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In the beverage industry, the detection of spoilage yeasts such as Wickerhamomyces anomalus and Brettanomyces bruxellensis can be labourious and time-consuming. In the present study, a simple and repeatable technique was developed for rapid yeast detection using a combination of patterned gold-coated surface-enhanced Raman spectroscopy (SERS) substrates and gold nanoparticles. W. anomalus and B. bruxellensis showed several characteristic peaks, enabling the discrimination of these yeasts without chemometric analysis. The control yeast used as an indicator yeast, Rhodotorula mucilaginosa, showed 7 cell wall-related peaks originating from lipids and haemoproteins. Analysing W. anomalus SERS spectra with differently sized and shaped gold nanoparticles revealed the benefit of using either large, spherical, chemically synthesised gold nanoparticles or small, laser-synthesised, gold-silicon …