Naslov (srp)

Enzimska modifikacija pšeničnog glutena u cilju smanjenja njegove alergenosti I poboljšanja funkcionalnih svojstava : doktorska disertacija

Autor

Gazikalović, Ivana, 1992-

Doprinosi

Vukašinović-Sekulić, Maja, 1969-
Knežević-Jugović, Zorica, 1969-
Jovanović, Jelena R., 1987-
Žilić, Slađana, 1969-
Mijin, Dušan, 1962-

Opis (eng)

Gluten proteins belong to highly nutritionally valuable proteins that make up the basicchemical structure of cereal grains, such as wheat, rye and barley. However, these proteins arecharacterized 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, byconsuming products rich in gluten proteins, large peptide fractions are formed that initiate animmunogenic reaction, thus making gluten proteins one of the most important food allergens. Theproblem 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 andunconventional thermal treatments, such as microwave radiation, for the production and modificationof non-allergenic gluten proteins. In general, within the framework of this dissertation, the parametersof non-thermal procedures for the modification of wheat gluten protein, such as microwave radiationunder controlled conditions in a suitable reactor, were defined, all with the aim of reducingallergenicity, i.e. the content of allergenic gluten epitopes. In addition, in order to reduce theallergenicity of wheat gluten, the development of a biotechnological procedure based on theapplication of highly specific proteolytic enzymes was also undertaken. This procedure wasoptimized with careful definition of all important process parameters, but it was also completed and/orsupplemented by combining it with microwave radiation. As a source of wheat gluten, two substrateswere used in the experiments:• the first, a model of gluten protein originating from wheat (rich in glutenin and gliadinfractions), and• the second, a commercially available raw material used in the daily diet of people, wheatwhite flour.In each phase of the experimental work, a detailed characterization of the modified wheatgluten, both the protein model and wheat flour, was carried out. The characterization involved testingthe allergenic properties of modified gluten proteins using a competitive ELISA test that contains R5monoclonal antibodies. Then, modified gluten was characterized from the spectrum of technologicalfunctionalproperties (foaming, emulsification, surface charge), structural changes (FTIR analysis,RP-HPLC analysis, SDS-PAGE analysis) and bioactive properties (antioxidant and antimicrobialactivity).In detail, in the first part of the research, the influence of microwave radiation parameters onthe structure of model gluten proteins originating from wheat, their allergenic properties, wasexamined. By the action of microwave radiation with a power of 200 to 800 W, changes in thedetection 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 severaldifferent temperatures was examined in order to more closely determine the changes caused by thesetreatments. Through the synergistic action of enzymes as biocatalysts, a controlled hydrolysis of thegluten protein model was performed after microwave irradiation. Commercial endopeptidaseAlcalase was used as a biocatalyst for the hydrolysis reaction of microwave-treated gluten. Theapplication of microwave pretreatment with a power of 200 W at a controlled temperature lastingonly 1 min, and in combination with enzymatic hydrolysis, proved to be very effective, because theproduced gluten protein hydrolysates showed an exceptional improvement in technologicalfunctionaland antioxidant activities compared to untreated gluten.The specific non-thermal effect of microwaves had an impact on the structure andallergenicity of gluten, which resulted in the improvement of enzymatic hydrolysis. This has beenconfirmed because the thermal treatment of gluten under completely identical conditions has showna different impact on the molecular structure, from which it can be concluded that microwavetreatment results in unique protein modifications. It is generally known that due to the reduction inthe length of peptide chains during hydrolysis, the technological and functional properties of proteinsalso change. The emulsifying stability of gluten hydrolysate with and without pretreatment wassignificantly improved compared to raw gluten, however, no significant improvement was notedamong the samples when testing the stability of the formed foam. The antioxidant properties of theproduced gluten hydrolysates, above all the ability to neutralize the radical cation ABTS˙+ and theDPPH˙ radical, were significantly improved in comparison to the original sample, while the abilityto chelate Fe2+ ions is particularly noteworthy. In this regard, the final hydrolysates with and withoutpretreatment show a significant difference in IC50 values (mg/ml) when examining the possibility ofmetal 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 definedrange of molecular masses, with the aim of finding the fraction with the most pronounced antioxidantactivity. When testing the ability to inhibit the ABTS radical cation, no significant differences werenoted among the peptide fractions. The contribution of the peptide fraction F3 (3–10 kDa) wasrecorded when testing the inhibitory activity against DPPH radicals. The measured values of thedegree 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 ofpeptide fractions obtained from microwave pretreated gluten proteins. Fractions that stood out inparticular were fraction 2 with peptides of molecular weight 10–30 kDa, fraction 3 (3–10 kDa) andfraction 4 (1–3 kDa).In the second part of the research within this dissertation, white wheat flour was used as a rawmaterial for gluten modification. Therefore, in order to modify the gluten from wheat flour, as wellas the flour itself, research was carried out in two parts. The first part of the research was based onthe application of previously adopted parameters of enzymatic hydrolysis with protease Alcalase ofmicrowave-pretreated gluten, while a subsequent study was carried out on the optimization of thewheat flour hydrolysis procedure in order to create a valid model that could be applied in the processof reducing the allergenic properties of gluten protein from flour. In this regard, during the hydrolysisof flour, different concentrations of the substrate were varied in order to examine the influence of thepresent starchy substances on the hydrolysis process itself, and then on the techno-functionalproperties of the obtained gluten hydrolysates. The greatest reduction in the content of allergenicepitopes of gluten was recorded in the sample prepared as a 15% (w/w) flour suspension, where thedifference in allergenicity between the samples was confirmed by the difference and absence ofprotein bands using SDS-PAGE electrophoresis. The emulsifying properties were significantlyimproved by the enzymatic hydrolysis procedure, while the stability of the formed emulsions was notsignificantly improved. Upon examining the foaming properties, significant differences were notedbetween the hydrolysates, revealing that careful control of enzymatic hydrolysis can yieldhydrolysates containing sufficiently long polypeptide chains that form good and stable foams can beproduced. The high values of antioxidant activity of the obtained wheat flour hydrolysate wereconfirmed with the ABTS and chelation test, thus making the preparation of the hydrolysate suitablefor further tests that can be carried out for the purposes of creating preparations with improvedantioxidant properties. In summary, in this part of the research, it was evident that the natural presenceof 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 thehydration of the components of gliadin and glutenin, the presence of starchy substances hinders theformation of the gluten network, which facilitates the access of enzymes to peptide bonds and theinterior of the substrate. Subsequent optimization of the wheat flour hydrolysis procedure analyzedthe influence of process parameters: pH, temperature, enzyme/substrate (E/S) ratio and amount ofsubstrate on the degree of hydrolysis, allergenic properties and antioxidant activity. By analyzing theobtained model based on 29 experimental points within the response surface method, it was concludedthat pH, E/S ratio and the amount of substrate (S) have the greatest influence on the hydrolysisprocess, while the allergenic properties were directly conditioned by the pH and temperature of theprocess. The antioxidant activity depends mostly on the applied pH during hydrolysis and theenzyme/substrate ratio, while chelation is additionally influenced by the amount of introducedsubstrate.In order to examine the possibility of even more intensive modification of wheat gluten, butalso of the application of the Pronase enzyme preparation (endo- and exopeptidase activity), primarilyfor 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, thisexperimental setup was carried out in order to theoretically test the possibility of applying Pronase asanother preparation that would reduce the allergenic properties of gluten. Namely, with an increasein the achieved degree of hydrolysis, a decrease in allergenic properties was noted, emulsifyingactivities improved, while the capacity and stability of foaming are inversely dependent on theachieved degree of hydrolysis. On the contrary, the hydrolysate sample with the lowest degree ofhydrolysis exhibited the highest foaming capacity and stability, due to the presence of polypeptidechains with smaller molecular masses. SDS-PAGE electrophoresis confirmed the absence of glutenfractions with large molecular masses (> 50 kDa). Additionally, differences in the antioxidantproperties of the produced hydrolysates were also evident. Namely, all the samples showed a certainlevel of antioxidant activity, where in relation to the hydrolysates obtained by Alcalase, the reducedactivities of the hydrolysates towards Fe2+ ions stand out.The obtained hydrolysate of white wheat flour with the lowest relative gluten content wasused to examine its influence on the rheological properties of gluten-free flour, whole buckwheatflour, on Mixolab. Namely, with the addition of hydrolysate, a change in rheological properties wasachieved in the form of a reduction in the weakening of the gluten network, more precisely, thedeterioration of the protein structure during heating. The addition of the hydrolysate also affected thechange in amylase activity, as well as the retrogradation of starch in the last phase of the analysis.Overall, summarizing the results obtained during the research conducted for the purposes ofthis dissertation, it was established that the use of Alcalase and Pronase can effectively, to a certainextent, reduce the presence of allergenic epitopes in raw gluten, which trigger an immune responsein sensitive population groups. The application of microwave pretreatment leads to structural changesthat are recorded in the secondary structure of the gluten protein, and additionally positively promotesthe further course of enzymatic hydrolysis. During the hydrolysis of wheat flour, it was establishedthat the presence of starch granules facilitates the enzymatic hydrolysis process because the starchgranules directly prevent the formation of protein aggregates. The resulting wheat flour hydrolysatescan be considered as hydrolysates rich in antioxidant peptides, with improved specific functionalproperties and reduced allergenicity. Therefore, based on everything presented, it is possible to takemodified gluten proteins into consideration when creating new products with reduced allergenicproperties. Finally, as a contribution to the research within this dissertation, the economy ofmicrowave pretreatment as well as the hydrolysis procedure in the presence of starch substances canbe attributed, because by applying such procedures it is possible to make a certain level of savingsduring the production process of modified gluten products with a reduced content of allergenicepitopes (~ 20 ppm).

Opis (srp)

Proteini glutena spadaju u visokonutritivno vredne proteine koji čine osnovnu hemijsku građuzrna žitarica, kao što su pšenica, raž i ječam. Međutim, ovi proteini odlikuju se visokim sadržajemglutamina i prolamina što ih čini otporne na enzime (Pepsin, Tripsin i Himotripsin) prisutne udigestivnom traktu ljudi. Kao posledica toga, konzumiranjem proizvoda bogatih glutenom obrazujuse velike peptidne frakcije koje iniciraju imunogenu reakciju, čineći tako ovaj protein jednim odnajznačajnih prehrambenih alergena. Upravo naveden problem poslužio je kao polazna osnova zakreiranje cilja ove doktorske disertacije, u kojoj su istraživanja bazirana na primeni enzima kaobiokatalizatora prirodnog porekla i nekonvencionalnih termičkih tretmana, poput mikrotalasnogzračenja, zarad proizvodnje i modifikacije nealergenih proteina glutena. Generalno, u okviru ovedisertacije pristupilo se definisanju parametara netermičkih postupaka za modifikaciju proteinaglutena iz pšenice kakav je mikrotalasno zračenje pod kontrolisanim uslovima u odgovarajućemreaktoru, a sve u cilju smanjenja alergenosti, odnosno sadržaja alergenih epitopa glutena. Osim toga,u cilju smanjenja alergenosti pšeničnog glutena pristupilo se i razvoju biotehnološkog postupkazasnovanog na primeni proteolitičkih enzima velike specifičnosti. Ovaj postupak je optimizovan uzpažljivo definisanje svih važnih procesnih parametara, ali je i upotpunjen i/ili dopunjenkombinovanjem sa mikrotalanim zračenjem. Kao izvor pšeničnog glutena, u okviru eksperimenata,korišćena su dva supstrata:• prvi, model protein glutena poreklom iz pšenice (bogat frakcijama glutenina igliadina), i• drugi, komercijalno dostupna sirovina koja se koristi u svakodnevnoj ishrani ljudi,pšenično belo brašno.U svakoj fazi eksperimentalnog rada vršena je detaljna karakterizacija modifikovanogpšeničnog glutena, kako model proteina, tako i pšeničnog brašna. Karaktertizacija je podrazumevalaispitivanje alergenih svojstava modifikovanih proteina glutena primenom kompetitivnog ELISA testakoji u sebi sadrži R5 monoklonska antitela. Potom, modifikovan gluten je okarakterisan sa aspektatehnološko-funkcionalnih svojstava (penjenje, emulgovanje, površnko naelektrisanje), strukturnihpromena (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 nastrukturu model proteina glutena poreklom iz pšenice, i njihova alergena svojstva. Delovanjemmikrotalasnog zračenja snage 200 do 800 W, utvrđene su promene u detekciji relativnog sadržajaalergenog epitopa glutena pomoću odabranog antitela. Poređenjem odabranog mikrotalasnogtretmana sa konvencionalnim zagrevanjem, ispitan je uticaj i više različitih temperatura kako bi sebliže odredile promene nastale usled ovih tretmana. Sinergističkim delovanjem enzima kaobiokatalizatora, izvršena je kontrolisana hidroliza model proteina glutena nakon mikrotalasnogzračenja. Korišćena je komercijalna endopeptidaza Alkalaza kao biokatalizator za reakciju hidrolizemikrotalasno tretiranog glutena. Primena mikrotalasnog pretretmana snage 200 W pri kontrolisanojtemperaturi u trajanju od svega 1 min, a u kombinaciji sa enzimskom hidrolizom se pokazala kaoveoma efikasna, jer su proizvedeni hidrolizati proteina glutena ispoljili izuzetno poboljšanjetehnološko-funkcionalnih svojstava i antioksidativnih aktivnosti u poređenju sa netretiranimglutenom.Specifični netermalni efekat mikrotalasa je imao uticaj na strukturu i alergenost glutena, usledčijeg dejstva je došlo do unapređenja enzimske hidrolize. Ovo je potvrđeno jer termički tretmanglutena pri potpuno istim uslovima je pokazao drugačiji uticaj na strukturu molekula, na osnovu čegase može zaključiti da mikrotalasni tretman rezultuje u jedinstvenim modifikacijama proteina. Opšteje poznato da se usled smanjenja dužine peptidnih lanaca tokom hidrolize menjaju i tehnološkofunkcionalnasvojstva proteina. Emulgujuća stabilnost hidrolizata glutena sa i bez pretretmanaznačajno je unapređena u odnosu na sirov gluten, međutim do značajnijeg unapređenja međuuzorcima nije došlo prilikom ispitivanja stabilnosti formirane pene. Antioksidativna svojstvaproizvedenih hidrolizata glutena, pre svega sposobnost neutralizacije radikalskog katjona ABTS˙+ iDPPH˙ radikala značajno su unapređene u odnosu na polazni uzorak, dok se posebno ističesposobnost heliranja jona Fe2+. S tim u vezi, krajnji hidrolizati sa i bez pretretmana pokazuju značajnurazliku u IC50 vrednostima (mg/ml) prilikom ispitivanja mogućnosti heliranja jona metala. Metodomdead-end ultrafiltracije, izvršeno je frakcionisanje krajnjih hidrolizata, mikrotalasno pretretiranog ikontrolnog, na frakcije peptida tačno definsanog opsega molekulskih masa, a u cilju pronalaženjafrakcije sa najizraženijom antioksidativnom aktivnošću. Prilikom ispitivanja sposobnosti inhibicijeABTS radikalskog katjona, nisu zabeležene značajne razlike među peptidnim frakcijama. Doprinospeptidne frakcije F3 (3–10 kDa) zabeležen je prilikom testiranja inhibitorne aktivnosti prema DPPHradikalima. Izmerene vrednosti stepena heliranja jona metala značajno se razlikuju među peptidnimfrakcijama istog uzorka, ali i među uzorcima. Naime, čak 10–25% veće aktivnosti su zabeležene uslučaju peptidnih frakcija dobijenih od mikrotalasno pretretiranih proteina glutena. Frakcije koje suse posebno istakle su frakcija 2 sa peptidima molekulske mase 10–30 kDa, frakcija 3 (3–10 kDa) ifrakcija 4 (1–3 kDa).U drugom delu istraživanja u okviru ove disertacije, kao sirovina za modifikaciju glutenakorišćeno 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 naprimeni prethodno usvojenih parametara enzimske hidrolize proteazom Alkalazom mikrotalasnopretretiranog glutena, dok je naknadno izvršena studija optimizacije postupka hidrolize pšeničnogbrašna u cilju kreiranja valjanog modela koji bi se mogao primeniti u postupku smanjenja alergenihsvojstava glutena iz brašna. S tim u vezi, prilikom hidrolize proteina brašna varirane su različitekoncentracije supstrata u cilju ispitivanja uticaja prisutnih skrobnih materija na sam postupakhidrolize, a potom i na tehno-funkcionalna svojstva dobijenih hidrolizata glutena. Najveće smanjenjesadržaja alergenih epitopa glutena zabeleženo je kod uzorka pripremljenog kao 15% (w/w) suspenzijabrašna, pri čemu je razlika u alergenosti među uzorcima potvrđena razlikom i odsustvom proteinskihtraka primenom SDS-PAGE elektroforeze. Emulgujuća svojstva su značajno unapređena postupkomenzimske hidrolize, dok stabilnost formiranih emulzija nije značajno unapređena. Ispitivanjemsvojstva penjenja zabeležene su značajne razlike među hidrolizatima, pri čemu je interesantno istaćida se pažljivom kontrolom enzimske hidrolize mogu proizvesti hidrolizati sa dovoljno dugačkimpolipeptidnim lancima koji formiraju dobre i stabilne pene. Visoke vrednosti antioksidativnihaktivnosti dobijenih hidrolizata proteina pšeničnog brašna potvrđene su u okviru ABTS i helatnogtesta, čineći tako pripremljenje hidrolizate podobne za dalja ispitivanja koja se mogu sprovoditi zapotrebe kreiranja preparata sa unapređenim antioksidativnim svojstvima. Sumarno, ovim delomistraživanja evidentno je bilo da prirodno prisustvo skrobnih materija u formiranim suspenzijama neometa sam postupak hidrolize, već ga „olakšava“ jer dovodi do sprečavanja formiranja agregata, tj.usled hidratacije sastavnih delova gliadina i glutetnina prisustvo skrobnih materija ometa formiranjeglutenske mreže čime se olakšava pristup enzima peptidnim vezama i unutrašnjosti supstrata.Naknadnom optimizacijom postupka hidrolize pšeničnog brašna analiziran je uticaj procesnihparametara: pH, temperature, enzim/supstrat (E/S) odnosa i količine supstrata na stepen hidrolize,alergena svojstva i antioksidativne aktivnosti. Analizom dobijenog modela baziranog na 29eksperimentalnih tačaka u okviru metode odzivnih površina, zaključeno je da najveći uticaj na tokhidrolize imaju pH, E/S odnos i količina supstrata (S), dok su alergena svojstva direktno bilauslovljena sa pH i temperaturom procesa. Antioksidativna aktivnost zavisi najviše od primenjenogpH tokom hidrolize i enzim/supstrat odnosa, dok na heliranje dodatno utiče i količina unetogsupstrata.Kako bi se ispitala mogućnost još intenzivnije modifikacije pšeničnog glutena, ali i primeneenzimskog preparata Pronaze (endo- i egopeptidazna aktvnost), prevashodno u svrhu smanjenjaalergenih svojstava, istraživanja su nastavljena u tom smeru. S obzirom na slabu dostupnostliteraturnih podataka u vezi delovanja ovog enzima na gluten, ova eksperimentalna postavka jeizvedena u cilju teorijskog ispitivanja mogućnosti primene Pronaze kao još jednog preparata kojimbi se smanjila alergena svojstva glutena. Naime, sa povećanjem postignutog stepena hidrolizezabeleženo je smanjenje alergenih svojstava, unapređene su emulgujuće aktivnosti, dok je kapaciteti stabilnost penjenja obrnuto zavistan od postignutog stepena hidrolize. Suprotno tome, najvećikapacitet i stabilnost penjenja pokazao je uzorak hidrolizata sa najmanjim stepenom hidrolize, usledprisustva polipeptidnih lanaca srednjh molekulskih masa. SDS-PAGE elektroforezom potvrđeno jeodsustvo frakcija glutena velikih molekulskih masa (> 50 kDa). Dodatno, razlike i u antioksidativnimsvojstvima proizvedenih hidrolizata bile su očigledne. Naime, svi uzorci su pokazali određeni nivoantioksidativne aktivnosti, pri čemu se u odnosu na hidrolizate dobijene Alkalazom, ističe smanjenaaktivnost hidrolizata prema jonima Fe2+.Dobijeni hidrolizat pšeničnog brašna sa najnižim relativnim sadržajem glutena iskorišćen jekako bi se ispitao njegov uticaj na reološke osobine bezglutenskog brašna, heljdinog integralnogbrašna, na Miksolabu. Naime dodatkom hidrolizata postignuta je promena reoloških osobina u vidusmanjenja slabljenja glutenske mreže tačnije propadanja proteinske strukture tokom zagrevanja.Dodatak hidrolizata uticao je i na promenu u aktivnosti amilaza, kao i na retrogradaciju skroba uposlednjoj fazi analize.Sveobuhvatno, sumiranjem rezultata dobijenih tokom istraživanja sprovedenih za potrebeizrade ove disertacije utvrđeno je da se primenom Alkalaze i Pronaze može efikasno i do određenemere smanjiti prisustvo alergenih epitopa u sirovom glutenu koji izazivaju imuni odogovor uosetljivim grupama populacije. Primena mikrotalasnog pretretmana dovodi do strukturnih promenakoje su zabeležene u sekundarnoj strukturi proteina glutena, a dodatno pozitivno pospešuju dalji tokenzimske hidrolize. Tokom hidrolize proteina pšeničnog brašna, ustanovljeno je da prisustvoskrobnih granula olakšava postupak enzimske hidrolize jer skrobne granule direktno sprečavajuformiranje proteinskih agregata. Dobijeni hidrolizati proteina pšeničnog brašna se mogu smatratihidrolizatima bogatim antioksidativnim peptidima, sa unapređenim određenim funkcionalnimsvojstvima i smanjenom alergenošću. Stoga, na osnovu svega izloženog moguće je modifikovaneproteine glutena uzeti u razmatranje prilikom kreiranja novih proizvoda sa smanjenim alergenimsvojstvima. Finalno, kao doprinos istraživanjima u okviru ove disertacije može se pripisati iekonomičnost mikrotalasnog pretretmana kao i postupka hidrolize u prisustvu skrobnih materija, jerprimenom ovakvih postupaka moguće je izvršiti određeni nivo uštede tokom postupka proizvodnjemodifikovanih proizvoda glutena sa smanjenim sadržajem alergenih epitopa (~ 20 ppm).

Opis (srp)

Tehnološko inženjerstvo - Biohemijsko inženjerstvo i biotehnologija / Technological engineering - Biochemical engineering and biotechnology Datum odbrane: 04.07.2024.

Jezik

srpski

Datum

2024

Licenca

Ovo delo je licencirano pod uslovima licence
Creative Commons CC BY-NC-ND 3.0 AT - Creative Commons Autorstvo - Nekomercijalno - Bez prerada 3.0 Austria License.

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Predmet

OSNO - Opšta sistematizacija naučnih oblasti, Prehrambena industrija. Industrijska mikrobiologija

wheat gluten, wheat flour, microwave radiation, enzymatic hydrolysis, allergenicity, functional properties, antioxidant properties

OSNO - Opšta sistematizacija naučnih oblasti, Prehrambena industrija. Industrijska mikrobiologija

pšenični gluten, pšenično brašno, mikrotalasno zračenje, enzimska hidroliza, alergenost, funkcionalna svojstva, antioksidativna svojstva