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Simple and fast HPLC-DAD method for determination of HCDC activity and formation of vinylphenol in Saccharomyces and non-Saccharomyces yeast
Jelena Topič, Lorena Butinar, Dorota Korte, Branka Mozetič Vodopivec, published scientific conference contribution abstract

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.
Found in: osebi
Keywords: yeast, hydroxycinnamate decarboxylase, 4-vinylphenol
Published: 18.06.2018; Views: 2382; Downloads: 0
.pdf Fulltext (4,46 MB)

Uporaba različnih kvasovk v pridelavi jabolčnega vina
Luka Koporec, 2018, undergraduate thesis

Abstract: Kemijske in senzorične lastnosti vina so poleg genetskih danosti ter geo-klimatskih okoliščin odvisne tudi od strategije tehnologije pridelave, ki jo izbere vinar. V to strategijo spada tudi izbira seva kvasovke. Vinski trg postaja vse bolj zahteven in željan nečesa novega, zato vse več vinarjev poskuša pridobiti drugačne lastnosti vin z uporabo drugačnih sevov kvasovk, oziroma z uporabo kombinacij različnih vrst kvasovk. V diplomski nalogi smo preučevali vpliv različnih kombinacij kvasovk na potek fermentacije ter na osnovne kemijske in senzorične lastnosti pridelanega jabolčnega vina. Poskus je bil izveden na laboratorijski skali. Rezultati poskusa so pokazali, da različne vrste kvasovk različno vplivajo na senzorične lastnosti vina, nekoliko manj pa na kemijske lastnosti. Rezultati nakazujejo, da različne kvasovke različno hitro porabljajo sladkorje, kar vpliva tudi na hitrost poteka fermentacije. Ob opazovanju osnovnih kemijskih lastnostih smo opazili razlike med kvasovkami oz. kombinacijami kvasovk pri parametrih kot so hlapne kisline in reducirajoči sladkorji, medtem ko v vsebnosti alkohola in pH vrednostih končnih jabolčnih vin med obravnavanimi fermentacijami nismo opazili razlik. Senzorična analiza vonja in barve jabolčnih vin je potrdila vpliv kvasovk na barvo, intenzivnost, trajnost in vrsto vonja.
Found in: osebi
Keywords: jabolčno vino, ne-Saccharomyces kvasovke, potek fermentacije, kvasovke, senzorična analiza
Published: 26.10.2018; Views: 2748; Downloads: 135
.pdf Fulltext (1,34 MB)

Characterization of lactic bacteria for biogenic amine formation
Branka Mozetič Vodopivec, Martina Bergant Marušič, Jelena Topić, Dorota Korte, Lorena Butinar, 2018, independent scientific component part or a chapter in a monograph

Abstract: Biogenic amines are compounds present in many different foods and beverages (wine, beer, dairy products, fermented vegetables and soy products, fish, etc.). Their presence in foodstuff is a result of a microbial action during storage and ageing. The most important are histamine, tryptamine, β-phenylethylamine and tryptamine, which can induce undesirable physiological effects in humans. They are formed through decarboxylation of corresponding amino acids, through the action of enzymes. Consumption of food containing biogenic amines can lead to food poisoning such as histamine poisoning. Histamine, the most studied biogenic amine, is known to cause headaches, oedema, vomiting, etc. [1]–[4]. Monitoring of the content of biogenic amines in foods is of concern for public health in their relation to the food safety, food spoilage and food intolerance. Because microorganisms are used in food productions as starters and biopreservers, characterization of microorganisms for their ability to produce biogenic amines is equally important. Lactic acid bacteria are often used as biopreservers as they can produce antimicrobial metabolites and antifungal peptides. Some strains can also produce undesirable biogenic amines [5]. In order to use lactic acid bacteria as starters or biopreservers, the selection of strains that would not produce biogenic amines is necessary. When considering studies of biogenic amines in foods, focus should be on developing new or improving analysis methods for biogenic amines detection. Secondly, the connections between microorganisms capable of producing biogenic amines and the content of biogenic amines in foods should be investigated [3]. The most widely technique used for quantification of biogenic amines in foodstuff is liquid chromatography, Alternatively to chromatographic techniques, other techniques such as enzymatic biosensors, ELISA and flow-injection analysis have also been employed. Sensors are interesting due to the fact that they do not require special instrumentations, and there is no need for sample clean-up and derivatization, which are the main drawback of chromatographic methods [4]. Detection of biogenic amines producing lactic bacteria is important due to the concerns for public health and there is a need for the early and rapid detection of such microorganisms. Most of the methods that are used for screening involved the measurement of amino acid-decarboxylase activity, although there were been some methods reported that used differential media and pH indicators. Nowadays, molecular methods are replacing culture methods. Molecular approaches are used to determine the presence or absence of genes responsible for biogenic amines formation. The main advantages of DNA hybridization and PCR methods are speed, simplicity, sensitivity and specificity as they allow detection of targeted genes. Culture independent methods which are based on PCR techniques are now regarded as most suitable methods for screening isolates [5]. [1] A. R. Shalaby, “Significance of biogenic amines to food safety and human health,” Food Res. Int., vol. 29, no. 7, pp. 675–690, Oct. 1996. [2] J. M. Landete, S. Ferrer, and I. Pardo, “Biogenic amine production by lactic acid bacteria, acetic bacteria and yeast isolated from wine,” Food Control, vol. 18, pp. 1569–1574, 2007. [3] F. B. Erim, “Recent analytical approaches to the analysis of biogenic amines in food samples,” TrAC - Trends in Analytical Chemistry, vol. 52. pp. 239–247, 2013. [4] J. L. Ordóñez, A. M. Troncoso, M. D. C. García-Parrilla, and R. M. Callejón, “Recent trends in the determination of biogenic amines in fermented beverages – A review,” Analytica Chimica Acta, vol. 939. pp. 10–25, 2016. [5] R. M. Elsanhoty and M. F. Ramadan, “Genetic screening of biogenic amines production capacity from some lactic acid bacteria strains,” Food Control, vol. 68, pp. 220–228, Oct. 2016.
Found in: osebi
Keywords: lactic bacteria, biogenic amines
Published: 13.12.2018; Views: 1679; Downloads: 0
.pdf Fulltext (504,99 KB)

Preliminarni rezultati študije mlečnokislinskih bakterij povezanih z vinom Teran
Jelena Topić, Lorena Butinar, unpublished conference contribution

Found in: osebi
Keywords: Mlečnokislinske bakterije, teran, biogeni amini
Published: 13.12.2018; Views: 1795; Downloads: 0
.pdf Fulltext (308,36 KB)

Modulation of Pinot Noir wine colour by yeast selection
Dorota Korte, Lorena Butinar, Jelena Topić Božič, Branka Mozetič Vodopivec, 2019, published scientific conference contribution abstract

Found in: osebi
Keywords: yeast, HCDC activity, wine colour, Pinot Noir, pyranoanthocyanins
Published: 27.05.2019; Views: 1596; Downloads: 0
.pdf Fulltext (157,36 KB)

Prisotnost biokontrolne aktivnosti pri kvasovkah, osamljenih iz vinogradniškega okolja
Urban Česnik, 2019, master's thesis

Abstract: Opravili smo dve hitri molekularni tehniki za identifikacijo enoloških kvasovk, ki so bile izolirane iz vinogradov z ekološko in intergrirano pridelavo grozdja. Najprej smo določili 15 Saccharomyces kvasovk s pomočjo analize multipleks PCR med zbranimi 131 izolati, nato smo s pomočjo ribotipizacije ITS določili približno 98 kvasovk do nivoja vrste, ki so bile potrjene tudi s sekvenciranjem regije ITS. Določili smo 16 vrst vinskih kvasovk: Hanseniaspora uvarum, H. valbyensis, H. opuntiae / H. uvarum, Lachancea thermotolerans, Candida tropicalis, Candida sp. / Starmerella sp., Metschnikowia pulcherrima, Pichia kudriavzevii, P. terricola, P. kluyveri, Wickerhamomyces anomalus, Kodamaea ohmeri, S. bacillaris, Saccharomyces cerevisiae, Saturnispora diversa in Torulaspora delbrueckii. Ugotovili smo, da so najdene kvasovke običajne naseljevalke grozdne mikroflore ter da je način pridelave grozdja vplival na pojavljanje določenih vrst v obravnavanih vinogradih. Nadalje smo testirali kvasovke na tvorbo sideroforov, tvorbo protiglivnih hlapnih organskih spojin (HOS), hidrolitično aktivnost in toleranco kvasovk na fungicide, kot so baker, iprodion, fluazinam ter kombinacijo ciprodinil / fludioksonil. Za kvalitativno detekcijo hidrolitične aktivnosti smo uporabili presejalne teste na osnovi trdnega gojišča z dodanim hitinom ali β-D-glukozidi kot substratom in ugotovili, da je veliko testiranih kvasovk sposobnih proizvajati litične encime, ki bi lahko bili odgovorni za razgradnjo celičnih sten fitopatogenih gliv in potencialno sposobni proizvajati HOS s hidrolizo grozdnih glikozidov zaradi prisotnosti β-glukozidaz. Prav tako smo opazili, da so te encimske aktivnosti običajno odvisne od seva. Na splošno so kvasovke tudi tolerantne na testirane fungicide. Ugotovili smo tudi, da je odpornost biofungicidnih kvasovk na fungicide koristna lastnost za razvoj biofungicidov v prihodnosti pri aplikacijah v strategijah z zmanjšanim vnosom pesticidov.
Found in: osebi
Keywords: Kvasovka, biološki nadzor, fitopatogen, Botrytis cinerea, vinogradništvo
Published: 01.12.2019; Views: 1788; Downloads: 63
.pdf Fulltext (1,57 MB)

Occurence of biogenic amine - producing lactic acid bacteria in Refošk grape and wine
Jelena Topić Božič, Jan Reščič, Martina Bergant Marušič, Klemen Lisjak, Branka Mozetič Vodopivec, Lorena Butinar, 2019, published scientific conference contribution abstract

Abstract: Lactic acid bacteria (LAB) are naturally present in grapes, musts and wines. During malolactic fermentation, besides the beneficial conversion of L-malic to L-lactic acid, LAB can form also other compounds like biogenic amines (BAs). BAs are formed through decarboxylation of corresponding amino acids, through the action of enzymes. Consumption of food containing BAs can lead to food poisoning such as histamine poisoning. Histamine, the most studied BA, is known to cause headaches, oedema, vomiting. In the last years, BAs associated pathways have been described as strain dependent and not as species dependent. Because of the high variability of microorganisms to decarboxylase amino acids, the detection of bacteria that have the possibility to transform precursor amino acid into BAs is very important in order to estimate the risk of BAs accumulation in wines. Since in previous years during project AGROTUR I we detected the higher BA content in some wines, we decided to focus on the presence of BA-producing LAB in Refošk grapes and wines. Therefore, during AGROTUR II project LAB collection of autochthonous lactic acid bacteria was set-up by isolating LAB from Refošk grapes and wines, originated from the grape growing cross-border region Karst (Slovenia). Over 600 isolates were obtained using MRS medium with added cycloheximide and 2% tomato juice. In parallel, also selective enrichment was performed using MRS medium described above with additionally added 5% ethanol. BA-producing LAB were primarily detected in microtiter-plate format using the decarboxylase screening medium with added amino acids (histidine, lysine, ornithine or tyrosine), which enable us to identify histamine-, cadaverine-, ornithine and tyrosine-producing LAB. Selected representatives from each BA-producing LAB groups were further on confirmed with chromatographic analysis (HPLC) and molecular methods (multiplex PCR method). BA-producing LAB were in majority represented by thyramin-producers, thereafter followed cadaverine-producers, with putrescine and histamine producers being the least presented. This screening of the BA-producing LAB is also incorporated in our on-going accurate selection of LAB starters for potential production of Refošk wines.
Found in: osebi
Keywords: biogenic amines, lactic acid bacteria, refošk, grape, wine
Published: 22.01.2020; Views: 1406; Downloads: 0
.pdf Fulltext (185,72 KB)

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