Non-Saccharomyces Wild Yeast

Effects of Wild Yeast Sources of Contamination Avoiding Contamination

‘Wild’ Yeasts
● Unwanted, not deliberately inoculated Contamination ● Raw materials - especially culture yeast
● Unclean vessels and equipment- brett
●
●
Pasteurisation
CIP- focus on pores, cracks, shadow
● Unicellular ● Changes in fermentation rate ● Bottling, canning, kegging operations- especially ● Separate vessels for using brettanomyces
● Potential spoilers ● Changes in final attenuation unpasteurised ● Air filter in sanitary packaging area
● Can compete with brewing yeast ● Altered flocculation characteristics ● Returned bottles ● Hygiene reinforced for personnel
● Detection is often difficult ● Flavour and texture defects- phenolic ● Air ● Analysis of culture yeast to ensure no
● Poor foam stability ● Personnel contaminants
Common Spoilers
Brettanomyces/Dekkera
B. anomalus
Methods For Detection Inhibiting Saccharomyces Yeast
● Suited to beer pH, ethanol ● When present, non-Saccharomyces wild yeast contaminants Lysine Medium
<18%, low oxygenation are minor components compared to deliberately inoculated ● Primary source of nitrogen is lysine
● In tanks saccharomyces yeasts ● Suppresses culture yeast growth along with most wild Saccharomyces
● Utilises glucose (non-fermentatively) and
● Detection methods must not be subject to interference by ● Incubate at 25 oC for 5 days
produces off-flavours such as acetic acid,
and turbidity brewing yeasts, or inhibit brewing yeasts ● Lower concentration provides more distinguishable colonies
● Most detection methods utilise a growth medium or ● Purity is required to ensure no other nitrogen sources are present
Candida microscopic analysis, along with suppression of brewing ● Particularly for detection of Torula, Candida, Pichia, Zygosaccharomyces which
C. ingens yeasts utilise lysine as sole nitrogen source
● Infection in aerobic
phase of fermentation
● Prevented by
Wallerstein Laboratory Nutrient MALDI-TOF MS and Biotyper Software
pasteurisation Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight Mass Spectrometry
● Some strains can exhibit anaerobic growth
● General brewery growth medium ● Novel Technique
● Can ferment glucose
● Bacterial growth inhibited using bactericide such as ● Dekkera/Brettanomyces Reference Mass Spectra used
Kluyveromyces tetracycline or chloramphenicol ● Spectra obtained from cytosolic ribosomal proteins
K. lactis ● Contains yeast extract for vitamins, amino acids ● Consistent and independent of environmental conditions
● Vigorous fermentation ● Enzymatic digest of Casein for nitrogen, amino acids, carbon ● Rapid (<30 min), reliable, cost effective analysis
of glucose, some strains ● KCl, CaCl2 , FeCl2 essential ions & for osmotic balance ● Detection and identification of wild yeasts
can also ferment other ● MgSO4, MnSO4 divalent cation sources ● Can be incorporated into routine QC analysis
sugars
● Bromcrescol Green pH indicator- changes colour for wild yeast ● 98.9% success (Turvey, Weiland, Meneses, Sterenberg & Hoffmann, 2016)
● Can tolerate temperatures
between 37oC and 43oC
(but also lager yeast) ● Pre-enrich using nutrient agar
● Produces lactobacillus, ● Identification of wild yeast is by physical appearance- colour,
other
contaminants
flavour
and
shape, texture
POF-Based Approach
turbidity
Pichia POF 1 gene regulates decarboxylation of hydroxycinnamic acids in beer
PCR- based Methods
● Only aerobic growth ●
P. membranaefaciens ● Most wild yeasts in brewing have the gene and are Pof+
● Mostly aerobes but can ● Brewing strains are Pof-
grow in anaerobic conditions
● Polymerase Chain Reaction ● Incubate yeast in hopped wort without other contaminants, with ferulic acid supplement
● Form surface pellicle in cask beer
● Non-fermentative ● Rapid, reliable, simple (of which decarboxylation occurs), at 27 oC for 24h
● Growth impaired by acidity and ethanol ● High temperature denaturation 95oC ● Check for presence of 4-vinylguaiacol, (with characteristic clove aroma)
● Ester and yeast off-flavours, and turbidity ● Primers hybridised 55 oC ● Beers containing 4-vinylguaiacol have a Pof+ yeast
DNA primers copied- 30-40 cycles, doubled each cycle, uses
Torulaspora ●
T. delbrueckii
●
DNA polymerase
Labelled with fluorescent dye and separated using gel
Key References
Lin, Y. (1975). DETECTION OF WILD YEASTS IN THE BREWERY EFFICIENCY OF DIFFERENTIAL MEDIA. Journal Of The Institute Of Brewing, 81(5),
● Ferment glucose,
maltose, sucrose electrophoresis to produce profile 410-417.
Walters, L., & Thiselton, M. (1953). UTILIZATION OF LYSINE BY YEASTS. Journal Of The Institute Of Brewing, 59(5), 401-404.
● Like Saccharomyces ● Visualisation using ethidium bromide or UV Briggs, D., Boulton, C., Stevens, R., & Brookes, P. (2004). Brewing (pp. 606-649). Cambridge: Woodhead.
Boulton, C., & Quain, D. (2013). Brewing Yeast and Fermentation. Hoboken: Wiley.
● Poor aerobic growth ● Distinguish wild yeast from brewing yeast Bokulich, N., & Bamforth, C. (2013). The Microbiology of Malting and Brewing. Microbiology And Molecular Biology Reviews, 77(2), 157-172.
Hornsey, I. (2007). Brewing (pp. 194-223). Royal Society of Chemistry.
● Unpasteurised draught beer ● Restriction Fragment Length Polymorphism and Randomly Hall, J. (1971). DETECTION OF WILD YEASTS IN THE BREWERY. Journal Of The Institute Of Brewing, 77(6), 513-516.
Amplified Polymorphic DNA methods particularly useful Turvey, M., Weiland, F., Meneses, J., Sterenberg, N., & Hoffmann, P. (2016). Identification of beer spoilage microorganisms using the MALDI
Biotyper platform. Applied Microbiology And Biotechnology, 100(6), 2761-2773. doi: 10.1007/s00253-016-7344-8