Spoilage Microorganisms: Yeasts Elizabeth Crawford Dept. of Food Analysis & Nutrition Institute of Chemical Technology, Prague, Czech Republic Industrial and Food Microbiology Course 1. April 2014 General Considerations for Yeasts . ‘Foods are considered habitats for microorganisms’ . Most susceptible foods/beverages for yeast spoilage have: • Low pH (5.0 or lower) which restricts the growth of competing bacteria • High sugar & organic acid content (easily metabolized carbon sources) . Compared with bacteria and molds, yeasts play a minor role in food spoilage Handbook of Food Spoilage Yeasts, Second Edition Tibor Deak (CRC Press 2007; Print ISBN: 978-1-4200-4493-5; eBook ISBN: 978-1-4200-4494-2) Growth Requirements for Yeasts What defines a ‘Spoilage Yeast’? . Yeasts responsible for undesirable changes to the sensory quality of the food. • Unwanted changes in the flavor, aroma and taste of the final products. In fermented alcoholic beverages, any yeast changing the “sensorial characteristics can be regarded as a spoilage yeast”. Food technologists define as…yeasts that spoil a food product despite following GMP standards. * Loureiro V., Malfeito-Ferreira M. (Review Paper) Spoilage yeasts in the wine industry. Int. J. of Food Microbiology 2003(86) 23-50. Commodities Susceptible to Yeast Spoilage . Fresh and processed fruits . Fruit juices and soft drinks . Vegetables . Fermented alcoholic beverages • Beer and wines . Diary products • Milk, cheeses and fermented milk Major Spoilage Yeasts in Foods & Beverages Loureiro V., Malfeito-Ferreira M. (Review Paper) Spoilage yeasts in the wine industry. Int. J. of Food Microbiology 2003(86) 23-50. (Fresh) Fruits Processed Fruits . High moisture content . Yeast associations are (high aW) directly reflective of . pH range of 3-5 harvesting and handling . High concentration of practices soluable carbohydrates . Contamination during growing season, injuries during harvesting and . Overall a very nice handling source for yeast growth Yeasts in Fruits Fruit Juices Soft Drinks . Low pH . Cause of spoilage not often from the . Low N2 and O2 content • Generally an adverse ingredients, but most environment for most often originates from the microorganisms, but manufacturing process amenable for yeast . Critical points of growth contamination are: . Fruit juices are higher in pumps, holding tanks, nitrogenous compounds bottle washers and & vitamins than soft bottling lines drinks, therefore they are more susceptible to yeast spoilage Yeasts in Fruit Juices & Soft Drinks Preventative Steps Against Yeast Spoilage . Chemical preservation • Addition of sulfur dioxide, sorbic acid, benzoic acid, acetic acid . Pasteurization . Freezing . Concentration (lowering of aW) . Irradiation Vegetables . Increased incidence of yeast spoilage in these commodities due to storage/packaging in plastics, minimal processing and stronger consumer demands for ready-to-eat vegetables. Spoilage is caused most frequently by Saccharomyces cerevisiae. Tomatoes are exceptional, in that yeasts represented nearly 17% of fungal isolates from ripe, damaged and decayed tomatoes. Ready-to-eat vegetable salads were P. fermentans, P. membranifaciens and unidentified Candida spp. and Trichosporon spp. Beer . Wild Yeasts - unwanted yeasts that enter into the beer during fermentation • Two kinds - Saccharomyces and non-Saccharomyces genera • Origin: Brewery environment and pitching yeast . Phenolic off-flavor - coming from some strains of S. cerevisiae wild yeasts that contain an enzyme that decarboxyates wort phenolic acids . Strains (Pichia and Candida species) producing zymocins (killer toxins) could completely eliminate pitching yeasts causing fermentation to end . Film forming: responsible yeasts P. membranifaciens, P. fluxuum and P. anomala. Yeasts in Beer Wine . Spoilage yeasts originating from the grapes are the primary source of Dek. (Bret.) bruxellensis leading to phenolic off-flavors . High concentrations of acetaldehyde can be achieved by several Candida species and S’codes ludwigii, P. anomala, and other Pichia species . Hanseniaspora (Kloeckera) species are also responsible for high levels of acetic acid and its esters. Common spoilage yeasts in bottled wine include Zygo. bailii, S. cerevisiae, C. rugosa, P. membranifaciens, and C. vini. Preventing Wine Spoilage: Rapid Screen & Quantification of Off-flavor Phenolics using Ambient Ionization coupled with High Resolution MS/MS Elizabeth Crawford1, Paola Domizio2,3, Brian Musselman1, C. M. Lucy Joseph2, Linda F. Bisson2, Bart C. Weimer4 and Richard Jeannotte4,5 1IonSense, Inc, Saugus, MA, USA 2Dept. of Viticulture & Enology, Univ. of California-Davis, Davis, CA, USA 3Dipart. di Gestione Sistemi Agrari, Alimentari e Forestali (GESAAF), Univ. degli Studi di Firenze, Italy 4Dept. of Health & Reproduction, School of Veterinary Med, Univ. of California-Davis 5 Facultad de Ciencias, Univ. de Tarapacá, Arica, Chile 47. Jahrestagung der Deutschen Gesellschaft für Massenspektrometrie (DGMS) 03. März 2014 Frankfurt am Main, Deutschland Brettanomyces Background: • Budding yeast found widely distributed in nature • Discovered in beer in 1904 (Claussen), in wine (Krumbholz & Tauschanoff,1930) and again in 1940 (Custers) • Produces a wide array of aromatic compounds • Wine cellar contamination was widespread • “Brett” characters can compete with varietal characters for dominance of wine profile “Brett” Wheel http://heysmartbeerdude.files.wordpress.com/2013/04/brett-aroma-wheel.jpeg (Access: 10 June 2013) When Is It Spoilage? • High concentration, dominating wine profile • Conflict with wine matrix characters • Suppression of varietal character • Enhancement of off-notes • Lactic acid bacteria often found in wines with Brettanomyces Recovery Thresholds: • Chatonnet* has defined spoilage as: • >426 ppb of 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) • >620 ppb of 4-EP • 50% of tasters can detect 605 ppb in wine or 440 ppb in water of 4-EP * Chatonnet, P.; Boidron, J. N.; Dubourdieu, D. Influence des conditions d’ élevage et de sulfitage des vins rouges en barriques sur leur teneur en acide acétique et en éthyl-phenols. J. Int. Sci. Vigne Vin. 2003, 27, 277-298. Slide courtesy of Dr. L. Bisson, Dept. of Viticulture & Enology, Univ. of California-Davis (GC-MS) Slide courtesy of Prof. Jana Hajšlová, ICT Prague, Czech Republic DART MS/MS Method: Figures of Merit 4-EP Conc. Low High (µg/L) 50 500 1 47.7 492.2 2 58.7 509.2 3 52.1 N/A 4 41.0 N/A Mean 49.9 500.7 %CV 14.9 2.4 %Bias -0.3 0.1 n 4 2 Compare: Calculated Levels of 4-EP & 4-EG Wine DART HRAM GC MS Sample MS/MS 4-EP 4-EG 4-EP 4-EG (µg/L) (µg/L) (µg/L) (µg/L) Sample 04 854 * 197 845 203 Sample 05 518 157 563 161 Sample 06 52 ND 129 14 Sample 09 ND ND 110 13 Sample 14 2774 * 492 2534 433 Yellow = Brettanomyces * Levels above selected calibration range .