Spent yeast from beer manufacturing is a cost-effective and nutrient-rich starting material for the production of yeast extracts. In this study, it is shown how physiologically important ingredients in a yeast extract are influenced by the composition of the spent yeast from the brewing process. In pilot fermentations, the time of cropping (primary fermentation, lagering) of the spent yeast and the original gravity (12 ˚P, 16 ˚P, 20 ˚P) of the fermentation medium was varied, and four alternative non-Saccharomyces yeast strains were compared with two commercial Saccharomyces yeast strains. In addition, spent yeast was contaminated with the beer spoiler Lactobacillus brevis. The general nutrient composition (total protein, fat, ash) was investigated as well as the proteinogenic amino acid spectrum, the various folate vitamers (5-CH₃-H₄folate, 5-CHO-H₄folate, 10-CHO-PteGlu, H₄folate, PteGlu) and the biological activity (reduction, antioxidative potential) of a mechanically (ultrasonic sonotrode) and an autolytically produced yeast extract. All the investigated ingredients from the yeast extract were influenced by the composition of the spent yeast from the brewing process. The biodiversity of the spent yeast from the brewing process therefore directly affects the content of physiologically valuable ingredients of a yeast extract and should be taken into consideration in industrial manufacturing processes.