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)  pH range of 3-5  High concentration of soluable carbohydrates 

 Overall a very nice source for yeast growth

directly reflective of harvesting and handling practices Contamination during growing season, injuries during harvesting and handling

Yeasts in Fruits

Fruit Juices

Soft Drinks

 Low pH  Cause of spoilage not often from the  Low N2 and O2 content

ingredients, but most often originates from the environment for most 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 • Generally an adverse

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 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 2Dept.

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

Low

High

1 2 3 4 Mean %CV

50 47.7 58.7 52.1 41.0 49.9 14.9

500 492.2 509.2 N/A N/A 500.7 2.4

%Bias

-0.3

0.1

n

4

2

4-EP Conc. (µg/L)

Compare: Calculated Levels of 4-EP & 4-EG Wine Sample

DART HRAM MS/MS

GC MS

4-EP (µg/L)

4-EG (µg/L)

4-EP (µg/L)

4-EG (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