Spoilage Microorganisms: Yeasts

Elizabeth Crawford

Industrial and Food Microbiology Course 1. April 2014

Dept. of Food Analysis & Nutrition Institute of Chemical Technology, Prague, Czech Republic

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 (high aW)

pH range of 3-5

High concentration of soluable carbohydrates

Overall a very nice source for yeast growth

Yeast associations are 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

Low N2 and O2 content

• Generally an adverse environment for most microorganisms, but amenable for yeast growth

Fruit juices are higher in nitrogenous compounds & vitamins than soft drinks, therefore they are more susceptible to yeast spoilage

Cause of spoilage not often from the ingredients, but most often originates from the manufacturing process

Critical points of contamination are: pumps, holding tanks, bottle washers and bottling lines

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.

Elizabeth Crawford1, Paola Domizio2,3, Brian Musselman1, C. M. Lucy Joseph2, Linda F. Bisson2, Bart C. Weimer4 and Richard Jeannotte4,5

47. Jahrestagung der Deutschen Gesellschaft für Massenspektrometrie (DGMS) 03. März 2014 Frankfurt am Main, Deutschland

Preventing Wine Spoilage: Rapid Screen & Quantification of Off-flavor Phenolics using Ambient Ionization coupled with High Resolution MS/MS

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 5Facultad de Ciencias, Univ. de Tarapacá, Arica, Chile

Brettanomyces

“Brett” Wheel http://heysmartbeerdude.files.wordpress.com/2013/04/brett-aroma-wheel.jpeg (Access: 10 June 2013)

• 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

Background:

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

Slide courtesy of Dr. L. Bisson, Dept. of Viticulture & Enology, Univ. of California-Davis

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 Prof. Jana Hajšlová, ICT Prague, Czech Republic

(GC-MS)

4-EP Conc.

(µg/L)

Low High

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

DART MS/MS Method: Figures of Merit

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

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

* Levels above selected calibration range