Cupuaçu (Theobroma grandiflorum Schum.) is an Amazonian fruit rich in antioxidants potential due to its phenolic compounds. Its seeds produce cupulate, a chocolate-like product, and the residue remains rich in biocompounds. This study aimed to optimize and compare microwave-assisted (MAE) and high pressure/temperature extraction (HPTE) of polyphenolic and flavonoid compounds from cupuaçu seed by-products. A Box-Behnken 33 factorial design assessed the influence of extraction time, solid/liquid ratio, and ethanol concentration. Responses included total polyphenol content, total flavonoid content, and anti-radical power via the ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) method. In optimum conditions, MAE had the highest total polyphenol content and HPTE for flavonoids. Optimal conditions for both methods were 65% (m/v) ethanol concentration, 45 min extraction time, and 0.03 g mL−1 solid/liquid ratio. MAE and HPTE achieved greater recovery of (−)-epicatechin and (−)-epigallocatechin-3-gallate compared to percolation. Both techniques proved viable for recovering polyphenols from cupuaçu seed by-products, in less time and with less solvent. Thus, cupuaçu by-products can be a sustainable and functional food source, with MAE and HPTE providing high yields and quality extracts rich in polyphenols. Theobroma Schum. Schum cupulate chocolatelike chocolate like product biocompounds microwaveassisted microwave assisted (MAE pressuretemperature pressure temperature (HPTE byproducts. byproducts by products. products BoxBehnken Box Behnken 3 solidliquid solid liquid ratio concentration antiradical anti radical 2,2azinobis3ethylbenzothiazoline6sulphonic 22azinobis3ethylbenzothiazoline6sulphonic azinobisethylbenzothiazolinesulphonic 2,2 azino bis ethylbenzothiazoline 6 sulphonic 2 acid method flavonoids 65 m/v mv m v (m/v 4 003 0 03 0.0 mL1 mL 1 mL− −epicatechin epicatechin − −epigallocatechin3gallate epigallocatechin3gallate epigallocatechingallate epigallocatechin gallate percolation byproducts, products, solvent Thus source 2azinobis3ethylbenzothiazoline6sulphonic azinobis 22 2, 00 0.

Abstract Cardiovascular diseases, resulting from the deposition of clots in blood vessels, are the leading cause of death worldwide. Fibrinolytic enzymatic activity can catalyze blood clot degradation. Findings show that 36 fungal isolates recovered from Caatinga soils have the potential to produce fibrinolytic protease under submerged conditions. About 58 % of the isolates displayed fibrinolytic activity above 100 U/mL, with Mucor subtilissimus UCP 1262 being the most active. The protease was biochemically and biophysically characterized, showing that the enzyme had a high affinity for SAApNA substrate and was significantly inhibited by fluoride methyl phenyl sulfonyl-C7H7FO2S, suggesting that it is a chymotrypsin-like serine protease. The highest enzyme activity was detected at pH 5.0 and 28 °C. This fibrinolytic protease’s far-UV circular dichroism (CD) showed that its secondary structure was primarily α-helical. The purified fibrinolytic enzyme may represent a novel therapeutic agent for treating thrombosis. At temperatures above 65 °C, the enzyme lost all its secondary structure. Its melting temperature was 58.1 °C, the denaturation enthalpy 85.1 kcal/mol, and the denaturation entropy 0.26 kcal/K∙mol. diseases vessels worldwide degradation 3 conditions 5 10 UmL U mL U/mL 126 active characterized sulfonylC7H7FO2S, sulfonylC7H7FO2S sulfonylCHFOS sulfonyl C7H7FO2S, C7H7FO2S C H FO S sulfonyl-C7H7FO2S chymotrypsinlike chymotrypsin like 50 0 5. 2 °C proteases s farUV far UV CD (CD αhelical. αhelical α helical. helical α-helical thrombosis 6 581 1 58. 851 85 85. kcalmol kcal mol kcal/mol 026 26 0.2 kcalKmol K kcal/K∙mol 12 sulfonylC CHFOS 8 02 0.

Abstract A fibrinolytic enzyme from the microalga Dunaliella tertiolecta was produced under mixotrophic conditions using different corn steep liquor (CSL) concentrations ( 0 ≤ CLS ≤ 0.75%), purified using a combination of salting out and ion-exchange chromatography, and then biochemical characterized. Cultivation of this microalga using 0.5% CSL led to the highest maximum cell concentration (1.960±0.010 mg L-1) and cell productivity (0.140g L-1 day-1), besides a high fibrinolytic activity of the extract obtained by the homogenization method (102 ±1 U mL-1). The enzyme extracted from the microalgal biomass was 5-fold purified with a 20% yield and was found to have a specific activity of 670 U mg-1. The enzyme, whose molecular weight determined by fibrin zymography was 10 kDa, was shown to be stable at pH 3.0–9.0 and up to 70°C with optimal pH and temperature values of 8.0 and 50°C, respectively. When compared to other fibrinolytic enzymes, this protease stood out for its high fibrinolytic activity, which was enhanced by Fe2+, inhibited by Zn2+, Cu2+, Mg2+, and Ca2+, and strongly inhibited by phenylmethylsulfonyl fluoride, suggesting that it belongs to the serine metalloprotease family. Moreover, thanks to its thermal stability, the enzyme may be easily preserved and activated under high-temperature conditions. (CSL 0.75%, 075 0.75% , 75 0.75%) ionexchange ion exchange chromatography characterized 05 5 0.5 1.960±0.010 19600010 1 960 010 (1.960±0.01 L1 L 0.140g 0140g g 140g L- day1, day1 day day-1) 102 (10 ± mL1. mL1 mL . mL-1) 5fold fold 20 67 mg1. mg1 1. mg-1 kDa 3090 3 9 3.0–9. 70C C 70 80 8 8. 50C 50 50°C respectively enzymes Fe2 Fe Fe2+ Zn2 Zn Zn2+ Cu2 Cu Cu2+ Mg2 Mg Mg2+ Ca2 Ca Ca2+ fluoride family Moreover stability hightemperature 07 0.75 7 0. 1.960±0.01 1960001 96 01 (1.960±0.0 day-1 (1 mL-1 2 6 mg- 309 3.0–9 0.7 1.960±0.0 196000 (1.960±0. day- mL- 30 3.0– 1.960±0. 19600 (1.960±0 3.0 1.960±0 1960 (1.960± 3. 1.960± 196 (1.960 1.960 19 (1.96 1.96 (1.9 1.9 (1.

Abstract Yellow mombin juice was microencapsulated by spray drying. Optimum operating conditions were selected from the outcomes of tests carried out according to a central composite rotatable design, using air inlet temperature (90 to 190 °C), feed flow rate (0.20 to 1.00 L/h) and 15 DE maltodextrin concentration (10 to 30%) as the independent variables, while powder moisture content, water activity, hygroscopicity, total carotenoids retention, and process yield as the responses. As expected, water activity, moisture content and yield of samples were significantly reduced by spray drying. The powder prepared under optimum conditions, whose reconstituted nectar was the most appreciated by panelists in sensory analysis, was partially characterized in terms of physicochemical properties. It exhibited an apparent density of 0.59 g/mL and a solubility of 81.49%. Particles generally occurred in clusters and exhibited spherical shape and smooth surface. The antioxidant activity was shown to be weak to intermediate.

Abstract This work deals with the application of microencapsulated extract of cupuassu seed by-product to functionally enrich a food multimixture. The methodological bases applied were those of the Official Methods of Analysis of AOAC. Multimixture enrichment with the extract led to 54 and 19% increases in total dietary fiber and total fat contents, to slight decreases in carbohydrate and protein contents, while the total energy value was reduced by 6.4% due to the increase in unsaturated fatty acids content. The enriched multimixture showed a significant antioxidant activity and high levels of Ca2+, Fe2+, Zn2+ and ω-9 and ω-6 unsaturated fatty acids. Nutritional and functional qualities of this multimixture, as well as its excellent stability against progressive temperature increases frequently occurring in food preparations, suggest its use as a basic ingredient for food supplementation. The results also indicate excellent technological properties of the multimixture to formulate new food products.

Abstract Cupuassu is a fruitful species from the Amazon basin with great economical potential, due to the multiple uses of its pulp and seeds in the food and cosmetic industries. This fruit generates large amounts of solid waste, which, despite of its high content of bioactive compounds, is discarded in the environment. Therefore, with the aim of reusing these wastes and adding economic value, we investigated the nutritional potential of the seed by-product resulting from industrial cupuassu oil extraction. The by-product was submitted to green extraction, and its contents of bioactive compounds were quantified by HPLC. The extract had a total polyphenol content (16.9 ± 1.8 mg/gDM) and an antioxidant activity, ABTS + (151.0 ± 5.5 mg/100 g) and DPPH. (85.4 ± 1.7 mmol/L). HPLC analysis of the extract identified epicatechin and glycosylated quercetin as the major flavonoids. This by-product shows great potential for being used as a source of the ingredients of high nutritional value, especially dietary fiber and polyphenols.

Abstract Aqueous two-phase systems (ATPS) are formed by mixtures of chemical species that, under certain conditions, separate into two immiscible phases, with water as the major component in global terms and, at lower concentrations, polymers, salts, ionic liquids and alcohols, depending on the system type. Different parameters influence ATPS equilibrium such as temperature, alcohol carbon chain size, salt type, pH, etc. The present work aimed to obtain the binodal curves for ATPS made up of ethanol, sodium citrate/ammonium sulfate and water at different temperatures (15, 25 and 35 °C) as well as to evaluate the potential of the ethanol/ammonium sulfate system in anthocyanins partition. The results showed that the increase in temperature did not alter the biphasic region in the temperature range investigated, not influencing the formation of phases. Nonlinear equations were satisfactorily fitted to binodal curves data, except for sodium citrate-containing ATPS at 15 °C. Higher concentration of the overall mixture resulted in longer tie-line of ammonium sulphate-containing ATPS. Higher values of partition coefficient and recovery yield of Syzygium cumini fruit anthocyanins were obtained at longer tie-lines.

Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.

ABSTRACT To select the best biosurfactant producer, Pseudomonas putida, Bacillus megatherium, Bacillus licheniformis and Bacillus subtilis were cultured in flasks on media with different salinity [low salinity (LS), Bushnell-Haas (BH) and artificial sea water (SW) media] supplemented or not with toluene as a model pollutant. Toluene inhibited the growth of all microorganisms and stimulated the biosurfactant production. B. subtilis exhibited the best performance, being able to lower the surface tension (ST) in the LS medium to 65.5 mN/min in the absence of toluene, and to 46.5 mN/min in the BH medium in the presence of toluene, corresponding to ST reductions of 13.0 and 27.5 mN/m, respectively. Scaling up the process to a bench-scale fermentor, the best results were obtained in the LS medium, where B. subtilis was able to reduce the toluene concentration from 26.0 to 4.3 g/L within 12 h and ST by 17.2 mN/m within 18 h. The results of this study point out that B. subtilis is an interesting biosurfactant producer, which could be used in the bioremediation of toluene-contaminated water.

The selection of new microorganisms able to produce antimicrobial compounds is hoped for to reduce their production costs and the side effects caused by synthetic drugs. Clavulanic acid is a β-lactam antibiotic produced by submerged culture, which is widely used in medicine as a powerful inhibitor of β-lactamases, enzymes produced by bacteria resistant to antibiotics such penicillin and cephalosporin. The purpose of this work was to select the best clavulanic acid producer among strains of Streptomyces belonging to the Microorganism Collection of the Department of Antibiotics of the Federal University of Pernambuco (DAUFPE). Initially, the strains were studied for their capacity to inhibit the action of β-lactamases produced by Klebsiella aerogenes ATCC 15380. From these results, five strains were selected to investigate the batch kinetics of growth and clavulanic acid production in submerged culture carried out in flasks. The results were compared with the ones obtained by Streptomyces clavuligerus ATCC 27064 selected as a control strain. The best clavulanic acid producer was Streptomyces DAUFPE 3060, molecularly identified as Streptomyces variabilis, which increased the clavulanic acid production by 28% compared to the control strain. This work contributes to the enlargement of knowledge on new Streptomyces wild strains able to produce clavulanic acid by submerged culture.

The aim of our study was to select the optimal operating conditions to permeabilize Kluyveromyces lactis cells using ethanol as a solvent as an alternative to cell disruption and extraction. Cell permeabilization was carried out by a non-mechanical method consisting of chemical treatment with ethanol, and the results were expressed as β-galactosidase activity. Experiments were conducted under different conditions of ethanol concentration, treatment time and temperature according to a central composite rotatable design (CCRD), and the collected results were then worked out by response surface methodology (RSM). Cell permeabilization was improved by an increase in ethanol concentration and simultaneous decreases in the incubation temperature and treatment time. Such an approach allowed us to identify an optimal range of the independent variables within which the β-galactosidase activity was optimized. A maximum permeabilization of 2,816 mmol L-1 oNP min-1 g-1 was obtained by treating cells with 75.0% v/v of ethanol at 20.0 °C for 15.0 min. The proposed methodology resulted to be effective and suited for K. lactis cells permeabilization at a lab-scale and promises to be of possible interest for future applications mainly in the food industry.

This work aimed to develop a new economic enzymatic process to treat the phenolic pollutants using pure tyrosinase in stirred vessel and adopting temperature (T), pH, rotational speed (N), initial phenol (C P,o) and enzyme (C T) concentrations as independent variables. Experimental data of the residual phenol concentration (C P) were used to calculate the oxidation efficiency (η), initial oxidation rate (-r o) and time required to reach the end of reaction (t) that were selected as the responses. Under the optimal conditions (T = 45°C, pH 6.6, N = 400 rpm, C P,o = 100 ppm and C T = 50 U/mL), η was 88.1%, -r o = 10.2 mg L-1 min-1, t = 40 min. These results suggested that tyrosinase-rich crude extracts from vegetable byproducts could be quite promising.

This study evaluated the effects of prebiotics on fermentation profile and growth of Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus rhamnosus, and Bifidobacterium lactis in co-cultures with Streptococcus thermophilus. Acidification rate and viability were positively influenced by the co-culture with B. lactis and by both inulin or oligofructose in low fat milk.

Clavulanic acid is a β-lactam antibiotic which has a potent β-lactamase inhibiting activity. In order to optimize its production by the new isolate Streptomyces DAUFPE 3060, the influence of two independent variables, temperature and soybean flour concentration, on clavulanic acid and biomass concentrations was investigated in 250 mL-Erlenmeyers according to a 2² central composite design. To this purpose, temperature and soybean flour (SF) concentration were varied in the ranges 26-34°C and 10-50 g/L, respectively, and the results evaluated utilizing the Response Surface Methodology. The experimental maximum production of clavulanic acid (629 mg/L) was obtained at 32°C and 40 g/L SF after 48 h, while the maximum biomass concentration (3.9 g/L) at 30°C and 50 g/L soybean flour, respectively. These values are satisfactorily close to those (640 mg/L and 3.75 g/L, respectively) predicted by the model, thereby demonstrating the validity of the mathematical approach adopted in this study.

A relatively complex network of reactions has been investigated, using as a network model the isothermal batch esterification of acetic acid with ethanol in n-heptane catalyzed by lyophilized mycelium of Aspergillus oryzae. The kinetic analysis was firstly carried out on the whole system, without any simplification, by means of the well-known integral method. Owing to the poor results obtained by this way, we developed an alternative approach, combining initial rates and integral analysis and reducing the number of empirical parameters to be determined by the use of equilibrium data. All the values of the parameters calculated according to this "composite" approach to kinetic analysis well correlate with experimental data.