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

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