Optimization of exogenous carbohydrases supplemented in broiler diets using in vitro simulated gastrointestinal digestion and response surface methodology.

The present study aimed to explore the optimal zymogram of combination of 6 carbohydrases (glucoamylase, pullulanase, maltase, thermostable α-amylase, medium temperature α-amylase, and cold-active α-amylase) supplemented in corn-soybean based diet of broilers aged 1 to 3 wk for the maximum starch digestibility, by using in vitro simulated gastrointestinal digestion and response surface method. The third generation of simulated monogastric animal digestion system was used for in vitro digestion experiment. By using single factor completely random design, the optimal supplement levels of single carbohydras were determined by the reducing sugar release amount and improved dry matter digestibility, which were the parameters representing the starch digestibility of the diet. Additionally, Box-Behnken response surface method was used to predict the optimal combination of 6 carbohydrases. The results showed that the optimistic zymogram of 6 carbohydrases in corn-soybean based diet for broilers aged 1 to 3 wk were 297.39 U/g glucoamylase, 549.72 U/g pullulanase, 3.01 U/g maltase, 1,455.73 U/g thermostable α-amylase, 278.64 U/g medium temperature α-amylase, and 1,985.97 U/g cold-active α-amylase, and the associated reduced sugar release amount and improved dry matter digestibility were 215.98 mg/g, and 6.23%, respectively. Furthermore, we conducted in vitro digestion experiments with diets supplemented with the predicted optimistic zymogram and found that the experimental reduced sugar release amount and improved dry matter digestibility were 219.26 mg/g and 6.31% respectively, whose errors to the predicted optimistic reducing sugar release amount and the improved dry matter digestibility were 1.05% and 1.02%. To sum up, the predicted optimal zymogram of 6 carbohydrases in the present study were capable to improve the starch digestibility in diet for broilers aged 1 to 3 wk, which were represented by increased reduced sugar release amount and improved dry matter digestibility.

Preclinical Evaluation of Recombinant Microbial Glycoside Hydrolases in the Prevention of Experimental Invasive Aspergillosis.

Aspergillus fumigatus is a ubiquitous mold that can cause invasive pulmonary infections in immunocompromised patients. Within the lung, A. fumigatus forms biofilms that can enhance resistance to antifungals and immune defenses. Aspergillus biofilm formation requires the production of a cationic matrix exopolysaccharide, galactosaminogalactan (GAG). In this study, recombinant glycoside hydrolases (GH)s that degrade GAG were evaluated as antifungal agents in a mouse model of invasive aspergillosis. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that although GHs have short half-lives, GH prophylaxis resulted in reduced fungal burden in leukopenic mice and improved survival in neutropenic mice, possibly through augmenting pulmonary neutrophil recruitment. Combining GH prophylaxis with posaconazole treatment resulted in a greater reduction in fungal burden than either agent alone. This study lays the foundation for further exploration of GH therapy in invasive fungal infections. IMPORTANCE The biofilm-forming mold Aspergillus fumigatus is a common causative agent of invasive fungal airway disease in patients with a compromised immune system or chronic airway disease. Treatment of A. fumigatus infection is limited by the few available antifungals to which fungal resistance is becoming increasingly common. The high mortality rate of A. fumigatus-related infection reflects a need for the development of novel therapeutic strategies. The fungal biofilm matrix is in part composed of the adhesive exopolysaccharide galactosaminogalactan, against which antifungals are less effective. Previously, we demonstrated antibiofilm activity with recombinant forms of the glycoside hydrolase enzymes that are involved in galactosaminogalactan biosynthesis. In this study, prophylaxis with glycoside hydrolases alone or in combination with the antifungal posaconazole in a mouse model of experimental aspergillosis improved outcomes. This study offers insight into the therapeutic potential of combining biofilm disruptive agents to leverage the activity of currently available antifungals.

Supplemental Bacillus subtilis DSM 29784 and enzymes, alone or in combination, as alternatives for antibiotics to improve growth performance, digestive enzyme activity, anti-oxidative status, immune response and the intestinal barrier of broiler chickens.

The present study investigated the effect of Bacillus subtilis DSM 29784 (Ba) and enzymes (xylanase and ß-glucanases; Enz), alone or in combination (BE) as antibiotic replacements, on the growth performance, digestive enzyme activity, immune response and the intestinal barrier of broiler chickens. In total, 1200 1-d-old broilers were randomly assigned to five dietary treatments, each with six replicate pens of forty birds for 63 d as follows: (a) basal diet (control), supplemented with (b) 1 × 109 colony-forming units (cfu)/kg Ba, (c) 300 mg/kg Enz, (d) 1 × 109 cfu/kg Ba and 300 mg/kg Enz and (e) 250 mg/kg enramycin (ER). Ba, Enz and BE, similar to ER, decreased the feed conversion rate, maintained intestinal integrity with a higher villus height:crypt depth ratio and increased the numbers of goblet cells. The BE group exhibited higher expression of claudin-1 and mucin 2 than the other four groups. BE supplementation significantly increased the α-diversity and ß-diversity of the intestinal microbiota and markedly enhanced lipase activity in the duodenal mucosa. Serum endotoxin was significantly decreased in the BE group. Compared with those in the control group, increased superoxide dismutase and glutathione peroxidase activities were observed in the jejunal mucosa of the Ba and BE groups, respectively. In conclusion, the results suggested that dietary treatment with Ba, Enz or BE has beneficial effects on growth performance and anti-oxidative capacity, and BE had better effects than Ba or Enz alone on digestive enzyme activity and the intestinal microbiota. Ba or Enz could be used as an alternative to antibiotics for broiler chickens.

Effect of cereal fermentation and carbohydrase supplementation on growth, nutrient digestibility and intestinal microbiota in liquid-fed grow-finishing pigs.

This study aimed to determine the impact of fermenting the cereal fraction of the diet (Cferm) and enzyme supplementation (ENZ) on the bacterial composition of the feed, nutrient digestibility, pig growth, feed efficiency (FE), intestinal volatile fatty acid (VFA) concentrations and intestinal microbiota composition. A total of 252 grow-finisher pigs (~ 40.4 kg; 7 pigs/pen) were randomly allocated to 4 diets in a 2 × 2 factorial arrangement for 55d. The diets were: (1) fresh liquid feed (Fresh); (2) Cferm liquid feed (Ferm); (3) Fresh + ENZ and (4) Ferm + ENZ. Cferm increased total tract nutrient digestibility, reduced caecal butyrate and propionate concentrations, and increased average daily gain (ADG). ENZ increased ileal and total tract nutrient digestibility, reduced caecal isobutyrate and propionate concentrations, and improved FE. Bacterial taxa positively correlated with pig growth (Lactobacillus kisonensis in the ileum and Roseburia faecis in the caecum) were more abundant in pigs fed ENZ diets, whereas most of the ileal bacterial taxa negatively correlated with growth (Megasphaera, Bifidobacterium and Streptococcus) had lower abundance in pigs fed Cferm diets. In conclusion, Cferm increased ADG and ENZ improved FE, with these improvements possibly mediated by increased nutrient digestibility, and beneficial modulation of the intestinal microbiota.

Valores energéticos e composição bromatológica do resíduo seco de fecularia associado a carboidrases para frangos de corte em fase de crescimento / [Energy value and bromatological composition of dry residue of cassava associated with carbohydrases for grower broilers]

Foram determinados os valores energéticos e a composição bromatológica do resíduo seco de fecularia (RSF) para frangos de corte, na fase de crescimento, utilizando ou não enzimas carboidrases. Os tratamentos foram distribuídos em esquema fatorial 2x4 + ração referência, sendo uma RR sem adição de RSF e quatro tratamentos experimentais com 10%, 20%, 30% e 40% de inclusão do RSF e a suplementação ou não com carboidrases. A composição química encontrada para o RSF, na MN, foi de 89,86% de matéria seca, 0,98% de proteína bruta, 3519kcal kg-1 de energia bruta, 0,19% de extrato etéreo, 27% de fibra em detergente neutro, 19,5% de fibra em detergente ácido, 0,33% de cálcio, 0,43% de fósforo, 0,46% de potássio e 0,12% de magnésio. O uso de carboidrases proporcionou um aumento de 173 e 213kcal kg-1 nos valores de EMA e EMAn, respectivamente, resultando em 1828kcal kg-1 EMA e 1840kcal kg-1 EMAn. Concluiu-se que os maiores níveis de EMA e EMAn foram encontrados para o nível de inclusão médio do RSF de 35% e que a suplementação enzimática pode promover aumento desses parâmetros em até 12% em dietas para frangos de corte na fase de crescimento.(AU)

The energetic values and the bromatological composition of the dry residue of cassava (DRC) were determined for growing broilers with or without carbohydrase enzymes. The treatments were distributed in a 2x4 + reference diet factorial scheme, with one RD without addition of DRC and four experimental treatments with 10, 20, 30 and 40% inclusion levels of RSF and supplementation or not with carbohydrases. The chemical composition found for DRC in natural matter was 89.86% dry matter, 0.98% crude protein, 3519kcal kg-1 gross energy, 0.19% ether extract, 27% neutral detergent fiber, 19.5% of acid detergent fiber, 0.33% of calcium, 0.43% of phosphorus, 0.46% of potassium and 0.12% of magnesium. The use of carbohydrase resulted in an increase of 173 and 213kcal kg-1 in EMA and EMAn values, respectively, resulting in 1828kcal kg-1 EMA and 1840kcal kg-1 EMAn. It was concluded that the highest levels of AME and AMEn were found for the mean inclusion level of the DRC of 35% and that enzymatic supplementation may promote the increase of these parameters by up to 12% in broiler diets in the growth phase.(AU)

Carcass characteristics and meat quality of slow-growing broilers fed diets containing dry residue of cassava, with or without the addition of carbohydrases.

This study evaluated the carcass and portion yield and meat quality of slow-growing broilers fed diets with increasing levels of dry residue of cassava (DRC), with or without the addition of carbohydrases. A total of 1100 Label Rouge male broilers were distributed in a completely randomized 2 × 5 factorial design (with or without the addition of carbohydrases and five levels of DRC [0, 2.5, 5.0, 7.5, and 10.0%]), with five replicates and 22 birds per experimental unit. No interaction (P > 0.05) was observed between enzyme addition and DRC levels for the carcass yield, portion yield (back, legs, wings, breast), or abdominal fat variables. There was an interaction (P < 0.05) between the addition of enzyme and DRC on breast pH 15 min post-mortem and the a* intensity of the breast in a colorimetry test. There was a quadratic effect on b* intensity independent of adding enzymes, with the lowest intensity predicted at 8.89% DRC. Regarding feet color, increasing DRC supplementation (independent of enzyme supplementation) led to an increase and decrease in L* and b* intensity, respectively (P < 0.05). Moreover, there was a quadratic effect on a* intensity, with a lower level of intensity predicted at 6.93% DRC (P < 0.05), regardless of the inclusion of enzymes. The inclusion of up to 10% dry residue of cassava in slow-growing broiler diets does not affect carcass and portion yield or the qualitative characteristics of the meat but does reduce the color of the breast meat and feet.

Effect of cereal soaking and carbohydrase supplementation on growth, nutrient digestibility and intestinal microbiota in liquid-fed grow-finishing pigs.

Soaking the cereal fraction of a liquid diet prior to feeding (Csoak), and/or carbohydrase enzyme supplementation (ENZ) are likely to modulate both feed and intestinal microbial populations and improve feed efficiency (FE) in pigs. To test this hypothesis, a total of 392 grow-finisher pigs (~33.4 kg, 7 pigs/pen) were randomly allocated to 4 treatments in a 2 × 2 factorial arrangement for 70 days as follows: (1) fresh liquid feed (Fresh); (2) Cereal soaked liquid feed (Soak); (3) Fresh + ENZ and (4) Soak + ENZ. An interaction between ENZ and Csoak was found for average daily gain (ADG) during the growing phase (day 0 to 21; P < 0.05) where pigs fed the Soak + ENZ diet had higher ADG than pigs fed the Fresh + ENZ diet. No treatment effect was found for ADG thereafter. Enzyme supplementation increased total tract nutrient digestibility (P < 0.05) and reduced caecal VFA concentrations (P < 0.05) but did not improve pig growth or FE. Both Csoak and ENZ modulated intestinal microbiota composition; increasing abundance of bacterial taxa that were negatively correlated with pig growth and reducing abundance of taxa positively correlated with pig growth and caecal butyrate concentration. In conclusion, both strategies (Csoak and ENZ) improved nutrient digestibility in pigs and modulated intestinal microbiota composition.

Development of Substrate Degradation Enzyme Therapy for Mucopolysaccharidosis IVA Murine Model.

Mucopolysaccharidosis IVA (MPS IVA) is caused by a deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Conventional enzyme replacement therapy (ERT) is approved for MPS IVA. However, the fact that the infused enzyme cannot penetrate avascular lesions in cartilage leads to minimal impact on the bone lesion. Moreover, short half-life, high cost, instability, and narrow optimal pH range remain unmet challenges in ERT. Thermostable keratanase, endo-ß-N-acetylglucosaminidase, has a unique character of a wide optimal pH range of pH 5.0-7.0. We hypothesized that this endoglycosidase degrades keratan sulfate (KS) polymer in circulating blood and, therefore, ameliorates the accumulation of KS in multiple tissues. We propose a novel approach, Substrate Degradation Enzyme Therapy (SDET), to treat bone lesion of MPS IVA. We assessed the effect of thermostable keratanase on blood KS level and bone pathology using Galns knock-out MPS IVA mice. After a single administration of 2 U/kg (= 0.2 mg/kg) of the enzyme at 8 weeks of age via intravenous injection, the level of serum KS was significantly decreased to normal range level, and this suppression was maintained for at least 4 weeks. We administered 2 U/kg of the enzyme to MPS IVA mice every fourth week for 12 weeks (total of 3 times) at newborns or 8 weeks of age. After a third injection, serum mono-sulfated KS levels were kept low for 4 weeks, similar to that in control mice, and at 12 weeks, bone pathology was markedly improved when SDET started at newborns, compared with untreated MPS IVA mice. Overall, thermostable keratanase reduces the level of KS in blood and provides a positive impact on cartilage lesions, demonstrating that SDET is a novel therapeutic approach to MPS IVA.

Influence of inclusion level of barley in wheat-based diets and supplementation of carbohydrase on growth performance, nutrient utilisation and gut morphometry in broiler starters.

1. The influence of barley inclusion level and supplementation of a multi-component non-starch polysaccharide degrading enzyme on performance and nutrient utilisation in broilers was investigated. Normal-starch hulled barley was evaluated with five levels of inclusion (0, 141, 283, 424 and 565 g/kg) in a wheat-based diet and two levels of enzyme supplementation (0 and 150 g/tonne of feed; a 5 × 2 factorial arrangement of 10 dietary treatments). All diets were equivalent in metabolisable energy and digestible amino acid contents. A total of 400, one-d old male broilers (five cages/treatment; eight birds/cage) were used in the experiment.2. Regardless of enzyme supplementation, weight gain (WG) increased up to 283 g/kg of barley and was reduced afterwards (P < 0.01). Increasing levels of barley resulted in greater (P < 0.001) gain per feed (G/F). Enzyme addition increased WG (P < 0.05) and G/F (P < 0.001) at each barley inclusion level.3. Birds fed diets with 0 and 565 g/kg barley showed the lowest and highest (P < 0.001to 0.05) digestibility for all nutrients measured, respectively. Digestibility of all nutrients was improved by enzyme supplementation at each barley inclusion level (P < 0.05). The nitrogen-corrected apparent metabolisable energy improved with increasing inclusion of barley (P < 0.001) and supplemental enzyme (P < 0.01). Increasing inclusion of barley increased the relative weight of gizzard (P < 0.001) and reduced jejunal digesta viscosity (P < 0.001). Supplemental enzyme (P < 0.001) reduced digesta viscosity.4. The optimum inclusion level of barley, with respect to growth performance, was 283 g/kg of diet. Increasing barley inclusion improved nutrient and energy utilisation, possibly through lowered digesta viscosity and better function of the gizzard. Feed efficiency and nutrient and energy utilisation can benefit from carbohydrase supplementation in barley-based diets, regardless of barley inclusion level.

Bioavailability of Sulforaphane Following Ingestion of Glucoraphanin-Rich Broccoli Sprout and Seed Extracts with Active Myrosinase: A Pilot Study of the Effects of Proton Pump Inhibitor Administration.

We examined whether gastric acidity would affect the activity of myrosinase, co-delivered with glucoraphanin (GR), to convert GR to sulforaphane (SF). A broccoli seed and sprout extract (BSE) rich in GR and active myrosinase was delivered before and after participants began taking the anti-acid omeprazole, a potent proton pump inhibitor. Gastric acidity appears to attenuate GR bioavailability, as evidenced by more SF and its metabolites being excreted after participants started taking omeprazole. Enteric coating enhanced conversion of GR to SF, perhaps by sparing myrosinase from the acidity of the stomach. There were negligible effects of age, sex, ethnicity, BMI, vegetable consumption, and bowel movement frequency and quality. Greater body mass correlated with reduced conversion efficiency. Changes in the expression of 20 genes in peripheral blood mononuclear cells were evaluated as possible pharmacodynamic indicators. When grouped by their primary functions based on a priori knowledge, expression of genes associated with inflammation decreased non-significantly, and those genes associated with cytoprotection, detoxification and antioxidant functions increased significantly with bioavailability. Using principal components analysis, component loadings of the changes in gene expression confirmed these groupings in a sensitivity analysis.

Fluoromethylcyclopropylamine derivatives as potential in vivo toxicophores - A cautionary disclosure.

Fluorination of metabolic hotspots in a molecule is a common medicinal chemistry strategy to improve in vivo half-life and exposure and, generally, this strategy offers significant benefits. Here, we report the application of this strategy to a series of poly-ADP ribose glycohydrolase (PARG) inhibitors, resulting in unexpected in vivo toxicity which was attributed to this single-atom modification.

Egg quality, fatty-acid composition and gastrointestinal morphology of layer hens fed whole flaxseed with enzyme supplementation.

1. Flaxseed is a rich source of α-linolenic acid (ALA, 18:3 n-3). Feeding flaxseed to hens can increase n-3 fatty acids (FA) in eggs. However, non-starch polysaccharides (NSP) in flaxseed decrease nutrient digestibility and can have a negative impact on egg n-3 FA incorporation. Addition of carbohydrase enzymes to flaxseed-based diets can decrease the anti-nutritive effects of NSP. 2. An experiment was conducted to investigate the effect of enzyme supplementation on FA composition and gastrointestinal morphology in hens fed flaxseed. A total of seventy-two, 51-week old brown layer hens were randomly assigned to one of the four dietary treatments (six replicates with three hens per replicate): corn-soybean based diet containing 0% flax (Control), 10% flax (Flax), Flax+0.05% enzyme (Flax+E1), or Flax+0.1% enzyme (Flax+E2) in a 120-day feeding trial. 3. Egg weight was highest in hens fed Flax+E1 (P < 0.05). Yolk weight was higher in Flax+E1 compared with the control and Flax+E2 and was not different from Flax treatment. ALA and total n-3 FA was highest in eggs from Flax+E2 hens (P < 0.05). Addition of enzyme has no effect of on docosahexaenoic acid (DHA), total long chain (>20-C FA), or n-6:n-3 FA ratio in eggs from hens fed flaxseed-based diets (P > 0.05). Over nine-fold increase in hepatic ALA was observed in the liver of hens fed flaxseed-based diets when compared with the control diet (P < 0.0001). No effect of enzyme supplementation was observed on liver ALA, DHA or long chain n-3 FA (P > 0.05). Enzyme supplementation reduced arachidonic acid, total n-6 and LC n-6 FA in liver tissue from hens fed flaxseed-based diets (P > 0.05). 4. Villi height and width was higher in the duodenum and jejunum of hens fed flax-based diets compared to the control (P < 0.05). Enzyme supplementation led to an increase in villi width in jejunum (P < 0.05) in hens fed Flax+E2 (P < 0.05). No effect of diet was observed in the crypt depth and villi height:crypt depth ratio in the jejunum (P > 0.05). 5. It was concluded that enzyme supplementation enhanced total n-3 FA deposition in eggs and liver and influence gastrointestinal morphology in layer hens fed flaxseed.

Dietary fiber sources and non-starch polysaccharide-degrading enzymes modify mucin expression and the immune profile of the swine ileum.

Due to their complex chemical and physical properties, the effects and mechanisms of action of natural sources of dietary fiber on the intestine are unclear. Pigs are commonly fed high-fiber diets to reduce production costs and non-starch polysaccharide (NSP)-degrading enzymes have been used to increase fiber digestibility. We evaluated the expression of mucin 2 (MUC2), presence of goblet cells, and ileal immune profile of pigs housed individually for 28 days and fed either a low fiber diet based on corn-soybean meal (CSB, n = 9), or two high fiber diets formulated adding 40% corn distillers' dried grains with solubles (DDGS, n = 9) or 30% wheat middlings (WM, n = 9) to CSB-based diet. Pigs were also fed those diets supplemented with a NSP enzymes mix (E) of xylanase, ß-glucanase, mannanase, and galactosidase (n = 8, 10, and 9 for CSB+E, DDGS+E and WM+E, respectively). Feeding DDGS and WM diets increased ileal MUC2 expression compared with CSB diet, and this effect was reversed by the addition of enzymes. There were no differences in abundance of goblet cells among treatments. In general, enzyme supplementation increased gene expression and concentrations of IL-1ß, and reduced the concentrations of IL-4, IL-17A and IL-11. The effects of diet-induced cytokines on modulating intestinal MUC2 were assessed in vitro by treating mouse and swine enteroids with 1 ng/ml of IL-4 and IL-1ß. In accordance with previous studies, treatment with Il-4 induced Muc2 and expansion of goblet cells in mouse enteroids. However, swine enteroids did not change MUC2 expression or number of goblet cells when treated with IL-4 or IL-1ß. Our results suggest that mucin and immune profile are regulated by diet in the swine intestine, but by mechanisms different to mouse, emphasizing the need for using appropriate models to study responses to dietary fiber in swine.

Effects of pea extrusion and enzyme and probiotic supplementation on performance, microbiota activity and biofilm formation in the broiler gastrointestinal tract.

1. The effects of supplementation of broiler chicken diets with pea meal, carbohydrase enzymes and a probiotic were investigated for potential performance improvement. 2. Raw or extruded pea meal (cv Model, grown in Poland) was included in a wheat-soybean meal-based diet at 250 g/kg. The diets were unsupplemented (control) or supplemented with either carbohydrase enzymes (200 U/kg xylanase and 10 U/kg ß-glucanase in feed) or a probiotic (Bacillus subtilis), or both. The diets were fed to Ross 308 broilers aged 9-28 days. 3. After two additional days, chick gastrointestinal tracts were excised and analysed for the presence of Bacillus subtilis biofilm; and the ileal and caecal digesta were analysed for bacterial enzyme activities and to determine the concentration of short-chain fatty acids (SCFAs). 4. Feeding the pea-based diet supplemented with the probiotic compromised feed utilisation, due to higher feed intake. The addition of enzymes to the raw, but not the extruded, pea containing diet partially ameliorated this effect (pea form × additives; P < 0.002). 5. In the ileal digesta, interactions between the dietary treatments were observed for the activities of all bacterial glycolytic enzymes and for SCFA concentrations. ß-glucosidase, α-galactosidase and ß-glucuronidase were highest in birds fed the diet containing extruded pea supplemented with the probiotic and enzymes (pea form x additives; P = 0.018 to P < 0.006). In the caecal digesta, interactions were observed for bacterial enzyme activities, but not for total SCFA concentration. Biofilm formation in the caecum indicated that the probiotic strain was metabolically active in the broiler gut. 6. In conclusion, supplementation of diets containing raw or extruded pea meal with enzymes and a Bacillus subtilis spore-based probiotic modulated microbiota activity but had no clear effects on broiler performance. Probiotic administration did not cause excessive fermentation in the ileum and caecum but enhanced Bacillus subtilis spp. biofilm formation in the caecum, which may be indicative of a beneficial effect on gut health.

The Consequences of Biofilm Dispersal on the Host.

Chronic infections are often associated with the presence of a biofilm, a community of microorganisms coexisting within a protective matrix of extracellular polymeric substance. Living within a biofilm can make resident microbes significantly more tolerant to antibiotics in comparison to planktonic, free-floating cells. Thus, agents that can degrade biofilms are being pursued for clinical applications. While biofilm degrading and dispersing agents may represent attractive adjunctive therapies for biofilm-associated chronic infections, very little is known about how the host responds to the sudden dispersal of biofilm cells. In this study, we found that large-scale, in vivo dispersal of motile biofilm bacteria by glycoside hydrolases caused lethal septicemia in the absence of antibiotic therapy in a mouse wound model. However, when administered prudently, biofilm degrading enzymes had the potential to potentiate the efficacy of antibiotics and help resolve biofilm-associated wound infections.

Supplementation of the Diet by Exogenous Myrosinase via Mustard Seeds to Increase the Bioavailability of Sulforaphane in Healthy Human Subjects after the Consumption of Cooked Broccoli.

SCOPE: Broccoli contains glucosinolate glucoraphanin, which, in the presence of myrosinase, can hydrolyze to isothiocyanate sulforaphane, reported to have anticarcinogenic activity. However, the myrosinase enzyme is denatured on cooking. Addition of an active source of myrosinase, such as from powdered mustard seed, to cooked Brassica vegetables can increase the release of health beneficial isothiocyanates; however, this has not previously been proven in vivo. METHODS AND RESULTS: The concentration of sulforaphane metabolite (sulforaphane N-acetyl-l-cysteine [SF-NAC]) in 12 healthy adults after the consumption of 200 g cooked broccoli, with and without 1 g powdered brown mustard, was studied in a randomized crossover design. During the 24-h period following the consumption of the study sample, all urine was collected. SF-NAC content was assayed by HPLC. When study subjects ingested cooked broccoli alone, mean urinary SF-NAC excreted was 9.8 ± 5.1 µmol per g creatinine, and when cooked broccoli was consumed with mustard powder, this increased significantly to 44.7 ± 33.9 µmol SF-NAC per gram creatinine. CONCLUSION: These results conclude that when powdered brown mustard is added to cooked broccoli, the bioavailability of sulforaphane is over four times greater than that from cooked broccoli ingested alone.

Digestibility, productive performance, and egg quality of laying hens as affected by dried cassava pulp replacement with corn and enzyme supplementation.

Two experiments were conducted to investigate the potential use of dried cassava pulp (DCP) supplemented with enzymes as an alternative feed ingredient in laying hen diets. In experiment 1, 45 laying hens (Isa Brown) aged 45 weeks were placed in individual cages to measure nutrient digestibility for 10 days. Nine dietary treatments were control and DCP as a replacement for corn at 20, 25, 30, and 35% supplemented with mixed enzymes (cellulase, glucanase, and xylanase) at 0.10 and 0.15%. Results showed that the use of DCP at 20-35% added with mixed enzymes had no negative effects on dry matter digestibility, while organic matter digestibility and nitrogen retention decreased with increased DCP up to 30-35% in diets. Both enzyme levels (0.10 and 0.15%) showed similar results on nutrient digestibility and retention. In experiment 2, a total of 336 laying hens aged 32 weeks were randomly allocated to seven dietary treatments (control and DCP-substituted diets at 20, 25, and 30%) supplemented with mixed enzymes (0.10 and 0.15%). Diets incorporated with 20-30% of DCP and supplemented with mixed enzymes at both levels had no significant effects on egg production, egg weight, feed intake, egg mass, feed conversion ratio, protein efficiency ratio, or egg quality, except for egg yolk color being decreased with an increase of DCP in diets (P < 0.05). In conclusion, it is suggested that DCP supplemented with enzymes can be used as an energy source in laying hen diets up to 30% without showing negative effects on nutrient digestibility, productive performance, or egg quality.

Effects of carbohydrase enzyme supplementation on performance, eggshell quality, and bone parameters of laying hens fed on maize- and wheat-based diets.

1. This study was conducted to determine the effects of enzyme supplementation of maize/wheat-based diets on the performance, egg quality, and serum and bone parameters of laying hens. 2. During the 12-week experimental period, a total of 72 laying hens aged 52 weeks were randomly distributed among 6 experimental groups. Each experimental group contained 4 replicates, each with three birds. The experiment was a randomised design consisting of a 3 × 2 factorial arrangement, with three levels of wheat substitution and two levels of enzyme (xylanase: 1500.00 U/kg, ß-glucanase: 100 000 U/kg, cellulase: 1 000 000 U/kg, α-amylase: 160 000 U/kg) inclusion in the diet. Wheat replaced 0, 50, or 100% of maize with or without 1.0 g/kg enzyme supplementation in iso-nitrogenous and iso-caloric experimental diets. 3. Body weight, egg production, egg weight, egg mass, eggshell thickness, and the feed conversion ratio were adversely affected by the wheat-based diet. The eggshell quality parameters decreased with enzyme supplementation to the diet. 4. Wheat-based diets adversely affected calcium and phosphorus concentrations in the tibia, but the addition of the enzymes to the wheat-based diet prevented the negative effects of wheat-based diets on tibia mineralisation in laying hens. The wheat-based diets tended to reduce plasma mineral contents, and the addition of enzymes tended to affect plasma minerals and biomechanical properties of the tibia positively in laying hens. 5. These results indicate that wheat-based diets in aged laying hens adversely affected the mineral metabolism compared with maize-based diets, and the negative effects of wheat on bone mineralisation can be prevented by enzyme supplementation to the diets in laying hens.

Performance, nutrient utilization, and energy partitioning in broiler chickens offered high canola meal diets supplemented with multicomponent carbohydrase and mono-component protease.

Two broiler chicken experiments were conducted to investigate the effects of canola meal (CM) replacing soybean meal (SBM) in diets supplemented with carbohydrase and protease on performance and partitioning of energy. First, a 2 × 2 × 2 factorial arrangement of treatments was employed to evaluate: protein meals (CM vs. SBM), carbohydrase (none or 300 mg/kg), protease (none or 200 mg/kg), and their interactions. Each treatment was fed to 6 replicated pens of 16 male broilers (Ross 308) from d 10 to 35. In the second experiment, 32 broiler chicks were used in a 2 × 2 factorial arrangement to investigate CM and carbohydrase effects on energy partitioning. Birds were transferred into 16 closed-circuit calorimeter chambers (4 chambers/diet; 2 birds/chamber) to measure heat production (HP), metabolizable and net energy (NE) by gaseous exchange, and total excreta collection from d 25 to 28. There were no 3-way interactions among experimental factors for any of the performance parameters measured. Birds given CM diets consumed less feed, had lower BW, and exhibited higher FCR compared to the control birds (P < 0.01). Both enzymes, alone or in combination, improved final BW and FCR (P < 0.05). There was an interaction between carbohydrase and protease for FCR over the grower period (P < 0.01), in which the combination of the enzymes resulted in further improvement of FCR. Energy, DM, and crude protein digestibility values were higher in control birds (P < 0.05). There was an interaction of protein meal and carbohydrase for HP, respiratory quotient (P < 0.05), and NE:ME ratio of the diets (P = 0.06). Inclusion of CM without carbohydrase increased HP and decreased NE and NE:ME ratio of the diets (P < 0.05). Carbohydrase decreased HP and increased retained energy (P = 0.06) and NE and NE:ME ratio (P < 0.05). In conclusion, high CM in the diet negatively affects growth performance through reduction in feed consumption, nutrient digestibility, and NE of the diet, which could partly be restored by enzyme supplementation.

Effects of multi-carbohydrase and phytase on standardized ileal digestibility of amino acids and apparent metabolizable energy in canola meal fed to broiler chicks.

Two assays were conducted to evaluate nutritive value of canola meal (CM) fed to broiler chicks without or with a multi-carbohydrase (MC) preparation (700 U α-galactosidase, 2,200 U galactomannanase, 30,000 U xylanase, and 22,000 U ß-glucanase per kg of diet) and phytase (Phy, 500 FTU per kg of diet). Assay 1 determined apparent total tract digestibility (ATTD) of nutrients and metabolizable energy (AME) by the difference method. Assay 2 determined apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of amino acids by the index method. Two reference diets (RD) - an 85% corn based and a 5% casein-cornstarch diet fortified with vitamins and minerals - were made for assays 1 and 2, respectively. For each assay, the test diets were made by mixing RD and CM 7:3 wt/wt basis and fed without or with MC or Phy or combination. A total of 245 day-old male broilers (Cobb 500) was allocated to 5 treatments to give 7 replicates (7 birds/cage). The birds were fed a commercial diet from day zero to 10 followed by assay 1 fed from d 11 to 18 and assay 2 fed from d 19 to 21. Excreta samples were collected on d 15 to18, and all birds were slaughtered on d 21 for ileal digesta. There was an interaction (P < 0.05) between MC and Phy on ATTD of DM, N, and P. There was no interaction (P > 0.05) between MC and Phy on AMEn; however, MC and Phy individually improved AMEn retention. Enzymes interacted (P < 0.05) on SID of Arg, His, Leu, Met, Thr, Ala, Asp, Gln, and Gly. In this context, feeding a combination of MC and Phy resulted in higher (P < 0.05) SID of Arg, His, Met, and Thr relative to single activity or control. Both enzymes improved (P < 0.05) SID of Lys independently. The combination of carbohydrase and Phy may be an effective strategy to improve amino acid utilization in CM for poultry.