Supercritical and ultrasound-assisted extracts from Pleurotus pulmonarius mushroom: chemical profiles, antioxidative, and enzyme-inhibitory properties.

BACKGROUND: This study provides an insight into the impact of ultrasound-assisted extraction with water as solvent (UAEW) and extraction by supercritical carbon dioxide (SC-CO2 ) with 5% EtOH on antioxidant and enzyme inhibitory activity in regard to the chemical profile of the edible and medicinal mushroom, Pleurotus pulmonarius. RESULTS: Extraction efficiency was between 0.36% and 63.32%, depending on the extraction technique. The main compounds in the extracts were fatty acids. Supercritical CO2 extraction with co-solvent was the most suitable method for obtaining extracts that were rich in ergosterol content, reaching a value of 40.1 mg g-1 . The UAEW of crude mushroom powder ensured the highest yield, as well as the extracts with best antioxidative activity. The measurements of enzyme inhibitory activity revealed that all types of investigated extracts exhibited only tyrosinase and amylase inhibition at a significant level. CONCLUSION: Based on our results, the extraction methods significantly affected the chemical profile and bioactivity of P. pulmonarius. © 2020 Society of Chemical Industry.

Simultaneous production of commercial enzymes using agro industrial residues by statistical approach.

BACKGROUND: Simultaneous production of commercial enzymes using agro-industrial residues by statistical approach is an important perspective in an industrial point of view. Despite the advantages of statistical methods optimization, the report on simultaneous production of pectinase and amylases are limited. The accumulation of agro-industrial residues causes serious environmental problems; however, citrus peel can be the important substrate for various enzymes production, including pectinase. These enzymes involving saccharification process and act as clarifying agent. RESULTS: In this study, orange peel and banana peel mixture were used as the suitable substrate for pectinase and amylase production using Bacillus pumilus in solid-state culture. The process parameters were optimized for simultaneous production of enzymes by a traditional-one-variable-at-a-time approach, a two level full factorial design, central composite design and response surface methodology. Among the selected variables, moisture content of the medium, pH and mineral supplement significantly influenced pectinase and amylase production. Pectinase production increased over 3-fold, whereas, 2-fold increase on amylase production was achieved after optimization by statistical approach. The purified pectinase exhibited maximal activity at pH 8.0, temperature of 60 °C and the molecular weight was 60 kDa. The purified amylase was highly active at pH 8.0, at 50 °C and the molecular weight was 37 kDa. The enzyme showed activity on fruit pulp in increasing clarity in orange and carrot juice and the saccharification of starch. CONCLUSION: Orange peel and banana peel mixture was effective as a solid medium for the simultaneous production of pectinase and amylase by Bacillus pumilus. Also, our statistical approach to optimize the medium components to yield more pectinase and amylase was fruitful and these enzymes showed appreciable results suitable for various applications. © 2018 Society of Chemical Industry.

Enzymatic fingerprinting of isomalto/malto-polysaccharides.

In this study, we present an enzymatic fingerprinting method for the characterization of isomalto/malto-polysaccharides (IMMPs). IMMPs are produced by the modification of starch with the 4,6-α-glucanotransferase (GTFB) enzyme and consist of α-(1→4), α-(1→6) and α-(1→4,6) linked glucoses. Enzymes were used separately, simultaneously or in successive order to specifically degrade and/or reveal IMMP substructures. The enzymatic digests were subsequently analysed with HPSEC and HPAEC to reveal the chain length distribution (CLD) of different IMMP substructures. The presence of amylose in the substrate resulted in the formation of linear α-(1→6) linked glycosidic chains (13.5 kDa) in the former amylopectin fraction. The length of these chains indicates that GTFB transferase activity on amylopectin is more likely to elongate single amylopectin chains than to provide an even distribution. Enzymatic fingerprinting also revealed that the GTFB enzyme is capable of introducing large (20 kDa) linear α-(1→6) linked glycosidic chains in the α-glucan substrate.

Application of a commercial digestive supplement formulated with enzymes and probiotics in lactase non-persistence management.

Strategies to avoid lactose malabsorption, which affects 70% of the world's population, are focused on the restriction of milk and dairy products or the use of non-human ß-galactosidases or probiotics endowed with ß-galactosidase activity added at mealtime. Our evaluation of a commercial blend of probiotics and enzymes (protease, lactase, lipase and amylase) and its potential application in lactase non-persistence management is described in this work. Recommended amounts (460-1000 mg) of the commercial probiotics-enzyme blend were shown to be adequate for performing in vitro lactose hydrolysis in standard solutions (0.25-5%) and commercial dairy products, namely milks (5% lactose) and yogurts (3% lactose), reaching hydrolysis values between 44 and 96%. According to these percentages, the use of the enzymatic preparation would guarantee the intake of less than 12 g, the recommendation of the EFSA for lactose intolerance. Furthermore, formation of prebiotic galactooligosaccharides was also detected, increasing the potential benefits of the enzymatic preparation in the gastrointestinal system.

Assessment of the potential of the multi-enzyme producer Bacillus amyloliquefaciens US573 as alternative feed additive.

BACKGROUND: Recently, probiotics have increasingly been used as feed additives in poultry diets as an alternative to antibiotic growth promoters fostering resistance development. RESULTS: This study was aimed at assessing the potential of Bacillus amyloliquefaciens US573 as a direct-fed microbial. The US573 strain was found to be free of harmful enzymatic activities and sensitive to antibiotics. In addition, it showed a good acid and bovine bile tolerance, high adhesion efficacy to chicken enterocytes, and an ability to form biofilms, which may favor its survival and persistence in the animal gastrointestinal tract. Moreover, besides the previously described extremely salt-tolerant and highly thermostable phytase, the US573 strain secretes xylanase, ß-glucanase and amylase activities useful in neutralizing antinutritional factors and maximizing the absorption of nutrients. The secretion of such enzymes may be responsible for the good performance of the US573 isolate in the digestibility of wheat in vitro. Indeed, using the vegetative cells, a yield of wheat dry matter digestibility of approximately 48% was achieved, which is slightly lower than the commercial feed additive Rovabio used as a reference (56.73% digestibility). CONCLUSION: The obtained results illustrate the potential of US573 strain as a promising direct-fed microbial candidate for application in the poultry industry. © 2017 Society of Chemical Industry.

Chemical modification with phthalic anhydride and chitosan: Viable options for the stabilization of raw starch digesting amylase from Aspergillus carbonarius.

The raw starch digesting type of amylase (RSDA) presents greater opportunities for process efficiency at cheaper cost and shorter time compared to regular amylases. Chemical modification is a simple and rapid method toward their stabilization for a wider application. RSDA from Aspergillus carbonarius was modified with either phthalic anhydride (PA) or chitosan. Activity retention was 87.3% for PA-modified and 80.9% for chitosan-modified RSDA. Optimum pH shifted from 5 to 7 after PA-modification. Optimum temperature changed from 30°C (native) to 30-40°C and 60°C for PA-modified and chitosan-modified, respectively. Activation energy (kJmol-1) for hydrolysis was 13.5, 12.7, and 10.2 while the activation energy for thermal denaturation was 32.8, 80.3, 81.9 for free, PA-modified and chitosan-modified, respectively. The specificity constants (Vmax/Km) were 73.2 for PA-modified, 63.1 for chitosan-modified and 77.1 for native RSDA. The half-life (h) of the RSDA at 80°C was increased from 6.1 to 25.7 for the PA-modified and 138.6 for the chitosan derivative. Modification also led to increase in D value, activation enthalpy and Gibbs free energy of enzyme deactivation. Fluorescence spectra showed that center of spectral mass decreased for the PA-modified RSDA but increased for chitosan modified RSDA.

Shedding light on the biological and chemical fingerprints of three Achillea species (A. biebersteinii, A. millefolium and A. teretifolia).

Representatives of the Achillea genus are widely used as foods or nutraceuticals. Considering the increasing demand for herbal dietary supplements with health promoting effects, the objective of this research was to evaluate the biological and chemical profiles of different extracts (ethyl acetate, methanol and water) obtained from three Achillea species (A. biebersteinii, A. millefolium and A. teretifolia). The antioxidant (free radical scavenging (DPPH and ABTS), reducing power (CUPRAC and FRAP), metal chelating and phosphomolybdenum), enzyme inhibitory (anti-cholinesterase, anti-tyrosinase, anti-amylase and anti-glucosidase) and antimicrobial (antibacterial and antifungal) effects were assessed to investigate their biological profiles. Moreover, the total phenolic and flavonoid contents were determined and LC-MS analysis was performed for the chemical profile of the investigated extracts. The LC-MS analysis revealed the presence of several caffeoylquinic acids in these extracts. Generally, the methanol and water extracts exhibited stronger antioxidant abilities, which correlated with the higher levels of phenolic compounds when compared to the ethyl acetate extracts. In addition, the best antimicrobial activities were obtained for the ethyl acetate and methanolic extracts. However, the ethyl acetate extract had remarkable enzyme inhibitory potential. On the basis of our results, Achillea species may be promoted as promising sources of natural agents and used for the development of nutraceuticals or functional food ingredients.

Characterisation of the faecal metabolome and microbiome of Thoroughbred racehorses.

REASONS FOR PERFORMING STUDY: The intestinal bacterial community of the horse is a key determinant of intestinal and whole body health. Understanding the bacterial community structure and function is an important foundation for studies of intestinal health and disease. OBJECTIVES: To describe the faecal bacterial community and volatile organic compounds (VOCs) of the faecal metabolome of healthy Thoroughbred racehorses and to characterise responses to dietary supplementation with amylase-rich malt extract. STUDY DESIGN: Intervention study. METHODS: Faecal samples were collected noninvasively before and 6 weeks after supplementation in 8 privately owned Thoroughbred racehorses in active race training. Faecal metabolome was characterised using thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS), with spectral analysis performed using AMDIS and compared against the NIST database. Taxonomic description of the faecal microbiota was achieved using error-corrected 454 pyrosequencing data from 16S rRNA gene amplicons. RESULTS: The faecal metabolome of our study population was dominated by organic acids, alcohols and ketones. We identified 81 different VOCs only 28 of which were present in >50% of samples indicating functional diversity. Faecal VOC profiles differed between first and second sampling point, some VOCs being significantly reduced post supplementation, consistent with a marked response to dietary amylase-rich malt extract. Faecal microbiota was characterised as highly diverse; samples demonstrated verifiable diversity in the range 1200-3000 operational taxonomic units (OTUs) per individual. The methods used also describe high levels of infrequent, low abundance OTUs. Faecal microbial community structure was found to be different following dietary supplementation. Differences in several low abundance bacterial taxa were detected and also some evidence of interhorse variation in response. CONCLUSIONS: The volatile faecal metabolome of Thoroughbred racehorses is dominated by organic acids, alcohols and ketones; this study demonstrates that dietary supplementation with amylase-rich malt extract may significantly alter the profile of VOCs. The faecal microbiome is highly diverse, dominated by Firmicutes and Bacteroidetes. Small but significant changes in microbial community structure were detected following dietary supplementation. This study describes the faecal metabolome and microbiome of healthy Thoroughbred racehorses against which future studies of disease and dietary intervention can be benchmarked.

Mechanism for starch granule ghost formation deduced from structural and enzyme digestion properties.

After heating in excess water under little or no shear, starch granules do not dissolve completely but persist as highly swollen fragile forms, commonly termed granule "ghosts". The macromolecular architecture of these ghosts has not been defined, despite their importance in determining characteristic properties of starches. In this study, amylase digestion of isolated granule ghosts from maize and potato starches is used as a probe to study the mechanism of ghost formation, through microstructural, mesoscopic, and molecular scale analyses of structure before and after digestion. Digestion profiles showed that neither integral nor surface proteins/lipids were crucial for control of either ghost digestion or integrity. On the basis of the molecular composition and conformation of enzyme-resistant fractions, it was concluded that the condensed polymeric surface structure of ghost particles is mainly composed of nonordered but entangled amylopectin (and some amylose) molecules, with limited reinforcement through partially ordered enzyme-resistant structures based on amylose (for maize starch; V-type order) or amylopectin (for potato starch; B-type order). The high level of branching and large molecular size of amylopectin is proposed to be the origin for the unusual stability of a solid structure based primarily on temporary entanglements.

[Effects of temperature and pH on digestive enzymes activities in Whitmania pigra].

OBJECTIVE: Current study was conducted to investigate and compare the impact of temperature and pH on the activities of amylase, protease and lipase in alimentary tract of Whitmania pigra. METHOD: The responses of amylase, protease, and lipase activities were determined over a wide range of temperatures (7-52 degrees C) and pH gradient (2.2-11.2). RESULT: The highest lipase activity was found under 37 degrees C, pH 8.2, and the highest amylase activity was detected under 37 degrees C, pH 5.2, while protease activity peaked at 42 degrees C, pH 3.2 or pH 9.2. CONCLUSION: The optimal temperature in alimentary tract of Wh. pigra for lipase and amylase was 37 degrees C, and the responding temperature for protease was 42 degrees C. The optimal pH value in alimentary tract of Wh. pigra for lipase and amylase was pH 8.2 and pH 5.2, respectively. While pH 3.2 or 9.2 seems to be both favorable for high protease activity.

Production of acid-stable and high-maltose-forming α-amylase of Bacillus acidicola by solid-state fermentation and immobilized cells and its applicability in baking.

Among matrices used for immobilizing Bacillus acidicola cells [calcium alginate, chitosan + alginate, scotch brite, and polyurethane foam (PUF)], α-amylase production was highest by PUF-immobilized cells (9.1 U ml(-1)), which is higher than free cells (7.2 U ml(-1)). The PUF-immobilized cells could be reused over seven cycles with sustained α-amylase production. When three variables (moisture, starch, and ammonium sulfate), which significantly affected enzyme production in solid-state fermentation (SSF), were optimized using response surface methodology, 5.6-fold enhancement in enzyme production was attained. The enzyme production in SSF is 3.8-fold higher than that in submerged fermentation. The bread made by supplementing dough with α-amylase of B. acidicola scored better than those with the xylanase of Bacillus halodurans and thermostable α-amylase of Geobacillus thermoleovorans.

Partial characterization of a novel amylase activity isolated from Tunisian Ficus carica latex.

CONTEXT: A large number of plants still need to be investigated through screening of amylases suitable for industry. In the present study, and for the first time, we describe the amylolytic activity of Saint Pedro Ficus carica L. (Moraceae) crude latex of Kahli and Bidhi varieties. OBJECTIVE: Effects of temperature, pH, metal ions, and inhibitors and compatibility with some commercial detergents were investigated for amylase activity. MATERIALS AND METHODS: Amylase activity was screened in crude latex using the DNS method and potato starch as a substrate. Analyses of amylolytic reaction products by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were performed. RESULTS: Bidhi and Kahli amylases were active in optimal pH of 6.5 and 7 at 45°C, respectively, displaying a half life of 85 and 60 min, respectively, at 80°C, and they were very stable in a wide range of pH (4-12). Bidhi amylase activity increased to 260% by addition of 10(-3) mM Fe(2+) or 10(-2) mM Cu(2+), and was strongly inhibited by Mg(2+) and EDTA. In the presence of Ca(2+) and Mg(2+), Kahli amylase activity was dramatically enhanced by 220 and 260%, respectively. The compatibility of both amylases with certain commercial detergents was also shown to be good as enzymes retained up to 98% of their activities after 30 min of incubation at 80°C. DISCUSSION AND CONCLUSION: Analysis of amylolytic reaction products by TLC and HPLC suggested that Kahli amylase was an amyloglucosidase and Bidhi amylase was ß-fructose, α(1-4) glucose. Bidhi amylase is a good choice for application in starch, food, detergents and medical industries.

Comparative effects of different triazole compounds on growth, photosynthetic pigments and carbohydrate metabolism of Solenostemon rotundifolius.

The effects of two triazole compounds, triadimefon and hexaconazole, on the growth and carbohydrate metabolism were studied in Solenostemon rotundifolius Poir., Morton plants under pot culture. Plants were treated with triadimefon at 15mg l(-1) and hexaconazole at 10mg l(-1) separately by soil drenching on 80, 110 and 140 days after planting (DAP). The plants were harvested randomly and growth parameters were studied on 90, 120 and 150 DAP for determining the effect of both the triazole on growth and chlorophyll pigments. These triazole compounds increased the chlorophyll pigments. However, both the treatments decreased the fresh and dry weights of shoot and leaf area. Both these triazole resulted in a marginal increase in starch content and decreased the sugar contents. The carbohydrate metabolizing enzymes alpha- and beta-amylase activities were reduced and invertase activity increased in S. rotundifolius under triadimefon and hexaconazole treatments.

Temperature impacts the multiple attack action of amylases.

The action pattern of several amylases was studied at 35, 50, and 70 degrees C using potato amylose, a soluble (Red Starch) and insoluble (cross-linked amylose) chromophoric substrate. With potato amylose as substrate, Bacillus stearothermophilus alpha-amylase (BStA) and porcine pancreatic alpha-amylase displayed a high degree of multiple attack (DMA, i.e., the number of bonds broken during the lifetime of an enzyme-substrate complex minus one), the fungal alpha-amylase from Aspergillus oryzae a low DMA, and the alpha-amylases from B. licheniformis, Thermoactinomyces vulgaris, B. amyloliquifaciens, and B. subtilis an intermediate DMA. These data are discussed in relation to structural properties of the enzymes. The level of multiple attack (LMA), based on the relation between the drop in iodine binding of amylose and the increase in total reducing value, proved to be a good alternative for DMA measurements. The LMA of the endo-amylases increased with temperature to a degree depending on the amylase. In contrast, BStA showed a decreased LMA when temperature was raised. Furthermore, different enzymes had different activities on Red Starch and cross-linked amylose. Hence, next to the temperature, the action pattern of alpha-amylases is influenced by structural parameters of the starch substrate.

Carbohydrases, protease, and phytase have an additive beneficial effect in nutritionally marginal diets for broiler chicks.

To investigate the additive effects of xylanase, amylase, protease, and phytase in the diets of broiler chickens, a study was conducted using 1,152 growing broiler chicks (8 treatments with 12 replicate pens of 12 chicks). The birds were fed a corn/soybean-based negative control (NC) diet that was formulated to be nutritionally marginal in terms of metabolizable energy, Ca, and P. A nutritionally adequate positive control (PC) diet was fed for comparison. The NC diet was supplemented with phytase; a cocktail of xylanase, amylase, and protease (XAP); or a combination of phytase and XAP at 100 or 200 mg of each enzyme/kg (200 mg of XAP/kg provided a guaranteed minimum of 300 U of xylanase, 400 U of amylase, and 4,000 U of protease/kg; 200 mg of phytase/kg provided a guaranteed minimum of 1,000 U of phytase/kg). Growth performance, ileal digestible energy (IDE), and the digestibility coefficients of N, Ca, P, and DM were calculated. Individually and in combination, both phytase and XAP improved (P < 0.05) gain-to-feed ratio compared with the NC diet, particularly at the highest inclusion concentration. Body weight gain followed a similar trend, showing an improvement of approximately 6 to 7% with either enzyme individually and a 14% improvement with a combination of phytase and XAP. The effect of enzymes on IDE and nutrient digestibility coefficients was not as marked, but a 165 kcal/kg reduction in IDE was noted between the NC and PC diets, and a combination of phytase and XAP improved IDE by >100 kcal/kg. It can be concluded that the use of phytase and XAP individually in a corn/soybean meal-based diet is effective in improving nutrient digestibility and performance of broilers fed nutritionally marginal diets. Furthermore, there may be an additive effect of phytase and XAP on broiler performance, giving a cost-effective nutritional strategy for the profitable production of poultry products.

Italian buckwheat (Fagopyrum esculentum) starch: physico-chemical and functional characterization and in vitro digestibility.

A study on the physico-chemical properties and structure together with the evaluation of starch digestibility was carried out on starch isolated from buckwheat (Fagopyrum esculentum) cultivated in different Italian areas. Results showed that buckwheat samples analysed were different among them and from wheat starch used as reference. Buckwheat granules were polygonal in shape and had a smaller diameter than the wheat starch granule. The starch obtained from buckwheat had a higher swelling power than the wheat one, probably as a consequence of the wheaker but more extensive bonding forces in the granule. During cooling, buckwheat samples showed a good paste stability.

[Properties of white sorghum diastatic malt]. / Propiedades de malta diastásica de sorgo blanco.

Diastatic malt enzymes have potential to hydrolize pregelatinized starches releasing soluble sugars, lowering viscosity of slurries and allowing the use of high nutrient densities for preparation of cereal-based creams, baby food and drinks. Determination of the extent in which sorghum malt is able to develop desirable functional properties such as viscoty, water solubility and nutritional quality is fundamental. In this work the characteristics of a white sorghum during germination and the resulting malt were evaluated. "Dorado" white sorghum was germinated at 28 degrees C and 95% RH during 6 days in complete darkness, dried at 55 degrees C and ground to produce diastatic malt flour. Physicochemical, chemical, diastatic and nutritional characteristics of malt and its ability to liquify precooked flour slurries were determined. Maximum diastatic activity occurred at 3-4 days germination. Dry matter loss increased during germination at a rate of 2.7 percent units per day. Longer germination resulted in increased dry matter loss and decreased diastatic activity. Water solubility index of sorghum increased linearly during the first 5 days of germination probably caused by the production of soluble sugars and free amino acids. In vitro protein digestibility, lysine content and C-PER increased during germination. Sorghum malt was able to liquify precooked rice, wheat, oats or millet pastes (20% solids) in 5 min mixing at 30 degrees C. Utilization of malts with maximum diastatic activity are useful to liquify precooked cereal pastes or drinks and to increase the total solid contents and nutrient density while keeping the liquid properties of the product.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxic effects of cyclosporin A on the exocrine pancreas in rats.

The current study was done to evaluate the subacute toxic effects of cyclosporin A (CS) on the exocrine pancreas and the protective effect of potent protease inhibitor camostate (FOY-305). CS administration (15 milligrams, twice a day) for 14 days caused a significant increase in serum amylase levels, pancreatic amylase and cathepsin B content and mild acinar cell vacuolization and interstitial edema. CS also caused the redistribution of cathepsin B activity from the lysosomal fraction to the zymogen fraction, indicating colocalization of lysosomal enzyme with pancreatic digestive enzymes. The administration of camostate (150 milligrams per kilogram, twice a day for 14 days) almost completely prevented the toxic changes induced by CS. These results indicate that CS induces exocrine pancreatic injury and that lysosomal enzymes play important roles in the pathogenesis of the injury. The results also suggest the usefulness of camostate in protecting the exocrine pancreas in patients treated with CS after organ transplantation, because it can inhibit some proteases that are closely involved in the fragility of the subcellular organelles.