The evaluation of the antioxidant potential during the oxidative polymerization of polyphenol compounds induced by laccase enzyme

The polymerization and crosslinking of various phenolic compounds induced by the laccase enzyme have been in recent years increasingly used in food industry, due to the emergence of products with improved properties, especially with increased antioxidant activity. In this regard, this study has explored the laccase-induced structural changes of two types of naturally occurring polyphenols, named gallic acid and epigallocatechin gallate, and their antioxidant activity. In the first part, the effect of the laccase of different origin - laccase of white rot fungi (Ganoderma spp.), previously grown on waste cereals and commercial laccase from Novozym® 51003, were investigated. Based on the results obtained after incubation (24 h, 50ºC), the sample containing a mixture of polyphenols, rather than individually, gave visible changes in the reading of the UV-Vis spectrum and increased antioxidant activity with small differences between commercial laccase and laccase of white rot fungi, which indicates that laccase obtained by growing fungi on waste cereals may be competitive for such a purpose. Moreover, the use of such an obtained laccase is more environmentally friendly and economically viable than use of commercial one. Further process optimization of polyphenols polymerization was performed within the method of multifactor statistical analysis (Design Expert), by using of commercial laccase, where by the effect of enzyme concentrations (0.1, 0.3 and 0.5 U/ml) and incubation time (4, 14 and 24 h) were monitored. The maximal antioxidant activity, measured by both DPPH (inhibition of 58.58% of radicals) and FRAP (176.57 mmol Fe2+/ml) was achieved by a sample containing a mixture of polyphenols and 0.3 U/ml laccase enzymes, after 14 h, at 50˚C. The results of this study revealed that careful optimization of process variables during polyphenols polymerization is extremely important for obtaining the product with desirable value added properties, which may be implemented in food and pharmaceutical industry

APPLICATION OF CRUDE FUNGAL LACCASE FROM GANODERMA SPP. IN DECOLORIZATION OF TRIPHENYLMETHANE DYE CRYSTAL VIOLET

Industrial dye effluents that contain triphenylmethane dyes are environment-threatening problem. The triphenylmethane dyes are one of the largest dye’s group that is discharged in large quantities to water bodies. Removing of them with fungal enzymes is big scientific challenge. The fungal laccases are promising tool for removing of these dyes from water bodies. In this study, the potential of crude fungal laccase from genus Ganoderma in decolorization of triphenylmethane dye, crystal violet was investigated. The crude fungal enzyme was produced using agroindustrial lignocellulosic waste, cereal mix. The effect of different substrate masses (15g, 25g and 50 g) and Cu2+ ions concentrations (0.25 mM, 0.75 mM, 1.0 mM, 2.5 mM и 5.0 mM) on lccase production were investigated. The obtained crude fungal laccase with the highest activity was used for decolorization of crystal violet at different concentrations from 20 mg/L to 50 mg/L. The crude fungal laccase from genus Ganoderma had the highest activity of 47.43 U/g, when the substrate mass of 50 g was used for enyzme production. The lower masses of substrate (15g and 25g) induced lower laccase activities of 45.07 U/g and 46.69 U/g, respectively. The initial addition of Cu2+ ions in concentration of 0.25 mM to agroindustrial waste led to decrease of laccase activity compared to the laccase activity of laccase obtained from agroindustrial waste that didn’t contain Cu2+ ions. On the other hand, the increase of Cu2+ concentration to 1.0 mM in the substrate led to increase the laccase activity. The highest laccase activity of 47.53 U/g was obtained when the 1.0 mM Cu2+ was added to substrate, while the decrease in laccase activity was observed when 5.0 mM Cu2+ was added to the agroindustrial waste. Decolorization of crystal violet at different concentrations (20 mg/L, 30 mg/L, 40 mg/L, 50 mg/L) was carred out at pH 5 and temperature of 50 ºC for 120 min. The highest decolorization efficiency of 14.42 % was obtained in the case of the lowest dye concentration (20 mg/L), while the lowest decolorization efficiency of 3.76 % was obtained when the highest dye concentration of 50 mg/L was decolorized with crude fungal laccase for 120 min. The obtained results show that fungal crude laccases can be used for decolorization of triphenylmethane dyes, but the detailed optimization is very important for obtaining relatively high decolorization efficiencies for short time

Photocatalytic and antimicrobial effects of zinc oxide nanoparticles prepared by thermal decomposition of zinc benzenepolycarboxylato complexes

Zinc oxide nanoparticles (ZnO-NPs) have been a subject of numerous researches owing to their multifunctional properties in many applications, such as solar cells, UV light-emitting devices, gas sensors and photocatalysts. ZnO-NPs can be synthesized through various methods and their features can be tailored by shape and size, resulting in new applications relevant to their structural properties. The synthesis of ZnO-NPs via controlled thermal decomposition of the single-source precursors represents a novel synthetic methodology. The basic goal of this study was to investigate the influence of precursors on crystallite size and morphology of the resulting ZnO-NPs. Four structurally characterized Zn complexes with different benzenepolycarboxylato ligands [1] were used as precursors for investigation of photocatalytic and antimicrobial effects of thermally obtained ZnO-NPs. The XRPD and FESEM analysis of ZnO-NPs, prepared by thermolysis of Zn precursors at 450 °C in the air atmosphere, revealed hexagonal wurtzite structure (space group P63mc, a ≈ 3.25 and c ≈ 5.21 Å) with an average crystallite size in the range of 39–47 nm and similar morphology. The best photocatalytic activity for degradation of Reactive orange 16 dye has been observed for ZnO-NPs where crystallites form the smallest agglomerates. All obtained ZnO-NPs showed excellent inhibitory effect against Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli

Decolorization of azo dye Methyl Orange with crude fungal laccase obtained by growing Ganoderma spp. on cereal mix

In recent years, one of the biggest environmental problems is the pollution of water with colored wastewater which has negative effect on the environment and human health. Wastewaters contain complex structural compounds, such as azo dyes that used today in many industrial fields. Removing of azo dyes from wastewater using traditional methods is an extremely complex and in many cases ineffective process. In recent decades, there is a tendency towards the application of environmentally acceptable methods of removing synthetic dyes from wastewater. Method which has proven to be very effective, is the degradation of synthetic dyes using various fungal enzymes. In this study, the crude fungal laccase (31,42 UmL-1) obtained by growing fungal mycelium Ganoderma spp. on cereal mix was used for decolorization of Methyl Orange. Decolorization procedure was carried out at different temperatures (30-70 ºC) and pH (3-8) in order to determine the optimal conditions for dye decolorization. The incubation time was 180 min and every 15 min during the incubation time, the change in color intensity was monitored spectrophotometrically at 472 nm and decolorization efficiency (DE) was calculated. The optimal pH was 5 with DE of 57 % at 30 ºC, while in the case of other pH values, DE was lower. The lowest DE (1,2 %) was in the case of pH 8, which indicates that laccase activity decreases in the alkaline medium. The optimal temperature of decolorization was 50 ºC with DE of 62 % at pH 5, while the DE was lower at higher and lower temperatures, which is in accordance with the literature data on the laccase activity optimal temperature of the Ganoderma spp. The lowest DE was 35 % at 70 ºC and pH 5. The obtained results show that laccase with good decolorization properties can be obtained using cheap agro-industrial wastes, such as cereal mix. The low cost of laccase production as well as the relatively high DE in a short time may further broaden its application in wastewater treatment

IMMOBILIZATION OF CRUDE FUNGAL LACCASE FROM GANODERMA SPP. ON MODIFIED TITANIUM DIOXIDE NANOPARTICLES

In recent decades, enzyme immobilization on different supports occupies an important place in the modern biotechnology, given that it allows the design of green and sustainable production processes. Nanoparticles are very efficient supports for enzyme immobilization. The enzyme immobilized on nanoparticles is more stable than its soluble form and can be used in several operative cycles. In this study, the potential of modified titanium dioxide nanoparticles (Degussa P25, TiO2) for crude fungal laccase (47.43 U/g) immobilization was investigated. The TiO2 nanoparticles were modified with (3-glycidyloxypropyl) trimetoxylane under different conditions in order to obtain support with an optimal concentration of epoxy groups for immobilization. The obtained TiO2 nanoparticles had different concentrations of epoxy groups on their surface, exactly 950 μmol/g (TiO2M1) and 500 μmol/g (TiO2M2), respectively. The immobilization was carried out at pH 5 and at room temperature for 4 h. The residual activity of immobilized laccase on TiO2M2 was 33.40 %, while the residual activity of immobilized laccase on TiO2M1 was 17.39 %. The immobilization efficiency was 26.75 % and 20.18 % for laccase immobilized on TiO2M2 and immobilized laccase on TiO2M1, respectively. The immobilization of crude fungal laccase on TiO2M2 was further optimized by testing the influence of different contact time between laccase and TiO2M2 (2 h ,3 h, 3,5 h and 4 h) as well as the effect of various pH values (pH 4, pH 5 and pH 6) on immobilization. The highest residual activity of 35.04 % and immobilization efficiency of 28.95 % were obtained for optimal contact time of 3.5 h between crude fungal laccase and TiO2M2. The lowest residual activity (30.38 %) and immobilization efficiency (20.18 %) were obtained when immobilization was carried out at room temperature and at pH 5 for 2 h. The pH values had significant effect on immobilization. The optimal pH value was pH 5 with highest residual activity of 35.04 %, while the lowest residual activity of 14. 83 % was at pH 6. The residual activity of 21.21 % and immobilization efficency of 19.52 % were obtained when crude fungal laccase immobilized at pH 4. The immobilized laccase on TiO2M2 was successfully used in 5 cycles of guaiacol oxidation. In the 2nd cycle, the immobilized laccase had residual activity of about 60%, while after 3rd cycle, immobilized laccase had residual activity of 40 %. The lowest residual activity of 14.83 %, immobilized laccase had after 5th cycle

The effect of phytohormones application on morphological and biological properties of Thymus pannonicus All.

This study has explored the effect of three phytohormones application, based on α-naphthyl acetic acid, named INCIT 1, INCIT 8 and INCIT K, on the root system growing potential and rooting stimulation of Thymus pannonicus All. cuttings. This plant is highly reputable not only in a folk medicine, but also recognized in the business sector, due to a multiple uses and beneficial effects on human health. The research was located in Veliko Središte, Vršac (Serbia) and conducted in May, 2021. The cuttings were dipped in a powdered formulation of phytohormones and placed in plastic containers pre-filled with commercial substrate. The phytohormone-free variant was taken as a control. After 2 months, the cuttings were removed and the morphological and biological properties (polyphenols content and antioxidant activity by DPPH radical and FRAP method) were measured. Based on the morphological results, all of treated samples showed satisfactory behaviour, whereby the most suitable phytohormone to stimulate the growth of cuttings was INCIT 8, in which the average weight of the aboveground vegetative part of the plant and root mass were higher for more than 60% in relation to the control; the number of roots were about 25% higher, whereby the mass of the whole plant was almost 3 times increased. Considering the biological activities in a 5% aqueous infusions of the samples, it was found that the polyphenols content and the antioxidant activity were lowered in treated samples, with relation to the control, excluding the sample INCIT 8 where the FRAP value was slightly increased, while the inhibition of DPPH radicals was higher by around 15%. The applied phytohormone treatment may ensure economically viable yields of standard or even improved plant quality. In the following period, it is expected that all of treated adult plants will have a higher content of biologically active substances.UNIFood2021 Conference, 24th-25th September 2021 University of Belgrade, 2nd International UNIfood Conferenc

Jednostavni postupci za dobijanje multifunkcionalnih tkanina od jute

This study aims to obtain multifunctional jute fabrics by using simple alkaliand oxidative protocols. Performed chemical treatments contributed to obtaining jute fabricswith enhanced electro-physical and sorption properties. Moreover, the jute fabrics werefunctionalized by incorporation of Ag+ (resulted in decreased volume electrical resistivityand excellent antibacterial activity) and in-situ synthesis of Cu-based nanoparticles (resultedin 38-88 times higher energy storage from an external electric field). Following the increasedfocus on the concept of circular economy, the possibility of jute fabric waste revalorizationfor wastewater purification (from Cu2+, Zn2+, Ni2+) was examined.Cilj ovog istraživanja je dobijanje multifunkcionalnih tkanina od juteupotrebom jednostavnih alkalnih i oksidativnih postupaka. Ovi hemijski tretmani omogućilisu dobijanje tkanina od jute sa poboljšanim elektro-fizičkim i sorpcionim svojstvima.Tkanine od jute su, takođe, bile funkcionalizovane jonima srebra (što je rezultovalosmanjenom specifičnom zapreminskom električnom otpornošću i odličnom antibakterijskomaktivnošću) i in-situ sintezom nanočestica na bazi bakra (što je rezultovalo 38-88 puta većimskladištenjem energije iz spoljašnjeg električnog polja). U skladu sa načelima cirkularneekonomije, ispitana je mogućnost revalorizacije otpada tkanina od jute za prečišćavanjeotpadnih voda (od Cu2+, Zn2+, Ni2+- jona)

TEMPO oksidacija u funkciji poboljsanja antibakterijskih svojstava viskozne tkanine funkcionalizovane nanočesticama na bazi hitozana

TEMPO oxidation of viscose fabric was used as a pretreatment 10 introduce carboxyl and aldehyde groups for irreversible binding of chilosan and to improve antibacterial activity and washing durability of viscose fabrics functionalized with chitosan nanoparticles withour (NCS) and with emt edded zine (NCS+ Zn). Carboxyl and aldehyde group content, electrokinetic properties, elemental analysis, inductively coupled plasma optical emission spectrometry and antibacterial testing were used to caluare the influence of TEMPO axidation on subseguent NCS and NCS+Zn binding and antibacterial properties of chitosan functionalized viscose fabrics. Anvibacterial activity of NCS and NC$+Zn functionalized viscose fabrics was preserved after multiple wasting

Decomposition mechanism and kinetics of zinc–isophthalate complex with 2,2’-dipyridylamine as a precursor for obtaining nanosized zinc oxide

Studies related to the synthesis of nanosized ZnO as the antibacterial agent have become an interdisciplinary area gathering chemists, physicists, biologists, and medics. The broad scope of materials based on ZnO resulted in the development of various techniques for its preparation. Considering the dependence of particle shape and size onto physical and chemical properties of ZnO, the synthesis procedure is of major importance. In this work, an unconventional methodology of synthesis is proposed for obtaining nanosized ZnO. Polymeric zinc complex containing 2,2’-dipyridylamine (dipya) and dianion of 1,3-benzenedicarboxylic acid (ipht), [Zn(dipya)(ipht)]n, was used as precursor. Besides the crystal structure of [Zn(dipya)(ipht)]n which was already published [1], the luminescent properties are presented in this work. Also, the amazing antibacterial activity of this precursor prompted us to investigate the relationship between the crystal structure and thermal properties, especially if we bear in mind the lack of similar studies in the literature. Therefore, the mechanism and kinetics of its degradation was investigated under non isothermal conditions in nitrogen and air atmospheres. Degradation enthalpies, thermodynamic activation parameters, pre-exponential factor, A, and the apparent activation energy, Ea, were determined for each step using Kissinger’s and Ozawa’s equations. The complexity of degradation steps has been analyzed using isoconversional methods. TG/DCS data were collected at four different heating rates: 10, 15, 20 and 25 ºC min –1 , while the formation of nanosized ZnO was confirmed using XRPD and FESEM techniques. The influence of precursor on the crystallite size and morphology of the resulting ZnO along with its antibacterial activity was examined. The obtained results will be discussed and compared. [1] L. Radovanović, J. Rogan, D. Poleti, M. Milutinović, M.V. Rodić, Polyhedron 112 (2016) 18

Poljoprivredni i industrijski otpad kao supstrat za proizvodnju celulaza i amilaza pomoću novog bakterijskog soja Paenibacillus chitinolyticus CKS1

The subject of this thesis is the utilization of waste materials of agricultural and industrial origin, for the production of enzymes cellulases and amylases using a novel isolated bacterial strain Paenibacillus chitinolyticus CKS1. Optimization of the process of production of hydrolytic enzymes cellulases and amylases was performed using commercial substrates and waste materials by varying the influence of individual parameters but also by employing the method of the planned experiment and mathematical-statistical processing of experimental data - Response Surface Methodology (RSM). The process of producing cellulase (CMC-ase and Avicelase) was optimized using various commercial substrates but also using waste materials.The strain CKS1 showed predominant exoglucanase (Avicelase) activity which is not typical for most bacterial species. Results showed that P.chitinolyticus CKS1 could be used for cellulase (CMC-ase and Avicelase) production, in a liquid medium with comercial substrates (with maximum CMCase and Avicelase activity of 0,532 U/mL and 1,83 U/mLrespectively), but also in a medium with waste substrates: waste medicinal herbs (with maximum CMCase and Avicelase activityof 0,203 U/mL and 1,94 U/mL, respectively) and sawdust (with maximum CMCase and Avicelase activity of 0,145 U/mL and 1,75 U/mL, respectively)...Predmet ove disertacije je iskorišćenje otpadnih sirovina, poljoprivrednog i industrijskog porekla, za proizvodnju enzima celulaza i amilaza pomoću novoizolovanog bakterijskog soja Paenibacillus chitinolyticus CKS1. Optimizacija procesa proizvodnje hidrolitiĉkih enzima celulaza i amilaza vršena je korišćenjem komercijalnih supstrata i otpadnih sirovina ivariranjem uticaja pojedinaĉnih parametara primenom metode planiranog eksperimenta i matematiĉko-statistiĉka obrade eksperimentalnih podataka – metode odzivnih površina (RSM). Proces proizvodnje celulalaza (endoglukanaza i egzoglukanaza) je optimizovan na razliĉitim komercijalnim podlogama ali i na otpadnim sirovinama. Soj CKS1 je pokazao predominantnu aktivnost egzoglukanaza (iskazanih kao Avicelazna aktivnost) što je netipiĉno za većinu bakterijskih vrsta. Rezultati su pokazali da se P. chitinolyticus CKS1 moţe koristiti za dobijanje celulazau komercijalnoj podlozi (maksimalna CMC-azna i Avicelazna aktivnost iznose 0,532 U/mL i 1,83 U/mL, pojedinaĉno), ali i na otpadnim sirovinama: otpadnom lekovitom bilju (maksimalna CMC-azna i Avicelazna aktivnost iznose 0,203 U/mL i 1,94 U/mL, pojedinaĉno) i piljevini (maksimalna CMC-azna i Avicelazna aktivnost iznose 0,145 U/mL i 1,75 U/mL, pojedinaĉno). Soj CKS1 je pokazao mogućnost rasta i proizvodnje dve vrste celulaza: endoglukanaza (iskazanih kao karboksimetil celulazna (CMC-azna) aktivnost i egzoglukanaza (iskazanih kao Avicelazna) aktivnost tokom svog rasta na ĉvrstom otpadnom supstratu- repinom rezancu (maksimalna CMC-azna i Avicelazna aktivnost iznosi 3,159 U/g i 4,840 U/g). Metodom odzivnih površina optimizovana je proizvodnja endoglukanaza i egzoklukanaza u podlozi obogaćenoj jeĉmenim mekinjama (maksimalna CMC-aza i Avicelazna aktivnost iznosi 0,405 U/mL i 0,433 U/mL, pojedinaĉno). Dobijena je nešto niţa vrednostAvicelazne aktivnosti, ali optimizovani procesi jasno ukazuju na mogućnost njihovog korišćenja, pošto su u skladu sa principima odrţive proizvodnje i brige o ţivotnoj sredini. P. chitinolyticus CKS1 soj je pokazao i amilolitiĉku aktivnost, netipiĉnu za vrstu kojoj pripada i stoga je metodom odzivnih površina optimizovana proizvodnja β-amilaza na podlozi dobijenoj korišćenjem otpadne vode iz industrije prerade kartonske ambalaţe (0,333 U/mL) i na podlozi baziranoj na melasi i otpadnom repinom rezancu (2,237 U/mL). U cilju što kompletnije analize podobnosti primene dobijenih enzima u industrijskim procesima, izvršena je karakterizacija proizvoda hidrolize nastalih primenom obe klase enzima.UtvrĊeno je da su proizvodi enzimske hidrolize dobijeni pomoću enizima P. chitinolyticus CKS1 vredni biotehnološki proizvodi – glukoza i maltoza. Primenljivost bakterijskih celulaza i amilaza, koje produkuje soj CKS1 se ogleda u mogućnosti hidrolize pamuĉnog materijala, kao celulozne sirovine i jeĉmenih mekinja,kao lignocelulozno-skrobne sirovine, pri ĉemu se oslobaĊa 0,589 mg/mL i 2,74 mg/mL redukujućih šećera pojedinaĉno.Time se otvaraju nove mogućnosti industrijske primene ovih enzima, prevashodno za saharifikaciju skrobnih sirovina i u industriji proizvodnje bioetanola..