microbial growth.(AU)