L. M. A. Ferreira

Effect of Dietary Dehydrated Pasture and Citrus Pulp on the Performance and Meat Quality of Broiler Chickens

Some feedstuffs containing significant levels of fiber may be a good source of bioactive compounds that may contribute to improving broiler meat quality. However, high fiber level can have a negative impact on broiler performance. A study was undertaken to investigate the impact of incorporating citrus pulp (5 or 10%) or dehydrated pasture (5 or 10%) on the performance, carcass yield, and characteristics of broiler chickens. A diet containing neither citrus pulp nor dehydrated pasture was used as control. The results on growth performances showed that daily weight gain was reduced by 26% in birds of the 10% citrus pulp treatment (P<0.05). Compared with the control treatment, increases in feed intake occurred in birds consuming diets with 5 or 10% citrus pulp, which resulted in significantly higher feed conversion rates with the 10% level. Under the same incorporation rate, dehydrated pasture had effects less evident on the performances of broiler chicken. In addition, diets containing citrus pulp, displaying higher percentages of soluble nonstarch polysaccharides, increased small intestine relative length, and reduced carcass yield. Inclusion of 10% dehydrated pasture in diets resulted in improved breast skin yellowness (P<0.05). Finally, the results revealed that incorporation of the nonstarch polysaccharide-rich feedstuffs had a major impact on the fatty acid profile (affected 16 of 21 fatty acids) of broiler meat. Polyunsaturated fatty acids content in meat was higher in birds consuming the highest levels of both citrus pulp and dehydrated pasture, leading to increased ratios of polyunsaturated to saturated fatty acids. Together, the results suggest that incorporation of moderate levels of dehydrated pastures in poultry diets has a minor impact on broiler performance and can contribute significantly to improve breast skin yellowness and fatty acid composition of meat.

A novel Cellvibrio mixtus family 10 xylanase that is both intracellular and expressed under non-inducing conditions

Hydrolysis of the plant cell wall polysaccharides cellulose and xylan requires the synergistic interaction of a repertoire of extracellular enzymes. Recently, evidence has emerged that anaerobic bacteria can synthesize high levels of periplasmic xylanases which may be involved in the hydrolysis of small xylo-oligosaccharides absorbed by the micro-organism. Cellvibrio mixtus, a saprophytic aerobic soil bacterium that is highly active against plant cell wall polysaccharides, was shown to express internal xylanase activity when cultured on media containing xylan or glucose as sole carbon source. A genomic library of C. mixtus DNA, constructed in lambdaZAPII, was screened for xylanase activity. The nucleotide sequence of the genomic insert from a xylanase-positive clone that expressed intracellular xylanase activity in Escherichia coli revealed an ORF of 1137 bp (xynC), encoding a polypeptide with a deduced M(r) of 43413, defined as xylanase C (XylC). Probing a gene library of Pseudomonas fluorescens subsp. cellulosa with C. mixtus xynC identified a xynC homologue (designated xynG) encoding XylG; XylG and xynG were 67% and 63% identical to the corresponding C. mixtus sequences, respectively. Both XylC and XylG exhibit extensive sequence identity with family 10 xylanases, particularly with non-modular enzymes, and gene deletion studies on xynC supported the suggestion that they are single-domain xylanases. Purified recombinant XylC had an M(r) of 41000, and displayed biochemical properties typical of family 10 polysaccharidases. However, unlike previously characterized xylanases, XylC was particularly sensitive to proteolytic inactivation by pancreatic proteinases and was thermolabile. C. mixtus was grown to late-exponential phase in the presence of glucose or xylan and the cytoplasmic, periplasmic and cell envelope fractions were probed with anti-XylC antibodies. The results showed that XylC was absent from the culture media but was predominantly present in the periplasm of C. mixtus cells grown on glucose, xylan, CM-cellulose or Avicel. These data suggest that C. mixtus can express non-modular internal xylanases whose potential roles in the hydrolysis of plant cell wall components are discussed.

Restricting the Intake of a Cereal-Based Feed in Free-Range-Pastured Poultry: Effects on Performance and Meat Quality

Pastures are assumed to be good sources of alpha-linolenic acid (ALA) and other bioactive compounds. In this study, we evaluated the effects of restricting the intake of a cereal-based feed on the consumption of a legume-based pasture, and consequently on poultry performance and meat quality. Broilers of the RedBro Cou Nu x RedBro M genotype were fed a cereal-based feed at different intake restriction levels (100, 75, or 50% of ad libitum intake) in portable floorless pens located on a subterranean clover (Trifolium subterraneum) pasture. Control birds were maintained at the same site in identical pens but had no access to pasture. The results revealed that, although the growth rate achieved was below the levels expected for the genotype, restriction of cereal-based feed intake had a significant impact on broiler weight gain and feed conversion while leading to an increase in relative leguminous pasture intake (from 1.6 to 4.9% of the total intake, on a DM basis). In addition, bird performance was positively influenced by pasture consumption. The capacity of ingested pasture to modulate carcass characteristics, broiler meat fatty acid profiles, and the meat content of total cholesterol, tocopherols, and to-cotrienols was investigated in broiler chickens slaughtered on d 64. Pasture intake decreased carcass yield (P < 0.05) and meat pH (P < 0.001) and improved breast skin pigmentation (P < 0.001). Consumption of the leguminous pasture had a marginal effect in the vitamin E profiles and cholesterol contents of broiler meat (P < 0.05), although it significantly affected the meat fatty acid profile. Although pasture intake did not influence the linoleic acid content of poultry meat, the levels of n-3 polyunsaturated fatty acids in breast meat [ALA (P < 0.001), eicosapentaenoic acid (P < 0.001), docosapentaenoic acid (P < 0.001), and docosahexaenoic acid (P < 0.001)] were significantly greater in birds consuming the leguminous biomass. Overall, the data suggest an important deposition of ALA and some conversion of ALA to its derivatives in pastured broilers subjected to a restriction of cereal-based feed.

A family 6 carbohydrate-binding module potentiates the efficiency of a recombinant xylanase used to supplement cereal-based diets for poultry

1. Cellulases and xylanases display a modular architecture that comprises a catalytic module linked to one or more non-catalytic carbohydrate-binding modules (CBMs). On the basis of primary structure similarity, CBMs have been classified into more than 30 different families. These non-catalytic modules mediate a prolonged and intimate contact of the enzyme with the target substrate, eliciting efficient hydrolysis of the insoluble polysaccharides. 2. Xylanases are very effective in improving the nutritive value of wheat- or rye-based diets for broiler chicks although the role of non-catalytic CBMs in the function of exogenous modular xylanases in vivo remains to be determined. 3. A study was undertaken to investigate the importance of a family 6 CBM in the function of recombinant derivatives of xylanase 11A (Xyn11A) of Clostridium thermocellum used to supplement cereal-based diets for poultry. 4. The data show that birds fed on a wheat-based diet supplemented with the modular xylanase display an increased final body weight when compared with birds receiving Xyn11A catalytic module or birds receiving the enzyme mixture Roxazyme® G. 5. Interestingly, the modular xylanase was truncated and transformed into its single domain counterpart on the duodenum of birds fed on the wheat-based diets, most possibly due to the action of pancreatic proteases. 6. Together the data point to the importance of CBMs in the function of feed xylanases and suggest, that in chicken fed on wheat-based diets, the main sites for exogenous enzymes action might be the gastrointestinal (GI) compartments preceding the duodenum, most probably the crop.

Possible roles for a non-modular, thermostable and proteinase-resistant cellulase from the mesophilic aerobic soil bacterium Cellvibrio mixtus

Abstract The widespread presence of cellulose-binding domains in cellulases from aerobic bacteria and fungi suggests the existence of a strong selective pressure for the retention of these non-catalytic modules. The complete nucleotide sequence of the cellulase gene, celA, from the aerobic soil bacterium Cellvibrio mixtus, was determined. It revealed an open reading frame of 1089 bp that encoded a polypeptide, defined as cellulase A (CelA), of Mr 41 548. CelA displayed features characteristic of an endo-β-1,4-glucanase, rapidly decreasing the viscosity of the substrate while releasing only moderate amounts of reducing sugar. Deletion studies in celA revealed that removal of 78 nucleotides from the 5′ end or 75 from the 3′ end of the gene led to the complete loss of cellulase activity of the encoded polypeptides. The deduced primary structure of CelA revealed an N-terminal signal peptide followed by a region that exhibited significant identity with the catalytic domains of cellulases belonging to glycosyl hydrolase family 5. These data suggest that CelA is a single-domain endoglucanase with no distinct non-catalytic cellulose-binding domain. Analysis of the biochemical properties of CelA revealed that the enzyme hydrolyses a range of soluble cellulosic substrates, but was inactive against Avicel, xylan or any other hemicellulose. CelA was resistant to proteolytic inactivation by pancreatic proteinases and surprisingly, in view of its mesophylic origin, was shown to be thermostable. The significance of these findings in relation to the role of single-domain cellulases in plant cell wall hydrolysis by aerobic microorganisms is discussed.

Understanding How Noncatalytic Carbohydrate Binding Modules Can Display Specificity for Xyloglucan

Background: Carbohydrate binding modules (CBMs) contribute to the enzymatic degradation of complex polysaccharide structures. Results: New CBMs display specificity for decorated glucans through an extensive hydrophobic platform that interacts with both backbone and side chain structures. Conclusion: CBMs that bind to complex β-glucans exploit different components of these ligands as specificity determinants. Significance: CBMs can utilize the side chains of decorated glucans as specificity determinants. Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind β-glucan chains often display broad specificity recognizing β1,4-glucans (cellulose), β1,3-β1,4-mixed linked glucans and xyloglucan, a β1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the β1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of β-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln106 is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans.

Improving the Lipid Nutritive Value of Poultry Meat Through the Incorporation of a Dehydrated Leguminous-Based Forage in the Diet for Broiler Chicks

Dehydrated forages are assumed to be good sources of alpha-linolenic acid (ALA) and lipid-soluble antioxidant compounds (vitamin E homologs and beta-carotene). The effects of including a dehydrated leguminous-based forage in a typical diet for broiler chicken, on performance, meat quality, and fatty acid composition were evaluated. One hundred sixty 1-d-old male commercial broiler chicks (Ross 308) were housed in 20 battery brooders. During the 28-d growth period, the animals were fed ad libitum with a typical maize-soybean high-energy feed having access or not to a dehydrated leguminous-based forage provided in a separate feeder. The results revealed that dehydrated forage intake (which was 11.1% of the total intake) had no impact in broiler performance (P > 0.05). The capacity of ingested forage to modulate broiler meat fatty acid profile and the meat content in total cholesterol, tocopherols, tocotrienols, and beta-carotene was investigated in broiler chicks slaughtered at d 28. Dehydrated forage consumption had no effect on the lipid-soluble antioxidant compounds and cholesterol contents of broiler meat but had a significant effect on meat fatty acid profile. Although forage intake did not affect the linoleic acid and ALA contents in poultry meat, the levels of n-3 long-chain polyunsaturated fatty acids [eicosapentaenoic (P = 0.004), docosapentaenoic (P = 0.010), and docosahexaenoic (P = 0.007)] in breast meat were significantly higher in animals consuming leguminous biomass, which suggest a higher conversion of ALA into its derivatives in these birds. Overall, the data confirms that incorporation of a dehydrated leguminous-based forage in the diet for broiler chicks results in more favorable polyunsaturated fatty acids/saturated fatty acids and n-6/n-3 nutritional ratios for animals slaughtered at earlier stages of grow.

Direct supplementation of diet is the most efficient way of enriching broiler meat with n-3 long-chain polyunsaturated fatty acids.

Abstract 1. Concentrations of beneficial omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs) in poultry meat can be improved by increasing the concentration of n-3 PUFA in poultry diets. 2. A decrease in flavour quality is, however, usually associated with the dietary supplementation with n-3 PUFA, which is due to the susceptibility of PUFA to oxidation. 3. This experiment was conducted to study the effects of introducing two different n-3 fatty acid sources (extruded linseed and DHA Gold™, a proprietary algal product rich in docosahexaenoic acid), either separately or together, on broiler productive performance, and meat quality, oxidative stability, sensory traits and LC-PUFA profile. 4. Birds given the algal product displayed better productive performances than animals from other groups. 5. The data revealed an improvement in the fatty acid nutritional value of meat from birds receiving the algal product and an inefficient conversion of α-linolenic acid (LNA) into LC-PUFA. 6. Metabolisation of LNA in vivo is not sufficient to improve meat quality in n-3 LC-PUFA and direct supplementation of the diet with n-3 LC-PUFA is a better alternative to modulate an increase in beneficial fatty acids of broiler meat. 7. The overall acceptability of meat was negatively affected by the dietary supplementation with 7.4% of DHA, in contrast to the supplementation with 3.7% of DHA, which showed to be efficient in improving LC-PUFA meat content without affecting its sensory properties.

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

Significance Plant cell wall (PCW) polysaccharide degradation is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Ruminococcus flavefaciens synthesizes a highly efficient PCW degrading apparatus. Here, six previously unidentified R. flavefaciens CBM families were identified that targeted β-glucans, β-mannans, and pectins. Crystal structures of these CBMs revealed that recognition of β-glucans and β-mannans was mediated by differences in the conformation of conserved aromatic residues in the ligand binding cleft. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan. This report shows that the expansion of protein modules in the cellulosome of R. flavefaciens contributes to an extended CBM profile that supports efficient PCW degradation. The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBM families that targeted β-glucans, β-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize β-glucans and β-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

Identification of tandemly repeated type VI cellulose-binding domains in an endoglucanase from the aerobic soil bacterium Cellvibrio mixtus.

Abstract Cellulose-binding domains (CBD) play a pivotal role during plant cell wall hydrolysis by cellulases and xylanases from aerobic soil bacteria. Recently we␣have reported the molecular characterisation of a single-domain endoglucanase from Cellvibrio mixtus, suggesting that some cellulases produced by this aerobic bacterium preferentially hydrolyse soluble cellulosic substrates. Here we describe the complete nucleotide sequence of a second cellulase gene, celB, from the soil bacterium C. mixtus. It revealed an open reading frame of 1863 bp that encoded a polypeptide, defined as cellulase B (CelB), with a predicted Mr of 66 039. CelB contained a glycosyl hydrolase family 5 catalytic domain at its N terminus followed by two repeated domains, which exhibited sequence identity with type VI CBD previously found in xylanases. Full-length CelB bound to cellulose while catalytically active truncated cellulase derivatives were unable to bind the polysaccharide, confirming that CelB is a modular enzyme and that the type VI CBD homologues were functional. Analysis of the biochemical properties of CelB revealed that the enzyme hydrolyses a range of cellulosic substrates, although it was unable to depolymerise Avicel. We propose that type VI CBD, usually found in xylanases, provide an additional mechanism by which cellulases can accumulate on the surface of the plant cell wall, although they do not potentiate cellulase activity directly. These results demonstrate that C. mixtus, in common with other aerobic bacteria, is able to produce cellulases that are directed to the hydrolysis of cellulose in its natural environment, the plant cell wall.