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Keywords: Beam deflection spectroscopy, diffusive gradients in thin-films, iron species, photothermal techniques, natural waters.
Published in RUNG: 09.11.2018; Views: 3981; Downloads: 0
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Abstract: V strokovni literaturi zasledimo encim acetilholinesterazo (AChE) v različnih raziskavah s področja okoljevarstva, medicine in veterine. Zmanjšana encimska aktivnost je lahko indikator onesnaženosti okolja z organofosfatnimi in karbamatnimi pesticidi, policikličnimi aromatskimi ogljikovodiki ter težkimi kovinami. Spremembe v encimski aktivnosti AChE pa so tudi biološki označevalec nekaterih nevrodegenerativnih bolezni pri človeku. V človeškem telesu se encim AChE primarno nahaja v živčnem sistemu, najdemo pa ga tudi v krvi, zlasti v membranah eritrocitov. V krvi je v večji meri prisoten tudi encim butirilholinesteraza (BChE), ki ravno tako spada v družino holinesteraz. Naš cilj je bil razviti metodo, ki bi omogočala specifično merjenje encimske aktivnosti AChE v okviru celotnih holinesteraz v krvnem serumu. Za merjenje encimske aktivnosti smo uporabili Ellmanovo metodo. Količino obarvanih produktov reakcije smo določali s spektroskopijo s toplotnimi lečami TLS in z UV-VIS spektrofotometrijo. Z različnimi koncentracijami čistega encima AChE smo najprej določili mejo zaznavnosti obeh metod (LOD). Detekcija s TLS je bila bolj občutljiva, saj je bil LOD 3,13-krat manjši kot pri detekciji z UV-VIS. Potem smo izmerili encimsko aktivnost celotnih holinesteraz AChE in BChE v serumskih vzorcih zdravih ljudi. Izračunana encimska aktivnost holinesteraz se je v analiziranih vzorcih gibala med 3004 mU mL-1 in 5698 mU mL-1. Po dodatku specifičnega inhibitorja BChE se je celotna encimska aktivnost znižala za najmanj 60 %, kar kaže, da večina holinesteraz v krvnem serumu pripada BChE. Preizkusili smo tudi metodo enojne imunske precipitacije, vendar se je le-ta izkazala za neuspešno pri ločevanju posameznih holinesteraz iz serumskih vzorcev. Na podlagi naših rezultatov smo prišli do zaključka, da je za merjenje encimske aktivnosti posameznih holinesteraz AChE in BChE v krvnem serumu najprimernejša uporaba specifičnih inhibitorjev, vendar bi bilo to metodo potrebno še dodatno optimizirati.
Keywords: acetilholinesteraza, krvni serum, nevrodegenerativne bolezni, spektroskopija s toplotnimi lečami, UV-VIS spektrofotometrija, imunska precipitacija.
Published in RUNG: 07.11.2018; Views: 6365; Downloads: 177
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Keywords: HPLC-DAD and UV-VIS spectrometry, vinylphenolic pyranoanthocyanins formation in synthetic Pinot Noir grape, sequential fermentations with non-Saccharomyces yeast
Published in RUNG: 16.07.2018; Views: 4135; Downloads: 0
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Keywords: flow injection analysis, iron concentration, ocean water, thermal lens spectroscopy
Published in RUNG: 16.07.2018; Views: 4376; Downloads: 0
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Abstract: Conventionally, alcoholic fermentation in the production of wine is performed by yeast species Saccharomyces cerevisiae. There are numerous starters available, however due to the growing demand for wines with specific characteristics, other Saccharomyces and non-Saccharomyces species are being investigated for potential use as starters. [1]. Yeast selection has involved the development of techniques for detecting strains that might improve wines in terms of aroma, structure, colour and other technological properties [2]. Colour of the wine can be affected as some metabolites produced by yeast during fermentation may react with grape anthocyanins to produce highly stable pyranoanthocyanins. For the facilitation of formation of vinylphenolic pyranoanthocyanins, yeast strains with high hydroxycinnamate decarboxylase activity are used (HCDC). The mechanism of reaction is decarboxylation of hydroxycinnamic acids and formation of vinylphenols that condense with grape anthocyanins and form stabile vinyphenolic pyranoanthocyanin adducts [3]. It has been demonstrated that some non-Saccharomyces strains (Pichia guillermondii, Schizosaccharomyces pombe) have positive HCDC activity and they can produce vinylphenolic pyranoanthocyanins in higher concentrations than S. cerevisiae. A simple way of determining whether the yeast strain has HCDC activity or not, is the use of fermentation media with the addition of hydroxycinnamic acids, such as p-coumaric acid. The degradation of p-coumaric acid and transformation into 4-vinylphenol (and possibly in 4-ethylphenol) can be checked by LC-DAD. Most of the published data has been done on smaller number of strains. The goal of our work was to develop simple method for the screening of Slovenian in-house yeast collection, comprising of native isolates that mostly originated from Vipava valley and Karst region, and therefore try to determine strains with high HCDC activity. These strains can be used for wine fermentations in order to produce more stable pyranoanthocyanins; which is especially important in wines that has less anthocyanin concentration already from the grape, such as Pinot Noir. 103 different yeast strains belonging to 28 species were selected for the assessment of HCDC activity. In some cases the difference in p-coumaric acid metabolism rate between two strains exceeded 90%. All tested S. paradoxus strains showed higher than 40% degradation rate of p-coumaric acid. HCDC activity of S. cerevisiae strains which is the species most commonly used in fermentation, varied between 5.1 and 66.1%. The commercial strains tested, FPC and EC118 showed 43.9 and 21.5% conversion rate, respectively. It was observed that some native strains had higher HCDC activity than commercial tested ones. Three strains produced vinylphenol in concentration higher than 50 ppm, two of them being P. guillermondii and another strain being S. paradoxus (Sut85). In general strain with high HCDC activity also produced high concentration of 4-vinylphenol. The results showed that HCDC activity is highly strain dependent, which correlates with the literature data available. The proposed method is very simple and does not require special sample preparation prior to HPLC analysis. Furthermore, the proposed fermentations in deep-well microtiter plates allow the screening of high number of strains. The method could be used for routine screening, to determine which strain has high HCDC activity and produces high concentration of vinylphenols and can therefore be used in future for determination of strains ability to synthesize vinylphenolic pyranoanthocyanins.
Keywords: yeast, hydroxycinnamate decarboxylase, 4-vinylphenol
Published in RUNG: 18.06.2018; Views: 4232; Downloads: 0
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