Impact of feed supplement with alfa-amylase and beta-glucanase on ingestive-related biomarkers registered with real-time sensors.

The aim of this study was to investigate the impact of feed supplements with alfa-amylase and beta-glucanase (Optipartum C+ 200) on ingestive-related behaviour biomarkers registered with real-time sensors: rumination behaviours and reticulorumen parameters (pH and temperature). Cows (n=20) in the treatment group (TG) were fed with Optipartum C+ 200 (Enzymes feed supplement: Alfa-Amylase 57 Units; Beta-Glucanase 107 Units) from 21 days before calving until 30 days after calving with a feeding rate of 200 g/cow/day. Cows (n=22) in the control group (CG) were fed a feed ration without feed supplement. Measurements started from 6 days before calving and continued until 21 days after calving. The following indicators were registered: with the RumiWatch System: Rumination time; Eating time; Drinking time; Rumination chews; Eating chews; Drinking gulps; Bolus; Chews per minute; Chews per bolus. With the SmaXtec system: the temperature, pH of the contents of the cows' reticulorumens, and cows' walking activity. According to our results, feed supplementation with alfa-amylase and beta-glucanase (Optipartum C+ 200) in the TG group resulted in increases in the following parameters: 9% rumination time and eating time, 19% drinking time, 11% rumination chews, 16% eating chews, 13% number of boluses per rumination, 5% chews per minute and 16% chews per bolus. The rumination time showed a strong, positive relation with rumination chews and bolus indicators in both groups (TG and CG) (p⟨0.001); while the rumination time in both groups of cows showed an opposite direction and was negatively related to eating time and eating chews (p⟨0.05). We found a 1.28 % lower reticulorumen pH and a 0.64 % lower reticulorumen temperature in cows fed with the supplement compared with cows in the control group. Cows in TG were 8.80% more active than those in the CG group. For improvement of ingestive-related behaviour we suggest adding a feed supplement with alfa-amylase and beta-glucanase (Optipartum C+ 200).

Effects of dietary exogenous fibrolytic enzymes on ruminal fermentation characteristics of beef steers fed high- and low-quality growing diets1.

The effects of dietary pretreatment with fibrolytic enzyme-based cocktail were evaluated in 2 studies: (1) in vitro true digestibility; and (2) intake, digestibility, feeding behavior, and ruminal fermentation of beef steers fed growing diets. For the in vitro assessment, the ruminal inoculum was collected from 2 steers (BW = 543 ± 45 kg; 4-h after feeding; growing diets) and enzymes included or not (Trichoderma reesei fermentation extract; 0.75 µL/g of substrate DM). Within in vitro batches (n = 4), 12 substrates were incubated and in vitro true nutrient digestibility was evaluated. For study 2, 5 ruminally cannulated beef steers (BW = 520 ± 30 kg) were used in a 5 × 4 unbalanced Latin square using a 2 × 2 factorial arrangement of treatments: (a) diet quality (high = HQ; and low = LQ) and (b) enzyme inclusion (0 or 0.75 mL/kg of diet DM). Steers were fed ad libitum during four 21-d periods consisting of 14-d of adaptation and 7-d of collections. An enzyme × substrate was observed (P < 0.01), in which DM, OM, and NDF disappearance of sorghum grain increased with enzymes addition. Addition of enzymes increased (P < 0.01) ADF digestibility for all substrates. No diet quality × enzyme (P ≥ 0.18) was observed for intake variables in study 2. Enzyme-fed steers increased (P ≤ 0.05) intake of DM, digestible DM, NDF, and ADF compared with steers not fed fibrolytic enzymes. Addition of enzyme did not affect (P ≥ 0.28) apparent total tract digestibility of beef steers. Steers fed HQ diets consumed more (P ≤ 0.04) DM, digestible DM and OM, and less (P ≤ 0.03) total and digestible fiber than steers fed LQ diets. Ruminal pH average decreased (P = 0.01) for steers fed HQ or enzyme-fed diets compared with other treatments. A tendency (P = 0.06) toward improved total VFA was observed on enzyme-fed steers with HQ diets, but not for LQ diets. The molar proportion of ruminal propionate increased (P = 0.01) when steers were fed enzyme. Steers fed HQ diets had greater (P < 0.01) propionate and valerate molar proportions, lower (P < 0.01) acetate and acetate:propionate ratio than steers fed LQ diets. In vitro methane and total gas production were not affected (P ≥ 0.50) by dietary treatments. Fibrolytic enzymes positively affected digestion of multiple roughage sources commonly fed to cattle and might have additional benefit when used on unprocessed sorghum grain. Fibrolytic enzymes in beef cattle growing diets stimulated intake and generated positive impacts on ruminal fermentation.

The impact of direct-fed microbials and enzymes on the health and performance of dairy cows with emphasis on colostrum quality and serum immunoglobulin concentrations in calves.

Thirty-six cows were blocked by calving date and randomly assigned to one of three treatments. Cows were on treatments 3 weeks prepartum through 8 weeks post-partum. Treatments were as follows: (i) no direct-fed microbial (DFM) or cellulase and amylase enzymes (C), (ii) 45.4 g/day of DFM (D) or (iii) 45.4 g/day of DFM and 18.2 g/day of enzyme (DE). Total mixed ration fed and refused were measured daily to determine dry matter intake (DMI). Blood samples were taken three times weekly and analysed for ß-hydroxybutyrate, glucose and non-esterified fatty acids. Body weight (BW) was measured weekly. Colostrum was weighed and analysed for IgA and IgG concentration. Calves were fed 4 L of colostrum within 2 hr of birth. Calf blood samples were taken at 0 and 24 hr for analysis of IgA and IgG concentrations and apparent efficiency of absorption. Milk yield was measured daily and samples collected weekly. Initial BW was different among treatments with D being lesser than C or DE treatments. Body weight, weight gain, efficiency of gain, DMI and blood parameters were unaffected. Treatment did not affect colostrum yield. Ash percentage of colostrum tended to increase with D and DE, while IgA and total solids yield decreased with D. Colostrum fat yield was decreased in D and DE. Treatments did not impact BW, serum IgA and IgG concentrations or apparent efficiency of absorption of calves. Post-partum BW, DMI, blood parameters, milk production and composition were unaffected by treatment. However, cows on D gained more BW and tended to have greater efficiency of gain compared to those on DE, but were similar to C. Somatic cell scores were greatest for D. Results indicate that DFM and enzyme supplementation did not improve health and performance of dairy cattle during the pre- and post-partum periods under conditions of this study.

Effect of wheat dried distillers grains with solubles and fibrolytic enzymes on ruminal fermentation, digestibility, growth performance, and feeding behavior of beef cattle.

Two experiments were conducted to evaluate the effect of wheat dried distillers grains with solubles (DDGS) and fibrolytic enzymes (FE) on ruminal fermentation, in situ ruminal and in vivo total tract digestibility, growth performance, and feeding behavior of growing beef cattle. In Exp. 1, 6 ruminally cannulated Angus heifers (average BW of 794 ± 44.2 kg) were used in a 6 × 6 Latin square design with 2 × 3 factorial arrangement of treatments. Treatments were a control diet consisting of 50% barley silage, 10% grass hay, and 40% barley grain-based concentrate (CON) and the CON with 15% DDGS substituted for barley grain (WDG) combined with either 0, 1, or 2 mL FE/kg diet DM, respectively. Inclusion of DDGS increased total tract digestibility of CP ( < 0.01), NDF ( = 0.04), and ADF ( = 0.03). Increasing FE linearly ( = 0.03) increased CP digestibility without affecting the digestibility of other nutrients. There were no effects of DDGS inclusion or FE on ruminal pH or VFA concentration except that propionate was greater ( = 0.04) with the WDG. In situ ruminal DM and NDF disappearance of barley silage was greater ( < 0.04) in heifers fed the WDG than in heifers fed the CON after 24 h of incubation. Increasing FE linearly ( = 0.03) increased in situ NDF disappearance of barley silage after 24 h of incubation. In Exp. 2, 120 weaned steers (initial BW of 289 ± 11.0 kg) were fed diets similar to those in Exp. 1. The steers fed the WDG had greater ( < 0.01) final BW, ADG, DMI, and G:F compared with steers fed the CON. Increasing FE did not alter ADG or G:F but tended ( < 0.07) to linearly decrease DMI. There were interactions ( < 0.02) between DDGS and FE on eating rate and the time spent at the feed bunk. Supplementing FE decreased ( < 0.01) time at the bunk and increased ( < 0.01) eating rate for steers fed the WDG but not for steers fed the CON. Eating rate ( < 0.01) and meal frequency ( = 0.02) were greater but eating duration was shorter ( < 0.01) for steers fed the WDG than for those fed the CON. These results indicate that inclusion of wheat DDGS in a growing diet increased total tract digestibility of NDF and CP and improved the feed efficiency of steers. Moreover, supplementation of FE in barley silage-based growing diets may also have the potential to increase profits, with the evidence of the trend for a decline on DMI without decreasing ADG when adding FE.

Nutrient value of spray field forages fed to pigs and the use of feed enzymes to enhance nutrient digestibility.

This study determined the DE, ME, apparent total tract digestibility (ATTD) of N, and N retention of spray field forages (Bermuda grass, forage sorghum, and sweet sorghum) fed to pigs and the effects of the supplemental feed enzymes on energy and N utilization. A basal diet was formulated with 96% corn and 4% amino acids, minerals, and vitamins. Test diets contained 85% basal diet + 15% Bermuda grass, forage sorghum, or sweet sorghum. Allzyme SSF (Alltech, Nicholasville, KY) was used as a feed enzyme, which was composed of cellulase, glucanase, xylanase, phytase, and protease. The basal diet and test diets were evaluated by using 4 sets of 2 × 2 Latin square designs consisting of 2 pigs and 2 periods with a total of 32 barrows (38.7 ± 7.9 kg). Each period (10-d adjustment and 4-d collection) had 2 Latin squares. The 2 treatments were levels of enzyme supplementation (0 or 200 mg/kg). Pigs received experimental diets twice daily (0700 and 1700 h) at a fixed amount based on BW of pigs (0.09 × BW0.75 kg). On d 10, chromic oxide (0.5%) was added to the diets at 1700 h as an external marker. Fecal and urine samples were collected during 4 consecutive days. The basal diet contained 3,850 kcal DE/kg, 3,769 kcal ME/kg, 86.06% ATTD of N, and 71.10% N retention and was not affected by enzyme supplementation. Bermuda grass contained 893 kcal DE/kg, 845 kcal ME/kg, -16.50% ATTD of N, and -37.49% N retention and tended to be improved by enzyme supplementation to 1,211 kcal DE/kg (P = 0.098), 1,185 kcal ME/kg (P = 0.081), and -10.54% N retention (P = 0.076). The ATTD of N of Bermuda grass increased (P < 0.05) to 0.08% by enzyme supplementation. The forage sorghum contained 1,520 kcal DE/kg, 1,511 kcal ME/kg, -0.72% ATTD of N, and -16.99% N retention. The sweet sorghum contained 1,086 kcal DE/kg, 1,061 kcal ME/kg, -75.47% ATTD of N, and -49.22% N retention. Enzyme supplementation did not improve energy digestibility of forage sorghum and sweet sorghum. Nitrogen in these forages was poorly utilized. In conclusion, spray field forages including Bermuda grass, forage sorghum, and sweet sorghum can partly be utilized in pig feed to provide energy, although N is rather poorly digested. Feed enzymes could enhance both energy and N utilization in Bermuda grass but not sorghum.

Screening exogenous fibrolytic enzyme preparations for improved in vitro digestibility of bermudagrass haylage.

Our objectives were to evaluate the effects of 12 exogenous fibrolytic enzyme products (EFE) on ruminal in vitro neutral detergent fiber digestibility (NDFD) and preingestive hydrolysis of a 4-wk regrowth of bermudagrass haylage (BH), to examine the accuracy of predicting NDFD with EFE activity measures, and to examine the protein composition of the most and least effective EFE at increasing NDFD. In experiment 1, effects of 12 EFE on NDFD of BH were tested. Enzymes were applied in quadruplicate to culture tubes containing ground BH. The suspension was incubated for 24 h at 25 °C before addition of rumen fluid media and further incubation for 24 h at 39 °C. The experiment was repeated twice. In addition, regression relationships between EFE activity measures and NDFD were examined. Compared with the values for the control, 9 EFE-treated substrates had greater NDFD (37.8 to 40.4 vs. 35.6%), 6 had greater total VFA concentration (59.1 to 61.2 vs. 55.4 mM), and 4 had lower acetate-to-propionate ratios (3.03 to 3.16 vs. 3.24). In experiment 2, EFE effects on preingestive fiber hydrolysis were evaluated by incubating enzyme-treated and untreated bermudagrass suspensions in quadruplicate for 24 h at 25 °C and examining fiber hydrolysis measures. Compared with values for the control, 3 EFE reduced neutral detergent fiber concentration (62.8 to 63.7 vs. 67.3%), 10 increased release of water-soluble carbohydrates (26.8 to 58.5 vs. 22.8 mg/g), and 8 increased release of ferulic acid (210 to 391 vs. 198 µg/g). Regression analyses revealed that enzyme activities accurately [coefficient of determination (R(2)) = 0.98] predicted preingestive hydrolysis measures (water-soluble carbohydrates, ferulic acid), moderately (R(2) = 0.47) predicted neutral detergent fiber hydrolysis, but poorly (R(2) ≤ 0.1) predicted dry matter and NDFD. In experiment 3, proteomic tools were used to examine the protein composition of the most and least effective EFE at improving NDFD. Relative to the least effective, the most effective EFE at increasing NDFD contained 10 times more endoglucanase III, 17 times more acetylxylan esterase with a cellulose-binding domain 1, 33 times more xylanase III, 25 times more ß-xylosidase, and 7.7 times more polysaccharide monooxygenase with cellulose-binding domain 1 and 3 times more swollenin. The most effective EFE had a much greater quantity of fibrolytic enzymes and key proteins necessary for hemicellulose and lignocellulase deconstruction. This study identified several EFE that increased the NDFD and in vitro fermentation of 4-wk BH and revealed why some EFE are more effective than others.

Dilute acid pretreatment and enzymatic hydrolysis of sorghum biomass for sugar recovery--a statistical approach.

Sorghum is one of the commercially feasible lignocellulosic biomass and has a great potential of being sustainable feedstock for renewable energy. As with any lignocellulosic biomass, sorghum also requires pretreatment which increases its susceptibility to hydrolysis by enzymes for generating sugars which can be further fermented to alcohol. In the present study, sorghum biomass was evaluated for deriving maximum fermentable sugars by optimizing various pretreatment parameters using statistical optimization methods. Pretreatment studies were done with H2SO4, followed by enzymatic saccharification. The efficiency of the process was evaluated on the basis of production of the total reducing sugars released during the process. Compositional analysis was done for native as well as pretreated biomass and compared. The biomass pretreated with the optimized conditions could yield 0.408 g of reducing sugars /g of pretreated biomass upon enzymatic hydrolysis. The cellulose content in the solid portion obtained after pretreatment using optimised conditions was found to be increased by 43.37% with lesser production of inhibitors in acid pretreated liquor.

Using a fibrolytic enzyme in barley-based diets containing wheat dried distillers grains with solubles: ruminal fermentation, digestibility, and growth performance of feedlot steers.

Two experiments were conducted to evaluate the effects of adding an exogenous fibrolytic enzyme (FE) on ruminal pH and fermentation, digestibility, and growth performance of feedlot beef cattle fed a finishing diet containing wheat dried distillers grains with solubles (DDGS). In Exp. 1, 4 ruminally cannulated Angus heifers (average BW of 807 ± 93.9 kg) were used in a replicated 4 × 4 Latin square design. Treatments were 1) control (CON; 10% barley silage and 90% barley grain-based concentrate), 2) CON diet substituting 30% wheat DDGS for barley grain (WDG), 3) WDG diet supplemented with low FE (WDGL), and 4) WDG diet supplemented with high FE (WDGH). Heifers fed WDG had less (P = 0.01) total tract DM digestibility than heifers fed CON. Increasing FE linearly (P < 0.05) increased starch digestibility without affecting digestibility of other nutrients. Addition of FE also reduced (P = 0.03) ruminal ammonia-N (NH3-N) concentration but did not affect VFA concentration. Moreover, application of FE to wheat DDGS linearly increased in situ ruminal DM (P < 0.01) and NDF (P = 0.02) disappearance after 48 h of incubation. In Exp. 2, 160 yearling steers (initial BW = 495 ± 37.9 kg) were fed the same diets as in Exp. 1. No differences in DMI, final BW, ADG, dietary NEg, or carcass characteristics were observed among diets. However, the steers fed WDG had less (P < 0.05) G:F and greater number of (P < 0.01) abscessed livers than steers fed CON. Increasing FE application in wheat DDGS diets did not affect DMI, final BW, or ADG but tended (P < 0.09) to linearly improve feed efficiency and decreased (P = 0.03) the incidence of abscessed livers. These results demonstrated adverse effects of including wheat DDGS in finishing diets on feed digestion, feed efficiency, and animal health. Application of FE in wheat DDGS-based diets potentially improved starch digestion, protein metabolism in the rumen, feed efficiency, and animal health.

Isolation and characterization of Bacillus subtilis strain BY-3, a thermophilic and efficient cellulase-producing bacterium on untreated plant biomass.

UNLABELLED: Bioconversion of biomass, particularly crop wastes, into biofuels is being developed as an alternative approach in meeting the high energy demand. In this study, a thermophilic bacterial strain BY-3 that exhibits cellulolytic potential was isolated from faecal samples of Tibetan pigs; this strain was identified as Bacillus subtilis. The strain can produce cellulase when grown on various substrates, including carboxymethyl cellulose, rice straw, corn stover, soluble starch and wheat bran. The maximum cellulase activity of the strain was up to 4·323 ± 0·065 U ml(-1) when cultivated in the medium containing corn stover (30 g l(-1) ) for 24 h. The results demonstrated that corn stover is the most suitable substrate for cellulase production by the strain BY-3. The crude cellulase of strain BY-3 was most active at pH 5·5 and 60°C, and the enzyme in acetate buffer (50 mmol l(-1) ) demonstrated a good stability at 60°C for at least 1 h. The crude cellulase exhibited a strong antibacterial activity against Staphylococcus aureus. The strain can be used in cost-efficient cellulase production for bioconversion of agricultural residual biomass into biofuels. SIGNIFICANCE AND IMPACT OF THE STUDY: The increased consumption of fossil fuels has caused serious energy crisis and environmental problem. Thus, an alternative energy source is necessary. Bioconversion of biomass, particularly agricultural residuals, into value-added bioproducts, such as biofuels and chemical solvents, has received considerable attention. In this study, the newly isolated thermophilic Bacillus subtilis strain BY-3 produces cellulase efficiently with the use of untreated corn stover as a sole carbon source. This strain possesses the thermostable cellulase that is active with diverse crop wastes with a broad pH range and is a highly promising candidate for agricultural waste management.

Ethanol production from oil palm trunks treated with aqueous ammonia and cellulase.

Oil palm trunks are a possible lignocellulosic source for ethanol production. Low enzymatic digestibility of this type of material (11.9% of the theoretical glucose yield) makes pretreatment necessary. An enzymatic digestibility of 95.4% with insoluble solids recovery of 49.8% was achieved after soaking shredded oil palm trunks in ammonia under optimum conditions (80°C, 1:12 solid-to-liquid ratio, 8h and 7% (w/w) ammonia solution). Treatment with 60 FPU of commercial cellulase (Accellerase 1000) per gram of glucan and fermentation with Saccharomyces cerevisiae D(5)A resulted in an ethanol concentration of 13.3g/L and an ethanol yield of 78.3% (based on the theoretical maximum) after 96 h. These results indicate that oil palm trunks are a biomass feedstock that can be used for bioethanol production.

Levels of endogenous ß-glucanase activity in barley affect the efficacy of exogenous enzymes used to supplement barley-based diets for poultry.

To improve the nutritive value of barley-based diet for broilers, 2 experiments using 2 different barley lots were performed to evaluate the capacity of a mesophilic cellulase when fused to a ß-glucan specific family 11 carbohydrate-binding module. The data revealed that the recombinant ß-glucanase derivatives were not appropriate for feed supplementation because of a lack of stability at acidic pH levels. However, under the same experimental conditions, a commercial enzyme mixture improved the nutritive value of 1 of the cereal lots used. Analysis of the nutritive value of the 2 barleys revealed intrinsic differences in the levels of endogenous ß-glucanase activity. These differences were extensively evident when the studies were expanded to a range of 64 barley lots. Thus, to clarify the effect of endogenous cellulases on the efficacy of exogenous ß-glucanases used to supplement barley-based diets for poultry, 2 barley lots presenting low and high levels of endogenous plant cell wall-degrading enzymes were selected. These lots were used to prepare 2 barley-based diets, which were supplemented with or without a commercial enzyme product and fed to broiler chicks. The data revealed that the exogenous enzymes were effective when the basal diet presented low levels of endogenous ß-glucanases but were unable to improve the nutritive value of the barley lot displaying higher ß-glucanase activity. Thus, these studies suggest that levels of endogenous ß-glucanases may affect the efficacy of exogenous enzymes used to improve the nutritive value of barley-based diets for broilers. The development of a quick ß-glucanase assay that could be applied for cereal-based feeds may help identify those barley-based diets that are more responsive to the action of feed enzymes.

Effects of cellulase from Aspergillus niger and solvent pretreatments on the extractability of organic green tea waste.

As green tea is being consumed in larger amounts, more green tea waste is being produced. Following extraction, several bioactive compounds may exist in the waste including polyphenols and amino acids. It was found that an Aspergillus niger cellulase treatment of green tea waste increased the extractability of various nutritional and functional components after pretreatments with various extraction solvents such as cold water (CW), hot water (HW), sulfuric acid (SA), hydrochloric acid (HA), and methanol (Me). After the residue was treated with cellulase from Aspergillus niger, the amounts of polyphenols, total catechins, and reducing sugars in the HW extract were increased by 64.6, 941.2, and 350.9%, respectively. In particular, levels of epigallocatechin, epicatechin, and gallic acid were significantly enhanced compared to those in the nontreated control. However, protein extraction was not significantly affected, and cellulase treatment was not more efficient for caffeine extraction compared to phenolic extraction. Among the four extraction solvents, HW and SA showed relatively higher extractabilities as compared to the other groups (CW, HA, and Me). These results indicate that cellulase from A. niger can increase the extractability of green tea waste when combined with certain solvent pretreatments. Consequently, the residual functional compounds and essential nutrients from cellulase-treated green tea waste have the potential to be applied in the production of new functional foods.

Effect of exogenous fibrolytic enzymes on ruminal fermentation and nutrient digestion in dairy cows.

The aim of the present study was to examine the effects of an exogenous fibrolytic enzyme product applied to a total mixed ration (TMR) prior to feeding on ruminal fermentation, microbial protein synthesis, nutrient digestion, and milk yield and composition. Six multiparous lactating Holstein cows (598 +/- 29 kg initial live weight and 98 +/- 30 days in milk) fitted with rumen and duodenal cannulae were allocated to two treatments in a crossover design over three consecutive 28-d periods. The TMR containing 50% concentrates, 30% corn silage and 20% grass silage on dry matter (DM) basis, was mixed once daily and fed twice a day. Treatments were TMR alone (Control) or TMR with an enzyme product containing primarily cellulase and xylanase activities (9000 U endo-1,4-beta glucanase, 24000 U endo-1,3(4)-beta glucanase and 40000 U 1,4-beta xylanase per ml). The enzyme product was applied at a rate of 6.2 ml/kg TMR (DM basis). It was diluted at a rate of 1:5 with water and applied daily to the TMR. During the control period the cows received a TMR supplemented with 36 ml water/kg TMR on DM basis. Duodenal digesta flow was measured using Cr2O3 as flow marker and microbial protein in the duodenal digesta was estimated by near-infrared spectroscopy (NIRS). There were no significant differences in ruminal pH-values, NH3-N concentrations, total SCFA concentrations and molar proportions of SCFA. No treatment effects on microbial N flow to the duodenum and efficiency of microbial protein synthesis were observed. The apparent ruminal digestibilities of DM, organic matter, NDF and ADF, milk yield and composition were also not affected by the enzyme supplementation. In this study the application of exogenous fibrolytic enzymes fed to dairy cows did not show a significant effect on any parameter tested.

Effect of exogenous fibrolytic enzymes supplementation on milk production and nutrient utilization in Murrah buffaloes.

The present study was conducted to evaluate the effect of commercial exogenous fibrolytic enzyme (EFE) mixture added at 1.5 and 3.0 g (cellulase 4,000 microM glucose/g/h + xylanase 7,990 microM xylose/g/min; 50:50 w/w) per kilogram of dry matter (DM) of feed on nutrient digestibility, milk production, milk composition, and some blood constituents in lactating Murrah buffaloes. Eighteen buffaloes were allotted to three dietary treatments, on the basis of milk yield (8.48, 8.52, and 8.53 kg/day) and days in lactation (68.5, 80.33, and 82.00) for 90 days. The buffaloes were fed a total mixed ration (TMR) comprising of 45% chaffed wheat straw, 15% chopped green maize, and 40% concentrate on DM basis (control group), the same TMR plus EFE at 1.5 g/kg DM (T-1 group) and the same TMR plus EFE at 3.0 g/kg DM (T-2 group) supplemented through the concentrate mixture. There was no effect of fortifying EFE mixture on DM intake and crude protein intake (grams per day) whereas total digestible nutrients intake (kilogram per day) was higher by (P < 0.05) 12.53% in T-1 group over that of control, and there was no significant difference between T-2 and control groups. The average daily milk yield and 4% fat-corrected milk (FCM) yield was higher (P < 0.05) by 12.99% and 15.17% in T-1 group as over that of control, and there was no difference between T-2 and control groups. There was no (P > 0.05) difference in blood glucose and blood urea nitrogen concentration in different experimental groups. It is concluded that supplementation of cellulase and xylanase mixture at 1.5 g/kg of DM of TMR containing wheat straw (45%), green maize (15%), and concentrate (40%) on DM basis significantly increased (P < 0.05) the average daily milk yield and FCM yield in Murrah buffaloes due to improved dietary fiber digestion.

Means to improve the nutritive value of flaxseed for broiler chickens: the effect of particle size, enzyme addition, and feed pelleting.

Three experiments were conducted to explore dietary means (particle size, enzyme addition, bile salts addition, and feed pelleting) of minimizing the antinutritive effects of cell wall-nonstarch polysaccharides of flaxseed. Broiler chickens were fed corn-soybean meal-based diets containing 15% of full-fat flaxseed from 5 to 18 d. The effects of 2 enzyme preparations containing viscosity-reducing or cell wall-degrading activities on growth performance and nutrient digestibility were evaluated in experiment 1. Enzyme addition had beneficial effects (P<0.05) in increasing nonstarch polysaccharide digestibility and reducing intestinal viscosity. However, no differences in growth performance or fat digestibility were observed between the enzyme types. Therefore, the enzyme supplement containing both viscosity-reducing and cell wall-degrading activities was used in subsequent studies. A 2x2x2 factorial arrangement was used in experiment 2 to investigate the effects of particle size (coarse vs. fine), enzyme supplementation, and bile salt addition on the nutritive value of flaxseed for broiler chickens. In experiment 3, a 4x2 factorial arrangement was used to further investigate the effects of feed processing (whole seed, coarsely ground seed, and finely ground seed in pelleted diets or finely ground seed in mash diets) and enzyme addition on growth performance and fat utilization. Bile salt addition did not improve fat digestibility. Particle size reduction via grinding had no significant effect on growth performance no matter if present in the mash or pelleted diets. When compared with grinding, feed pelleting showed more pronounced and beneficial effects on growth performance particularly when whole, intact seeds were used, indicating a potential for using whole flaxseed in the pelleted diets. Enzyme addition resulted in an increase in total tract fat digestibility by 3 to 6%, which was reflected in an improved feed conversion ratio by 1 to 3%, regardless of the processing method used (P<0.05). In conclusion, enzyme addition and feed pelleting offer practical solutions to improve the nutritive value of flaxseed for broiler chickens.

Soluble fiber viscosity affects both goblet cell number and small intestine mucin secretion in rats.

We examined the role of soluble fiber viscosity in small intestinal mucin secretion. Viscosities were defined as the area under the viscosity curve (VAUC). Rats were fed a control diet or diets containing konjac mannan (KM) [low, medium, or high molecular weight (LKM, MKM, HKM), respectively] at 50 g/kg diet for 10 d. Luminal mucin content and goblet cell number increased in proportion to the molecular weight of KM. Such effects with the HKM diet were nullified by the concurrent ingestion of 2 g cellulase/kg diet. Diet containing LKM, MKM, HKM, guar gums (high or low molecular weight; HGG, LGG), psyllium (PS), or pectin (PC) at 50 g/kg was fed to rats. Fibers with higher VAUC (MKM, HKM, HGG, and PS) increased goblet cell numbers, but not those with lower VAUC (LKM, LGG, and PC). Luminal mucins were greater in rats fed HKM, PC, and PS diets. Goblet cell numbers and VAUC were correlated (r = 0.98; P < 0.01). In rats fed the HKM diet, ileal Muc2 gene expression was not affected, but that of Muc3 was lower than in those fed the control diet, indicating that the increase in luminal mucins after ingestion of HKM diet occurred independently of enhanced Muc gene expression. An incorporation study of 5'-bromo-deoxyuridine (BrdU) showed the position of the uppermost-BrdU labeled cell along the villi was higher in rats fed the HKM diet than in those fed the control diet. The results suggest that soluble fibers, except PC, upregulate baseline secretion of luminal mucins by increasing goblet cell numbers in proportion to fiber VAUC.

Effect of a cellulase treatment on extraction of antioxidant phenols from black currant (Ribes nigrum L.) pomace.

The effect of a commercial cellulase preparation on phenol liberation and extraction from black currant pomace was studied. The enzyme used, which was from Trichoderma spp., was an effective "cellulase-hemicellulase" blend with low ß-glucosidase activity and various side activities. Enzyme treatment significantly increased plant cell wall polysaccharide degradation as well as increasing the availability of phenols for subsequent methanolic extraction. The release of anthocyanins and other phenols was dependent on reaction parameters, including enzyme dosage, temperature, and time. At 50 °C, anthocyanin yields following extraction increased by 44% after 3 h and by 60% after 1.5 h for the lower and higher enzyme/substrate ratio (E/S), respectively. Phenolic acids were more easily released in the hydrolytic mixture (supernatant) and, although a short hydrolysis time was adequate to release hydroxybenzoic acids (HBA), hydroxycinnamic acids (HCA) required longer times. The highest E/S value of 0.16 gave a significant increase of flavonol yields in all samples. The antioxidant capacity of extracts, assessed by scavenging of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation, the oxygen radical absorbance capacity, and the ferric reducing antioxidant potential depended on the concentration and composition of the phenols present.

Crop beta-glucanase activity limits the effectiveness of a recombinant cellulase used to supplement a barley-based feed for free-range broilers.

1. The supplementation of diets rich in soluble polysaccharides with microbial cellulases and hemicellulases decreases digesta viscosity and promotes broiler performance. 2. In contrast, recent experiments suggest that polysaccharidases are ineffective for improving the nutritive value of pasture biomass used by free-range broilers. However, the feasibility of using cellulases and hemicellulases to improve the utilisation of cereal-based feeds by pastured poultry remains to be established. 3. A study was undertaken to investigate the capacity of a recombinant cellulase from Clostridium thermocellum to improve the nutritive value of a barley-based feed for free-range pastured broilers of the RedBro Cou Nu x RedBro M genotype. 4. The results show that supplementation of a barley-based diet with a recombinant beta-glucanase had no effect on the performance of free-range broilers, foraging in legume-based diets from d 28 to 56. In addition, the results confirm that the lack of effect of the recombinant enzyme in improving the nutritive value of the barley-based feed does not result from enzyme proteolysis or inhibition in the gastrointestinal tract. 5. Significantly, beta-glucanase activity was identified in the crop of non-supplemented animals. The data suggest that endogenous cellulases originated both from the barley-based feed and from the crop microflora. 6. The results presented here suggest that in older birds of slow-growing genotypes associated with free-range production systems, previously unknown sources of beta-glucanases, such as the feed and microbial symbiotic microflora, can affect the effectiveness of exogenous enzymes added to the feed.

Effects of exogenous cellulase supplementation on microbial growth and ruminal fermentation of a high-forage diet in Rusitec fermenters.

Two incubation runs were carried out with a Rusitec system to investigate the effects of 2 exogenous pure cellulases on ruminal microbial growth and fermentation of a 70:30 grass hay:concentrate (DM basis) substrate. The substrate was sprayed with buffer (control; pH = 6.5), a cellulase from Trichoderma longibrachiatum (TRI), a cellulase from Aspergillus niger (ASP), or a 1:1 mixture of both cellulases (MIX) 24 h before being placed in the fermenters. Enzymes were applied at a rate of 30 endoglucanase units/g of substrate DM. Treating the substrate with enzymes reduced substrate NDF and ADF content (P < 0.001 to P = 0.002) and increased DM, NDF, and ADF disappearance after 6 and 24 h of incubation (P < 0.001 to P = 0.004) but not after 48 h of incubation. Daily VFA production was increased (P = 0.004) by 15, 9, and 15% for TRI, ASP, and MIX, respectively, with half of the increase being due to production of acetate. All enzyme treatments augmented (P = 0.009) methane production, but none of them altered the methane:VFA ratio (P = 0.70). There were no differences (P = 0.80) among treatments in the daily flow of solid-associated microorganisms, as measured using 15N as a microbial marker. Although the TRI and MIX treatments increased (P < 0.05) the daily flow of liquid-associated microorganisms and the proportion of microbial N in the solid residue after 48 h of incubation, no effects were observed (P = 0.92 and P = 0.95, respectively) for the ASP treatment. The results show that the TRI and MIX treatments enhanced in vitro fermentation by increasing substrate fiber degradation, VFA production, and ruminal microbial growth. The lack of differences between TRI and MIX in most of the measured variables indicates that treating the substrate with a mixture of both cellulases did not further improve the effects of the TRI treatment.

Enzymatic recovery and purification of polyhydroxybutyrate produced by Ralstonia eutropha.

Polyhydroxybutyrate (PHB) is the most studied among a wide variety of polyhydroxyalkanoates, bacterial biodegradable polymers known as potential substitutes for conventional plastics. This work aimed at evaluating the use of enzymes to recover and purify the PHB produced by Ralstonia eutropha DSM545. Screening experiments allowed the selection of trypsin, bromelain and lysozyme among six enzymes, based on their efficiency in lysing cells of a non-PHB producing R. eutropha strain. Then, process conditions for high efficiency in PHB purification from the DSM545 cells were searched for the enzymes previously selected. The best result was achieved with 2.0% of bromelain (enzyme mass per biomass), equivalent to 14.1 U ml(-1), at 50 degrees C and pH 9.0, resulting in 88.8% PHB purity. Aiming at improving the process efficiency and reducing the enzyme cost, experiments were carried out with pancreatin, leading to 90.0% polymer purity and an enzyme cost three times lower than the one obtained with bromelain. The molecular mass analysis of PHB showed no polymer degradation. Therefore, this work demonstrates the potential of using enzymes in order to recover and purify PHB and bacterial biopolymers in general.