Suplementação nitrogenada com ureia comum ou encapsulada sobre parâmetros ruminais de novilhos alimentados com feno de baixa qualidade

Foi realizado um experimento de suplementação com novilhos fistulados no rúmen com o objetivo de verificar a utilização de ureia encapsulada como fonte de nitrogênio de liberação mais lenta e uniforme ao longo do tempo, bem como seu efeito sobre a degradabilidade da parede celular do feno. Os tratamentos foram: Feno + sal mineralizado (SM); Feno + suplemento proteico com ureia comum (SU); Feno + suplemento proteico com ureia encapsulada fórmula 1 (UE1); e Feno + suplemento proteico com ureia encapsulada fórmula 2 (UE2). O volumoso utilizado foi feno de Tifton (Cynodon dactylon L.) de baixa qualidade (PB: 4,62% e FDN: 83,46%). Foram realizadas medidas de pH e N-NH3 ruminais e parâmetros de degradação ruminal da FDN do volumoso. Verificou-se efeito (P<0,05) da suplementação nitrogenada sobre a concentração de nitrogênio amoniacal no rúmen; no entanto, a ureia encapsulada não foi diferente (P>0,05) da ureia comum. Os valores de pH e degradabilidade in situ não foram afetados pelos tratamentos (P>0,05), ao serem comparados os suplementados ou não suplementados com proteína degradável no rúmen e ao serem comparadas fontes de nitrogênio não proteico. A ureia encapsulada não demonstrou superioridade sobre a ureia comum, provavelmente pela baixa eficiência da sua proteção. A utilização de ureia encapsulada e a suplementação de proteína degradável não foram eficientes em aumentar a degradabilidade da parede celular do volumoso utilizado.

An Evaluation of Different Bioreactor Configurations with Immobilized Yeast for Bioethanol Production

The bioethanol industry expects a huge expansion and new technologies are being implemented with the aim of optimizing the fermentation process. The behavior of cells of Saccharomyces cerevisiae immobilized in PVA-LentiKats, during the production of bioethanol in two reactor systems, was studied. The entrapped cell in LentiKats lenses showed a different profile using stirred tank reactor (STR) and packed column reactor (PCR). Low free cells accumulation in the medium was observed for the STR after 72 h of fermentation. On the other hand, no free cells accumulation was observed, probably due to the absence of mechanical agitation in PCR configuration. Better fermentation results were obtained working with STR (final cellular concentration = 13 g.L-1, Pf = 28 g.L-1, Qp = 1.17 g.L-1.h-1,and Yp/s = 0.3 g.g-1) in comparison to PCR (final cellular concentration = 11.4 g.L-1, Pf = 20 g.L-1, Qp = 0.83 g.L-1.h-1,and Yp/s = 0.25 g.g-1). Such results are probably due to the mechanical agitation of the medium provided by STR configuration, which permitted a better heat and mass transference.

Production of Cellulolytic Enzymes by Anaerobic Fungi Cultivated in Different Conditions

The present paper studies the influence of different nutrients for the production of two cellulolytic enzymes: endo beta-1.4 glucanase and exo beta-1.4 glucanase by anaerobic fungi taken from cow rumen, that were fed a diet of corn silage and Brachiaria decumbens grass hay. During the enzymatic degradation assays, it was observed that the addition of some essential nutrients in the formulation of the culture medium contributed positively in the cellulolytic enzyme production, with exception of riboflavin. Such results contributed in the establishment of an effective method for the evaluation of enzymatic activities in anaerobic fibrolytic fungi. In this work, nutrients added to enrich the culture medium have successfully proven that they can be used as inoculating agents (inductors) in diets rich in ensilage with law nutritive value.

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus

Background: In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties. Results: The highest GOX expression levels (1552 units of secreted protein per gram dry cell weight) were achieved using an episomal system, in which the INU1 promoter and terminator were used to drive heterologous gene expression, together with the INU1 prepro sequence, which was employed to drive secretion of the enzyme. In all cases, GOX was mainly secreted, remaining either in the periplasmic space or in the culture supernatant. Whereas the use of genetic elements from Saccharomyces cerevisiae to drive heterologous protein expression led to higher expression levels in K. lactis than in K. marxianus, the use of INU1 genetic elements clearly led to the opposite result. The biochemical characterization of GOX confirmed the correct expression of the protein and showed that K. marxianus has a tendency to hyperglycosylate the protein, in a similar way as already observed for other yeasts, although this tendency seems to be smaller than the one of e. g. K. lactis and S. cerevisiae. Hyperglycosylation of GOX does not seem to affect its affinity for the substrate, nor its activity. Conclusions: Taken together, our results indicate that K. marxianus is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins.

Behavior and viability of spontaneous oxidative stress-resistant Lactococcus lactis mutants in experimental fermented milk processing

Previously, we isolated two strains of spontaneous oxidative (SpOx2 and SpOx3) stress mutants of Lactococcus lactis subsp cremoris. Herein, we compared these mutants to a parental wild- type strain (J60011) and a commercial starter in experimental fermented milk production. Total solid contents of milk and fermentation temperature both affected the acidification profile of the spontaneous oxidative stress- resistant L. lactis mutants during fermented milk production. Fermentation times to pH 4.7 ranged from 6.40 h (J60011) to 9.36 h (SpOx2); V(max) values were inversely proportional to fermentation time. Bacterial counts increased to above 8.50 log(10) cfu/mL. The counts of viable SpOx3 mutants were higher than those of the parental wild strain in all treatments. All fermented milk products showed post-fermentation acidification after 24 h of storage at 4 degrees C; they remained stable after one week of storage.

Increased CLA content in organic milk fermented by bifidobacteria or yoghurt cultures

This study investigates the kinetics of acidification, fatty acid (FA) profile and conjugated linoleic acid (CLA, C18:2 c9, t11) content in fermented milks prepared from organic and conventional milk. Fermented milks were manufactured with five mixed cultures: four different strains of Bifidobacterium animalis subsp. lactis (BL04, B94, BB12 and HN019) and Lactobacillus delbrueckii subsp. bulgaricus LB340, in co-culture with Streptococcus thermophilus TA040. The composition of milk was evaluated, and the kinetics of acidification was followed by continuous pH measurement using the Cinac system. The profile of FA, including CLA, was analyzed by gas chromatography. The chemical composition of conventional and organic milk was similar, with the exception of protein and Fe, the concentrations of which were higher in the organic milk. The rate of acidification was significantly influenced by the type of milk and the bacterial strain used. Co-cultures St-HN019 and St-BB12 showed higher maximal acidification rates in both milks. Final counts of S. thermophilus (9.0-10.1 log(10) colony forming units (CFU) . mL(-1), L)actobacillus bulgaricus (8.2-8.5 log(10) CFU . mL(-1)) and B. animalis subsp. lactis strains (8.3-9.3 log(10) CFU . mL(-1)) did not differ significantly in either milk. Unexpectedly, all fermented organic milks contained significantly higher amounts of CLA than the same milk before fermentation, whereas CLA amounts did not change during fermentation of conventional milk. Regardless of the type of milk, CLA was found to be significantly positively correlated with trans-vaccenic acid and negatively correlated with linoleic acid. Moreover, the CLA contents were significantly higher in fermented milks showing shorter fermentation times.

Use of Cassava Peel as Carbon Source for Production of Amylolytic Enzymes by Aspergillus niveus

Aspergillus niveus produced high levels of alpha-amylase and glucoamylase in submerged fermentation using the agricultural residue cassava peel as a carbon source. In static conditions, the amylase production was substantially greater than in the agitated condition. The optimized culture conditions were initially at pH 5.0, 35 degrees C during 48 hours. Amylolytic activity was still improved (50%) with a mixture of cassava peel and soluble starch in the proportion 1:1 (w/w). The crude extract exhibited temperature and pH optima approximately 70 degrees C and 4.5, respectively. Amylase activity was stable for 1 h at 60 degrees C, and at pH values between 3.0 and 7.0. The enzyme hydrolysed preferentially maltose, starch, penetrose, amylose, isomaltose, maltotriose, glycogen and amylopectin, and not hydrolysed cyclodextrin (alpha and beta), trehalose and sucrose. In the first hour of reaction on soluble starch, the hydrolysis products were glucose and maltose, but after two hours of hydrolysis, glucose was the unique product formed, confirming the presence in the crude extract of an alpha-amylase and a glucoamylase.

Influence of a polyclonal antibody preparation on the in situ degradability of three energy sources

The objective of this study was to evaluate the effect of a polyclonal antibody preparation (PAP) against specific ruminal bacteria on the in situ degradability of dry-grounded maize grain (DMG), high moisture maize silage (HMMS) starch and citrus pulp (CiPu) pectin. Nine ruminally cannulated cows were used in a 3 x 3 Latin square design, replicated three times in a factorial arrangement of treatments of two rumen modifiers represented by monensin and PAP plus a control group, and the three energy sources (DMG, HMMS and CiPu). Each period had 21 days, where 16 were used for adaptation to treatment and five for data collection. The group treated with PAP showed an effect on the soluble fraction (""a"") of DMG starch, decreasing it by respectively 45.3% and 45.4% compared to the CON and MON groups. No effect of PAP was observed for any in situ degradability parameters of starch from HMMS or pectin of CiPu. It was concluded that the polyclonal antibody preparation had limited effect on the in situ degradability of the tested energy sources.

Influence of palm fatty acid distillate on rumen degradability and protozoa population in buffaloes

Oil and fat as energy sources at low cost are relevant in ruminant nutrition. The aim of this study was to evaluate the effects of palm fatty acid distillate (PFAD) on the degradability and ciliate protozoa population in buffalo. Four rumen fistulated buffaloes were fed a basal diet in a Latin square (4x4) design trial. Treatments were designed with four of different levels of PFAD added directly into the rumen: 0; 200; 420 and 500 g/animal/d. High levels of PFAD (420 and 500 g/d) promoted higher degradation of the soluble fraction and lower in potentially degradable fraction of dry matter (DM) and neutral detergent fibre (NDF) with lower values of potential and effective degradability in two evaluated grasses, bermudagrass and brachiariagrass. Significant decreases in the total number of protozoa/mL of rumen content, Entodinium and ciliates belonging to subfamily Diplodiniinae were observed at higher level of PFDA addition in the rumen. Also, Epidinium and Holotrich ciliates disappeared from the rumen. Significant correlations were observed of the ciliate concentration and composition as a function of dietary lipids content. Entodinium composition increased from 68.0% to 99.6% and Diplodiniinae reduced from 30.4% to 0.4% with increasing PFAD level indicating higher fat toxicity effect on the Diplodiniinae ciliates than Entodinium species and direct action of the larger ciliates on the fibre degradation.

Complete Genome Sequence of Mycoplasma suis and Insights into Its Biology and Adaption to an Erythrocyte Niche

Mycoplasma suis, the causative agent of porcine infectious anemia, has never been cultured in vitro and mechanisms by which it causes disease are poorly understood. Thus, the objective herein was to use whole genome sequencing and analysis of M. suis to define pathogenicity mechanisms and biochemical pathways. M. suis was harvested from the blood of an experimentally infected pig. Following DNA extraction and construction of a paired end library, whole-genome sequencing was performed using GS-FLX (454) and Titanium chemistry. Reads on paired-end constructs were assembled using GS De Novo Assembler and gaps closed by primer walking; assembly was validated by PFGE. Glimmer and Manatee Annotation Engine were used to predict and annotate protein-coding sequences (CDS). The M. suis genome consists of a single, 742,431 bp chromosome with low G+C content of 31.1%. A total of 844 CDS, 3 single copies, unlinked rRNA genes and 32 tRNAs were identified. Gene homologies and GC skew graph show that M. suis has a typical Mollicutes oriC. The predicted metabolic pathway is concise, showing evidence of adaptation to blood environment. M. suis is a glycolytic species, obtaining energy through sugars fermentation and ATP-synthase. The pentose-phosphate pathway, metabolism of cofactors and vitamins, pyruvate dehydrogenase and NAD(+) kinase are missing. Thus, ribose, NADH, NADPH and coenzyme A are possibly essential for its growth. M. suis can generate purines from hypoxanthine, which is secreted by RBCs, and cytidine nucleotides from uracil. Toxins orthologs were not identified. We suggest that M. suis may cause disease by scavenging and competing for host nutrients, leading to decreased life-span of RBCs. In summary, genome analysis shows that M. suis is dependent on host cell metabolism and this characteristic is likely to be linked to its pathogenicity. The prediction of essential nutrients will aid the development of in vitro cultivation systems.

Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianum

The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source.

A new approach for in vitro bioassay to measure tannin biological effects based on a gas production technique

The aim of this paper was to study a method based on gas production technique to measure the biological effects of tannins on rumen fermentation. Six feeds were used as fermentation substrates in a semi-automated gas method: feed A - aroeira (Astronium urundeuva); feed B - jurema preta (Mimosa hostilis), feed C - sorghum grains (Sorghum bicolor); feed D - Tifton-85 (Cynodon sp.); and two others prepared mixing 450 g sorghum leaves, 450 g concentrate (maize and soybean meal) and 100 g either of acacia (Acacia mearnsii) tannin extract (feed E) or quebracho (Schinopsis lorentzii) tannin extract (feed F) per kg (w:w). Three assays were carried out to standardize the bioassay for tannins. The first assay compared two binding agents (polyethylene glycol - PEG - and polyvinyl polypirrolidone - PVPP) to attenuate the tannin effects. The complex formed by PEG and tannins showed to be more stable than PVPP and tannins. Then, in the second assay, PEG was used as binding agent, and this assay was done to evaluate levels of PEG (0, 500, 750, 1000 and 1250 mg/g DM) to minimize the tannin effect. All the tested levels of PEG produced a response to evaluate tannin effects but the best response was for dose of 1000 mg/g DM. Using this dose of PEG, the final assay was carried out to test three compounds (tannic acid, quebracho extract and acacia extract) to establish a curve of biological equivalent effect of tannins. For this, five levels of each compound were added to I g of a standard feed (Lucerne hay). The equivalent effect showed not to be directly related to the chemical analysis for tannins. It was shown that different sources of tannins had different activities or reactivities. The curves of biological equivalence can provide information about tannin reactivity and its use seems to be important as an additional factor for chemical analysis. (C) 2007 Elsevier B.V. All rights reserved.

A novel use for sugarcane bagasse hemicellulosic fraction: Xylitol enzymatic production

The excess of sugarcane bagasse (SCB) from the sugar-alcohol industry is considered a by-product with great potential for many bioproducts production. This work had as objective to verify the performance of sugarcane bagasse hemicellulosic hydrolysate (SCBHH) as source of sugars for enzymatic or in vitro xylitol production. For this purpose, xylitol enzymatic production was evaluated using different concentrations of treated SCBHH in the commercial reaction media. The weak acid hydrolysis of SCB provided a hydrolysate with 18 g L(-1) and 6 g L(-1) of xylose and glucose, respectively. Considering the reactions, changes at xylose xylitol conversion efficiency and volumetric productivity in xylitol were not observed for the control experiment and using 20 and 40% v.v (1) of SCBHH in the reaction media. The conversion efficiency achieved 100% in all the experiments tested. The results showed that treated SCBHH is suitable as xylose and glucose source for the enzymatic xylitol production and that this process has potential as an alternative for traditional xylitol production ways. (C) 2011 Published by Elsevier Ltd.

Profiles of xylose reductase, xylitol dehydrogenase and xylitol production under different oxygen transfer volumetric coefficient values

BACKGROUND: Xylitol is a sugar alcohol (polyalcohol) with many interesting properties for pharmaceutical and food products. It is currently produced by a chemical process, which has some disadvantages such as high energy requirement. Therefore microbiological production of xylitol has been studied as an alternative, but its viability is dependent on optimisation of the fermentation variables. Among these, aeration is fundamental, because xylitol is produced only under adequate oxygen availability. In most experiments with xylitol-producing yeasts, low oxygen transfer volumetric coefficient (K(L)a) values are used to maintain microaerobic conditions. However, in the present study the use of relatively high K(L)a values resulted in high xylitol production. The effect of aeration was also evaluated via the profiles of xylose reductase (XR) and xylitol clehydrogenase (XD) activities during the experiments. RESULTS: The highest XR specific activity (1.45 +/- 0.21 U mg(protein)(-1)) was achieved during the experiment with the lowest K(L)a value (12 h(-1)), while the highest XD specific activity (0.19 +/- 0.03 U mg(protein)(-1)) was observed with a K(L)a value of 25 h(-1). Xylitol production was enhanced when K(L)a was increased from 12 to 50 h(-1), which resulted in the best condition observed, corresponding to a xylitol volumetric productivity of 1.50 +/- 0.08 g(xylitol) L(-1) h(-1) and an efficiency of 71 +/- 6.0%. CONCLUSION: The results showed that the enzyme activities during xylitol bioproduction depend greatly on the initial KLa value (oxygen availability). This finding supplies important information for further studies in molecular biology and genetic engineering aimed at improving xylitol bioproduction. (C) 2008 Society of Chemical Industry

Batch cooling crystallization of xylitol produced by biotechnological route

BACKGROUND: This work deals with the xylitol production by biotechnological routes emphasizing the purification process using crystallization. RESULTS: Xylitol volumetric productivity of 0.665 g L(-1) h(-1) and yield of 0.7024 g g(-1) were obtained after 92 h fermentation. The fermented broth (61.3 g L(-1) xylitol) was centrifuged, treated and concentrated obtain a syrup (745.3 g L(-1) xylitol) which was crystallized twice, xylitol crystals with 98.5-99.2% purity being obtained. CONCLUSION: The hypothetical distribution obtained permits the determination of modeling parameters, which make possible the estimation of crystal dominant size from different initial experimental conditions. (C) 2008 Society of Chemical Industry