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Given their high protein content and widespread use, by-products of the oil processing sector, such as pumpkin leaves, are promising alternative protein sources. The most abundant protein on the planet is found in the soluble protein fraction of leaves, commonly referred to as white food protein. This protein is mainly composed of the photosynthetic enzyme RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase). Food proteins are intriguing GRAS (Generally Recognized As Safe) components for nanoparticle delivery systems due to their unique functional properties, enabling the encapsulation of hydrophilic and lipophilic bioactive substances. These traits include the ability to produce gels and emulsions. Furthermore, food proteins can bind bioactive compounds offering opportunities for improved protection until their release. This research serves as proof of concept that the cold gelation protocol, after optimisation, can be effectively employed for the creation of novel protein nanoparticles. The white protein fraction from leaves was isolated using a three-step process, consisting of screw pressing, thermal coagulation, and acid precipitation. Protein nanoparticles (50–150 nm) were prepared by employing a cold gelation protocol with calcium as the cross-linking cation. Nanoparticle characteristics including size, surface charge, and hydrophobicity, were adjusted by changing the cross-linking cation concentration (1–5 mmol/dm3), environmental pH (7–9), and temperature of the alkaline treatment (70–100 °C at pH 12). The yield of protein nanoparticle and their morphology characteristics, determined via SEM imaging, were the unique parameters for validating and selecting the optimal ultracentrifugation conditions for protein suspensions. For this purpose, time (10–60 min) and rotation speed (40000–80000 rpm) were varied using the OptimaTM XPN-100 ultracentrifuge. The obtained nanoparticles exhibited a uniform size distribution and spherical shape. A lower pH value and

RuBisCO (ribulose-1,5-diphosphate carboxylase/oxygenase), an enzyme crucial for plant photosynthesis, is the most widespread protein in the living world. It is predominantly found in the leaves of plants, which mostly remain unused after harvesting. Despite current efforts, the RuBisCO protein isolated from leaves has not yet found significant application as a supplement in human nutrition, due to the complexity and cost-effectiveness of the production process and the low yield. Two ultrafiltration methods were applied for isolation of RuBisCO protein. Initially, cross-flow ultrafiltration was used, employing a membrane module with a membrane’s pore size 10 kDa. The second method involved dead-end ultrafiltration with a feed flow under a nitrogen stream at a defined pressure, using a membrane with a pore size of 100 kDa. The obtained ultrafiltration fractions were used for the analysis of soluble protein content and protein yield, reducing sugars and polyphenols. The efficiency of the ultrafiltration process and the protein purity were determined by SDS-PAGE electrophoresis. Also, the obtained protein powders were analyzed for the total and free sulfhydryl group contents and surface hydrophobicity. These protein powders were characterized in terms of their technological and functional properties, including solubility, emulsifying properties, and water/oil binding capacities. The results were then compared with those of protein powder obtained from pumpkin leaves using the most common method, isoelectric precipitation. The different techniques for isolating RuBisCO protein showed certain variations in protein yield as well as in its structural and technological-functional properties. Different isoelectric point values were shown for all three protein powders, including variations in the content of free and total sulfhydryl groups. Furthermore, surface hydrophobicity and oil binding capacity showed lower values for the RuBisCO protein obtained by ultrafiltration metho

Ensuring enough protein for growing world population while respecting sustainability goals puts alternative proteins in the spotlight of food science research. Green leaf biomass represents prospective plant-based alternative source of proteins. The use of leafy biomass as protein source has long research history, but recent increase in protein demand highlighted its potential for food applications. This is mostly because of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), major fraction of leaf water-soluble (white) proteins. RuBisCO is ideal for human consumption due to its low allergenicity, high digestibility and excellent amino acid composition. In addition to the acknowledged nutritionally valuable RuBisCo protein, other leaf proteins, namely membrane (green) leaf proteins, could be used more in human nutrition. However, leafy biomass remains underutilized due to the complexity of protein extraction and purification. Insufficiently purified proteins compromise their functionality and applicability in food formulations. Therefore, the development of scalable processes remains an imperative for the cost efficient and sustainable production of leaf-based proteins. One of the key features for the efficient process is the right choice of the starting material. The starting material should be rich in protein, available in excessive amount, and preferably without other applications. Pumpkin leaves represent an appropriate choice for production of leaf-based proteins. These leaves are rich in proteins and they are obtained as waste (or by-product) during pumpkin production, hence their usage is in accordance with the trend of making agroindustry more sustainable. Our results showed that the highest yield of pumpkin leaf proteins is obtained when twin-screw press was used for tissue disruption with subsequent repressing. Usage of alternative extraction (ultrasound-assisted) and purification (membrane separation) methods positively affected protein

Processing conditions for the fabrication of soy protein concentrates (SPCs) have a profound impact on the tightly packed, globular structure of soy proteins, which is reflected in the weakening of structural and functional properties, limiting their use in food systems. Many scholars have investigated the modification of soy protein, but this is the first time that high-intensity ultrasound technology has been used to address its limitations through improvement of the physicochemical properties of SPC. Therefore, the aim of this study was to develop an ultrasound-based method capable of producing SPC with improved functional properties making it a multifunctional ingredient for food systems intended for human consumption. The effects of high-intensity ultrasonication (20 kHz; 30% for 0.5; 2; 5 or 10 min) on the solubility, emulsifying properties, hydrophobicity, oil and water binding capacities and color of commercially available SPC were investigated. Ultrasonic cavitation induced the restructuring of SPC, which was confirmed by significant changes in functional and structural properties. After ultrasonic treatment (30% amplitude for 5 min), the most significant shifts in solubility were observed. The emulsion fabricated with this restructured SPC was firm, stable, without perceptible phase separation, with emulsifying activity and emulsion stability of 1024.4 ± 10.6 m2/g and 836.3 ± 12.2 h, correspondingly. Ultrasonic treatment of 30% amplitude for 2 min enabled SPC with best oil (3.26 ± 0.4 goil/gprotein) and water binding capacity (5.04 ± 0.9 gwater/gprotein). Furthermore, the results additionally revealed that with the increase in sonication time the surface hydrophobicity of SPC increased first and then decreased. The value of a* and b* decreased significantly with the ultrasonic treatment time increment, while lengthened ultrasonic cavitation increased the L* value. In conclusion, the functional and structural improvement of SPC endorsed the adequacy of ultr

Soybean, a protein-rich leguminous oilseed, is often unacceptable due to its taste, appearance, and smell. Enzymatic technologies offer an alternative to conventional chemical procedures for protein modification, allowing for accurate oversight and adjustment of reaction flow to desired nutritional and bioactive properties due to the high sensitivity of protease under mild reaction conditions. Herein, the feasibility of implementing a one- or two-step biotechnological process, triggered by commercial endo- and exo-peptidases, to enhance the nutritional and bioactive characteristics of soy protein concentrates (SPC), was investigated. Two nutritionally valuable fractions, the hydrolysate (liquid stream-fraction) and the ocara (solid stream-fraction), have been separated. The hydrolysates were characterized by examining the crude protein content, protein recovery, and free amino group content. The antioxidant activity was quantified by measuring superoxide radical inhibition, and metal-ion chelation. Commercial peptidases led to different reaction kinetics and protein recovery, resulting in different peptide profiles determined by dead-end ultrafiltration (3, 10, and 30 kDa) and subsequent gel-filtration chromatography using the Toyopearl HW40F resin. Enzymatic hydrolysis seemed to enhance the hydrolyzate's protein content while decreasing the ocara's protein content. The percentage of soluble protein recovered from SPC ranged between 68 and 82%, revealing that Flavourzyme was most suitable to solubilize SPC. The highest yield of hydrolyzed peptide bonds, correlated with higher antioxidant activity, was shown by Alcalase- Flavourzyme (~20%) as well as Neutrase-Flavourzyme (~21.5%), with a tendency to favor Neutrase due to more sensory-acceptable product. Each produced hydrolysate's amino acid content, sulfhydryl groups, and surface hydrophobicity have been examined, and substantial correlations with antioxidant activity have been found. Because the quantity of phytic

Utilization of agro-industrial byproducts represents a way to broaden the palette of products derived from the less attractive and usually cheaper sources. The underutilized agroindustrial byproducts have attracted attention in recent years due to their effect on the environment. Wheat bran (WB) represents a byproduct of wheat industry and is a good source of fiber. Valorization of industry byproducts leads to reduction in production cost of enzymes due to high expenditure of expensive substrates required for microbial growth during the production process. Hydrolytic enzyme xylanase which transforms xylan into xylose is possible to produce using various microbial strains. Production of xylanase was investigated via submerged fermentation by Penicillium chrysogenum using a defined fermentation broth supplemented with adequate amount of WB (1 and 2.5% (w/v)). Submerged fermentation was carried out in a shaking incubator (100 rpm, 30 °C, n=2) over the course of 10 days. Crude enzyme samples were investigated each day for xylanase enzyme activity via 3,5-dinitrosalicylic acid (DNS) assay for reducing sugars using 1% (w/v) beechwood xylan solution in 50 mM Sodium Citrate buffer, pH 5.0 at 37 °C. Maximum activity was achieved on the 4th (1% (w/v) WB) and 5th (2.5% (w/v) WB) day of fermentation, 3.93±0.31 IU/ml and 4.48±0.067 IU/ml, respectively. pH (4–10) and temperature (25–60 °C) optima were determined via DNS method, and the optimal conditions were determined to be pH 5.0–5.5 and 37 °C. The influence of the addition of different metal ions and reagents on the enzyme activity was determined. The most beneficial effect was noted for the addition of MnCl2 and dithiothreitol. The increase of enzyme activities recorded was 22–29% for MnCl2 and 53–59% for dithiothreitol. It was concluded that P. chrysogenum can be further exploited in an enzyme production optimization process to increase xylanase activity.

This paper reviews the potential applications of soybean hulls and their conversion into value-added products. In recent years, importance has been given to solving the problems of negative effects of fossil fuels, general pollution and the growing demand for environmentally friendly and sustainable alternatives. The increased worldwide demand for animal feed and plant protein directly affects the increase in soybean production and the generation of large amounts of by-products. By valorizing by-products, it is possible to reduce the impact of waste on the environment, solve the problem of malnutrition and close the circular economy loop. The main uses of soybean hulls are in the production of biofuels, application in animal feed, production of industrially important enzymes, synthesis of biomaterials and functional food ingredients., Ovaj rad daje pregled potencijalnih primena sojinih ljuski i njihove konverzije u proizvode sa dodatom vrednošću. Poslednjih godina se značaj pridaje rešavanju problema negativnih efektata fosilnih goriva, opšteg zagađenja i porasta potražnje za ekološki prihvatljivim i održivim alternativama. Povećani zahtevi za hranom za životinje i proteina biljnog porekla širom sveta direktno utiču na povećanje proizvodnje soje i generisanje velikih količina nusproizvoda. Valorizacijom nusproizvoda moguće je smanjiti uticaj otpada na životnu sredinu, rešiti problem neuhranjenosti i zatvoriti petlju cirkularne ekonomije. Glavne upotrebe sojinih ljuski su u proizvodnji biogoriva, primeni u stočnoj hrani, proizvodnji industrijski značajnih enzima, sintezi biomaterijala i funkcionalnih sastojaka hrane.

Incorporation of alternative plant protein concentrates or isolates into cereal-based matrices can lead to the production of nutritionally improved products such as bread with high protein content and an improved essential amino acid profile, especially lysine. Green leaves, as the richest protein source containing soluble – white and insoluble – green proteins, seem to be an attractive and sustainable source for obtaining alternative proteins in line with the concept of green biorefinery, compared to the cultivation of soybeans for protein production. Thus, the overall objective of this study was to evaluate the quality of white and green proteins from various leaf sources for inclusion in a library of alternative proteins and for use in the development of plant protein-based products. A food-grade, sustainable three-step process was used to produce the protein concentrate from the pumpkin and spinach leaves, including mechanical pressing of the leaves with screw presses, thermal coagulation, and acid precipitation. The functional properties of the white and green protein concentrates, such as solubility, emulsifying activity/stability, and water/oil holding capacity, were investigated. The rheological behaviour of wheat flour enriched with leaf protein was studied using the Mixolab Chopin. The functional properties of the leaf protein concentrates, as well as, the rheological properties of the leaf protein-enriched flour were compared with a soy protein concentrate that served as a reference. The leaf protein concentrates showed comparable or better functional properties than the currently used soy protein concentrates. The changes in the rheological behaviour were observed in terms of water absorption, development and stability time, mechanical resistance during mixing, and behaviour of the protein-enriched flour during heating. This study has shown that leaf protein has the potential for the production of leaf protein-rich baked goods.

The projection that the world's population will reach nearly 10 billion people by 2050 serve as a clear signal for the urgent need to find sustainable solutions that can provide enough food for the growing population, along with reducing environmental impact. To achieve this, a global transition towards sustainable nutrition that fulfills all nutritional requirements is essential. Currently, the primary sources of protein, essential macronutrients in our diet, are of animal origin. This presents a significant challenge for the environment, given that the livestock system is responsible for emitting significant amount of greenhouse gases (GHG). As a result, finding alternative protein sources becomes imperative goal of circular bioeconomy in order to meet the demands for this crucial macronutrient while respecting the principles of sustainable development. Leafy biomass presents a good alternative protein source with respect to both nutritive and sustainability demands., Prognoza da će do 2050. godine na planeti biti gotovo 10 milijardi ljudi predstavlja signal za hitno pronalaženje održivih rešenja koja bi omogućila dovoljno hrane za rastuću populaciju uz smanjenje ekološkog otiska. Da bi se ovo postiglo neophodna je globalna tranzicija prema održivoj ishrani koja pritom ispunjava sve nutritivne zahteve. Osnovni izvori proteina, esencijalnih makronutrijenata, u ishrani su životinjskog porekla, što predstavlja ozbiljan problem za životnu sredinu, jer je samo stočarska industrija odgovorna za značajnu emisiju gasova staklene baste. Pronalaženje alternativnih izvora proteina tako postaje imperativ cirkularne bioekonomije kako bi se zadovoljile potrebe za ovim makronutrijenom uz istovremeno poštovanje principa održivog razvoja. Lisna biomasa, predstavlja dobar alternativni izvor proteina i sa nutritivnog i održivog aspekta.

The potential of food biodiversity and its production streams to generate additional features for isolating high-value products has been underutilized. Even so, the recovery of valuable nutritional compounds like proteins from waste streams and by-products has been identified as a key strategy for enhancing production sustainability in order to open up new market potential. In accordance with those facts, the primary aim of this research was to utilize a relatively novel extraction technique, high-intensity ultrasound, in order to achieve a reduction in time, energy, and extraction solvent consumption while at the same time improving the extraction yield and nutritional value of extracted leaf proteins. For this purpose, an ultrasound probe system with a low frequency (20±0.2 kHz) was utilized to extract the white proteins from the pumpkin leaf biomass, and the effects of different sonication amplitudes (20, 30, 40, 50, 60 and 70%) and duration periods (0, 1, 3, 5, 7.5, 10, 12.5, 15, 17.5, and 20 min) on the yield, solubility and emulsifying qualities, antioxidant properties, and structural characteristics of proteins were studied. In the functional properties evaluated, leaf proteins isolated using ultrasound outperformed those extracted using the conventional extraction method, maceration. High-intensity ultrasound resulted in a slight but gradual decline in solubility with an increase in amplitude, but a significant increase in solubility in an acidic environment was observed with the decrease of cavitation periods. Ultrasound-extracted proteins exhibit nearly 12, 1.5, and 3-fold greater solubility compared to the maceration-extracted sample at pH 3, 4, and 5, respectively. The emulsifying activity diminishes with increasing sonication amplitude and duration but increases in emulsifying stability as sonication periods are extended. The ultrasound extraction provided pumpkin proteins with high radical scavenging activities (i.e., good electron donors) and chelatin

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.

Proteins are an essential component of the human diet and effectively are provided from both animal and plant sources. Sourcing ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from green biomass such as pumpkin leaves, a by-product of the oil industry, is a promising tool for obtaining protein with an excellent nutritional profile under the green biorafinery concept, that can be used as a substitute for animal proteins. This study provides the results regarding the implementation of ultrasound-assisted processing (UAP) in the protein extraction from pumpkin leaves. UAP for recovery of RuBisCO proteins was optimized by varing the solvent-to-sample ratio, solvent pH, ultrasound amplitudes and time. UAP appears to have a strong potential for recovering RuBisCO protein from pumpkin leaves, as it was highly influenced by the solvent-to-sample ratio along with UAP amplitude and time., Proteini životinjskog i biljnog porekla čine osnovne namirnice ljudske ishrane. Prema konceptu zelene biorafinerije, izolovanje RuBisCO proteina (ribuloza-1,5-bisfosfat karboksilaza/oksigenaze) iz zelene biomase kakva je lišće bundeve, nusproizvod industrije prerade uljarica, je obećavajući pristup za dobijanje proteina sa visokom nutritivnom vrednošću koje je moguće koristiti kao zamenu za proteine životinjskog porekla. U ovom radu, ultrazvučno potpomognutim procesom ekstrakcije izolovani su proteini iz lišća bundeve. Ultrazvučni proces ekstrakcije RuBisCO proteina optimizovan je variranjem odnosa rastvarač-uzorak, pH rastvarača, vremena trajanja i amplitude ultrazvuka. Ultrazvuk je ispoljio veliki pozitivan efekat na izolovanje RuBisCO proteina iz lišća bundeve, na šta je u najvećoj meri tokom optimizacije procesa uticao odnos rastvaračuzorak, kao i amplituda i vreme ultrazvuka.

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 degrees 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 (k(2)) 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.

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.

In modern times wheat gluten has drawn attention to many research groups. Wheat gluten represents one of the most widely used proteins in the food industry. It is a byproduct of the starch industry and has a higher percentage of protein content compared to other plant-based protein sources. In order to help reduce the allergenicity of wheat gluten, bacterial enzymes have been proven to have beneficial results in wheat gluten treatment. In search for an extracellular peptidase producing strain we have tested Bacillus subtilis TMF-1 isolate, which has previously been proven to have several enzyme activities. B. subtilis TMF-1 isolate has a food grade status, making it safe for application in the food industry. Thus, the aim of this research was to examine the possibility of utilizing mentioned strain in terms of gluten-degrading enzyme production. Tested strain was first streaked onto several agar plates in order to detect extracellular peptidase activity. Bacterial isolate has then been sequentially transferred to the same growth medium several times. Conditions varied for the submerged fermentation in 25 mL flasks were pH value of fermentation broth, concentration of gluten powder (0-10 g/L) in fermentation broth and concentration of peptone (0-1 g/L). Shaking flasks containing the fermentation broth with the bacterial strain were kept for 48 h at 370C. The results obtained show that the isolate has the possibility of thriving in low acidic to neutral pH values of the fermentation broth. Varied gluten concentrations showed that even 1 g/L of gluten powder was sufficient for the bacterial strain to manifest extracellular proteolytic enzyme activity. Peptone concentrations were also varied, but even the minimal presence of peptone has proven beneficial for bacterial growth and proteolytic activity. This research show that the B. subtilis TMF-1 isolate has proteolytic activity specific for wheat gluten as substrate and that it may be used in further research in order t

Constant world population growth imposes the need for the production of more food, and as a consequence, has an increase in the organic-rich wastes. These agro-industrial wastes represent problem from the environmental viewpoint, and their utilization as the raw materials for the production of value-added products has become one of the main topics of the biorefinery. The aim of this research was to use soybean meal, co-product of dietary oil production, as a model agro-industrial by-product for optimization of phenolic compounds extraction. In this paper, ethanol influenced microwave-assisted extraction is chosen since it is recognized as eco-friendly and food-safe. Optimization of the extraction included finding the optimal liquid/solid ratio, extraction duration, and microwave power. The same method was applied for the extraction of phenolics from apple pomace, apple pomace mixed with soybean meal, ultrasound-treated soybean meal, and wheat bran. Antioxidative potential of all extracts was evaluated using a fixed reaction time 2,2-diphenyl-1-picrylhydrazyl (DPPH) method as well as a steady-state measurement DPPH method. 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) i.e., ABTS and ferric reducing antioxidant power (FRAP) methods were applied as well. Under optimized conditions (liquid/solid ratio of 15 mL/g for 70 s at a microwave power of 300 W) 3.87 mg gallic acid equivalent (GAE)/g was extracted from soybean meal, which represented a substantial increase compared to 2.46 mg GAE/g obtained before optimization. Results showed that extracts had different behavior towards different radicals depending on the type of the extracted phenolics. Apple pomace extract was the most potent towards DPPH• radical, while the most effective extract towards ABTS•+ radical was obtained from apple pomace and soybean meal mixture. The soybean meal extract showed the highest reducing power assessed via the FRAP method. Results obtained in this study show that optimization of

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.

Constant world population growth imposes the need for the production of more food, and as a consequence, has an increase in the organic-rich wastes. These agro-industrial wastes represent problem from the environmental viewpoint, and their utilization as the raw materials for the production of value-added products has become one of the main topics of the biorefinery. The aim of this research was to use soybean meal, co-product of dietary oil production, as a model agro-industrial byproduct for optimization of phenolic compounds extraction. In this paper, ethanol influenced microwave-assisted extraction is chosen since it is recognized as eco-friendly and food-safe. In this paper, we used soybean meal, co-product of dietary oil production, as a model agro-industrial by-product for optimization of phenolic compounds extraction. Ethanol influenced microwave-assisted extraction is chosen since it is recognized as eco-friendly and food-safe. Optimization of the extraction included finding the: optimal liquid/solid ratio, extraction duration, and microwave power. The same method was applied for the extraction of phenolics from apple pomace, apple pomace mixed with soybean meal, ultrasound-treated soybean meal, and wheat bran. Antioxidative potential of all extracts was evaluated using a fixed reaction time 2,2-diphenyl-1- picrylhydrazyl (DPPH) method as well as a steady-state measurement DPPH method. 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) i.e., ABTS and ferric reducing antioxidant power (FRAP) methods were applied as well. Under optimized conditions (liquid/solid ratio of 15 mL/g for 70 s at a microwave power of 300 W) 3.87 mg gallic acid equivalent (GAE)/g was extracted from soybean meal, which represented a substantial increase compared to 2.46 mg GAE/g obtained before optimization. Results showed that extracts had different behavior towards different radicals depending on the type of the extracted phenolics. Apple pomace extract was the most potent towa

In recent years, there has been an unprecedented de-mand by both consumers and industry, for inexpensive plant-derived protein hydrolysates as potential dietary sources of antioxidants. This is a particular refers to the protein from agricultural crop, such as soybean flour and/or flakes, especially from the soy protein concentrate (SPC). However, insolubility of SPC in water limits its more extensive use as food-protein ingredient, but it can be overcome by applying various enzymatic and non-enzymatic treatments. In order to expand the application of SPC, the influence of the one-step enzyme modification by using the commercial food-grade en-doprotease from Bacillus licheniformis on the antiox-idant and functional properties has been considered. For this purpose, SPC under the various dry matter contents (DM) were pre-incubated at the optimal temperature and hydrolyzed in a mechanically stirred batch reactor. The bioactivity was evaluated using rad-ical-scavenging and metal-chelating assays. The ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sul-phonic acid)) radical-scavenging ability reached higher values at DM 8% and smaller hydrolysis degree (DH) 12.85%, then a decreasing for higher DH and the minimum reducing power was observed after hydrolysis of SPC with DM 2% and DH~18% (64.2%). It appeared that the all prepared hydrolysates had the high yields of bioactive peptides which had the ability to strong-ly chelating prooxidant ferrous metal ions ( gt 95%). The techno-functionalities of SPC hydrolysate (DM 8%) with relatively higher antioxidant activities were remarkably higher compared to the untreated SPC. Namely, this hydrolysate had excellent solubility over a pH range of 2-12 which was improved compared to untreated SPC-8%. The oil holding capacity was also enhanced, while enzyme treatment was damaging to both of emulsifying properties, activity and stability, and water holding capacity. The results obviously show that enzyme treated SPCs were superior to the

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.

In recent years, there has been an unprecedented de-mand by both consumers and industry, for inexpensive plant-derived protein hydrolysates as potential dietary sources of antioxidants. This is a particular refers to the protein from agricultural crop, such as soybean flour and/or flakes, especially from the soy protein concentrate (SPC). However, insolubility of SPC in water limits its more extensive use as food-protein ingredient, but it can be overcome by applying various enzymatic and non-enzymatic treatments. In order to expand the application of SPC, the influence of the one-step enzyme modification by using the commercial food-grade en-doprotease from Bacillus licheniformis on the antiox-idant and functional properties has been considered. For this purpose, SPC under the various dry matter contents (DM) were pre-incubated at the optimal temperature and hydrolyzed in a mechanically stirred batch reactor. The bioactivity was evaluated using rad-ical-scavenging and metal-chelating assays. The ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sul-phonic acid)) radical-scavenging ability reached higher values at DM 8% and smaller hydrolysis degree (DH) 12.85%, then a decreasing for higher DH and the minimum reducing power was observed after hydrolysis of SPC with DM 2% and DH~18% (64.2%). It appeared that the all prepared hydrolysates had the high yields of bioactive peptides which had the ability to strong-ly chelating prooxidant ferrous metal ions ( gt 95%). The techno-functionalities of SPC hydrolysate (DM 8%) with relatively higher antioxidant activities were remarkably higher compared to the untreated SPC. Namely, this hydrolysate had excellent solubility over a pH range of 2-12 which was improved compared to untreated SPC-8%. The oil holding capacity was also enhanced, while enzyme treatment was damaging to both of emulsifying properties, activity and stability, and water holding capacity. The results obviously show that enzyme treated SPCs were superior to the

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).

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 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 esterification is well-established type of reaction in organic synthesis used for manufacturing various valuable esters used prevalently as ingredients of food, skincare products, pharmaceuticals and fuels. In recent decades biocatalytic synthetic routes based on using enzymes as catalysts have received a lot of attention in scientific researches and industry. Main advantages of enzymatic esterifications are high selectivity (regioselectivity and enantioselectivity), performing reaction at milder conditions (moderate temperature and atmospheric pressure) and the fact that enzymatically synthesized product gain label of natural product, which increases its market appeal. The purpose of this review is to point out the most important advances in lipase-catalyzed ester synthesis in non-conventional reaction media, with special focus on several most important types of ester. This part of review starts with outcomes in synthesis of aroma esters, usually obtained using monofunctional aliphatic alcohols and acids, which have been investigated for decades and industrial processes are already established. However, later sections are dedicated to enzymatic esterification of more complex molecules (carbohydrates, vitamin C, flavonoids) having more than one functional groups, which are subject of numerous studies in recent times because in these reactions regioselectivity of biocatalysts can be fully explored and processes are still subject of optimization, especially with respect to selection of reaction media and media engineering.

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

L-Ascorbyl oleate is recognized as efficient liposoluble antioxidant and biosurfactant which can be synthesized by using immobilized lipases as catalysts in organic media. However, development of economical production process is still a challenge, particularly in (semi)continuous systems. Hereby, we present application of novel immobilized lipase preparation, lipase B from Candida antarctica hydrophobically adsorbed onto styrene divinylbenzene based support, for the synthesis of L-ascorbyl oleate in batch bioreactor (BB) and fluidized bed bioreactor (FBB) with recirculation of reaction medium. Reaction kinetics was described by Ping Pong bi bi mechanism with low acyl donor inhibition. Considerably higher reaction rates (similar to 40%) were accomplished in FBB. Hydrodynamic study revealed that reaction is kinetically controlled and unhindered external and internal mass transfer was enabled in both systems. However, much higher shear stress occurred in BB (10.46 Pa) comparing to FBB (5 x 10(-2) Pa). After 12 consecutive reaction cycles lasting 24h, 23.13 g of product per gram of biocatalyst was synthesized in a FBB. Within this study, usage of new immobilized enzyme in semicontinuous system enabled high reaction rates and satisfying operational stability in the synthesis of L-ascorbyl oleate, offering good prospect for further process development.

The effects of some relevant process parameters for the Alcalase-catalyzed wheat gluten hydrolysis such as gluten concentration (X1; 1-9% w/v), temperature (X2; 40-60 oC), pH (X3; 7-9) and enzyme/substrate ratio, [E]/[S] ratio (X4; 0.25– 0.75 AU/g of protein) were investigated by the means of an experimental design. The second-order models developed for the degree of hydrolysis, DH, DPPH and ABTS radical scavenging activity of gluten hydrolysates were significant (p<0.01) with a high value of coefficients of determination (0.981-0.992). 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. It seemed there was not a correspondence between degree of hydrolysis and ABTS activity at different protein concentrations, suggesting that there were peptides with considerable size presenting a good antioxidant capacity.

Fenolne kiseline su sekundarni metaboliti biljaka i kao takve čine deo svakodnevne čovekove ishrane. Poznato je da ova jedinjenja poseduju niz fizioloških svojstava koja ih čine atraktivnim u naučnom svetu, među kojima je antioksidativna aktivnost najbolje ispitana. Ovi prirodni antioksidansi još više dobijaju na značaju zbog indikacija da razgradnjom komercijalnih sintetskih antioksidanasa nastaju kancerogena jedinjenja. Međutim, zbog hidrofilne prirode njihova efikasnost u nepolarnim sredinama je ograničena, a lipofilizacija esterifikacijom je jedan od načina da se ovaj problem prevaziđe. Enzimska sinteza fenolnih estara predstavlja ekološki prihvatljiv način sinteze estara, a sami proizvodi dobijeni na ovaj način spadaju u zelene proizvode, što je od izuzetnog značaja kod proizvodnje namirnica namenjenih za ljudsku upotrebu. Kako bi enzimska sinteza estara bila konkurentna hemijskoj, odnosno kako bi se sintetisale velike količine estra uz male utroške enzima, neophodno je izvršiti optimizaciju ključnih reakcionih parametara (koncentracije enzima i supstrata, temperature, intenziteta mešanja i reakcionog vremena), što je bio i cilj ove disertacije. Pre svega pristupilo se pronalaženju adekvatnog biokatalizatora, koji bi sintetisao fenolne estre u visokom prinosu. Ispitani su komercijalni imobilisani preparati lipaze B iz Candida antarctica i lipaze iz Rhizomucor miehei, Novozyme 435 i Lipozyme RM IM, redom, kao i nekoliko lipaza izolovanih i prečišćenih na Katedri za Biohemijsko inženjerstvo i biotehnologiju. Od ispitanih enzima, Novozyme 435 se pokazao kao superioran u sintezi svih sintetisanih estara. U nastavku je pokazano da specifičnost ovog preparata zavisi od strukture fenolne kiseline i da najveći afinitet pokazuje prema fenolnim kiselinama sa zasićenim bočnim nizom, p-hidroksifenilpropionskom i dihidrokafenom kiselinom. Dvostruka veza konjugovana sa aromatičnim prstenom kod cimetne kiseline, dovela je do blagog pada prinosa reakcije, dok je uvođenje elekt, As secondary plant metabolites phenolic acids are common components of daily human diet. It is well established that these compounds have several physiological activities, amongst which the antioxidant activity is the most studied one. Indications that degradation of commercial synthetic antioxidants leads to formation of toxic and carcinogenic compounds have made phenolic compounds even more attractive. Nevertheless, their hydrophilic nature limits their efficiency in non polar environments. One of the ways to overcome this obstacle is lipophilization of these compounds via esterification. Enzymatic synthesis seems to be eco-friendly way of producing lipophilic phenols, and the products obtained on this way are considered as green which is of utter importance for the products intended for human consumption. For enzymatic synthesis to prevail over the chemical synthesis, the process has to be efficient i.e. high ester yields should be produced using minimal amounts of enzyme. In order to achieve this it is necessary to optimize key process parameters (enzyme and substrate concentrations, temperature, mixing intensity and reaction time), that was set as the main goal of this thesis. The first aim was to find adequate biocatalyst which would synthesize phenolic esters with high yields. Commercial immobilized preparations of lipase B from Candida antarctica and lipase from Rhizomucor miehei, Novozyme 435 and Lipozyme RM IM, respectively, were tested along with several lipases isolated at the Department of Biochemical Engineering and Biotechnology, University of Belgrade. Amongst all tested enzymes, Novozyme 435 was proved as the superior biocatalyst for all synthesized esters. Further, it was shown that specificity of Novozyme 435 towards phenolic acids was highly dependent upon their structure. The highest conversions were achieved with p-hydroxyphenyl propionic and dihydrocaffeic acids, which both have saturated side chain. Double bond conjugated with aromatic ring o

As secondary plant metabolites phenolic acids are common components of daily human diet. It is well established that these compounds have several physiological activities, amongst which the antioxidant activity is the most studied one. Indications that degradation of commercial synthetic antioxidants leads to formation of toxic and carcinogenic compounds have made phenolic compounds even more attractive. Nevertheless, their hydrophilic nature limits their efficiency in non polar environments. One of the ways to overcome this obstacle is lipophilization of these compounds via esterification. Enzymatic synthesis seems to be eco-friendly way of producing lipophilic phenols, and the products obtained on this way are considered as green which is of utter importance for the products intended for human consumption. For enzymatic synthesis to prevail over the chemical synthesis, the process has to be efficient i.e. high ester yields should be produced using minimal amounts of enzyme. In order to achieve this it is necessary to optimize key process parameters (enzyme and substrate concentrations, temperature, mixing intensity and reaction time), that was set as the main goal of this thesis. The first aim was to find adequate biocatalyst which would synthesize phenolic esters with high yields. Commercial immobilized preparations of lipase B from Candida antarctica and lipase from Rhizomucor miehei, Novozyme 435 and Lipozyme RM IM, respectively, were tested along with several lipases isolated at the Department of Biochemical Engineering and Biotechnology, University of Belgrade. Amongst all tested enzymes, Novozyme 435 was proved as the superior biocatalyst for all synthesized esters. Further, it was shown that specificity of Novozyme 435 towards phenolic acids was highly dependent upon their structure. The highest conversions were achieved with p-hydroxyphenyl propionic and dihydrocaffeic acids, which both have saturated side chain. Double bond conjugated with aromatic ring o, Fenolne kiseline su sekundarni metaboliti biljaka i kao takve čine deo svakodnevne čovekove ishrane. Poznato je da ova jedinjenja poseduju niz fizioloških svojstava koja ih čine atraktivnim u naučnom svetu, među kojima je antioksidativna aktivnost najbolje ispitana. Ovi prirodni antioksidansi još više dobijaju na značaju zbog indikacija da razgradnjom komercijalnih sintetskih antioksidanasa nastaju kancerogena jedinjenja. Međutim, zbog hidrofilne prirode njihova efikasnost u nepolarnim sredinama je ograničena, a lipofilizacija esterifikacijom je jedan od načina da se ovaj problem prevaziđe. Enzimska sinteza fenolnih estara predstavlja ekološki prihvatljiv način sinteze estara, a sami proizvodi dobijeni na ovaj način spadaju u zelene proizvode, što je od izuzetnog značaja kod proizvodnje namirnica namenjenih za ljudsku upotrebu. Kako bi enzimska sinteza estara bila konkurentna hemijskoj, odnosno kako bi se sintetisale velike količine estra uz male utroške enzima, neophodno je izvršiti optimizaciju ključnih reakcionih parametara (koncentracije enzima i supstrata, temperature, intenziteta mešanja i reakcionog vremena), što je bio i cilj ove disertacije. Pre svega pristupilo se pronalaženju adekvatnog biokatalizatora, koji bi sintetisao fenolne estre u visokom prinosu. Ispitani su komercijalni imobilisani preparati lipaze B iz Candida antarctica i lipaze iz Rhizomucor miehei, Novozyme 435 i Lipozyme RM IM, redom, kao i nekoliko lipaza izolovanih i prečišćenih na Katedri za Biohemijsko inženjerstvo i biotehnologiju. Od ispitanih enzima, Novozyme 435 se pokazao kao superioran u sintezi svih sintetisanih estara. U nastavku je pokazano da specifičnost ovog preparata zavisi od strukture fenolne kiseline i da najveći afinitet pokazuje prema fenolnim kiselinama sa zasićenim bočnim nizom, p-hidroksifenilpropionskom i dihidrokafenom kiselinom. Dvostruka veza konjugovana sa aromatičnim prstenom kod cimetne kiseline, dovela je do blagog pada prinosa reakcije, dok je uvođenje elekt