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- Novel combination of feed enzymes to improve the degradation of Chlorella vulgaris recalcitrant cell wallPublication . Coelho, Diogo; Lopes, Paula A.; Cardoso, Vânia; Ponte, Patricia; Brás, Joana; Madeira, Marta S.; Alfaia, Cristina M.; Fontes, Carlos M.G.A.; Prates, José A.MABSTRACT - In this study, a rational combination of 200 pre-selected Carbohydrate-Active enzymes (CAZymes) and sulfatases were tested, individually or combined, according to their ability to degrade Chlorella vulgaris cell wall to access its valuable nutritional compounds. The disruption of microalgae cell walls by a four enzyme mixture (Mix) in comparison with the control, enabled to release up to 1.21g/L of reducing sugars (p<0.001), led to an eight-fold increase in oligosaccharides release (p<0.001), and reduced the fuorescence intensity by 47% after staining with Calcofuor White (p<0.001). The Mix treatment was successful in releasing proteins (p<0.001), some MUFA (p<0.05), and the benefcial 18:3n-3 fatty acid (p<0.05). Even if no variation was detected for chlorophylls (p>0.05), total carotenoids were increased in the supernatant (p<0.05) from the Mix treatment, relative to the control. Taken together, these results indicate that this four-enzyme Mix displays an efective capacity to degrade C. vulgaris cell wall. Thus, these enzymes may constitute a good approach to improve the bioavailability of C. vulgaris nutrients for monogastric diets, in particular, and to facilitate the cost-efective use of microalgae by the feed industry, in general.
- Impact of Chlorella vulgaris as feed ingredient and carbohydrases on the health status and hepatic lipid metabolism of finishing pigsPublication . Coelho, Diogo; Alfaia, Cristina; Lopes, Paula Alexandra; Pestana, José M.; Costa, Monica M.; Pinto, Rui M.; Almeida, Joao M.; Moreira, Olga; Fontes, Carlos M.G.A.; Prates, José A.MThe implication of high dietary level of Chlorella vulgaris, individually and supplemented with two carbohydrase mixtures, on pigs' health and liver metabolism was assessed in this study. Forty crossbred (Large White × Landrace sows crossed with Pietrain boars) entire male pigs were randomly allocated to the following feeding treatments (n = 10): cereal-soybean meal basal diet (control); basal diet with 5% C. vulgaris; basal diet with 5% C. vulgaris supplemented with 0.005% Rovabio® Excel AP; and basal diet with 5% C. vulgaris supplemented with 0.01% of a preselected four-CAZyme mixture. The trial lasted from 59.1 ± 5.69 kg of initial live weight to 101 ±1.9 kg of slaughter weight. Data indicate that this high dietary level of C. vulgaris has impact on several blood parameters of finishing pigs. However, the most relevant health outcome observed was a strong immunosuppressive effect promoted by the microalga, which increases pigs' susceptibility to infection diseases. In addition, the dietary incorporation of C. vulgaris reduced the systemic antioxidant capacity of pigs. In turn, the dietary supplementation with the four-CAZyme mixture promoted a clear decrease on some blood parameters compared with the control group. Regarding hepatic lipids, pigs fed C. vulgaris diets, had an increased hepatic content of n-3 PUFA, with a consequent decrease on the n-6/n-3 ratio. In conclusion, the use of C. vulgaris as feed ingredient appears to be safe under controlled experimental conditions. However, it is imperative test it in industrial production systems, with more stressful and less hygienic environments.
- Recalcitrant cell wall of Ulva lactuca seaweed is degraded by a single ulvan lyase from family 25 of polysaccharide lyasesPublication . Costa, Monica; Pio, Luís Bernardo; Bule, Pedro; Duarte, Marlene; Alfaia, Cristina; Coelho, Diogo; Bras, Joana; Fontes, Carlos M.G.A.; Prates, José A.M; Cardoso, VâniaABSTRACT - Green macroalgae, e.g., Ulva lactuca, are valuable bioactive sources of nutrients; but algae recalcitrant cell walls, composed of a complex cross-linked matrix of polysaccharides, can compromise their utilization as feedstuffs for monogastric animals. This study aimed to evaluate the ability of pre-selected Carbohy- drate-Active enZymes (CAZymes) and sulfatases to degrade U. lactuca cell walls and release nutritive compounds. A databank of 199 recombinant CAZymes and sulfatases was tested in vitro for their action towards U. lactuca cell wall polysaccharides. The enzymes were incubated with the macroalga, either alone or in combination, to release reducing sugars and decrease fluorescence intensity of Calcofluor White stained cell walls. The individual action of a polysaccharide lyase family 25 (PL25), an ulvan lyase, was shown to be the most efficient in cell wall disruption. The ulvan lyase treatment, in triplicate measures, promoted the release of 4.54 g/L (P < 0.001) reducing sugars, a mono- and oligosaccharides release of 11.4 and 11.2 mmol/100 g of dried alga (P < 0.01), respectively, and a decrease of 41.7% (P < 0.001) in cell wall fluorescence, in comparison to control. The ability of ulvan lyase treatment to promote the release of nutritional compounds from alga biomass was also evaluated. A release of some monounsaturated fatty acids was observed, particularly the health beneficial 18:1c9 (P < 0.001). How- ever, no significant release of total fatty acids (P > 0.05), proteins (P ¼ 0.861) or pigments (P > 0.05) was found. These results highlight the capacity of a single recombinant ulvan lyase (PL25 family) to incompletely disrupt U. lactuca cell walls. This enzyme could enhance the bioaccessibility of U. lactuca bioactive products with promising utilization in the feed industry.
- An individual alginate lyase is effective in the disruption of Laminaria digitata recalcitrant cell wallPublication . Costa, Monica; Pio, Luís Bernardo; Bule, Pedro; Cardoso, Vânia; Alfaia, Cristina; Coelho, Diogo; Brás, Joana; Fontes, Carlos M.G.A.; Prates, José A.MIn the present study, 199 pre-selected Carbohydrate-Active enZymes (CAZymes) and sulfatases were assessed, either alone or in combination, to evaluate their capacity to disrupt Laminaria digitata cell wall, with the consequent release of interesting nutritional compounds. A previously characterized individual alginate lyase, belonging to the family 7 of polysaccharide lyases (PL7) and produced by Saccharophagus degradans, was shown to be the most efcient in the in vitro degradation of L. digitata cell wall. The alginate lyase treatment, compared to the control, released up to 7.11 g/L of reducing sugars (p< 0.001) and 8.59 mmol/100 g dried alga of monosaccharides (p< 0.001), and reduced cell wall fuorescence intensity by 39.1% after staining with Calcofuor White (p= 0.001). The hydrolysis of gel-forming polymer alginate by the alginate lyase treatment could prevent the trapping of fatty acids and release benefcial monounsaturated fatty acids, particularly 18:1c9 (p < 0.001), to the extracellular medium. However, no liberation of proteins (p > 0.170) or pigments (p > 0.070) was observed. Overall, these results show the ability of an individual alginate lyase, from PL7 family, to partially degrade L. digitata cell wall under physiological conditions. Therefore, this CAZyme can potentially improve the bioavailability of L. digitata bioactive compounds for monogastric diets, with further application in feed industry.
- Impact of dietary Chlorella vulgaris and carbohydrate-active enzymes incorporation on plasma metabolites and liver lipid composition of broilersPublication . Coelho, Diogo; Alfaia, Cristina; Pestana, JMP; Costa, Mónica; Pinto, Rui; Fontes, Carlos M.G.A.; Lordelo, Madalena; Prates, José A.MBackground: Chlorella vulgaris has been proposed as a sustainable green feedstock in poultry nutrition due to its ease of cultivation, minimal environmental impact and balanced nutritional composition. However, the majority of studies documents the use of C. vulgaris as a dietary supplement in broilers instead of a feed ingredient. To the best of our knowledge, no report has shown the effect of a high-level incorporation (>2 % in the diet) of C. vulgaris on plasma metabolites and hepatic lipid composition of broilers. One hundred and twenty Ross 308 male birds were housed in 40 wired-floor cages and randomly distributed by the following experimental diets at 22 days of age (n = 10) during 15 days: (1) a corn-soybean meal based diet (control); (2) based diet with 10% of C. vulgaris; (3) diet 2 supplemented with 0.005% Rovabio® Excel AP; and (4) diet 2 supplemented with 0.01% of a pre-selected four-CAZyme mixture. Results: The inclusion of C. vulgaris at 10% in the diet, regardless of the presence of exogenous CAZymes, changed plasma metabolites but did not compromise broilers growth. Plasma total lipids increased in broilers fed C. vulgaris combined with the two feed CAZymes (p < 0.001) compared with the control diet. Moreover, the supplementation with Rovabio® increased total cholesterol and LDL-cholesterol, while the addition of the four-CAZyme mixture increased triacylglycerols, VLDL-cholesterol and ALP activity. In opposition, HDL-cholesterol levels decreased in broilers fed microalga alone (p = 0.002). Regarding hepatic composition, the inclusion of C. vulgaris in broiler diets, individually or combined with exogenous CAZymes, had a minor effect on fatty acids but improved the n-6/n-3 ratio and total carotenoids. Conclusions: In summary, the inclusion of a high level (10%) of C. vulgaris in broiler´s diet, regardless of the presence of exogenous CAZymes, improved hepatic antioxidant composition and did not impair broiler’s performance. In addition, the feed supplementation with CAZymes increased broilers lipemia. Therefore, dietary C. vulgaris at this incorporation level seems to be safe for animal health and do not compromise performance traits, with no need of CAZymes supplementation.