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The aim of this study was to investigate covalent immobilization of horseradish peroxidase (HRP) on magnetic nanoparticles (Mag) encapsulated in calcium alginate beads (MABs) for color degradation, combining easy and fast removal of biocatalyst from the reaction mixture due to its magnetic properties and strong binding due to surface alginate functional groups. MABs obtained by extrusion techniques were analyzed by optical microscopy, FEG-SEM and characterized regarding mechanical properties, magnetization and HRP binding. HRP with initial concentration of 10 mg/gcarrier was successfully covalently bonded on MABs (diameter ~1 mm, magnetite/alginate ratio 1:4), with protein loading of 8.9 mg/gcarrier, immobilization yield 96.9% and activity 32.8 U/g. Immobilized HRP on MABs (HRP-MABs) was then used to catalyze degradation of two anthraquinonic dyes, Acid Blue 225 (AB225) and Acid Violet 109 (AV109), as models for wastewater pollutants. HRP-MABs decolorized 77.3% and 76.1% of AV109 and AB225, respectively after 15 min under optimal conditions (0.097 mM H2O2, 200 mg of HRP-MABs (8.9 mg/gcarrier), 0.08 and 0.1 g/mg beads/dye ratio for AV109 and AB225, respectively). Biocatalyst was used for 7 repeated cycles retaining 75% and 51% of initial activity for AB225 and AV109, respectively, showing potential for use in large scale applications for colored wastewater treatment. Supplementary information: [https://hdl.handle.net/21.15107/rcub_dais_13156]

The possibility to utilize Alcalase to hydrolyze soft wheat flour (SWF) containing high concentration of solids (i.e. starch) and the effect of its presence was investigated. At a degree of hydrolysis of 23.49% +/- 0.64%, sample prepared as 15% (w/w) showed greatest reduction in gliadin content, resulting in 9.42 +/- 1.5 ppm of gluten, tested by competitive ELISA test. SDS-PAGE electrophoresis confirmed the differences in gliadin content for all of the prepared wheat flour hydrolysates (WFHs). All WFHs had improved radical-scavenging (>60%), metal-ion chelating (>80%) and technofunctional properties after enzymatic hydrolysis compared to SWF. FTIR spectra confirmed structural changes in the Amide I (1,700-1,600 cm(-1)) region. Changes in total zeta-potential and average particle size were observed. The hydrolysis reaction was facilitated by the increase of the solids content since it interferes with the aggregation of gluten proteins, therefore less available water was proven beneficial for the enzymatic reaction. Novelty impact statement: Gluten originating from soft wheat flour can be efficiently hydrolyzed by endo-protease Alcalase (DH similar to 24%), without introducing steps that include the extraction and isolation of gluten. By preparing the wheat flour hydrolysates, the significant reduction of adverse allergenicity of wheat flour has been achieved; gluten content was less than 10 ppm. Improvement of techno-functional properties, zeta-potential and particle size distribution, as well as antioxidant potential of soft wheat flour was observed upon enzymatic hydrolysis for all samples.

In this study, we assessed the effects of microwave irradiation of wheat gluten proteins as a pretreatment performed in a microwave reactor that could accurately control process parameters as a function of power and temperature, as well as comparing it with conventional heat treatment. The aim was to identify suitable combinations of partial enzymatic hydrolysis and microwave pretreatment parameters to produce gluten hydrolysates with reduced allergenicity and conserved techno-functional features for food application. FTIR analysis, and total and reactive SH group contents confirmed that the microwave-controlled heating can significantly change the secondary structure and conformation of gluten protein. The microwave treatment had the largest effect at 200 W and 100 °C, at which the content of gluten has been reduced by about 2.5-fold. The microwave pretreatment also accelerated the enzymatic hydrolysis of gluten, changing the kinetic profile. The apparent hydrolysis rate constants (k2) were 1.00, 3.68, 3.48, 4.64 and 4.17 min−1 for untreated gluten, and those pretreated with microwave power of 200, 400, 600 and 800 W, respectively. Compared to the heat treatment, it appeared that microwave specific non-thermal effects had a significant influence on the gluten structure and allergenicity and, in combination with the enzymatic hydrolysis, ultimately yielded protein hydrolysates with enhanced antioxidant and functional properties.

Soy proteins known for their high nutritional value and pronounced techno-functional properties, can be hydrolyzed by using proteolytic enzymes and thus converted into hydrolysates rich in di-, tri- and oligopeptides. The resulting peptides are carriers of valuable biological activities, which make the soy hydrolysates very important in functional food applications as techno-functional and bioactive ingredients. However, commercial incorporation and application of soy protein hydrolysates can be hinderedby their low bioavailability and instability, bitter taste, hygroscopicity and possibility to interact with the food matrix. The aim of this research is encapsulation of the soy protein hydrolysate in liposomes in order to overcome the stated shortcomings, while preserving the biological activities that protein hydrolysates exhibit. The soy hydrolysate was prepared by a two-step enzymatic hydrolysis of a soy protein concentrate using commercial food-grade proteases, endoprotease from Bacillus amyloliquefaciens (Neutrase?) and egzo- and endoprotease from Aspergillus oryzae (Flavourzyme?) and encapsulated within liposomes. The liposomes were produced by a thin film method using a commercial lipid mixture (Phospolipon? 90G) containing mainly phosphatidylcholine. Next, the obtained multilamellar vesicles (MLV) with the soy protein hydrolysate were treated by high-intensity ultrasound waves generated by using (1) an ultrasonic probe at a frequency of 20 kHz and (2) an ultrasonic bath with a frequency 40 kHz. The smallest (310 nm) and uniform (unimodal size distribution) liposomes with the highest efficiency of peptide encapsulation (19 %) were obtained by the probe sonication. The presented results showed that incorporation of the soy protein hydrolysates was achieved within the liposome membrane and caused an increase in the liposome size in all tested formulations, namely: from 297 to 310 nm by using the ultrasonic probe, from 722 to 850 nm by using the ultrasonic bath, while in formulations without the ultrasonic treatments the increase from 2818 to 3464 nm was recorded. The entrapped peptides caused enlargement of all liposomes and the increase in negative charge of zeta potential values, which in the case of MLV liposomes was below -30 mV, indicating high stability of these liposomes. Significant antioxidant activity of the probe-sonicated liposomal formulation was confirmed by the ABTS scavenging ability and iron-chelating activity. Release studies conducted under simulated gastrointestinal conditions confirmed that liposomes provide prolonged release of encapsulated soy protein hydrolysates as compared to diffusion of the free hydrolysate. In the first 75 min, only 20 % of liposome encapsulated soy peptides diffused, which is 2.2-fold lower as compared to the diffusion of the non-encapsulated soy hydrolysate. Liposome encapsulated soy protein hydrolysates may provide the possibility for application in the areas such as food science and technology, with the aim to enhance the nutritional value and shelf life of food products, and develop functional foods.

Stillage is the main by-product of bioethanol production and the cost of its treatment significantly affects the economy of bioethanol production. A process of thermal sterilization before lactic acid fermentation (LAF) is energy demanding and is causing deterioration of valuable compounds in stillage. In this study, ultrasound (UT) and plasma (PT) treatments were used for microbial inactivation, and a significant reduction in the number of viable microorganisms in the stillage after PT and UT was observed. After application of treatment, LAF by Lactobacillus rhamnosus ATCC 7469 was initiated. The concentration of LA is used to quantify the efficiency of the stillage revalorization. The highest LA productivity of 1.21 g/Lh and yield of 0.82 g/g were obtained after PT, while UT of 10 min provided productivity of 1.02 g/Lh and LA yield of 0.69 g/g. The results were benchmarked against closed LAF. Around 20% better revalorization of stillage by PT was achieved when compared with conventional sterilization. In addition, an excellent L (+) LA stereoselectivity of 95.5% was attained after PT. From the aspect of energy efficiency, that of PT was three times lower than UT and almost ten times lower than thermal sterilization, but it is the most expensive due to the high consumption of gas which could reduce application of closed Ar atmosphere on larger scales. This way, a simpler and energy efficient process for LA production on stillage was accomplished by "open" fermentation.

Enzymatic hydrolysis of food proteins is convenient method to improve their functional properties and physiological activity. Herein, the successful covalent attachment of alcalase on alginate micron and submicron beads using the carbodiimide based chemistry reaction and the subsequent application of the beads for egg white and soy proteins hydrolysis were studied. In addition to the electrostatic extrusion technique (EE) previously used by others, the potential utilization of a novel ultrasonic spray atomization technique without drying (UA) and with drying (UAD) for alginate submicron beads production has been attempted. The immobilization parameters were optimized on microbeads obtained by EE technique (803 ± 23 µm) with respect to enzyme loading and alcalase activity. UA and UAD techniques resulted in much smaller particles (607 ± 103 nm and 394 ± 51 nm in diameter, respectively), enabling even higher enzyme loading of 671.6 ± 4 mg g−1 on the carrier and the highest immobilized alcalase activity of 2716.1 IU g−1 in the standard reaction. The UAD biocatalyst exhibited also better performances in the real food system based on egg white or soy proteins. It has been shown that the immobilized alcalase can be reused in seven successive soy protein hydrolysis cycles with a little decrease in the activity.

This paper aims to reduce the gap between intensified innovative process designs and the application of enzyme technologies at an industrial scale that often take full advantage of microreactor systems. Soybean and potato processing waste was used as the source of peroxidase in both the free and immobilized form for the biodegradation of the Acid Violet 109 anthraquinone dye (AV109) in a PTFE (polytetrafluoroethylene) microreactor which is 0.5 mm in diameter and 6 m in length. High biodegradation efficiencies of 95 and 76% were achieved by soybean and potato peroxidase respectively after 3 min of residence in the microreactor, with a noticeable lack of hydrogen peroxide inhibition. The reactor’s design, with regard to its diameter and length, significantly affected the biodegradation efficiency. The polymer membrane formation by the cross-linking approach with oxidized pectin and soybean or potato peel peroxidase on the inner PTFE wall was SEM confirmed. The cross-linking efficiency with oxidized pectin of both peroxidases is inversely correlated with the activity, hence the highest recorded activity of immobilized peroxidase was 11 and 19μmol/min of soybean and potato peroxidase, respectively, at an initial pectin concentration of 0.05%. A promising reusability for AV109 biodegradation in the microreactor with immobilized soybean and potato peroxidase was confirmed, since the immobilized peroxidases retained 65 and 35% of the initial activity after the tenth cycle, respectively. The LC–MS and GC–MS techniques confirmed the biodegradation of the AV 109 dye, and the intermediates with a lower molecular weight were separated and identified. Supplementary material: [http://technorep.tmf.bg.ac.rs/handle/123456789/4730]

The number of different technologies available for colored wastewater treatment shows the vastness of the issue. Finding the most feasible one, yet taking into account the cost, the efficiency and eco-friendliness is like searching needle in a haystack. Here, we have taken the eco-friendliness to a level above, using one bio-waste material for treatment of another waste. Potato peel and soybean hull are agroindustrial waste that is at the same time abundant source of potent enzymes such as peroxidases that can be used in oxidation reactions. In this study, the anthraquinone dye Acid Violet 109 is used as a model for simulation of colored wastewater and for oxidation by peroxidase from two bio-waste sources: potato peel and soybean hull. The operational conditions were optimized and a kinetic study was performed in order to obtain detailed information about the behavior of the enzymes when faced with extreme substrate concentration. Under the optimal conditions, 72.78 +/- 3.13% and 66.12 +/- 2.51% biodegradation was achieved with potato peel peroxidase, and soybean hull, respectively. As most of the industrial wastewater treatments require higher temperatures, both soybean hull and potato peel peroxidase were subjected to and proved to be able to operate at significantly higher temperatures up to 70 degrees C.

This work aims to characterize a novel system for thyme essential oil delivery based on the combination of natural emulsifiers (soy protein and soy lecithin) and alginate, produced using the extrusion technique. The formulations are optimized concerning alginate and soy protein concentrations (both 1 to 1.5 wt.%), and consequently lecithin amount, in order to achieve spherical beads in the range 2.0 to 2.3 mm and 1.2 to 1.4 mm, wet and dry, respectively. Fourier-transform infrared analysis was performed, proving that there are interactions between all components. Lecithin-soy protein synergistic combination improved entrapment efficiency of total polyphenols (for nearly 12%) and decreased thymol release in a simulated gastric solution for nearly 35%, in comparison with beads without lecithin. The addition of lecithin enhances the thermal properties of the polysaccharide-protein systems at 50 degrees C after 3 hr of heating. The mechanical stability of the biopolymer carriers is improved with lecithin addition and the elastic modulus varied from 80.06 to 123.7 kPa, depending on the formulation. Alginate/soy protein/lecithin are effective carriers for the encapsulation, protection, and controlled release of thyme essential oil. Practical Application There is unfortunately growing human resistance to antibiotics. This work offers a novel system for effective protection and controlled release of thyme essential oil in the small intestine. The mechanical and thermal properties of the carrier were estimated as they indicate how the beads will be able to resist stress during their incorporation into food (i.e. cookies-mixing, baking). The proposed approach offers ''green advantage'' as arises from all-natural materials.

Study on potential of different agro-industrial waste residues for supporting the growth of newly isolated Bacillus sp. TMF-1 strain under solid-state fermentation (SSF) was conducted aiming to produce several industrially valuable enzymes. Since the feasibility of the initial study was confirmed, further objectives included evaluation of several pretreatments of the studied agricultural by-products (soybean meal, sunflower meal, maize bran, maize pericarp, olive oil cake and wheat bran) on the microbial productivity as means of enhancing the yields of produced proteases, alpha-amylases, cellulases and pectinases. Among acid/alkaline treatment, ultrasound and microwave assisted methods, chemical treatments superiorly affected most of the studied substrates. Highest yields of produced proteases (50.5 IU g (1)) and alpha-amylases (50.75 IU g (1)) were achieved on alkaline treated corn pericarp. Alkaline treatment also promoted the secretion of cellulases on maize bran (1.19 IU g (1)). Highest yield of pectinases was obtained on untreated soybean meal (64.90 IU g (1)).

Horseradish peroxidase (HRP) is a highly specific enzyme with great potential for use in the decolorization of synthetic dyes. A comprehensive study of HRP immobilization using various techniques such as adsorption and covalent immobilization on the novel carrier Purolite (R) A109 with a special focus on enzymatic decolorization and toxicity of artificially colored wastewater. The immobilized preparations with an activity of 156.21 +/- 1.41 U g(-1) and 85.71 +/- 1.62 U g(-1) after the HRP adsorption and covalent immobilization, respectively, were obtained. Stability and reusability of the immobilized preparations were also evaluated. A noteworthy decolorization level (similar to 90%) with immobilized HRP was achieved. Phytotoxicity testing using Mung bean seeds and acute toxicity assay with Artemia salina has confirmed the applicability of the obtained immobilized preparation in industrial wastewater plants for the treatment of colored wastewater.

Wheat bran is a by-product of wheat flour production used as a nutritious and economic feed ingredient, but since it possesses several anti-nutritional factors that entrap nutrients and interfere with digestion and absorption, its application is limited. The utilization of organic-rich wastes for the production of value-added products and nutritional improvement of food is a means to an end – sustainability. Here we propose addressing these challenges via fermentation by Bacillus sp. TMF–2, a strain that produces several enzymes and simultaneously, wheat bran of higher nutritional quality. Following solid-state fermentation, the soluble phenolic content of wheat bran nearly triplicated compared to the raw bran. The total proportion of polyphenols along with the antioxidant capacity and free radical scavenging rate were significantly improved, particularly the ability to reduce Fe3+ increased by 10-fold. Additionally, the activity of hydrolytic enzymes: amylase, cellulase, pectinase, mannanase, protease, and phytase increased during fermentation, whereas the level of anti-nutrient, phytic acid decreased. Among all produced enzymes, there is a special emphasis on the protease prone to degrade gluten thus resulting in gluten-free wheat bran. The results of this research could contribute to a beneficial procedure for the natural modification of wheat bran, which further augments the health benefits and utilization of wheat bran.

The impact of ultrasound waves generated by probe-type sonicator and ultrasound cleaning bath on egg white protein susceptibility to hydrolysis by alcalase compared to both thermal pretreatment and conventional enzymatic hydrolysis was quantitatively investigated. A series of hydrolytic reactions was carried out in a stirred tank reactor at different substrate concentrations, enzyme concentrations, and temperatures using untreated, and pretreated egg white proteins (EWPs). The kinetic model based on substrate inhibition and second-order enzyme deactivation successfully predicts the experimental behavior providing an effective tool for comparison and optimization. The ultrasound pretreatments appear to greatly improve the enzymatic hydrolysis of EWPs under different conditions when compare to other methods. The apparent reaction rate constants for proteolysis (k2 ) are 0.009, 0.011, 0.053, and 0.045 min-1 for untreated EWPs, and those pretreated with heat, probe-type sonicator, and ultrasound cleaning bath technologies, respectively. The ultrasound pretreatment also decreases hydrolysis activation (Ea ) and enzyme deactivation (Ed ) energy, enthalpy (ΔH), and entropy (ΔS) of activation and for the probe-type sonication this decrease is 61.7%, 61.6%, 63.6%, and 32.2%, respectively, but ultrasound has little change in Gibbs free energy value in the temperature range of 318 to 338 K. The content of sulfhydryl groups and ζ potential show a significant increase (P < 0.05) for both applied ultrasound pretreatments and the reduction of particle size distribution are achieved, providing some evidence that the ultrasound causes EWP structural changes affecting the proteolysis rate.

Recovery of protein from soy grit, its functional properties and possibility for the reduction of time of conventional alkaline extraction by the assistance of enzymes were studied. Enzymatic treatment was performed by commercial preparations of cellulase (NS22086), xylanase (NS22083) and pectinase (Vinozym) (applied separately or in combination) as well as by commercial carbohydrases cocktail (Enzyme complex, NS22119). Three different extractions were investigated - alkaline (at pH 8 for 1 h, 2 h or 3 h), enzyme-assisted aqueous (at pH 5.5 for 3 h) and enzyme-assisted alkaline extractions (enzymatic extraction for 1 h followed by alkaline extraction for 1 h or 2 h) at 50 °C and solid:liquid ratio 1:10 (w/v). The highest enhancement of recovery of protein was achieved by pretreatment of soy grit with enzyme cocktails. Treatment with Enzyme complex followed by 1 h alkaline extraction increased protein yield for 21% compared to 2 h alkaline extraction. Treatment by combination of individual cellulase, xylanase and pectinase followed by 2 h alkaline extraction enhanced protein yield for 13% in comparison to 3 h alkaline extraction. So, reduced time of alkaline extraction was attained by the assistance of carbohydrases cocktails with even positive effect on protein yield. In addition, protein from enzyme-assisted alkaline extraction exhibited ameliorated solubility, emulsifying and whipping properties compared to alkaline extracted protein.

Protease, cellulase, and alpha-amylase producing Bacillus subtilis strain was cultivated by solid-state fermentation technique using soybean meal as a substrate. The aim of the present study was to establish a highly efficient enzymes' extraction method as a first stage in downstream processing. The conventional extraction procedure was optimized by determining pH, stirring rate, solid/liquid ratio, and time of extraction on enzymes' recoveries from fermented soybean meal. Yields of leached enzymes were compared to the amounts of enzymes that are achieved with ultrasound-assisted extraction (UAE). UAE was established to be superior method for obtaining higher yields of proteases (up to 330 IU) and alpha-amylases (825 IU), under significantly shorter extraction time and gaining more concentrated product. However, the obtained model predicts that conventional process led to a product with a higher cellulolytic activity ( gt = 7.5 IU).

Polyphenols present in different plant cell organelles increase the resistance of plants to various types of environmental stresses. We investigated the possibility of increasing the content of bioactive compounds in the seed of yellow soybean variety Laura. The soybean was treated during vegetation with five products based on plant extracts, on the assumption of enrichment of plants with various nutrients. Soybean flour extracts were screened spectrophotometrically for total phenolic content and antioxidant activity. The antioxidant activity was evaluated using three methods. The content of phenolic acids was determined by HPLC, and the raw protein content was estimated by the Kjeldahl method. Depending on the treatment, variations in the quantity of individual phenolic acids with up to 90% higher concentration as compared to the control were observed. Controlled usage of certain plant extracts can increase the concentration of the target group of bioactive compounds in the samples. The synergistic effect of proteins and phenolic compounds on the antioxidant activity of extracts was detected. The results of this study are not only important from the aspect of plant resistance to various types of stress, but also when considering soybean as a functional food. https://doi.org/10.2298/ABS190123024D Received: January 23, 2019; Revised: March 15, 2019; Accepted: April 8, 2019; Published online: April 9, 2019 How to cite this article: Đurovic S, Dragicevic V, Waisi H, Pagnacco M, Lukovic N, Kneževic-Jugovic Z, Nikolic B. Enhancement of antioxidant activity and bioactive compound contents in yellow soybean by plant-extract-based products. Arch Biol Sci. 2019;71(3):425-34.

α-Arbutin (4-hydroquinone-α-D-glucopyranoside), an effective skin-lightening agent due to its considerable inhibitory effect on human tyrosinase activity, is widely used in the pharmaceutical and cosmetic industries. Recently, α-arbutin was prepared through transglucosylation of hydroquinone using microbial glycosyltransferases as catalysts. However, the low yield and prolonged reaction time of the biotransformation process of α-arbutin production limited its industrial application. In this work, an amylosucrase (ASase) from Xanthomonas campestris pv. campestris str. ATCC 33913 (XcAS) was expressed efficiently in Escherichia coli JM109. The catalytic property of the purified XcAS for the synthesis of α-arbutin was tested. The recombinant strain was applied for highly efficient synthesis of α-arbutin using sucrose and hydroquinone as glucosyl donor and acceptor, respectively. By optimizing the biotransformation conditions and applying a fed-batch strategy, the final production yield and conversion rate of α-arbutin reached 60.9 g/L and 95.5%, respectively, which is the highest reported yield by engineered strains. Compared to the highest reported value (&lt, 1.4 g/L/h), our productivity (7.6 g/L/h) was improved more than five-fold. This work represents an efficient and rapid method for α-arbutin production with potential industrial applications.

Chitosan–glutaraldehyde microparticles were produced by emulsion crosslinking method to be used as drug delivery system for polyphenols from Thymus serpyllum L. aqueous extract. The effect of preparation conditions, chitosan concentration (1.5–3% w/v), and glutaraldehyde/chitosan (GA/Ch) mass ratio (0.15–1.20) on water and polyphenols transport properties was investigated. Swelling ratio of dry particles (68–230 µm) in water ranged from 280% to 530%, depending on the formulation. The decrease in swelling was observed with increased GA/Ch mass ratio (i.e. crosslinking degree) at the same chitosan concentration, or with increased chitosan concentration at the same GA/Ch mass ratio. The increase in GA/Ch mass ratio was also manifested by increased particle compactness i.e. decreased size and reduced surface roughness. The sorption capacity for polyphenols seems to be a complex interplay of swelling behaviour and interactions chitosan–glutaraldehyde–polyphenols identified by Fourier transmission infrared analysis. An increase in crystallinity of chitosan was observed upon crosslinking with glutaraldehyde and encapsulation of polyphenols, as observed by X-ray diffraction analysis. The results obtained from release kinetics of selected polyphenolic compounds (caffeic acid, rosmarinic acid, total flavonoids, and total phenol content) showed that polyphenols were released at a lower amount (2–4 times) in water, but more rapidly (45–120 min) in comparison with the release in gastric followed by intestinal simulated fluid (SGF-SIF) (120–240 min). The experimental results of the time-dependent swelling in water and polyphenols release in both, water and SGF-SIF, were analyzed with several mathematical models. The results depicted Fickian diffusion as the water transport mechanism. In the case of polyphenols, only empirical Weibull model could be suggested for describing release kinetics.

A lipase derived from an indigenous extremophile Pseudomonas aeruginosa strain isolated from rancid metalworking fluid was evaluated as a detergent additive. Applicability of the obtained enzyme as an additive in detergent formulations was confirmed by its implementation in the formulations of several new products differing in surfactant type and concentrations, demonstrating satisfactory performance in terms of degreasing efficiency and composition of the washing wastewater. The degreasing efficiency of different enzyme-containing detergent formulations was studied on cotton fabric samples stained with triolein and compared to that of formulations containing only surfactant. The highest efficiency of the fatty soil removal in formulations with a low content of surfactants (0.4 %) was noted in the enzyme formulation containing Lutensol(A (R)) XP-80 (degreasing efficiency gt 80 %) and Triton(A (R)) X-100 (degreasing efficiency gt 60 %). An attempt was then made to optimize the composition of the enzyme formulation on the basis of one or both of these surfactants using statistically planned experiments and response surface methodology (RSM). Taking into consideration the environmental aspects and the shown detergency, it appeared that rather high degreasing effects were achieved in formulations based on a low quantities of Lutensol(A (R)) XP-80 (0.4 %) at all pH values. However, pH seemed to have a notable effect since the degreasing efficiency significantly increased with increasing pH and the amount of the enzyme. Formulations having a moderate alkaline pH profile and higher amount of enzyme exhibited a high cleaning performance of fatty soil even at a low concentration of the surfactant.

The objective of this study was to discover the relationship between the ultrasound probe treatment (UPT) on egg white proteins (EWPs) before EWPs hydrolysis by different proteases, and the functional properties of the obtained hydrolysates. To fulfill this goal, the protein solubility, foaming, and emulsifying properties were studied as a function of the UPT time and then related to the surface characteristics and structural properties. The changes in the hydrolysates microstructures and macromolecular conformation, induced by the UPT, were followed using scanning electron microscope analyzis (SEM) and Fourier transforms infrared spectroscopy (FTIR). The results showed that UPT influenced (P lt 0.05) the proteolysis of egg white proteins for all examined treatment times. Alcalase hydrolysates (AHs) and papain hydrolysates (PHs) were found to have a higher solubility, as a consequence of their relatively higher foaming, and emulsifying properties compared to the untreated hydrolysates. The changes in surface hydrophobicity, sulfhydryl content and surface charge of AHs and PHs indicated unfolding of EWPs affected by ultrasound. SEM analyzis showed that UPT destroyed the microstructures of AHs and PHs, while FTIR spectra indicated remarkable changes in the macromolecular conformation of AHs and PHs after UPT. This study revealed that by combining ultrasound pre-hydrolysis treatment under controlled conditions with thoughtful proteases selection, hydrolysates with improved functional properties could be produced, enhancing utilization of EWPs in food products.

The aim of the study was to investigate and compare several extraction protocols like 1) high-power ultrasound probe assisted solvent extraction; 2) microwave assisted solvent extraction; 3) direct acid hydrolysis; 4) direct alkali hydrolysis, and 5) two step extraction consisting of ultrasound or microwave assisted solvent extraction followed by alkaline and acid hydrolysis in terms of efficiency of the extraction of phenolic acids from the yellow soybean seed variety Laura. These extracts were screened for their total phenol content (TPC), and for their antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH) assay, as well as on content of some individual phenolic acids. It appeared that the acetone containing mixtures gave much higher TPC than methanol containing ones, but the presence of acid in the methanol solvent significantly improved the extraction of phenolic compounds. To further improve the extraction, an ultrasound lab-scale probe at 20 kHz was used, with 15 and 30% of the maximum amplitude, and the extraction time was varied from 2 to 15 min. Microwave assisted extraction was performed varying the temperature from 55 to 85 degrees C, microwave power from 25 to 100 W and extraction time from 2 to 10 min. Changes in the content of six phenolic acids were examined: gallic, trans cinnamic, chlorogenic, caffeic, p-coumaric and ferulic acid. The separation and quantification of phenolic acids was accomplished by high-performance liquid chromatography-diode array detection (DAD) procedure. The results suggested that microwaves contributed to more efficient extraction of phenolic acids from the seed of yellow soybean. The amount of phenolic acids varied from 65.52 mu g/g of dry matter (d.m.) for caffeic acid, to 581.84 mu g/g d.m. for p-coumaric acid. Both, ultrasound and microwaves contributed to more efficient extraction of total phenol compounds and enhanced antioxidant activity of soybean seed extracts. TPC varied from 12.48 to 18.77 mg GAE/g d.m. and antioxidant activity varied from 244.58 to 345.21 mu mol TROLOX eq/g d.m.

This study focuses on the influence of operating conditions on Alcalase-catalyzed egg white protein hydrolysis performed in a continuously stirred tank reactor coupled with ultrafiltration module (10 kDa). The permeate flow rate did not significantly affect the degree of hydrolysis (DH), but a significant increase in process productivity was apparent above flow rate of 1.9 cm(3) min(-1). By contrast, an increase in enzyme/substrate (E/S) ratio provided an increase in DH, but a negative correlation was observed between E/S ratio and productivity. The relationship between operating conditions and antioxidant properties of the hydrolysates, measured by three methods, was studied using Box-Behnken experimental design and response surface methodology. The statistical analysis showed that each variable (impeller speed, E/S ratio, and permeate flow rate) had a significant effect on the antioxidant capacity of all tested systems. Nevertheless, obtained response functions revealed that antioxidative activity measured by DPPH, ABTS and FRAP methods were affected differently by the same operating conditions. High impeller speeds and low permeate flow rates favor ABTS while high impeller speeds and high permeate flow rates had a positive effect on the DPPH scavenging activity. On the other hand, the best results obtained with FRAP method were achieved under moderate operating conditions. The integration of the reaction and ultrafiltration membrane separation in a continuous manner appears to be a right approach to improve and intensify the enzymatic process, enabling the production of peptides with desired antioxidant activity.

The objective of this study was to investigate the effects of different ultrasound pretreatment on enzymatic hydrolysis of egg white proteins (EWPs) by Alcalase as well as evaluating some functional and antioxidant properties of hydrolysates obtained by various proteases treatment and ultrasound technology. The effects of chosen ultrasound pretreatment parameters including frequency of ultrasonic waves (35 and 40 kHz), temperature (25 and 55 A degrees C), time of pretreatment (15-60 min) and pH of egg white solution (7.00-10.00) were examined. It appeared that controlled ultrasound treatment can improved the hydrolysis process compared with untreated samples, but optimization of the power and length of sonication was important. The optimal ultrasound pretreatment at calorimetric power of 21.3 W and frequency of 40 kHz for 15 min at 25 A degrees C and with naturally basic egg white (pH 9.25) resulted in increased initial rate and equilibrium degree of Alcalase hydrolysis by about 139.8 and 13.86 % compared with the control, respectively. EWP hydrolysates with a parts per thousand 27.0 % degree of hydrolysis obtained with heat pretreatment and ultrasound pretreatments under optimal conditions were further separated by sequential ultrafiltration into 4 hydrolysate fractions ( lt 1, 1-10, 10-30 and gt 30 kDa) which were investigated for protein content, peptide yield and antioxidant activity. The hydrolysis after heat pretreatment generated more peptides lt 1 kDa (19.04 +/- A 1.02 %) than ultrasound pretreatment did (11.90 +/- A 0.53 %), whereas the proportion of peptides lt 10 kDa were higher in the second case (28.80 +/- A 0.07 vs. 20.46 +/- A 0.39 %). The fraction obtained by the ultrasound pretreatment containing peptides with a molecular weight between 1 and 10 kDa demonstrated the strongest ABTS radical scavenging efficacy among the fractions (97.54 +/- A 0.30) with IC50 value of 4.31 mg/mL. Compared with single-enzyme processes, the two-stage enzymatic processes did not significantly improve both antioxidant and functional hydrolysates' properties.

In this study, alcalase (protease from Bacillus licheniformis) immobilization by adsorption, enzyme crosslinking and covalent enzyme binding to activated chitosan microbeads were examined. The biocatalysts highest activity was obtained by covalent immobilization of alcalase onto a solid support. The alcalase covalent immobilization onto different types of chitosan beads obtained by inverse emulsion technique and electrostatic extrusion was studied. Parameters examined under different conditions were beads diameter, enzyme loading, enzyme capacity yield, and biocatalyst activity. The highest activity and enzyme loading of 23.6 IU/mg protein and 340.2 mg/g, respectively, were achieved by the enzyme immobilized onto chitosan microbeads obtained by the electrostatic extrusion technique. FT-IR analysis was used to confirm formation of alcalase-chitosan conjugates. The activity of optimally produced alcalase-chitosan microbeads was then verified in the industrially feasible reaction systems of egg white and soy protein hydrolysis. The high degree of hydrolysis of 29.85 +/- 0.967% after 180 min and five successive reuses obtained under real conditions (50 A degrees C, pH 8) verified the covalently bound alcalase to chitosan beads a promising candidate for use in industrial egg white protein hydrolysis process.

The objective of this research was to investigate the impact of high-intensity ultrasound (HIU) generated by a probe-type sonicator (frequency 20 +/- 0.2 kHz and an amplitude of 40%) for 2-20 min on the selected functional and structural properties of egg white proteins (EWPs) and their susceptibility to hydrolysis by alcalase. The protein solubility, foaming, and emulsifying properties were studied as a function of ultrasonication time and related to protein particle and structural properties. The length of ultrasonication exhibited important effect on EWP particle size, uniformity, and charge, affecting also the protein conformation and susceptibility to alcalase hydrolysis and determining functional properties. There was a linear correlation between the particle size decrease and the solubility while a two-step linear correlation between the foam capacity (FC)/foam stability (FS) and particle size was apparent. Specifically, FC and FS sharply increased with decreasing particle size for range from similar to 370 to similar to 260 nm, and below this range from 260.6 to 68.4 nm, the changes were not that substantial. Besides, the solubility, FC, and FS were directly and linearly related with the absolute value of the particle zeta potential. The overall emulsifying properties were also improved with an increase of sonication time, through both the decrease of the mean particle diameter and the increase of zeta potential, but there was no direct correlation between the emulsion activity/stability index and protein particle size and/or charge. Analysis of EWP structure by Raman spectroscopy revealed that the HIU leads to changes in the secondary structure, while heat and ultrasound generated by the ultrasound bath were not sufficient to exhibit this effect.

The application of immobilized enzyme is strictly related to economic benefits of immobilization. Regardless of numerous patents and published articles, application of immobilized enzymes in food industry is relatively limited to several processes. Immobilization of enzyme onto inorganic carriers is a well-established technique, especially in the field of industrial applications. Zeolites are another group of inorganic materials that have been applied in enzyme immobilization. Cysteine was also found to be suitable as a binding agent for enzyme immobilization on to the glass surface. The covalent immobilization of enzymes onto epoxy-activated carriers has drawn considerable interest. A new process for synthesis of carriers, such as electrochemical synthesis of polyaniline, was found to be a promising procedure for production of stable enzyme support. Natural and synthetic polymer hydrogels have been frequently used for encapsulation of cells, enzymes and food compounds.

Lipase B from Candida antarctica was immobilized by adsorption onto laboratory prepared metakaolin. This support has great potential for enzyme immobilization since it has good mechanical characteristics and thermal stability and it is easily obtained from naturally abundant raw kaolin, as presented in this paper. Immobilization process was studied as a function of pH, ionic strength and protein concentration in terms of immobilization and activity yield. It was shown that immobilization occurred via the Langmuir model, and that electrostatic forces were major, but not exclusive contributor for the binding process. The optimum conditions were achieved in 10 mM acetic buffer pH 5. Biocatalyst obtained under optimum conditions was further successfully used in the synthesis of lipophilic antioxidants, where conversion yields as high as 100% were achieved.

The aim of the study was to find the optimal operational and process parameters for the enzymatic hydrolysis of wheat gluten in a batch stirred bioreactor regarding both degree of hydrolysis and antioxidant capacity of the obtained hydrolysates. It appeared that impeller geometry and agitation speed influenced the mass transfer resulting in enhanced gluten hydrolysis. The highest initial reaction rate (0.83 ± 0.02 min−1) and degree of hydrolysis (30.47%) were achieved with the pitched four-bladed impeller and agitation speed of 350–450 rpm, conditions which provided proper balance between requirements for adequate mass/heat transfer and low shear stress. The impact of other process conditions including gluten concentration, temperature, pH and enzyme-gluten (E/S) ratio on the enzymatic reaction was investigated by applying a Box-Behnken experimental design from the viewpoint of the degree of hydrolysis (DH) and antioxidant activity. Three models obtained allowed calculation of the hydrolysis degree, and both 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulpfonic acid) radical scavenging activity from a given set of reaction conditions with good predictability. The statistical analysis showed that each variable had a significant effect on degree of hydrolysis and the antioxidant capacity of both tested systems. Hydrolysis up to around DH 15% improved DPPH radical scavenging activity, while excessive hydrolysis worsened it. The ABTS activity of the hydrolysates was not associated with the DPPH activity nor with the DH, revealing that it was not possible to fulfill all desirable quality requirements (maximum degree of hydrolysis and protein yield, maximum DPPH and ABTS scavenging activities) by using the same reaction conditions. Overall, the study might contribute to approve wheat gluten, a by-product of wheat starch industry, as an accessible and cheap source of bioactive compounds for the development of novel nutraceuticals, cosmetics and drugs.

A selection of best combination of adequate immobilization support and efficient immobilization method is still a key requirement for successful application of immobilized enzymes on an industrial level. Eupergit® supports exhibit good mechanical and chemical properties and allow establishment of satisfactory hydrodynamic regime in enzyme reactors. This is advantageous for their wide application in enzyme immobilization after finding the most favorable immobilization method. Methods for enzyme immobilization that have been previously reported as efficient considering the obtained activity of immobilized enzyme are presented: direct binding to polymers via their epoxy groups, binding to polymers via a spacer made from ethylene diamine/glutaraldehyde, and coupling the periodate-oxidized sugar moieties of the enzymes to the polymer beads. The modification of the conventionally immobilized enzyme with ethylenediamine via the carbodiimide route seems to be a powerful tool to improve its stability and catalytic activity.

The series of poly(N-isopropylacrylamide-co-itaconic acid) hydrogels, with lipase from Candida rugosa as a model protein, were synthesized by free radical copolymerization. The composition of hydrogels was varied by monomers ratio, crosslinking agent concentration and amounts of lipase, which was loaded by in situ polymerization. All samples were characterized regarding morphology. The investigation of hydrogel swelling properties revealed their pH and temperature sensitive character. Protein loading efficiency, release profiles and the specific activity yield of the released lipase were also investigated as a function of hydrogel composition, protein content and pH, at the physiological temperature of 37 degrees C. Copolymers of N-isopropylacrylamide and itaconic acid presented high lipase loading efficiency. Another very important feature of these copolymers was that the protein release kinetic strongly depended on the pH value of the medium. The diffusion exponents values around 1 denoted that these hydrogel compositions could be adjusted to follow near zero-order kinetics. Namely, hydrogel formulations released low amounts of lipase at pH 2.20, but much higher released protein quantities were observed at pH 6.80 enabling these copolymers to be attractive candidates as site specific protein oral drug delivery systems.

A macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA), with various surface characteristics and mean pore size diameters ranging from 44 to 200 nm was synthesized, modified with 1,2-diaminoethane, and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35 U g(-1) dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameters of 200 and 120 nm by the periodate method. A study of deactivation kinetics at 65 °C and in 80 % dioxane revealed that periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with a mean pore diameter of 120 nm by periodate method showing not only the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in the stabilization of enzyme over a broader pH range while the Michaelis constant value (K (m)) of the immobilized HRP was 10.8 mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in a batch reactor for pyrogallol oxidation, the immobilized HRP retained 45 % of its original activity.

A commercial Candida rugosa lipase was immobilized into poly(N-isopropylacrylamide-co-itaconic acid) hydrogel under different pH immobilization conditions, characterized and evaluated for both hydrolytic activity in an aqueous medium and esterolytic activity in an organic medium. Although the apparent enzyme activity in the hydrolytic reaction was acceptable, the immobilized lipase appeared to be more suitable for application in ester synthesis in low aqueous system based on n-hexane. The esterification reaction parameters, such as temperature, biocatalyst amount, added water content and substrate concentration was evaluated and optimized by response surface analysis. An adequate statistical quadratic model was obtained, making it possible to predict ester yields from known values of the four main factors. Under optimized conditions, the yield of ester > 90% could be achieved after 48 h of reaction, corresponding to the volumetric activity of 4.75 mmol L−1 h−1. The operational stability of the immobilized system in esterification reaction proved to be highly attractive with 15 consecutive 48 h uses with a residual activity of 67.4%, implying that the developed hydrogel and immobilized system could provide a promising solution for the flavor ester synthesis at the industrial scale.

An indigenous Pseudomonas aeruginosa strain has been studied for lipase and protease activities for their potential application in detergents. Produced enzymes were investigated in order to assess their compatibility with several surfactants, oxidizing agents and commercial detergents. The crude lipase appeared to retain high activity and stability in the presence of several surfactants and oxidizing agents and it was insusceptible to proteolysis. Lutensol (R) XP80 and Triton (R) X-100 strongly activated the lipase for a long period (up to 40 and 30% against the control after 1 h) while the protease activity was enhanced by the addition of Triton (R) WR1339 and Tween (R) 80. The washing performance of the investigated surfactants was significantly improved with the addition of the crude enzyme preparation. Studies were further undertaken to improve enzymes production. The optimization of fermentation conditions led to an 8-fold increase of lipase production, while the production of protease was enhanced by 60%.

Olive oil cake is a by-product from the olive oil processing industry and can be used for the lipase and protease production by Candida utilis in solid state fermentation. Different carbon and nitrogen sources were evaluated, and the results showed that the supplementation of the substrate with maltose and starch as carbon sources and yeast extract as a nitrogen source significantly increased the lipase production. The best results were obtained with maltose, whereas rather low lipase and protease activities were found with glucose and oleic acid. Response surface methodology and a five-level-three-factor central composite rotatable design were used to evaluate the effects of the initial moisture content, inoculum size and fermentation time on both lipase and protease activity levels. A lipase activity value of approximate to 25 Ug(-1) and a protease activity value of 110 Ug(-1) were obtained under the optimized fermentation conditions. An alkaline treatment of the substrate appeared to be efficient, leading to increases of 39% and 133% in the lipase and protease production, respectively. The results showed that the olive cake could be a good source for enzyme production by solid state fermentation.

The objective of this paper was the investigation of a suitable Sepabeads(A (R)) support and method for immobilization of lipase from Candida rugosa. Three different supports were used, two with amino groups, (Sepabeads(A (R)) EC-EA and Sepabeads(A (R)) EC-HA), differing in spacer length (two and six carbons, respectively) and one with epoxy group (Sepabeads(A (R)) EC-EP). Lipase immobilization was carried out by two conventional methods (via epoxy groups and via glutaraldehyde), and with periodate method for modification of lipase. The results of activity assays showed that lipase retained 94.8% or 87.6% of activity after immobilization via epoxy groups or with periodate method, respectively, while glutaraldehyde method was inferior with only 12.7% of retention. The immobilization of lipase, previously modified by periodate oxidation, via amino groups has proven to be more efficient than direct immobilization of lipase via epoxy groups. In such a way immobilized enzyme exhibited higher activity at high reaction temperatures and higher thermal stability.

Polyaniline (PANI) enzyme electrode was formed by immobilisation of Glucose oxidase (GOx) via glutaraldehyde into electrochemically polymerised PANI on graphite electrode. Electrochemical polymerisation of PANI on graphite was performed from aqueous solution of 1.0 mol dm(-3) HCl and 0.25 mol dm(-3) aniline at constant current density of 2.0 mA cm(-2). Hronopotentiometric curves of the PANI enzyme electrode obtained at current density of 10 mu A cm(-2) were recorded in different glucose concentrations. The linearity response range was between 1.0 and 5.0 mmol dm(-3) of glucose concentration. The estimated apparent Michaelis-Menten constant, was K-m(') = 0.30 mmol dm(-3), which is significantly lower than that of free enzyme.

Temperature- and pH-sensitive hydrogels based on N-isopropylacrylamide (NiPAAm) and itaconic acid (IA) were applied for immobilization of lipase from Candida rugosa (CRL). The hydrogels were synthesized by free radical crosslinking copolymerization in the presence of lipase. Characterization of samples by swelling studies, at pH 2.20 and 6.80 at a temperature of 37 degrees C, scanning electron microscopy (SEM) and Fourier transform infrared analysis (FT-IR) confirmed that the degree of crosslinking, the nonionic/ionic (NiPAAm/IA) ratio and the enzyme content had impacts on the hydrogel structure, mechanical properties, morphology and swelling kinetics. All hydrogels demonstrated protein loading efficiencies as high as 95 wt.%. A specific activity of the immobilized lipase of around 38 IU/g was attained for an enzyme loading of 20.0 wt.%. As a result, improved pH and temperature optima values were obtained for the immobilized systems in relation to those for the free lipase.