Non-purified Extract Research Articles

Purification of bioactive phenolics from Phanerochaete chysosporium biomass extract on selected macroporous resins

In this study, two different types of macroporous resins known as XAD-7HP and HP-20 were evaluated for the adsorption and desorption properties against bioactive phenolics extracted from Phanerochaete chrysosporium. From the previous static sorption studies, it was found that the adsorption capacity for both resins had has no significant difference. Then, the kinetic adsorption data were analyzed with both pseudo-first-order and pseudo-second-order equations and the later performed better. The adsorption isotherm data were fitted well by both Langmuir and Freundlich models. Meanwhile in desorption study, HP-20 and XAD-7HP gave 90.52% and 88.28% recoveries, respectively. Considering the desorption results of the macroporous resins, HP-20 and XAD-7HP were packed in chromatography column to further purify the phenolics. For dynamic adsorption, breakthrough capacity of HP-20 (0.522) was found to be higher than XAD-7HP (0.131). Different ethanol concentrations (30% to 50% (v/v)) were investigated at fixed flowrate (1 ml/min) on phenolics recovery from both types of resins. The highest recovery of bioactive phenolics was 94.3% using XAD-7HP resins at 50% (v/v) of ethanol. Only 77.1% of bioactive phenolics were recovered using HP-20 resin at the same experimental conditions. The purified extract subsequently was analyzed using HPLC. The results showed that three phenolics (gallic acid 3,4-dihydroxybenzoic acid and 4-hydroxybenzoic acid) were identified with higher concentrations as compared to non-purified extract. Finally, the purified extract was tested for scavenging activity against DPPH, and it showed that the activity increased significantly to 90.80% from 59.94% in non-purified extract.

Regioselective hydroxylation of cholecalciferol, cholesterol and other sterol derivatives by steroid C25 dehydrogenase

Steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S is a molybdenum oxidoreductase belonging to the so-called ethylbenzene dehydrogenase (EBDH)-like subclass of DMSO reductases capable of the regioselective hydroxylation of cholesterol or cholecalciferol to 25-hydroxy products. Both products are important biologically active molecules: 25-hydroxycholesterol is responsible for a complex regulatory function in the immunological system, while 25-hydroxycholecalciferol (calcifediol) is the activated form of vitamin D3 used in the treatment of rickets and other calcium disorders. Studies revealed that the optimal enzymatic synthesis proceeds in fed-batch reactors under anaerobic conditions, with 6-9% (w/v) 2-hydroxypropyl-β-cyclodextrin as a solubilizer and 1.25-5% (v/v) 2-methoxyethanol as an organic co-solvent, both adjusted to the substrate type, and 8-15mMK3[Fe(CN)6] as an electron acceptor. Such thorough optimization of the reaction conditions resulted in high product concentrations: 0.8g/L for 25-hydroxycholesterol, 1.4g/L for calcifediol and 2.2g/L for 25-hydroxy-3-ketosterols. Although the purification protocol yields approximately 2.3mg of pure S25DH from 30g of wet cell mass (specific activity of 14nmolmin-1mg-1), the non-purified crude extract or enzyme preparation can be readily used for the regioselective hydroxylation of both cholesterol and cholecalciferol. On the other hand, pure S25DH can be efficiently immobilized either on powder or a monolithic silica support functionalized with an organic linker providing NH2 groups for enzyme covalent binding. Although such immobilization reduced the enzyme initial activity more than twofold it extended S25DH catalytic lifetime under working conditions at least 3.5 times.

Experimental and Simulation Study on Formulation of Clove Essential Oil Products Using Alternative Surfactant

Abstract The formulation of clove essential oil as an emulsion using nonpurified aqueous extract from Pfaffia glomerata roots as surfactant has been experimentally demonstrated. Firstly, a commercial surfactant (OSA-modified starch) was used to evaluate the formulation of nano-emulsions in our devices. Results show that, the method of emulsification and its duration had an insignificant influence on all response variables. On the other hand, the influence of OSA-modified starch concentration was very significant (P<0.01) regarding droplet size (d 32 and d 43 ), specific surface area and percentage of demulsified oil, while volume fraction of oil influenced very significantly (P<0.01) the droplet size (d 43 ), polidispersity, specific surface area and percentage of demulsified oil. Secondly, considering these results, it was selected a condition to perform the evaluation of the use of nonpurified aqueous extract from Pfaffia glomerata roots as an alternative surfactant. The properties of the emulsions prepared with this alternative surfactant were quite different to the properties obtained with OSA-modified starch. On the other hand, a stable emulsion with a droplet size in the range of 900-1046 nm was obtained. At the end, using SuperPro Designer software we compared in terms of utilities consumption and total utility cost different technological scenarios for the production of clove essential oil as an emulsion or as dry microcapsules. Ultrasonication method was more convenient during emulsification, while for encapsulation purposes promising results were obtained by carbon dioxide assisted nebulisation with a bubble dryer (CAN-BD) process since besides its low energy consumption, part of the available infrastructure used for obtaining the essential oil could be shared.

Optimization of the immobilization of sweet potato amylase using glutaraldehyde-agarose support. Characterization of the immobilized enzyme

A simplified procedure for the preparation of immobilized beta-amylase using non-purified extract from fresh sweet potato tubers is established in this paper, using differently activated agarose supports. Beta-amylase glutaraldehyde derivative was the preparation with best features, presenting improved temperature and pH stability and activity. The possibility of reusing the amylase was also shown, when this immobilized enzyme was fully active for five cycles of use. However, immobilization decreased enzyme activity to around 15%. This seems to be mainly due to diffusion limitations of the starch inside the pores of the biocatalyst particles. A fifteen-fold increase in the Km was noticed, while the decrease of Vmax was only 30% (10.1Umg−1 protein and 7.03Umg−1 protein for free and immobilized preparations, respectively).

The VISYT trial: Venom Immunotherapy Safety and Tolerability with purified vs nonpurified extracts

Venom immunotherapy (VIT) is a highly effective treatment but can induce systemic adverse effects. To evaluate the safety and tolerability of VIT with purified and nonpurified extracts for treating yellow jacket and honeybee allergy. Ninety-four patients (mean age, 46 years) with a history of insect venom allergy were randomly allocated to undergo purified extract VIT (group A [44 patients]) or nonpurified extract VIT (group B [50 patients]). Fifty-six patients were allergic to yellow jacket venom (group A: 25; group B: 31) and 38 to honeybee (19 per group). The induction phase was performed using a 2- or 7-day ultrarush scheme. The maintenance phase lasted 11 weeks. Local and systemic reactions were recorded after each injection. A total of 1,401 VIT injections were performed. Six systemic reactions were observed in 4 patients (honeybee-allergic patients only) (4% of patients; 0.4% of injections): 1 patient in group A (2%) and in 3 in group B (6%) (P = .57). Local extensive reactions were recorded after 5 injections in 4 patients (9%) in group A (2 yellow jacket- and 2 honeybee-allergic patients) and after 17 injections in 12 patients (24%) in group B (8 yellow jacket- and 4 honeybee-allergic patients) (P = .02). Total reactions (systemic and large local) numbered 6 in group A (0.9% of injections; 11% of patients) and 20 in group B (2.7% of injections; 30% of patients) (P = .001). In patients with honeybee or yellow jacket venom allergy, VIT with purified extracts has a significantly lower propensity toward severe local reactions compared with VIT with nonpurified extracts.

Renewable enzyme reactors based on beds of artificial gel antibodies

A novel approach is described for the synthesis of beds for enzyme reactors. The method is based on the use of artificial antibodies in the form of polyacrylamide gel particles with diameters around 0.1–0.3 mm. These gel particles mimic protein antibodies, raised in experimental animals, in the sense that they selectively recognize and adsorb only the protein present during the preparation of the “antibodies”. The gel antibodies have several advantages over conventional protein antibodies, which can be taken advantage of in the design of enzyme reactors; for instance, if upon prolonged use the immobilized enzyme loses its activity it can easily be replaced by an active enzyme, which is not possible when the enzyme is immobilized via a conventional protein antibody (a new bed with immobilized protein antibodies must be prepared); and equally or more remarkable: the enzyme can be applied in the form of a non-purified extract since the selectivity of the artificial gel antibodies is so high that they will “fish-out” the enzyme, but no other proteins in the extract. In addition, no preconcentration of the enzyme solution is required prior to the immobilization, since the enzyme is enriched at the top of the column upon the application. These unique properties make enzyme reactors based on artificial gel antibodies very attractive, also in process chromatography. The potential application range of the artificial gel antibodies is enormous since the same method for their synthesis can be used independent of the structure and the size of the “antigen”; for instance, renewable biosensors based on gel antibodies for the selective detection of protein biomarkers, as well as pathogenic viruses, bacteria, and spores (for instance Anthrax) should not be difficult to design.

Overcoming the drawbacks of natural coagulants for drinking water treatment

A number of natural coagulants from plants or animal origin can be effectively used for the treatment of drinking water. Some are used at household levels in traditional systems using crude (non-purified) extract. In the crude extract form, natural coagulants release organic and nutrient loads to the water resulting in poor treated water quality. In such cases it becomes necessary to purify the coagulant component from the crude extract before using it for water treatment. This paper discusses an effective and simple purification of the coagulant from Moringa oleifera seed using an ion exchange method that would overcome the drawbacks of natural coagulants. The impact of the purification process on reducing organic and nutrient release was studied. Adsorption and elution parameters of the ion exchange process were optimised. The study indicated that the purified protein has effective coagulation activity and it did not release organic and nutrient loads to the water. Compared to other purification methods used in previous studies, this is simple and of comparatively lower cost. This purification method can be readily scaled up and applied even in remote parts of developing countries. This work suggests that the concerns of natural coagulants can be overcome by simple purification and this is believed to promote their use in drinking water treatment.

Phenylalanine Ammonia-Lyase Activity in Soybean Roots Extract Measured by Reverse-Phase High Performance Liquid Chromatography

Abstract: The high performance liquid chromatography (HPLC) technique was applied to measure phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity in soybean (Glycine max L. Merril cv. BR16) roots. t-Cinnamate, the catalytic product of the PAL reaction was quantified at 275nm by isocratic elution with methanol: water through an ODS(M) column. Comparative experiments were carried out with 1.0 mM ferulic acid, an inducer of PAL activity. The results suggest that liquid chromatography is a rapid and sensitive method to analyze PAL activity in non-purified extract.

Primer dependent and independent forms of soluble starch synthetase from developing barley endosperms

The activity of soluble starch synthetase (ADP-glucose: α-1,4-glucan α-4-glucosyltransferase) in the non-purified extract from 16 day-old Bomi barley endosperms (Hordeum vulgare L.) was low and the reaction was non-linear when plotted against protein concentration. Starch synthetase was purified by ammonium sulfate precipitation and DEAE-cellulose chromatography and separated into four fractions. In the absence of an added carbohydrate primer two of the four fractions catalized the synthesis of a methanol-precipitable α-glucan when high concentrations of sodium citrate and bovine serum albumim were added. The rate of α-glucan synthesis by the unprimed reaction was higher than for the primed reaction. The four enzyme fractions were active with ADP-Glc, but not with UDP-Glc, both in the primed and in the unprimed reaction.