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Layered zinc−2,2'-bipyrimidine complex with anion of mellitic acid
No full textБезбојни кристали дводимензионалног комплекса цинка {[Zn2(bipym)2(H2mell)(H2O)2]·10H2O}n, 1, добијени су спорим упаравањем на собној температури (bipym = 2,2’-бипиримидин, mell6– = хексаанјон мелитне киселине) и окарактерисани рендгенском структурном и квантно-хемијском анализом. Атоми Zn су у деформисаном октаедарском окружењу и повезани су мостовним bipym- и H2mell4–-лигандима, тако да формирају таласасте слојеве паралелне ac-равни (Слика 1). Слојеви су повезани сложеном мрежом водоничних веза које граде молекули воде, аква-лиганди и некоординирани О-атоми из H2mell4– и додатно су стабилизовани нековалентним C–H∙∙∙O, O–H···π and C–O···π интеракцијама. Интеракција између две октаедарске јединице (Слика 2а) је веома јака (−114,56 kcal/mol), што је у сагласности са израженом комплементарношћу електростатичких потенцијала на одговарајућим молекулским површинама (Слика 2б). Слика 1. Пројекција таласастих слојева комплекса 1 на ac-раван. Одабрани кристалографски подаци: C36H18N12O12Zn2, моноклинични, P21/c, a = 10,530(2), b = 17,920(4), c = 21,160(4) Å, β = 96,74(3) °, V = 3965,1(14) Å3, Z = 4, R1 = 0,0449, wR2 = 0,1078, S = 1,09 за 633 параметара и 5914 рефлексија.Colourless single crystals of two-dimensional zinc complex {[Zn2(bipym)2(H2mell)(H2O)2]·10H2O}n, 1, were prepared by slow evaporation method at room temperature (bipym = 2,2’-bipyrimidine, mell6– = hexaanion of mellitic acid) and characterized by the single-crystal X-ray diffraction and quantum chemical calculations. Zn atoms are in a deformed octahedral environment and connected by bridging bipym and H2mell4– ligands into wavy-like layers parallel to the ac plane (Figure 1). The layers are interconnected by a complex hydrogen bond network involving free water molecules, aqua ligands, and noncoordinated O atoms from H2mell4– and additionally stabilized by C–H∙∙∙O, O–H···π and C–O···π non-covalent interactions. Interaction between two octahedral units (Figure 2a) in this crystal structure is very strong (−114.56 kcal/mol), which is in agreement with the pronounced complementarity of electrostatic potentials on the corresponding molecular surfaces (Figure 2b). Figure 2. Interaction between two octahedral units of 1 (а) and electrostatic potentials of a unit plotted at the surface defined by electron density of 0.001 a. u. (b). Selected crystallographic data: C36H18N12O12Zn2, monoclinic, P21/c, a = 10.530(2), b = 17.920(4), c = 21.160(4) Å, β = 96.74(3) °, V = 3965.1(14) Å3, Z = 4, R1 = 0.0449, wR2 = 0.1078, S = 1.09 for 633 parameters and 5914 reflections
Simple, cost effective and eco-friendly method for synthesis of hydrogels based on poly (methacrylic acid
No full textPredmetnim pronalaskom postignuto je da se hidrogelovi na bazi poli(metakrilne kiseline) dobiju jednostavnom, ekonomski isplativom i ekološkom metodom. Takođe, predmetnim pronalaskom je postignuto da se ovi hirogelovi dobiju korišćenjem sistema vitamin C/vodonik peroksid kao inicijatora i da se sinteza izvede u ambijentalnim uslovima. Predmetni pronalazak obezbeđuje i da hidrogelovi na bazi poli(metakrilne kiseline) dobijeni ovom metodom imaju optimalna svojstva.Broj prijave: P-2022/103
Natural plant dye inks set new challenges: analysing the interaction of anthocyanin-rich dye with modern calcium carbonate containing substrates
No full textPlant dyes are increasingly finding applications across a broad spectrum of print technologies, leading to replacement of conventional synthetic dyes and pigmented inks for a range of print media. Despite technical advances, industrial application faces some fundamental challenges of achieving the necessary critical print properties demanded when using such dyes. These include maintaining runnability, colour definition and fastness while retaining functional stability, the latter being particularly challenging since many prints are based on digital patterning adopting inkjet or flexographic methods. This study explores the fundamental interactions between an example pure dye ink, derived from Aronia melanocarpa, a member of the family Rosaceae commonly known as chokeberry, and specific substrate filler and coating components. Key interactive factors include ink formulation, the nature of dye chemistry in relation to substrate structure, its optical properties and constituent components. The acidity of the juice-based ink is mainly dependent on the amount of anthocyanin (ANC), a water-soluble phytochemical plant protective flavonoid, occurring together with other phenolic compounds. Novel experiments are reported in which interactive substrate components are isolated and studied directly in contact with the naturally acidic anthocyanin-rich ink. Coloration of the dye is confirmed to be pH-dependent, and, as a result, major challenges arise when acidic ink contacts alkaline substrate, which covers the majority of paper, board and cellulose-based packaging materials today, due to the dominance of calcium carbonate as the filler and coating pigment of choice. In parallel, dye imbibed into substrate pores surrounded by materials of contrasting refractive index lead to effective colour gamut changes as the ratio of transmitted light through dye and scattered light from surrounding materials changes. This effect is exemplified comparing high refractive index titanium dioxide (TiO2) versus lower refractive index calcium carbonate (CaCO3). Finally, a strategy is proposed aimed at controlling the interaction and enhancing the overall printing performance
Solidification of flotation tailings in zeolite and fly-ash-based geopolymers
No full textMining tailings are fine-grained remains of ore processing with an inhomogeneous and complex
composition. Its improper disposal has a negative impact on the environment (acid mine
drainage, leaching of toxic elements, and dust generation). However, the physicochemical
properties of tailings determine their sustainable disposal methods and potential industrial
application. The high content of aluminosilicate minerals (60-90%) is a good prerequisite for
the reuse of tailings in the production of geopolymer materials. In this paper, the solidification
of flotation tailings in the presence of natural zeolite (NZ), sodium-modified natural zeolite
(NaZ), and fly ash (FA) by the geopolymerization process was investigated. The influence of
basic parameters (molar ratios of Ca/Si, Si/Al, Na/Al, and H2O/Na2O) on the unconfined
compressive strength (UCS) was analysed to ensure efficient solidification of tailings. Sodium
hydroxide solution (10M) was used as an alkaline activator. Three proportions of flotation
tailings (FT) (20%, 35 and 50%) dry mix and NaZ, NaZ or FA, respectively, were used as raw
materials. The results showed that the value of UCS decreases in zeolite-based geopolymers
with a higher content of flotation tailings. The highest UCS (44.3 MPa) was measured in a fly
ash-based geopolymer with 50% flotation tailings and optimal basic parameters of 0.4 Ca/Si,
4.7 Si/Al, and 1.3 Na/Al. It was observed that the mentioned parameters have a strong influence
on the mechanical strength of geopolymer based on fly ash and natural zeolite. Finally, the
geopolymerization process has been confirmed as a potentially safe technological approach to
tailings treatment and immobilization of heavy metals
Sustainability assessment of geopolymer synthesized from the automotive industry by-products
No full textThe use of different industrial by-products in the development of geopolymers as alkalineactivated
materials based on reactive silicates and aluminum is encouraged. This article
addresses some of the automotive industrial by-products that contain organic components
(coatings, emulsions, and vacuum distillation sludge) and aluminosilicate (dust and casting
sand) with the potential to be used in the production of geopolymers. This process involved the
addition of fly ash and bentonite, followed by alkaline activation using a 10 M KOH solution
and thermal activation at 70 °C. The effectiveness of the proposed technique was assessment
based on XRPD and SEM-EDS analysis of hardened geopolymers. The measured compressive
strength of the geopolymer indicated an efficient immobilization process of undesirable
industrial by-products which can be optimized by additional activation of aluminosilicate
components. The results of the standard leaching test (EN 12457) confirmed that the content of
organic components can be controlled by geopolymerization
Kinetic Study of the Anthraquinone Dyes Degradation by Immobilized Peroxidase on Magnetite/Alginate Beads
No full textThis study explores the immobilization of horseradish peroxidase (HRP) on magnetitealginate beads for their use in the removal of anthraquinone dyes from wastewater. Magnetic particles were synthesized by the co-precipitation of FeCl3 and FeSO4 in an excess of NH4OH and coated with alginate by electrostatic extrusion method. The surface morphology of MABs was examined using scanning electron microscopy (FEG–SEM), while the magnetic sizes of the magnetic particles and MAB beads were measured using Quantum Design MPMS (Magnetic Property Measurement System) XL-5 SQUID magnetometer. The immobilization of HRP was carried out by covalent bonding, with EDAC as the activation agent through amide bond between the amino group of enzyme and carboxyl group of alginate. Furthermore, the study evaluated the potential of HRP immobilized on magnetic alginate beads (HRP-MABs) for degrading two anthraquinone dyes, AB225 and AV109. Kinetics of the reaction with native and immobilized enzyme was examined by monitoring the reaction rate at different initial substrate concentrations in initial conditions, while kinetic parameters were determined using the Enzyme Kinetics application of the OriginPro software package. The kinetic results reveal that the immobilized HRP decolorization follows the Ping Pong Bi–Bi mechanism. Previous results have shown that immobilization reduces the initial reaction rate for both colors, thereby reducing the inhibitory effect of the dye while increasing the effect of hydrogen peroxide on inhibiting the reaction. HRP-C has shown a greater affinity for removing AV109 dye. Overall, HRP-MABs demonstrated promising results, achieving significant removal percentages (>75%) of two target dyes under optimized conditions
Advanced thermodynamic approach to adsorption of charged adsorbates from aqueous electrolyte solutions
No full textThis study present estimation of the thermodynamic parameters and the influence of ionic activity coefficient on the thermodynamic equilibrium constant of adsorption of phosphates on fly ash. The adsorption was conducted over a wide range of initial phosphate concentrations at different pH values pH = (3; 7; 10). The adsorption results were treated using the Langmuir, Freundlich and Sips adsorption isotherms, which provide information about the maximum adsorption capacity. The Langmuir and Sips isotherms were a satisfactory fit to the adsorption data, especially at pH = 3, with an acceptable regression coefficient over the entire concentration range, Langmuir (r2 = 0.9737) and Sips (r2 = 0.9969). The estimated maximum phosphate sorption capacity of fly ash was 6.21 ± 0.68 (mmol·g−1) according to Langmuir and 4.19 ± 0.16 (mmol·g−1) according to the Sips model, at pH = 3.0. However, there is no data in the published literature for estimating the thermodynamic parameters of the phosphate adsorption process using thermodynamic models for activity coefficients. Novel approach of this paper was determination of the thermodynamic equilibrium constant and Gibbs free energy, using the Pitzer ion-interaction model to predict the nature of adsorption. The Pitzer-ion interaction model was used for the mixed ionic systems, taking into consideration the effect of other present ions besides phosphates in the equilibrium solution resulting from fly ash desorption. The procedure for comprehensive estimation of the ion activity coefficient at maximum adsorption capacity and the dimensionless thermodynamic equilibrium constant using Langmuir's and Sips's constants was presented. The calculated value of the phosphate activity coefficient in the equilibrium solution was γH = 0.7003 ± 0.0027 and the converted molar activity coefficient was γH = 0.6903 ± 0.0027. The estimated values of Gibbs free energy were: ΔGL = −6.788 ± 0.521 kJ·mol−1 based on Langmuir equilibrium constant and ΔGa = −7.707 ± 0.527 kJ·mol−1 based on activity and thermodynamic equilibrium constant. According to Sips model, the adsorption process is even more spontaneous, with the Gibbs free energy calculated using the phosphate activity coefficient and the thermodynamic equilibrium constant, ΔGa = −9.707 ± 0.617 kJ·mol−1. Consideration of the ionic activity coefficient is particularly important for large, charged adsorbates at higher concentrations, as the absolute difference in free energy for adsorption is app. 12 %. The scientific contribution is reflected in obtaining the necessary and more accurate information for the improvement of adsorption processes and possibly for the upgrading of fly ash in overall wastewater treatment technology.Supplementary information: [https://technorep.tmf.bg.ac.rs/handle/123456789/7332
Enzymatic modification of wheat gluten in order to decrease its allergenicity and improve its functional properties
No full textProteini glutena spadaju u visokonutritivno vredne proteine koji čine osnovnu hemijsku građu zrna žitarica, kao što su pšenica, raž i ječam. Međutim, ovi proteini odlikuju se visokim sadržajem glutamina i prolamina što ih čini otporne na enzime (Pepsin, Tripsin i Himotripsin) prisutne u digestivnom traktu ljudi. Kao posledica toga, konzumiranjem proizvoda bogatih glutenom obrazuju se velike peptidne frakcije koje iniciraju imunogenu reakciju, čineći tako ovaj protein jednim od najznačajnih prehrambenih alergena. Upravo naveden problem poslužio je kao polazna osnova za kreiranje cilja ove doktorske disertacije, u kojoj su istraživanja bazirana na primeni enzima kao biokatalizatora prirodnog porekla i nekonvencionalnih termičkih tretmana, poput mikrotalasnog zračenja, zarad proizvodnje i modifikacije nealergenih proteina glutena. Generalno, u okviru ove disertacije pristupilo se definisanju parametara netermičkih postupaka za modifikaciju proteina glutena iz pšenice kakav je mikrotalasno zračenje pod kontrolisanim uslovima u odgovarajućem reaktoru, a sve u cilju smanjenja alergenosti, odnosno sadržaja alergenih epitopa glutena. Osim toga, u cilju smanjenja alergenosti pšeničnog glutena pristupilo se i razvoju biotehnološkog postupka zasnovanog na primeni proteolitičkih enzima velike specifičnosti. Ovaj postupak je optimizovan uz pažljivo definisanje svih važnih procesnih parametara, ali je i upotpunjen i/ili dopunjen kombinovanjem sa mikrotalanim zračenjem. Kao izvor pšeničnog glutena, u okviru eksperimenata, korišćena su dva supstrata:
• prvi, model protein glutena poreklom iz pšenice (bogat frakcijama glutenina i gliadina), i
• drugi, komercijalno dostupna sirovina koja se koristi u svakodnevnoj ishrani ljudi, pšenično belo brašno.
U svakoj fazi eksperimentalnog rada vršena je detaljna karakterizacija modifikovanog pšeničnog glutena, kako model proteina, tako i pšeničnog brašna. Karaktertizacija je podrazumevala ispitivanje alergenih svojstava modifikovanih proteina glutena primenom kompetitivnog ELISA testa koji u sebi sadrži R5 monoklonska antitela. Potom, modifikovan gluten je okarakterisan sa aspekta tehnološko-funkcionalnih svojstava (penjenje, emulgovanje, površnko naelektrisanje), strukturnih promena (FTIR analiza, RP-HPLC analiza, SDS-PAGE analiza) i bioaktivnih svojstava (antioksidativna i antimikrobna aktivnost).
Detaljnije, u prvom delu istraživanja ispitan je uticaj parametara mikrotalasnog zračenja na strukturu model proteina glutena poreklom iz pšenice, i njihova alergena svojstva. Delovanjem mikrotalasnog zračenja snage 200 do 800 W, utvrđene su promene u detekciji relativnog sadržaja alergenog epitopa glutena pomoću odabranog antitela. Poređenjem odabranog mikrotalasnog tretmana sa konvencionalnim zagrevanjem, ispitan je uticaj i više različitih temperatura kako bi se bliže odredile promene nastale usled ovih tretmana. Sinergističkim delovanjem enzima kao biokatalizatora, izvršena je kontrolisana hidroliza model proteina glutena nakon mikrotalasnog zračenja. Korišćena je komercijalna endopeptidaza Alkalaza kao biokatalizator za reakciju hidrolize mikrotalasno tretiranog glutena. Primena mikrotalasnog pretretmana snage 200 W pri kontrolisanoj temperaturi u trajanju od svega 1 min, a u kombinaciji sa enzimskom hidrolizom se pokazala kao veoma efikasna, jer su proizvedeni hidrolizati proteina glutena ispoljili izuzetno poboljšanje tehnološko-funkcionalnih svojstava i antioksidativnih aktivnosti u poređenju sa netretiranim glutenom.
Specifični netermalni efekat mikrotalasa je imao uticaj na strukturu i alergenost glutena, usled čijeg dejstva je došlo do unapređenja enzimske hidrolize. Ovo je potvrđeno jer termički tretman glutena pri potpuno istim uslovima je pokazao drugačiji uticaj na strukturu molekula, na osnovu čega se može zaključiti da mikrotalasni tretman rezultuje u jedinstvenim modifikacijama proteina. Opšte je poznato da se usled smanjenja dužine peptidnih lanaca tokom hidrolize menjaju i tehnološko-funkcionalna svojstva proteina. Emulgujuća stabilnost hidrolizata glutena sa i bez pretretmana značajno je unapređena u odnosu na sirov gluten, međutim do značajnijeg unapređenja među uzorcima nije došlo prilikom ispitivanja stabilnosti formirane pene. Antioksidativna svojstva proizvedenih hidrolizata glutena, pre svega sposobnost neutralizacije radikalskog katjona ABTS˙+ i DPPH˙ radikala značajno su unapređene u odnosu na polazni uzorak, dok se posebno ističe sposobnost heliranja jona Fe2+. S tim u vezi, krajnji hidrolizati sa i bez pretretmana pokazuju značajnu razliku u IC50 vrednostima (mg/ml) prilikom ispitivanja mogućnosti heliranja jona metala. Metodom dead-end ultrafiltracije, izvršeno je frakcionisanje krajnjih hidrolizata, mikrotalasno pretretiranog i kontrolnog, na frakcije peptida tačno definsanog opsega molekulskih masa, a u cilju pronalaženja frakcije sa najizraženijom antioksidativnom aktivnošću. Prilikom ispitivanja sposobnosti inhibicije ABTS radikalskog katjona, nisu zabeležene značajne razlike među peptidnim frakcijama. Doprinos peptidne frakcije F3 (3–10 kDa) zabeležen je prilikom testiranja inhibitorne aktivnosti prema DPPH radikalima. Izmerene vrednosti stepena heliranja jona metala značajno se razlikuju među peptidnim frakcijama istog uzorka, ali i među uzorcima. Naime, čak 10–25% veće aktivnosti su zabeležene u slučaju peptidnih frakcija dobijenih od mikrotalasno pretretiranih proteina glutena. Frakcije koje su se posebno istakle su frakcija 2 sa peptidima molekulske mase 10–30 kDa, frakcija 3 (3–10 kDa) i frakcija 4 (1–3 kDa).
U drugom delu istraživanja u okviru ove disertacije, kao sirovina za modifikaciju glutena korišćeno je belo pšenično brašno. Tom prilikom, u cilju modifikacije glutena iz pšeničnog brašna, ali i samog brašna, istraživana su sprovedena u dva dela. Prvi deo istraživanja je bio baziran na primeni prethodno usvojenih parametara enzimske hidrolize proteazom Alkalazom mikrotalasno pretretiranog glutena, dok je naknadno izvršena studija optimizacije postupka hidrolize pšeničnog brašna u cilju kreiranja valjanog modela koji bi se mogao primeniti u postupku smanjenja alergenih svojstava glutena iz brašna. S tim u vezi, prilikom hidrolize proteina brašna varirane su različite koncentracije supstrata u cilju ispitivanja uticaja prisutnih skrobnih materija na sam postupak hidrolize, a potom i na tehno-funkcionalna svojstva dobijenih hidrolizata glutena. Najveće smanjenje sadržaja alergenih epitopa glutena zabeleženo je kod uzorka pripremljenog kao 15% (w/w) suspenzija brašna, pri čemu je razlika u alergenosti među uzorcima potvrđena razlikom i odsustvom proteinskih traka primenom SDS-PAGE elektroforeze. Emulgujuća svojstva su značajno unapređena postupkom enzimske hidrolize, dok stabilnost formiranih emulzija nije značajno unapređena. Ispitivanjem svojstva penjenja zabeležene su značajne razlike među hidrolizatima, pri čemu je interesantno istaći da se pažljivom kontrolom enzimske hidrolize mogu proizvesti hidrolizati sa dovoljno dugačkim polipeptidnim lancima koji formiraju dobre i stabilne pene. Visoke vrednosti antioksidativnih aktivnosti dobijenih hidrolizata proteina pšeničnog brašna potvrđene su u okviru ABTS i helatnog testa, čineći tako pripremljenje hidrolizate podobne za dalja ispitivanja koja se mogu sprovoditi za potrebe kreiranja preparata sa unapređenim antioksidativnim svojstvima. Sumarno, ovim delom istraživanja evidentno je bilo da prirodno prisustvo skrobnih materija u formiranim suspenzijama ne ometa sam postupak hidrolize, već ga „olakšava“ jer dovodi do sprečavanja formiranja agregata, tj. usled hidratacije sastavnih delova gliadina i glutetnina prisustvo skrobnih materija ometa formiranje glutenske mreže čime se olakšava pristup enzima peptidnim vezama i unutrašnjosti supstrata. Naknadnom optimizacijom postupka hidrolize pšeničnog brašna analiziran je uticaj procesnih parametara: pH, temperature, enzim/supstrat (E/S) odnosa i količine supstrata na stepen hidrolize, alergena svojstva i antioksidativne aktivnosti. Analizom dobijenog modela baziranog na 29 eksperimentalnih tačaka u okviru metode odzivnih površina, zaključeno je da najveći uticaj na tok hidrolize imaju pH, E/S odnos i količina supstrata (S), dok su alergena svojstva direktno bila uslovljena sa pH i temperaturom procesa. Antioksidativna aktivnost zavisi najviše od primenjenog pH tokom hidrolize i enzim/supstrat odnosa, dok na heliranje dodatno utiče i količina unetog supstrata.
Kako bi se ispitala mogućnost još intenzivnije modifikacije pšeničnog glutena, ali i primene enzimskog preparata Pronaze (endo- i egopeptidazna aktvnost), prevashodno u svrhu smanjenja alergenih svojstava, istraživanja su nastavljena u tom smeru. S obzirom na slabu dostupnost literaturnih podataka u vezi delovanja ovog enzima na gluten, ova eksperimentalna postavka je izvedena u cilju teorijskog ispitivanja mogućnosti primene Pronaze kao još jednog preparata kojim bi se smanjila alergena svojstva glutena. Naime, sa povećanjem postignutog stepena hidrolize zabeleženo je smanjenje alergenih svojstava, unapređene su emulgujuće aktivnosti, dok je kapacitet i stabilnost penjenja obrnuto zavistan od postignutog stepena hidrolize. Suprotno tome, najveći kapacitet i stabilnost penjenja pokazao je uzorak hidrolizata sa najmanjim stepenom hidrolize, usled prisustva polipeptidnih lanaca srednjh molekulskih masa. SDS-PAGE elektroforezom potvrđeno je odsustvo frakcija glutena velikih molekulskih masa (> 50 kDa). Dodatno, razlike i u antioksidativnim svojstvima proizvedenih hidrolizata bile su očigledne. Naime, svi uzorci su pokazali određeni nivo antioksidativne aktivnosti, pri čemu se u odnosu na hidrolizate dobijene Alkalazom, ističe smanjena aktivnost hidrolizata prema jonima Fe2+.
Dobijeni hidrolizat pšeničnog brašna sa najnižim relativnim sadržajem glutena iskorišćen je kako bi se ispitao njegov uticaj na reološke osobine bezglutenskog brašna, heljdinog integralnog brašna, na Miksolabu. Naime dodatkom hidrolizata postignuta je promena reoloških osobina u vidu smanjenja slabljenja glutenske mreže tačnije propadanja proteinske strukture tokom zagrevanja. Dodatak hidrolizata uticao je i na promenu u aktivnosti amilaza, kao i na retrogradaciju skroba u poslednjoj fazi analize.
Sveobuhvatno, sumiranjem rezultata dobijenih tokom istraživanja sprovedenih za potrebe izrade ove disertacije utvrđeno je da se primenom Alkalaze i Pronaze može efikasno i do određene mere smanjiti prisustvo alergenih epitopa u sirovom glutenu koji izazivaju imuni odogovor u osetljivim grupama populacije. Primena mikrotalasnog pretretmana dovodi do strukturnih promena koje su zabeležene u sekundarnoj strukturi proteina glutena, a dodatno pozitivno pospešuju dalji tok enzimske hidrolize. Tokom hidrolize proteina pšeničnog brašna, ustanovljeno je da prisustvo skrobnih granula olakšava postupak enzimske hidrolize jer skrobne granule direktno sprečavaju formiranje proteinskih agregata. Dobijeni hidrolizati proteina pšeničnog brašna se mogu smatrati hidrolizatima bogatim antioksidativnim peptidima, sa unapređenim određenim funkcionalnim svojstvima i smanjenom alergenošću. Stoga, na osnovu svega izloženog moguće je modifikovane proteine glutena uzeti u razmatranje prilikom kreiranja novih proizvoda sa smanjenim alergenim svojstvima. Finalno, kao doprinos istraživanjima u okviru ove disertacije može se pripisati i ekonomičnost mikrotalasnog pretretmana kao i postupka hidrolize u prisustvu skrobnih materija, jer primenom ovakvih postupaka moguće je izvršiti određeni nivo uštede tokom postupka proizvodnje modifikovanih proizvoda glutena sa smanjenim sadržajem alergenih epitopa (~ 20 ppm).Gluten proteins belong to highly nutritionally valuable proteins that make up the basic chemical structure of cereal grains, such as wheat, rye and barley. However, these proteins are characterized by a high content of glutamine and prolamin, which makes them resistant to enzymes (Pepsin, Trypsin and Chymotrypsin) present in the human digestive tract. As a consequence, by consuming products rich in gluten proteins, large peptide fractions are formed that initiate an immunogenic reaction, thus making gluten proteins one of the most important food allergens. The problem just mentioned served as the starting point for creating the goal of this doctoral dissertation, in which research is based on the application of enzymes as biocatalysts of natural origin and unconventional thermal treatments, such as microwave radiation, for the production and modification of non-allergenic gluten proteins. In general, within the framework of this dissertation, the parameters of non-thermal procedures for the modification of wheat gluten protein, such as microwave radiation under controlled conditions in a suitable reactor, were defined, all with the aim of reducing allergenicity, i.e. the content of allergenic gluten epitopes. In addition, in order to reduce the allergenicity of wheat gluten, the development of a biotechnological procedure based on the application of highly specific proteolytic enzymes was also undertaken. This procedure was optimized with careful definition of all important process parameters, but it was also completed and/or supplemented by combining it with microwave radiation. As a source of wheat gluten, two substrates were used in the experiments:
• the first, a model of gluten protein originating from wheat (rich in glutenin and gliadin fractions), and
• the second, a commercially available raw material used in the daily diet of people, wheat white flour.
In each phase of the experimental work, a detailed characterization of the modified wheat gluten, both the protein model and wheat flour, was carried out. The characterization involved testing the allergenic properties of modified gluten proteins using a competitive ELISA test that contains R5 monoclonal antibodies. Then, modified gluten was characterized from the spectrum of technological-functional properties (foaming, emulsification, surface charge), structural changes (FTIR analysis, RP-HPLC analysis, SDS-PAGE analysis) and bioactive properties (antioxidant and antimicrobial activity).
In detail, in the first part of the research, the influence of microwave radiation parameters on the structure of model gluten proteins originating from wheat, their allergenic properties, was examined. By the action of microwave radiation with a power of 200 to 800 W, changes in the detection of the relative content of toxic gluten epitopes using the selected antibody were determined. By comparing the selected microwave treatment with conventional heating, the influence of several different temperatures was examined in order to more closely determine the changes caused by these treatments. Through the synergistic action of enzymes as biocatalysts, a controlled hydrolysis of the gluten protein model was performed after microwave irradiation. Commercial endopeptidase Alcalase was used as a biocatalyst for the hydrolysis reaction of microwave-treated gluten. The application of microwave pretreatment with a power of 200 W at a controlled temperature lasting only 1 min, and in combination with enzymatic hydrolysis, proved to be very effective, because the produced gluten protein hydrolysates showed an exceptional improvement in technological-functional and antioxidant activities compared to untreated gluten.
The specific non-thermal effect of microwaves had an impact on the structure and allergenicity of gluten, which resulted in the improvement of enzymatic hydrolysis. This has been confirmed because the thermal treatment of gluten under completely identical conditions has shown a different impact on the molecular structure, from which it can be concluded that microwave treatment results in unique protein modifications. It is generally known that due to the reduction in the length of peptide chains during hydrolysis, the technological and functional properties of proteins also change. The emulsifying stability of gluten hydrolysate with and without pretreatment was significantly improved compared to raw gluten, however, no significant improvement was noted among the samples when testing the stability of the formed foam. The antioxidant properties of the produced gluten hydrolysates, above all the ability to neutralize the radical cation ABTS˙+ and the DPPH˙ radical, were significantly improved in comparison to the original sample, while the ability to chelate Fe2+ ions is particularly noteworthy. In this regard, the final hydrolysates with and without pretreatment show a significant difference in IC50 values (mg/ml) when examining the possibility of metal ion chelation. Using the dead-end ultrafiltration method, fractionation of the final hydrolysates, microwave pretreated and the control, was performed into peptide fractions with a precisely defined range of molecular masses, with the aim of finding the fraction with the most pronounced antioxidant activity. When testing the ability to inhibit the ABTS radical cation, no significant differences were noted among the peptide fractions. The contribution of the peptide fraction F3 (3–10 kDa) was recorded when testing the inhibitory activity against DPPH radicals. The measured values of the degree of chelation of metal ions differ significantly among the peptide fractions of the same sample, but also between samples. Namely, even 10–25% higher activities were recorded in the case of peptide fractions obtained from microwave pretreated gluten proteins. Fractions that stood out in particular were fraction 2 with peptides of molecular weight 10–30 kDa, fraction 3 (3–10 kDa) and fraction 4 (1–3 kDa).
In the second part of the research within this dissertation, white wheat flour was used as a raw material for gluten modification. Therefore, in order to modify the gluten from wheat flour, as well as the flour itself, research was carried out in two parts. The first part of the research was based on the application of previously adopted parameters of enzymatic hydrolysis with protease Alcalase of microwave-pretreated gluten, while a subsequent study was carried out on the optimization of the wheat flour hydrolysis procedure in order to create a valid model that could be applied in the process of reducing the allergenic properties of gluten protein from flour. In this regard, during the hydrolysis of flour, different concentrations of the substrate were varied in order to examine the influence of the present starchy substances on the hydrolysis process itself, and then on the techno-functional properties of the obtained gluten hydrolysates. The greatest reduction in the content of allergenic epitopes of gluten was recorded in the sample prepared as a 15% (w/w) flour suspension, where the difference in allergenicity between the samples was confirmed by the difference and absence of protein bands using SDS-PAGE electrophoresis. The emulsifying properties were significantly improved by the enzymatic hydrolysis procedure, while the stability of the formed emulsions was not significantly improved. Upon examining the foaming properties, significant differences were noted between the hydrolysates, revealing that careful control of enzymatic hydrolysis can yield hydrolysates containing sufficiently long polypeptide chains that form good and stable foams can be produced. The high values of antioxidant activity of the obtained wheat flour hydrolysate were confirmed with the ABTS and chelation test, thus making the preparation of the hydrolysate suitable for further tests that can be carried out for the purposes of creating preparations with improved antioxidant properties. In summary, in this part of the research, it was evident that the natural presence of starchy substances in the formed suspensions does not hinder the hydrolysis process itself, but "facilitates" it because it leads to the prevention of the formation of aggregates, i.e. due to the hydration of the components of gliadin and glutenin, the presence of starchy substances hinders the formation of the gluten network, which facilitates the access of enzymes to peptide bonds and the interior of the substrate. Subsequent optimization of the wheat flour hydrolysis procedure analyzed the influence of process parameters: pH, temperature, enzyme/substrate (E/S) ratio and amount of substrate on the degree of hydrolysis, allergenic properties and antioxidant activity. By analyzing the obtained model based on 29 experimental points within the response surface method, it was concluded that pH, E/S ratio and the amount of substrate (S) have the greatest influence on the hydrolysis process, while the allergenic properties were directly conditioned by the pH and temperature of the process. The antioxidant activity depends mostly on the applied pH during hydrolysis and the enzyme/substrate ratio, while chelation is additionally influenced by the amount of introduced substrate.
In order to examine the possibility of even more intensive modification of wheat gluten, but also of the application of the Pronase enzyme preparation (endo- and exopeptidase activity), primarily for the purpose of reducing allergenic properties, research was continued in that direction. Considering the poor availability of literature data regarding the action of this enzyme on gluten, this experimental setup was carried out in order to theoretically test the possibility of applying Pronase as another preparation that would reduce the allergenic properties of gluten. Namely, with an increase in the achieved degree of hydrolysis, a decrease in allergenic properties was noted, emulsifying activities improved, while the capacity and stability of foaming are inversely dependent on the achieved degree of hydrolysis. On the contrary, the hydrolysate sample with the lowe
Green Leaf Biomass as an Alternative Protein Source
No full textEnsuring 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 functionality
Degradation Effect of Moisture on Mechanical Properties of Kevlar/PVB Composites with TiO2 Nanoparticles
No full textKevlar fibers are widely used for industrial and military purposes due to their remarkable mechanical properties, such as their high tenacity and high strength-to-weight ratio. In this study, two-layered Kevlar composite specimens were impregnated with 10 wt.% poly (vinyl butyral)/ethanol solution which contained TiO2 nanoparticles as reinforcement. The concentrations of the nanoparticles were 1 wt.% or 2 wt.% with respect to the poly (vinyl butyral), PVB. The single-axial tensile test and three-point bending test of the Kevlar/PVB composites have been performed according to the ASTM D 3039 and ASTM D 790-03 standards, respectively. The tensile and bending properties of the dry and wet Kevlar/PVB composite specimens after a 56-day immersion are examined in this work. Upon the addition of the 2 wt.% TiO2 nanoparticles, the tensile strength and modulus of the dry specimens without reinforcement were increased by 39.8% and 24.3%, respectively. All the submerged specimens’ tensile and flexural property values were lower than those of the dry specimens. After comparing the wet composite specimens to their dry counterparts, the percentage decrease in tensile strength was approximately 20%. The wet Kevlar/PVB specimens with no TiO2 reinforcement showed the greatest reduction in bending strength, 61.4% less than for the dry Kevlar/PVB specimens, due to the degradation of the PVB matrix. In addition, a numerical simulation of the three-point bending test was carried out in Abaqus