Izolovanje funkcionalne ukupne RNK iz lišća i polena lipe (Tilia cordata)

The conditions required for the isolation of high quality total RNA from European linden (Tilia cordata) leaves and pollen were determined. Pure total RNA was isolated from linden leaves utilizing a Qiagen plant mini kit, while the total RNA isolated from linden pollen using this method was degraded. Successful isolation of total RNA from both linden pollen and leaves, however, was achieved following TRIzol (TM) preparation of the total RNA. The total RNA isolated using TRIzol (TM) was contaminated with genomic DNA but treatment with the enzyme DNase, in solution or on-column, efficiently removed the genomic DNA. Furthermore, the conditions for the elimination of genomic DNA contamination on-column and isolation of pure total RNA from leaves were optimized. The isolated total RNA from both leaves and pollen was used successfully in first-and second-strand cDNA synthesis reactions and in a reverse transcription polymerase chain reaction (RT-PCR), demonstrating that the total RNA isolated using this method was functional. In conclusion, pure and functional total RNA from T. cordata leaves and pollen (27.8 +/- 7.9 mu g g(-1) leaves; 25.7 +/- 1.1 mu g g(-1) pollen) could be obtained and was suitable for application in further molecular biology studies.Uspostavljeni su uslovi za izolovanje ukupne RNK iz lišća i polena evropske lipe (Tilia cordata). Korišćenjem komercijalno dostupnog pribora za izolovanje RNK iz biljaka izolovana je čista ukupna RNK iz lišća lipe, dok je korišćenjem iste metode dobijena degradirana RNK iz polena lipe. Uspešno izolovanje RNK iz lišća i polena je dobijeno korišćenjem TRIzol reagensa. RNK izolovana ovim metodom je kontaminirana genomskom DNK, koja je uspešno eliminisana korišćenjem enzima DNaze. Dalje su optimizovani i uslovi uklanjanja genomske DNK pomoću DNaze. Izolovana ukupna RNK iz oba izvora je dalje uspešno iskorišćena za sintezu prvog i drugog lanca klonske DNK, kao i u reverzno-transkriptivnoj PCR reakciji, dokazujući time da je korišćenjem ovog metoda izolovana funkcionalna ukupna RNK. U zaključku, dobijena je čista i funkcionalna RNK iz lišća i polena T. cordata (27,8±7,9 μg g-1 lišća; 25,7±1,1 μg g-1 polena) koja se može koristiti u daljim molekularno-biološkim istraživanjima

Isolation of functional total RNA from Tilia cordata leaves and pollen

The conditions required for the isolation of high quality total RNA from European linden (Tilia cordata) leaves and pollen were determined. Pure total RNA was isolated from linden leaves utilizing a Qiagen plant mini kit, while\ud the total RNA isolated from linden pollen using this method was degraded. Successful isolation of total RNA from both linden pollen and leaves, however, was achieved following TRIzol™ preparation of the total RNA. The total RNA isolated using TRIzol™ was contaminated with genomic DNA but treatment with the enzyme DNase, in solution or on-column, efficiently removed the genomic DNA. Furthermore, the conditions for the elimination of genomic DNA contamination on-column and isolation of pure total RNA from leaves were optimized. The isolated total RNA from both leaves and pollen was used successfully in first- and second-strand cDNA synthesis reactions and in a reverse transcription polymerase chain reaction (RT-PCR), demonstrating that the total RNA isolated using this method was functional. In conclusion, pure and functional total RNA from T. cordata leaves and pollen (27.8±7.9 μg g-1 leaves; 25.7±1.1 μg g-1 pollen) could be obtained and was suitable for application in further molecular biology studies

Digestibilnost β-laktoglobulina nakon njegovog unakrsnog povezivanja dejstvom lakaze iz Trametes versicolor i polifenola iz jabuke

beta-Lactoglobulin (BLG) is an important nutrient of dairy products and an important allergen in cow's milk allergy. The aim of this study was to investigate the potential of laccase to cross-link BLG in the presence of an apple phenolic extract (APE) and to characterize the obtained products for their digestibility by pepsin and pancreatin. The composition of the apple phenolics used for cross-linking was determined by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The apple phenolic extract contained significant amounts of quercetin glycosides, catechins and chlorogenic acid. The laccase cross-linked BLG in the presence of apple phenolics. The polymerization rendered the protein insoluble in the reaction mixture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the cross-linking reaction mixture revealed a heterogeneous mixture of high molecular masses (cross-linked BLG), with a fraction of the BLG remaining monomeric. Enzymatic processing of BLG by laccase and apple polyphenols as mediators can decrease the biphasal pepsin pancreatin digestibility of the monomeric and cross-linked protein, thus decreasing its nutritional value. In addition, reduced BLG digestibility can decrease its allergenic potential. Apple polyphenols can find usage in the creation of new, more functional food products, designed to prevent obesity and hypersensitivity-related disorders.β-Laktoglobulin (BLG) je važan nutrijent mlečnih proizvoda i važan alergen kod alergija na kravlje mleko. Cilj ove studije je bilo ispitivanje potencijala lakaze da unakrsno poveže BLG u prisustvu fenolnog ekstrakta jabuke (APE), kao i karakterizacija dobijenih proizvoda sa aspekta njihove digestibilnosti pepsinom i pankreatinom. Kompozicija fenola jabuke korišćenih za unakrsno povezivanje određena je pomoću LC-ESI-MS. Fenolni ekstrakt jabuke sadrži znatne količine glikozida kvercetina, katehine i hlorogensku kiselinu. BLG je unakrsno povezan lakazom u prisustvu fenola jabuke, pri čemu je polimerizacija učinila BLG nerastvornim u reakcionoj smeši. SDS-PAGE analiza pokazala je da reakciona smeša sadrži heterogenu smešu velikih molekulskih masa (unakrsno povezan BLG), kao i deo zaostalog monomernog BLG. Enzimsko procesovanje BLG lakazom, u prisustvu polifenola jabuke kao medijatora, može smanjiti bifaznu pepsin-pankreatinsku digestibilnost kako monomernog, tako i unakrsno povezanog BLG, i na taj način smanjiti njegovu nutritivnu vrednost. Takođe, smanjena digestibilnost BLG može smanjiti njegov alergeni potencijal. Polifenoli jabuke mogu se koristiti za kreiranje novih, funkcionalnijih prehrambenih proizvoda, dizajniranih za prevenciju kako gojaznosti, tako i bolesti vezanih za preosetljivost

Isolation of functional total RNA from Tilia cordata leaves and pollen

Conditions for isolation of high quality total RNA from European linden (Tilia cordata) leaves and pollen were determined. Pure total RNA was isolated from linden leaves utilizing Qiagen plant mini kit while total RNA isolated from linden pollen using this method was degraded. Successful isolation of total RNA from both linden pollen and leaves, however, was achieved following TRIZOL™ preparation of total RNA. Total RNA isolated using TRIZOL™ was contaminated with genomic DNA but treatment with enzyme DNase, in solution or on-column, efficiently removed the genomic DNA. Furthermore, conditions for elimination of genomic DNA contamination on-column and isolation of pure total RNA from leaves were optimized. Isolated total RNA from both leaves and pollen was used successfully in firstand second-strand cDNA synthesis reactions, as well as, in RT-PCR, demonstrating that the total RNA isolated using this method is functional. In conclusion, pure and functional total RNA from Tilia cordata leaves and pollen (27.8 ± 7.9μg/g leaves; 25.7 ± 1.1μg/g pollen) can be obtained and applicable for further molecular biology studies

Digestibility and allergenicity of beta-lactoglobulin following laccase-mediated cross-linking in the presence of sour cherry phenolics

beta-Lactoglobulin (BLG) is an important nutrient of dairy products, but it represents a serious health risk in patients allergic to milk. Sour cherry (Prunus cerasus L) extract (SCE) is frequently added as a natural food colour in composite foods, such as fruit yogurt, ice creams, frappes and milkshakes. The aim of this study was to investigate the potential of laccase to cross-link BLG in the presence of an SCE and to characterise the obtained products for their bioactivity. Laccase cross-linked BLG in the presence of sour cherry phenolics. In a basophil-activation assay, the allergenicity of the cross-linked protein was shown to decrease in all nine cow's milk-allergic patients, while digestibility of the remaining monomeric BLG in simulated conditions of the gastrointestinal tract increased. Tryptic peptides became immediately available in BLG treated by laccase and laccase/SCE. The hydrolysates obtained by trypsin digestion of BLG/laccase/SCE showed an increase of 57% in radical-scavenging activity, compared to the control BLG. Enzymatic processing and usage of natural phenolic extracts as mediators of enzymatic reaction may improve BLG safety and the availability of peptides following digestion, while conserving its bioactivity

Green tea catechins of food, supplements facilitate pepsin digestion of major food allergens, but hampers their digestion if oxidized by phenol oxidase

The in vitro gastric digestion of several food allergens (beta-lactoglobulin (BLG), alpha-lactalbumin (LA) and peanut allergens (PE)) in the presence of a catechin-enriched polyphenol extract of green tea (GTC), oxidized polyphenols and phenol oxidase processed food allergens and GTC was investigated. Pepsin-resistant proteins, such as BLG, major peanut allergens, Ara h 1 and Ara h 2, degrade faster in the presence of catechin-enriched green tea polyphenols. Phenol oxidase polymerized GTC affected adversely protein digestion of BLG and LA, but not digestion of PE proteins. Protecting effect of polyphenols correlated well with the ability of proteins to form insoluble complexes with oxidized catechins. Cross-linking of proteins and polyphenols further extended the half-lives of BLG and LA in the in vitro digestion by pepsin. Catechin-enriched green tea polyphenols of food supplements facilitate pepsin digestion of major food allergens, but hamper their digestion if oxidized and polymerized by phenol oxidase. (c) 2012 Elsevier Ltd. All rights reserved