Preparation of zein nanoparticles and their application as functional colloids

Biljni proteini, odnosno belančevine izolovane iz različitog biljnog materijala, poseduju odličan potencijal za primenu u proizvodnji, ali i pakovanju hrane. Oni poseduju različite funkcionalne osobine, kao što su sposobnost emulgovanja, stabilizovanja pene, geliranja, formiranja omotača i filmova itd., što im omogućava primenu u prehrambenoj i farmaceutskoj industriji. Zein je biljni protein, koji se može naći u žitaricama, a osnovni izvor ovog proteina je kukuruz (Zea mays). Zein nije rastvoran u vodi, a za njegovo rastvaranje najčešće se koristi razblaženi etanol. Smanjenjem koncentracije etanola u rastvoru zeina, bilo da se to postiže dodatkom nerastvarača ili uklanjanjem etanola (isparavanjem), zein može formirati različite koloidne strukture, koje pokazuju veliki potencijal za primenu u prehrambenoj industriji. Osobine ovih struktura zavise od načina pripreme, ali se na njih može uticati i dodatkom drugih materija u toku procesa pripreme. Stoga, osnovni cilj istraživanja u okviru ove disertacije bio je da se ispita mogućnost i uslovi formiranja nanočestica zeina i kompozitnih nanočestica zein/prirodna smola, kao i mogućnost primene takvih nanočestica za inkapsulaciju lipofilne aktivne supstance, odnosno za formiranje filmova zeina. Nanočestice (NČ) zeina (Z) i kompozitne nanočestice zein/šelak (Z/Š) i zein/kalafonijuim (Z/K) pripremane su metodom (ko)precipitacije usled smanjenja rastvorljivosti, gde su se etanolni rastvori makromolekula mešali sa vodom kao nerastvaračem. Ispitani su uslovi pripreme nanočestica zeina na njihove koloidne osobine. Pripremljene nanočestice karakterisane su primenom različitih metoda (dinamičko rasipanje svetla, elektroforetska pokretljivost, SEM). Takođe, ispitane su interakcije gume arabike (GA) sa Z i Z/K nanočesticama, upotrebom različitih tehnika, kao što su dinamičko rasipanje svetla i izotermalna mikrokalorimetrija. Ispitana je mogućnost inkapsulacije lipofilne aktivne supstance u Z i Z/K nanočesticama, primenom metode koprecipitacije usled smanjenja rastvorljivosti, na isti način kao što su pripremane same nanočestice. Filmovi zeina pripremani su izlivanjem disperzija nanočestica zeina i sušenjem na povišenoj tempreturi. Pripremljeni su filmovi iz disperzija sa nanočesticama različitog prosečnog prečnika ZDNČ90 i ZDNČ180 bez plastifikatora, kao i uz dodatak plastifikatora ZDPGNČ90 i ZDPGNČ180. U svrhu poređenja osobina filmova, pripremani su i filmovi iz rastvora zeina bez plastifikatora, ZR, i sa dodatkom plastifikatora, ZRPG filmovi. Ispitan je uticaj veličine čestica zeina kao i uticaj plastifikatora na morfologiju, barijerna i optička svojstva filmova (rastvorljivost, propustljivost vodene pare, boju, itd.). Utvrđeno je da se metodom precipitacije u nerastvaraču mogu dobiti zeinske i kompozitne nanočestice zein/smola. Na veličinu nanočestica može se uticati koncentracijom etanolnog rastvora biopolimera. Takođe, pH vrednost disperzije nanočestica utiče na njihov zeta potencijal, površinsku gustinu naelektrisanja, veličinu i stabilnost. Dodatak GA, u disperziju NČ utiče na zeta potencijal i veličinu nanočestica, odnosno nastalih aglomerata nanočestica. Mikrokalorimetrijskim merenjima utvrđeno je da su interakcije Z i Z/K nanočestica sa GA egzotermne, te da su najinteinzivnije na pH = 4. Merenja su pokazala da do zasićenja površine kod Z/K NČ dolazi pri masenom odnosu GA/NČ od 0,7. Kod Z NČ promena entalpije postaje konstantna nakon masenog odnosa GA/NČ 1. Takođe, utvrđeno je da se moguće pripremiti kontinualne zeinske filmove iz disperzija nanočestica, sušenjem na vazduhu. U poređenju sa filmovima dobijenim iz rastvora zeina, filmovi dobijeni iz disperzija su bili hrapaviji i krtiji. Takođe, hrapavost površine ZDNČ180 filmova bila je veća nego kod ZDNČ90 filmova. Dodatkom propilen–glikola kao plastifikatora smanjuje se krtost i hrapavost ZDNČ filmova. Svi ispitivani filmovi su, u ispitivanim uslovima, apsorbovali manje od 8% vlage, dok je rastvorljivost i Zr i Zd filmova u vodi bila je ispod 5%. Propustljivost vodene pare se nije značajno razlikovala (p < 0,05) za ZR, ZD i ZDPG filmove, nezavisno od veličine čestica. Optička zamućenost ZD filmova bila je veća nego kod ZR filmova, dok je najmanja bila kod ZDPG filmova, s obzirom da je dodatak plastifikatora uticao na smanjenje optičke zamućenosti. Pokazano je da udeo bele u boji filmova raste po redosledu ZR < ZRPG < ZDPG < ZD. Takođe je utvrđeno da žuta boja preovlađuje kod svih ispitivanih filmova. Statistička analiza pokazala je da ne postoji značajna razlika u mehaničkim svojstvima između filmova izlivenih iz disperzija nanočestica i filmova izlivenih iz etanolnog rastvora zeina.Plant proteins, i.e. proteins isolated from various plant materials, have excellent potential for use in food production, but also in food packaging. They possess various functional properties, such as emulsifying, foam stabilizing, gelling, coating, and film-forming properties, which allow them to be used in the food and pharmaceutical industries. Zein is a plant protein, which can be found in cereals, and the main source of this protein is corn (Zea mays). Zein is not soluble in water, and dilute ethanol is most often used to dissolve it. By reducing the concentration of ethanol in the zein solution, whether this is achieved by adding non–solvents or removing ethanol (evaporation), zein can form various colloidal structures, which show great potential for use in the food industry. The properties of these structures depend on the method of preparation, but they can also be influenced by the addition of other substances during the preparation process. Therefore, the main goal of the research within this dissertation was to examine the possibility and conditions of formation of zein nanoparticles and zein/natural resin composite nanoparticles, as well as the possibility of using such nanoparticles for encapsulation of lipophilic active substance, as well as for zein film formation. Zein (Z) nanoparticles and zein/shellac (Z/Š) and zein/rosin (Z/K) composite nanoparticles were prepared by the antisolvent (co)precipitation method, where ethanolic solutions of macromolecules were mixed with water as a non-solvent. The influence of the zein nanoparticles' preparation parameters on their colloidal properties was examined. Prepared nanoparticles were characterized by the application of different methods (dynamic light scattering, electrophoretic mobility, scanning electron microscopy). Also, interactions of gum arabic (GA) with Z and Z/K nanoparticles were investigated, using various techniques, such as dynamic light scattering and isothermal microcalorimetry. The possibility of encapsulation of lipophilic active substance in Z and Z/K nanoparticles was investigated, using the method of antisolvent coprecipitation in the same way as the nanoparticles themselves were prepared. Zein films were prepared by casting dispersions of zein nanoparticles and air drying. Films were prepared from dispersions with nanoparticles of different average diameters ZDNČ90 and ZDNČ180 without plasticizers, as well as with the addition of plasticizers ZDPGNČ90 and ZDPGNČ180. In order to compare their properties, films were also prepared from a solution of zein without a plasticizer, ZR, and with the addition of a plasticizer, ZRPG films. The influence of zein particle size, as well as the influence of plasticizers on morphology, barrier properties, and optical properties of films (solubility, water vapor permeability, color, etc.), were examined. It has been found that zein and composite zein/resin nanoparticles can be obtained by the antisolvent precipitation method. Nanoparticle size can be affected by the concentration of ethanol biopolymer solution. Also, the pH value of the dispersion of nanoparticles affects their zeta potential, surface charge density, size, and stability. The addition of GA to the dispersion of NP affects the zeta potential and size of nanoparticles, i.e. the formed agglomerates of nanoparticles. Microcalorimetric measurements showed that the interactions of Z and Z/K nanoparticles with GA are exothermic and that they are most intense at pH = 4. Measurements showed that surface saturation of Z/K NP occurs at a GA/NP mass ratio of 0.7. For Z NP, the change in enthalpy becomes constant after the mass ratio GA / NC 1. Also, it was found that it is possible to prepare continuous zein films from nanoparticle dispersions, by air drying. Compared with films obtained from zein solution, films obtained from dispersions were rougher and more brittle. Also, the surface roughness of ZDNČ180 films was higher than that of ZDNČ90 films. The addition of propylene–glycol as a plasticizer reduces the brittleness and roughness of ZDNČ films. All tested films, absorbed less than 8% of moisture, under the tested conditions, while the solubility of both Zr and Zd films in water was below 5%. Water vapor permeability did not differ significantly (p <0.05) for ZR, ZD, and ZDPG films, regardless of the particle size. The opacity of ZD films was higher than that of ZR films, while the lowest was in ZDPG films, since the addition of plasticizers had the effect of reducing opacity. It was shown that the share of white in the color of films increases in the order ZR < ZRPG < ZDPG < ZD. It was also determined that yellow color predominates in all examined films. Statistical analysis showed that there was no significant difference in mechanical properties between films cast from nanoparticle dispersions and films cast from ethanol zein solution