Bioactive compounds guided diversity of endophytic fungi from Baliospermum montanum and their potential extracellular enzymes.

Baliospermum montanum (Willd.) Muell. Arg, a medicinal plant distributed throughout India from Kashmir to peninsular-Indian region is extensively used to treat jaundice, asthma, and constipation. In the current study, 203 endophytic fungi representing twenty-nine species were isolated from tissues of B. montanum. The colonization and isolation rate of endophytes were higher in stem followed by seed, root, leaf and flower. The phytochemical analysis revealed 70% endophytic isolates showed alkaloids and flavonoids, 13% were positive for phenols, saponins and terpenoids. Further, these endophytes produced remarkable extracellular enzymes such as amylase, cellulase, phosphates, protease and lipase. The most promisive three endophytic fungi were identified by ITS region and secreted metabolites were identified by gas chromatography-mass spectrometry (GC-MS/MS). The GC-MS profile detected twenty-five bioactive compounds from ethyl acetate extracts. Among endophytic fungi, Trichoderma reesei isolated from flower exhibited nine bioactive compounds namely, 2-Cyclopentenone, 2-(4-chloroanilino)-4-piperidino, Oxime-methoxy-Phenyl, Methanamine N-hydroxy-N-methyl, Strychane, Cyclotetrasiloxane, Octamethyl and 1-Acetyl-20a-hydroxy-16-methylene. The endophyte, Aspergillus brasiliensis isolated from root and Fusarium oxysporum isolated from seed produced nine and seven bioactive compounds, respectively. Overall, a significant contribution of bioactive compounds was noticed from the diverse endophytic fungi associated with B. montanum and could be explored for development of novel drug with commercial values.

A simplified and miniaturized glucometer-based assay for the detection of ß-glucosidase activity.

ß-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of ß-glucosidase activity. Single-factor experiments showed that optimum ß-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of ß-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from ß-dextranase, snailase, ß-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for ß-glucosidase activity. The easy-to-operate method was successfully used to detect a series of ß-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of ß-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

A magnetic tri-enzyme nanobiocatalyst for fruit juice clarification.

The major complications in fruit juice quality improvement are the presence of polysaccharides components in the form of disrupted fruit cell wall and cell materials. Hence, breakdown of cellulose along with pectin and starch is important for the juice processing. In this context, magnetic tri-enzyme nanobiocatalyst was prepared by simultaneously co-immobilizing three enzymes; α-amylase, pectinase and cellulase onto amino-functionalized magnetic nanoparticle by 60mM glutaraldehyde concentration with 10h cross-linking time for one pot juice clarification. The prepared nanobiocatalyst was characterized by FT-IR, SEM and XRD. The thermal (50-70°C) and pH (3-6) stability studies indicated more than two folds increment in half-life and enhanced tolerance to lower pH. The immobilized enzymes retained up to 75% of residual activity even after eight consecutive cycles of reuse. Finally, the clarification of apple, grapes and pineapple juices using magnetic tri-enzyme showed 41%, 46% and 53% respective reduction in turbidity till 150min treatment.

Cellulase activity screening using pure carboxymethylcellulose: application to soluble cellulolytic samples and to plant tissue prints.

Reliable, rapid and inexpensive detection of cellulolytic enzymes that can be used for a wide variety of biological and environmental samples are currently in high demand. Here, a new cellulase detection protocol is described that circumvents problems observed with popular agar-based methods by exploiting the ability of carboxymethylcellulose (CMC) to form gel-like surfaces on its own. These pure CMC-layers are sensitive to cellulolytic degradation and stainable by Gram's iodine without showing unwelcome reactions with other enzymes. The staining intensity negatively correlates with the enzyme activity and can be used for quantification. Cellulase activities are not obstructed by high sugar contents (e.g., in plant material) which limit the applicability of other quantification methods, making our new method particularly attractive for screening of plant extracts. A useful variant of this new method is its applicability to plant tissue prints for spatial mapping of the cellulolytic activity in a zymogram-like fashion.

Enzyme activities of Pleurotustuber-regium (Fries) Singer, cultivated on selected agricultural wastes.

Studies were carried out on the enzyme activities of Pleurotus tuber-regium fruitbodies and sclerotia cultivated in various agro-wastes. Higher activities of proteinase, total amylase, and glucose-6-phosphatase were observed in the sporophores compared to the sclerotia. Cellulase, carboxymethylcellulase and lipase values were higher in fruitbodies grown on cotton waste, sawdust of Khaya ivorensis and rice straw (2.4, 0.4 and 3.0 mg/h/mg protein, respectively). Sclerotia propagated on groundnut shells and cocoyam peels had lipase and phenoloxidase levels of 5.8 and 2.6 mg/h/mg protein, respectively. The peroxidase, alpha-amylase and catalase activities were also determined. The implication of these findings in relation to shelf-life, food nutrient, and flavour of this mushroom are discussed.

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.

Pre- and postweaning attributes in faunated and ciliate-free calves fed calf starter with or without fish meal.

In a 2 x 2 factorial design, 24 newborn, crossbred (Bos indicus x Bos taurus) calves were distributed in 4 equal groups involving dietary treatments of prestarter diets with (FM) or without fish meal (NFM) in a faunated (F) or ciliate-free (D) ruminal environment to study the ruminal fermentative development in pre-and postweaning periods. Defaunation was achieved by rearing calves in isolation and its effect was studied after first appearance of ciliate protozoa (observed after 8 wk of age) in the faunated animals. Calves were fed colostrum for 24 h and whole milk until weaning at 8 wk of age. Ruminal content samples were collected on d 4, 1 wk, weekly to 8 wk, and then biweekly at 9, 11, and 13 wk of age. The samples were analyzed for fermentation products [pH, total volatile fatty acids (VFA) and ammonia N] and enzyme [carboxymethyl (CM) cellulase, xylanase, beta-glucosidase, alpha-amylase, beta-galactosidase, proteases, and urease] activities. Weekly feed intake increased with age, but was similar in both groups. Ruminal pH declined steadily during 0 to 4 wk of age and then stabilized. The total VFA concentration increased with the age. The ammonia N (mg/dL) concentration increased from 14.9 on d 4 to 32.4 at 4 wk, decreased to 17.6 at 8 wk, and then steadied during the postweaning period. Samples collected on d 4 had no fibrolytic activity. Xylanase (U/dL) appeared first (1 wk) followed by beta-glucosidase (U/dL) and CM cellulase (U/dL), which increased steadily from a low of 4.69, 0.08, and 2.95 to 31.8 (6 wk), 5.92 (7 wk), and 19.8 (8 wk), respectively, and the concentrations showed nonsignificant alterations during postweaning periods. The concentration of alpha-amylase (U/dL) increased from 34.3 on d 4 to 87.2 at 8 wk, and then decreased to 56.6 (13 wk). beta-Galactosidase increased up to 6 wk then decreased to trace level (0.20 U/dL) at 13 wk of age. The concentrations of proteases and urease reached a steady state after 1 wk of age. The effect of diet type on ruminal fermentation products and enzyme parameters was nonsignificant. However, a steady and proportional alteration in both parameters in response to dry feed intake with the advancement of age was seen in all calves. Defaunation increased total VFA (97.3 vs. 75.8 mM/L) and alpha-amylase activity (80.3 vs. 61.4 U/dL) and decreased ammonia N (16.4 vs. 21.1 mg/dL), whereas the effect on other parameters was nonsignificant. Ruminal fermentative changes responded to dry feed intake, but did not differ in response to animal protein in prestarter diet.

Effect of cereal brans on Lentinula edodes growth and enzyme activities during cultivation on forestry waste.

AIMS: To develop strategies for increasing the growth of Lentinula edodes in eucalyptus residues. To this end, we have examined the effects of cereal brans additions on production of mycelial biomass and enzymes. METHODS AND RESULTS: Three isolates of the mushroom shiitake, L. edodes (Berk. Pegler), were evaluated for enzyme and ergosterol production on eucalyptus residue supplemented with 5, 10, 15 and 20% (w/w) of soya, wheat or rice brans. Nitrogen imput on eucalyptus residues accelerated mycelial growth by supplying the L. edodes with this limiting nutrient. High levels of enzymes activities were produced in eucalyptus residues supplemented by soya bran. Comparison of cellulose and xylanase production with manganese peroxidase (MnP) at 20% soya bran indicated that hydrolytic enzymes, but oxidative enzymes were reduced. CONCLUSIONS: Mycelial growth measurements revealed that eucalyptus residues supplemented with cereal brans supported fast growth of L. edodes, indicating that mycelium extension is related to the bioavailability of nitrogen. The type and concentration of nutrient supplement has a considerable effect both on substrate colonization and on the type of hydrolytic and oxidative enzymes produced. These characteristics may be useful for mushroom growing. SIGNIFICANCE AND IMPACT OF THE STUDY: Lentinula edodes is commercially important for edible mushroom production and supplements which enhance growth and enzymes production might also be beneficial for mushroom yields.

Quantification of supplemental enzymes in animal feedingstuffs by radial enzyme diffusion.

Methods are described which facilitate quantification of supplemental cellulase, protease and alpha-amylase when added to animal feedingstuffs at normal industrial inclusion levels. The methods entail extraction of the enzymes from the feedingstuffs by agitation in buffer followed by quantification of extract activity using radial diffusion techniques. A linear relationship between the diameter of the zone of hydrolyzed substrate and the log of the enzyme activity applied is observed over a broad activity range. Assay of a feedingstuff supplemented with 1 kg t(-1) cellulase, protease and alpha-amylase yielded net supplemental activity recoveries of 104+/-11.7%, 91.3+/-6.74% and 126+/-29.5%, respectively. A similar assay method did not prove sufficiently sensitive to facilitate detection of xylanase at typical in-feed inclusion levels. The levels of endogenous cellulase, protease and alpha-amylase activity detected in the unsupplemented feedingstuffs were equivalent to 6.4+/-0.47%, 6.6+/-0.82% and 29.0+/-14.1%, respectively, of a 1 kg t(-1) supplement. The methods are technically straightforward and will facilitate determination of enzyme stabilities during processes such as high-temperature pelleting of feedingstuffs, as well as allowing more rigorous quality control related to enzyme-supplemented animal feedingstuffs.

Invertebrate biomarkers: links to toxicosis that predict population decline.

The application of biochemical measurements that can be used as individual biomarkers of impaired biological function in invertebrates is reviewed to evaluate whether biochemical biomarkers of aquatic invertebrates can predict changes in natural populations. Biomarkers that measure toxic effects at the molecular level (e.g., the inhibition of brain acetylcholinesterase activity by organophosphorus pesticides) have been shown to provide rapid quantitative predictions of a toxic effect upon individuals in laboratory studies. Such biomarkers should not be used as a replacement for conventional aquatic monitoring techniques, but should be applied as supplementary approaches for demonstrating links between sublethal biochemical and adverse effects in natural populations in field studies. The research challenge for using biomarker measurements in aquatic invertebrates is to predict effects at the population level from effects at the individual level measured upon individuals collected in the field.

[Study on extraction process of flavone from Ginkgo biloba leaves by enzymic treatment].

The extraction technology of flavonoids from Ginkgo biloba leaves was studied in this paper. When the crude leaves used the cellulase enzyme pretreatment before extraction process, the extraction yield of total flavone was up to 2.01%. The extraction conditions were also determined experimentally as following: concentration of enzyme in the liquor being 0.125 g/L, mixture ratio between enzyme and substrate being 1:1200, treating 2 hours, at 45 degrees C, under natural pH value.

Bioefficacy of enzyme preparations containing beta-glucanase and xylanase activities in broiler diets based on barley or wheat, in combination with flavomycin.

The objective of the study was to determine the effects of two enzyme preparations containing beta-glucanase and xylanase activities on barley- and wheat-based diets, respectively, for broilers, in combination with flavomycin. In addition, the stability of the enzyme preparations after pelleting was measured. Temperatures recorded during the pelleting process reached 75 to 80 C, and the activities recovered with respect to the amounts present in the mash feed before pelleting were 80% or higher. Two performance experiments were conducted simultaneously under the same conditions over 6 wk. In addition, intestinal viscosity and incidence of vent pasting were measured and carcasses were eviscerated to determine abdominal fat, carcass yield, and percentage weight of intestines and viscera. Twenty-four pens (12 per sex), each containing 75 chickens were used in each experiment. Wheat- or barley-based diets were supplemented with flavomycin and a xylanase or a beta-glucanase preparation, respectively, in a 2 x 2 factorial arrangement of treatments. In the wheat diets, xylanase and flavomycin improved feed efficiency, in parallel with a reduction of intestinal viscosity. Xylanase reduced the incidence of vent pasting and the percentage viscera, especially of intestines, and increased abdominal fat. In the barley diets, beta-glucanase and flavomycin improved feed conversion. beta-Glucanase also reduced intestinal viscosity and vent pasting. Both beta-glucanase and flavomycin reduced percentage intestines, but the effects were not additive. In general, the effects of the enzyme preparations and flavomycin were independent, except for percentage intestines with beta-glucanase.

Production, characterization and application of the cellulase-free xylanase from Aspergillus niger.

The effects of carbon source on xylanase and cellulase production were studied. The extract of steam-exploded corn stover was found to be the best raw material for producing cellulase-free xylanase. The xylanolytic enzymes of Aspergillus niger An-76 were purified by chromatography, and the properties of the four purified components were analyzed. When the enzyme was used to treat the birch Kraft pulp, and followed by a subsequent CEH bleaching, a brightness of 6.8% SBD more than that of the untreated one using the same chlorine dosage, or a saving of nearly 50% of chlorine consumption with the same brightness, was realized.