Enzimska modifikacija pšeničnog glutena u cilju smanjenja njegove alergenosti i poboljšanja funkcionalnih svojstava

Abstract

Proteini 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 lowest degree of hydrolysis exhibited the highest foaming capacity and stability, due to the presence of polypeptide chains with smaller molecular masses. SDS-PAGE electrophoresis confirmed the absence of gluten fractions with large molecular masses (> 50 kDa). Additionally, differences in the antioxidant properties of the produced hydrolysates were also evident. Namely, all the samples showed a certain level of antioxidant activity, where in relation to the hydrolysates obtained by Alcalase, the reduced activities of the hydrolysates towards Fe2+ ions stand out. The obtained hydrolysate of white wheat flour with the lowest relative gluten content was used to examine its influence on the rheological properties of gluten-free flour, whole buckwheat flour, on Mixolab. Namely, with the addition of hydrolysate, a change in rheological properties was achieved in the form of a reduction in the weakening of the gluten network, more precisely, the deterioration of the protein structure during heating. The addition of the hydrolysate also affected the change 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 of this dissertation, it was established that the use of Alcalase and Pronase can effectively, to a certain extent, reduce the presence of allergenic epitopes in raw gluten, which trigger an immune response in sensitive population groups. The application of microwave pretreatment leads to structural changes that are recorded in the secondary structure of the gluten protein, and additionally positively promotes the further course of enzymatic hydrolysis. During the hydrolysis of wheat flour, it was established that the presence of starch granules facilitates the enzymatic hydrolysis process because the starch granules directly prevent the formation of protein aggregates. The resulting wheat flour hydrolysates can be considered as hydrolysates rich in antioxidant peptides, with improved specific functional properties and reduced allergenicity. Therefore, based on everything presented, it is possible to take modified gluten proteins into consideration when creating new products with reduced allergenic properties. Finally, as a contribution to the research within this dissertation, the economy of microwave pretreatment as well as the hydrolysis procedure in the presence of starch substances can be attributed, because by applying such procedures it is possible to make a certain level of savings during the production process of modified gluten products with a reduced content of allergenic epitopes (~ 20 ppm).