Potencijalna primjena hladne plazme za kontrolu kvasca Brettanomyces bruxellensis u vinu

The aim of this work was to investigate the effect of high voltage electrical discharge (HVED) plasma on inactivation of Brettanomyces bruxellensis yeast in red wine. Research was conducted in two phases. Firstly, the efficacy of two main plasma parameters was examined - frequency (60, 90 and 120 Hz) and treatment duration (3, 5, 10, 20 and 30 min). Second phase included monitoring long-term effect of HVED plasma using optimal frequency during selected treatment durations, where culturability of B. bruxellensis was determined after 30, 60 and 90 days of storage. Results show that frequency of 120 Hz caused slightly higher inactivation than the lower ones. Furthermore, longer treatment durations resulted in significantly higher inactivation, wherein the treatment of 30 min showed the best results. Finally, during storage period, the inactivation effect of HVED plasma was reversed and B. bruxellensis yeast recovered almost completely in treated wine.Cilj ovog istraživanja bio je utvrditi utjecaj visokonaponskog električnog pražnjenja - hladne plazme na inaktivaciju kvasca Brettanomyces bruxellensis u crnom vinu. Istraživanje je provedeno u dvije faze. U prvoj fazi je istražena učinkovitost dva glavna procesna parametra - frekvencije (60, 90 i 120 Hz) i trajanja tretmana (3, 5, 10, 20 i 30 min). Druga faza je obuhvatila praćenje dugoročnog učinka hladne plazme, uz primjenu optimalne frekvencije tijekom različitih vremena tretiranja pri čemu je prisutna populacija kvasca B. bruxellensis utvrđena praćenjem rasta kolonija kvasca na selektivnoj podlozi nakon 30, 60 i 90 dana skladištenja. Utvrđeno je kako je primjena frekvencije od 120 Hz rezultirala blago povećanom inaktivacijom kvasca B. bruxellensis u usporedbi s primjenom nižih frekvencija. Nadalje, duža vremena tretiranja rezultirala su značajnijom inaktivacijom, pri čemu se tretman u trajanju od 30 min ističe kao najbolji. Tijekom skladištenja uočeno je obnavljanje rasta populacije kvasca B. bruxellensis prisutne u crnom vinu odnosno smanjenje inaktivacijskog učinka tretmana hladnom plazmom

Characterization of a S-adenosyl-l-methionine (SAM)-accumulating strain of Scheffersomyces stipitis

S-adenosyl-l-methionine (SAM) is an important molecule in the cellular metabolism of mammals. In this study, we examined several of the physiological characteristics of a SAM-accumulating strain of the yeast Scheffersomyces stipitis (M12), including SAM production, ergosterol content, and ethanol tolerance. S. stipitis M12 accumulated up to 52.48 mg SAM/g dry cell weight. Proteome analyses showed that the disruption of C-24 methylation in ergosterol biosynthesis, a step mediated by C-24 sterol methyltransferase (Erg6p), results in SAM accumulation by S. stipitis M12 compared to the wild-type strain. A comparative proteome-wide analysis identified 25 proteins that were differentially expressed by S. stipitis M12. These proteins are involved in ribosome biogenesis, translation, the stress response, ubiquitin-dependent catabolic processes, the cell cycle, ethanol tolerance, posttranslational modification, peroxisomal membrane stability, epigenetic regulation, the actin cytoskeleton and cell morphology, iron and copper homeostasis, cell signaling, and energy metabolism. [Int Microbiol 2015; 18(2):117-125]Keywords: Scheffersomyces stipitis · S-adenosyl- l-methionine (SAM) · SAM accumulating yeast · C-24 sterol methyltransferase (Erg6p

S-adenosyl-L-methionine production by an ergosterol-deficient mutant of Scheffersomyces stipitis

S-adenosyl-L-methionine (SAM, SAMe; AdoMet) is an important methyl donor in many reactions, and recently has attracted much attention due to its expanding application in pharmacy. Efficient ways of its production have been widely researched. In this study it was demonstrated that erg6 mutant of yeast Scheffersomyces stipitis (former Pichia stipitis), obtained by UV mutagenesis and selected by resistance to nystatin, could be used for production of SAM. The mutant strain showed higher ability to accumulate SAM than its parental strain. The experiments demonstrated different dependence of erg6 mutant strain on several cultivation conditions in comparison to parental strain, its higher sensibility to oxidative stress, as well as strong dependence of SAM accumulation on the cell growth phase and supplementation of growth media with methionin

Monitoring the influence of high power ultrasound treatment and thermosonication on inactivation of Brettanomyces bruxellensis in red wine

Brettanomyces bruxellensis is one of the most important spoilage microorganisms in winemaking. It is harmful for wine industry because it produces volatile phenols, compounds primarily responsible for off-odours in wine. One of the possible solutions for preventing its growth is using new non-thermal processing technologies. The aim of this study was to investigate the application of one non-thermal processing technology, high power ultrasound and its combination with heating (thermosonication) on the inactivation of B. bruxellensis in red wine in batch systems. Various parameters, such as treatment duration (1, 2, 3, 6, 10 and 15 minutes), temperature (25, 35, 40 and 43 ºC) and probe diameter (12.7 mm and 19.1 mm), were examined. The combination of high power ultrasound and heating (thermosonication) proved to be a better method compared to solely using high power ultrasound. However, the production of volatile phenols by B. bruxellensis was also reduced after high power ultrasound treatment. The optimal treatment of 3 min at 43 ºC with high power sonicator at ultrasound frequency of 20 kHz with 12.7 mm diameter ultrasonic probe for complete inactivation of B. bruxellensis was determined

Monitoring the influence of high power ultrasound treatment and thermosonication on inactivation of Brettanomyces bruxellensis in red wine

Brettanomyces bruxellensis is one of the most important spoilage microorganisms in winemaking. It is harmful for wine industry because it produces volatile phenols, compounds primarily responsible for off-odours in wine. One of the possible solutions for preventing its growth is using new non-thermal processing technologies. The aim of this study was to investigate the application of one non-thermal processing technology, high power ultrasound and its combination with heating (thermosonication) on the inactivation of B. bruxellensis in red wine in batch systems. Various parameters, such as treatment duration (1, 2, 3, 6, 10 and 15 minutes), temperature (25, 35, 40 and 43 ºC) and probe diameter (12.7 mm and 19.1 mm), were examined. The combination of high power ultrasound and heating (thermosonication) proved to be a better method compared to solely using high power ultrasound. However, the production of volatile phenols by B. bruxellensis was also reduced after high power ultrasound treatment. The optimal treatment of 3 min at 43 ºC with high power sonicator at ultrasound frequency of 20 kHz with 12.7 mm diameter ultrasonic probe for complete inactivation of B. bruxellensis was determined

Lipid Composition of Sheffersomyces stipitis M12 Strain Grown on Glycerol as a Carbon Source

Pozadina istraživanja. U ovom je istraživanju određen sadržaj i sastav lipida u soju kvasca Sheffersomyces stipitis M12 s narušenom biosintezom ergosterola, uzgojenom na glicerolu kao izvoru ugljika. Zaustavljanje biosintetskog puta ergosterola jedna je od metoda koja se u novije vrijeme primjenjuje za povećanje udjela S-adenozil-L-metionina (SAM) u biomasi kvasca. Eksperimentalni pristup. Šaržni je uzgoj kvasca M12 proveden aerobno u laboratorijskom bioreaktoru, s glicerolom kao izvorom ugljika i uz pulsni dodatak metionina. Koncentracija glicerola i maseni udjel SAM-a određeni su tekućinskom kromatografijom visoke djelotvornosti, dok je masnokiselinski sastav svih klasa lipida, odijeljenih ekstrakcijom na čvrstoj fazi, utvrđen plinskom kromatografijom. Rezultati i zaključci. Usprkos manjem udjelu ergosterola u kvascu M12, zbog reorganizacije metabolizma lipida ostvareni su veliki prirast biomase i prinos SAM-a. U tom soju kvasca prevladavali su neutralni lipidi (više od 75 % ukupnih lipida), no njihov sadržaj i sastav bio je značajno izmijenjen u odnosu na divlji tip kvasca. Nezasićene i C18 masne kiseline prevladavale su u obje vrste kvasca. U svim je frakcijama, osim u frakciji slobodnih masnih kiselina, indeks nezasićenosti bio niži u soju M12 u usporedbi s divljim tipom kvasca. Soj M12 je u promijenjenim uvjetima rasta nesmetano funkcionirao i zadovoljio sve svoje energetske potrebe mijenjanjem sastava lipida (osobito fosfolipida, sterola i sterolnih estera) i ugađanjem njihove nezasićenosti. Novina i znanstveni doprinos. U radu je prikazan proces reorganizacije sastava lipida kvasca S. stipitis s narušenim metaboličkim putem biosinteze ergosterola. Konstruiran je i shematski prikazan aktualni metabolizam lipida tijekom aktivne proizvodnje SAM-a u kvascu S. stipitis uzgojenom na glicerolu kao izvoru ugljika. Ove spoznaje o metabolizmu lipida mogu se iskoristiti kao vrijedan alat u daljnjem unapređivanju primjene kvasca S. stipitis kao modelnog organizma, te omogućiti njegovu uporabu u primijenjenim istraživanjima.Research background. In this study the content and composition of lipids in ergosterol-reduced Sheffersomyces stipitis M12 strain grown on glycerol as a carbon source is determined. Blocking the ergosterol synthesis route in yeast cells is a recently proposed method for increasing S-adenosyl-l-methionine (SAM) production. Experimental approach. The batch cultivation of M12 yeast was carried out under aerobic conditions in a laboratory bioreactor with glycerol as carbon source, and with pulsed addition of methionine. Glycerol and SAM content were monitored by high-performance liquid chromatography, while fatty acid composition of different lipid classes, separated by solid phase extraction, was determined by gas chromatography. Results and conclusion. Despite the reduced amount of ergosterol in yeast cells, thanks to the reorganized lipid metabolism, M12 strain achieved high biomass yield and SAM production. Neutral lipids prevailed (making more than 75 % of total lipids), but their content and composition differed significantly in the two tested types of yeast. Unsaturated and C18 fatty acids prevailed in both the M12 strain and wild type. In all fractions except free fatty acids, the index of unsaturation in M12 strain was lower than in the wild strain. Our tested strain adjusts itself by changing the content of lipids (mainly phospholipids, sterols and sterol esters), and with desaturation adjustments, to maintain proper functioning and fulfil increased energy needs. Novelty and scientific contribution. Reorganization of S. stipitis lipid composition caused by blocking the metabolic pathway of ergosterol synthesis was presented. A simple scheme of actual lipid metabolism during active SAM production in S. stipitis, grown on glycerol was constructed and shown. This fundamental knowledge of lipid metabolic pathways will be a helpful tool in improving S. stipitis as an expression host and a model organism, opening new perspectives for its applied research

Production of S-adenosyl-L-methionine by yeast Pichia stipitis

Cilj ovog rada je bio ispitati mogućnost proizvodnje S-adenozil-L-metionina (SAMe) pomoću kvasca Pichia stipitis CBS 5776 (P. stipitis). Primjenom UV-mutageneze i nistatina selekcioniran je soj M12 kvasca P. stipitis koji ne eksprimira C-24 sterol metiltransferazu (Erg6p) čime je narušena biosinteza ergosterola te povećana proizvodnja SAMe. Soju M12 kvasca P. stipitis određen je genetski i proteomski profil te optimalan sastav podloge (vrsta osnovnog izvora ugljika, količina i oblik metionina, prisutnost soli) kao i optimalni uvjeti uzgoja (aeracija, temperatura i pH-vrijednost) za proizvodnju SAMe. Uzgojem soja M12 kvasca P. stipitis na glicerolu kao osnovnom izvoru ugljika uz pulsni dodatak metionina na početku stacionarne faze rasta ostvareno je povećanje sadržaja SAMe u biomasi soja M12 kvasca P. stipitis u odnosu na divlji soj kvasca P. stipitis.The aim of this work was to evaluate the possibility of S-adenosyl-L-methionine (SAM) production using yeast Pichia stipitis CBS 5776 (P. stipitis). P. stipitis M12 strain was selected using UV-mutagenesis and nystatin. M12 strain does not express C-24 sterol metyltransferase (Erg6p), and thus has decreased ergosterol biosynthesis and increased production of SAM. Genetic and proteomic profile for P. stipitis M12 strain, as well as optimal medium composition (a type of the main carbon source, amount and form of methionine, presence of salts) and growth conditions (aeration, temperature and pH) for SAM production were determined. An increase in SAM content in the biomass by growing M12 strain on glycerol as the main carbon source with a pulse addition of methionine at the beginning of the stationary growth phase, compared to the P. stipitis wild strain was achieved

Production of S-adenosyl-L-methionine by yeast Pichia stipitis

Cilj ovog rada je bio ispitati mogućnost proizvodnje S-adenozil-L-metionina (SAMe) pomoću kvasca Pichia stipitis CBS 5776 (P. stipitis). Primjenom UV-mutageneze i nistatina selekcioniran je soj M12 kvasca P. stipitis koji ne eksprimira C-24 sterol metiltransferazu (Erg6p) čime je narušena biosinteza ergosterola te povećana proizvodnja SAMe. Soju M12 kvasca P. stipitis određen je genetski i proteomski profil te optimalan sastav podloge (vrsta osnovnog izvora ugljika, količina i oblik metionina, prisutnost soli) kao i optimalni uvjeti uzgoja (aeracija, temperatura i pH-vrijednost) za proizvodnju SAMe. Uzgojem soja M12 kvasca P. stipitis na glicerolu kao osnovnom izvoru ugljika uz pulsni dodatak metionina na početku stacionarne faze rasta ostvareno je povećanje sadržaja SAMe u biomasi soja M12 kvasca P. stipitis u odnosu na divlji soj kvasca P. stipitis.The aim of this work was to evaluate the possibility of S-adenosyl-L-methionine (SAM) production using yeast Pichia stipitis CBS 5776 (P. stipitis). P. stipitis M12 strain was selected using UV-mutagenesis and nystatin. M12 strain does not express C-24 sterol metyltransferase (Erg6p), and thus has decreased ergosterol biosynthesis and increased production of SAM. Genetic and proteomic profile for P. stipitis M12 strain, as well as optimal medium composition (a type of the main carbon source, amount and form of methionine, presence of salts) and growth conditions (aeration, temperature and pH) for SAM production were determined. An increase in SAM content in the biomass by growing M12 strain on glycerol as the main carbon source with a pulse addition of methionine at the beginning of the stationary growth phase, compared to the P. stipitis wild strain was achieved

Kiselo tijesto – tradicionalna i prirodna metoda za povećanje kvalitete pekarskih proizvoda

Bread has been an essential part of human nutrition and culture for thousands of years. Every step of it’s production, from careful raw material selection to dough preparation, processing and baking, could be considered as art that ensure unlimited abundance of color, taste and aroma. Diversity of bakery products is result of the diversities in tradition, culture and geographic origin. All around the world alluring and aromatic bakery products always contain sourdough. Sourdough is a complex microbial ecosystem that is composed of lactic acid bacteria and yeasts. The use of lactic acid bacteria in the bread production has long tradition, from spontaneous fermentations (leavening the dough only), to the development and use of defi ned and functional starter cultures. This review covers the sourdough technology, with major emphasis on production of wheat bread with enhanced nutritional value, prolonged shelf life, better taste and aroma.Kruh je od pamtivijeka neizostavni dio ljudske prehrane i kulture. Svaki korak u njegovoj proizvodnji, od pažljivog izbora sirovina do pripreme i obrade tijesta te procesa pečenja, predstavlja dio umjetnosti koja u konačnici osigurava neograničeno bogatstvo boje, okusa i arome kruha. Raznolikost pekarskih proizvoda rezultat je različitosti tradicije, kulture te geografskog podrijetla. Međutim, san svakog pekara, primamljivi i mirisni pekarski proizvodi diljem svijeta, uvijek sadržavaju kiselo tijesto. Kiselo tijesto je kompleksni mikrobni ekosustav kojeg čine bakterije mliječne kiseline i kvasci. Upotreba bakterija mliječne kiseline u pripremi kruha ima dugu tradiciju, od spontanih fermentacija gdje je njihova uloga bila samo dizanje tijesta, preko defi niranih starter kultura, do razvoja i primjene funkcionalnih starter kultura. U ovom radu dat je prikaz tehnologije proizvodnje kiselog tijesta s naglaskom na proizvodnju pšeničnog kruha povećane nutritivne vrijednosti i trajnosti, bogatijeg okusa i arome