Fundamental investigation and applied studies on non-Saccharomyces yeasts in non-alcoholic and low alcohol beer brewing

Non-alcoholic and low alcohol beer (NABLAB) is enjoying growing popularity owing to consumer lifestyle changes, improved production methods and stricter legislation. Among the biological methods for their production, particularly research into non-Saccharomyces yeasts has gained momentum in recent years in order to produce NABLAB with novel flavor characteristics in an easy-to-apply manner. In a proof-of-concept study, five selected non-Saccharomyces species isolated from kombucha showed to perform just as well in laboratory-scale trials in wort as commercially applied species Saccharomycodes ludwigii. In a subsequent study, species of the Cyberlindnera genus were found to produce a pleasant, fruity flavor in wort. Fermentation parameters were optimized by means of response surface methodology (RSM) and the resulting non-alcoholic beer (NAB; 0.36% ABV) produced with Cyberlindnera subsufficiens on pilot-scale (60 L) had a significantly more fruity and significantly less wort-like aroma compared to two commercial NABs. Regarding low alcohol beer (LAB), the yeast species Lachancea fermentati was introduced to create LAB by harnessing the species’ uncommon ability to produce significant amounts of lactic acid (LA) during alcoholic fermentation. Compared to a Saccharomyces cerevisiae brewers’ yeast, L. fermentati produced less ethanol (–15%) while producing 1.3 g/L lactic acid, giving the beer a sour taste. In a follow-up study, four L. fermentati isolated from individual kombucha cultures were investigated in detail. The strains genotypes and phenotypes where shown to be diverse, correlating with the strains’ geographical origin. LA production was optimized via RSM, where low pitching rate, high fermentation temperature, and a high initial glucose concentration resulted in the highest LA concentrations (max. 1.6 g/L). LAB (1.26 %ABV) produced with L. fermentati by stopped fermentation showed to have a balanced ratio of acidity from lactic acid to residual wort sweetness. In conclusion, the results of this thesis give prospect to future studies with non-Saccharomyces yeasts and strengthen their position as a serious and applicable alternative to established methods in NABLAB brewing

Lachancea fermentati strains isolated from Kombucha: fundamental insights, and practical application in low alcohol beer brewing

With a growing interest in non-alcoholic and low alcohol beer (NABLAB), researchers are looking into non-conventional yeasts to harness their special metabolic traits for their production. One of the investigated species is Lachancea fermentati, which possesses the uncommon ability to produce significant amounts of lactic acid during alcoholic fermentation, resulting in the accumulation of lactic acid while exhibiting reduced ethanol production. In this study, four Lachancea fermentati strains isolated from individual kombucha cultures were investigated. Whole genome sequencing was performed, and the strains were characterized for important brewing characteristics (e.g., sugar utilization) and sensitivities toward stress factors. A screening in wort extract was performed to elucidate strain-dependent differences, followed by fermentation optimization to enhance lactic acid production. Finally, a low alcohol beer was produced at 60 L pilot-scale. The genomes of the kombucha isolates were diverse and could be separated into two phylogenetic groups, which were related to their geographical origin. Compared to a Saccharomyces cerevisiae brewersâ yeast, the strainsâ sensitivities to alcohol and acidic conditions were low, while their sensitivities toward osmotic stress were higher. In the screening, lactic acid production showed significant, strain-dependent differences. Fermentation optimization by means of response surface methodology (RSM) revealed an increased lactic acid production at a low pitching rate, high fermentation temperature, and high extract content. It was shown that a high initial glucose concentration led to the highest lactic acid production (max. 18.0 mM). The data indicated that simultaneous lactic acid production and ethanol production occurred as long as glucose was present. When glucose was depleted and/or lactic acid concentrations were high, the production shifted toward the ethanol pathway as the sole pathway. A low alcohol beer (<1.3% ABV) was produced at 60 L pilot-scale by means of stopped fermentation. The beer exhibited a balanced ratio of sweetness from residual sugars and acidity from the lactic acid produced (13.6 mM). However, due to the stopped fermentation, high levels of diacetyl were present, which could necessitate further process intervention to reduce concentrations to acceptable levels

Screening and Application of Cyberlindnera Yeasts to Produce a Fruity, Non-Alcoholic Beer

Non-alcoholic beer (NAB) is enjoying growing demand and popularity due to consumer lifestyle trends and improved production methods. In recent years in particular, research into the application of non-Saccharomyces yeasts to produce NAB via limited fermentation has gained momentum. Non-Saccharomyces yeasts are known to produce fruity aromas, owing to a high ester production. This trait could be harnessed to mask the often-criticized wort-like off-flavor of NAB produced via limited fermentation. Six Cyberlindnera strains were characterized and screened in wort extract. Four of the six strains produced a pleasant, fruity aroma while exhibiting low ethanol production. The strain Cyberlindnera subsufficiens C6.1 was chosen for fermentation optimization via response surface methodology (RSM) and a pilot-scale (60 L) brewing trial with subsequent sensory evaluation. A low fermentation temperature and low pitching rate enhanced the fruitiness and overall acceptance of the NAB. The NAB (0.36% ABV) produced on pilot-scale was significantly more fruity and exhibited a significantly reduced wort-like off-flavor compared to two commercial NABs. This study demonstrated the suitability of Cyberlindnera subsufficiens to produce a fruity NAB, which can compete with commercial NABs. The outcome strengthens the position of non-Saccharomyces yeasts as a serious and applicable alternative to established methods in NAB brewing

Investigation into the potential of Lachancea fermentati strain KBI 12.1 for low alcohol beer brewing

In brewing research, non-Saccharomyces yeasts have gained attention in recent years, owing to their potential to influence the characteristics and flavor of beer. The Lachancea genus possesses an uncommon trait, the production of significant amounts of lactic acid during alcoholic fermentation. This trait could potentially be harnessed for brewing purposes, particularly for the production of low alcohol beer. In this study, the potential of Lachancea fermentati strain KBI 12.1 was investigated for the production of low alcohol beer in low gravity wort. KBI 12.1 was characterized for sugar utilization, hop sensitivity, phenolic off-flavor (POE) production, and propagation performance. Lab scale fermentation trials in diluted wort (6.6 degrees P) were conducted and compared to a brewers' yeast, Saccharomyces cerevisiae WLP001. Fermentations were monitored for lactic acid and ethanol production, pH drop, and sugar consumption. In the final beers, amino acid and free amino nitrogen (FAN) content were determined and secondary metabolites were quantified. Lachancea fermentati KBI 12.1 showed to be unable to utilize maltotriose. The strain exhibited no POF production, minor hop sensitivity, and excellent propagation performance. Amino acid and FAN consumption were much lower compared to that of the brewers' yeast. In the final beer fermented with KBI 12.1, the lactic acid concentration reached 1.3 g/L, giving the beer a sour taste. During sensory analysis, the beer was additionally described to have a fruity character. In conclusion, Lachancea fermentati KBI 12.1 proved to be a suitable strain for brewing purposes, with promising traits with regard to nonalcoholic and low alcohol beer brewing

Application of Non-Saccharomyces Yeasts Isolated from Kombucha in the Production of Alcohol-Free Beer

Alcohol-free beer (AFB) is no longer just a niche product in the beer market. For brewers, this product category offers economic benefits in the form of a growing market and often a lower tax burden and enables brewers to extend their product portfolio and promote responsible drinking. Non-Saccharomyces yeasts are known for their flavor-enhancing properties in food fermentations, and their prevailing inability to ferment maltose and maltotriose sets a natural fermentation limit and can introduce a promising approach in the production of AFB (≤0.5% v/v). Five strains isolated from kombucha, Hanseniaspora valbyensis, Hanseniaspora vineae, Torulaspora delbrueckii, Zygosaccharomyces bailii and Zygosaccharomyces kombuchaensis were compared to a commercially applied AFB strain Saccharomycodes ludwigii and a Saccharomyces cerevisiae brewer’s yeast. The strains were characterized for their sugar utilization, phenolic off-flavors, hop sensitivity and flocculation. Trial fermentations were analyzed for extract reduction, ethanol formation, pH drop and final beers were analyzed for amino acids utilization and fermentation by-products. The performance of non-Saccharomyces strains and the commercial AFB strain were comparable during fermentation and production of fermentation by-products. An experienced sensory panel could not discriminate between the non-Saccharomyces AFB and the one produced with the commercial AFB strain, therefore indicating their suitability in AFB brewing