Screening for new brewing yeasts in the non‐Saccharomyces sector with Torulaspora delbrueckii as model

This study describes a screening system for future brewing yeasts focusing on non‐Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, polymerase chain reaction fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off‐flavour tests and trial fermentations. Trial fermentations were analysed for extract reduction, pH drop, yeast concentration in bulk fluid and fermentation by‐products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavour and a beer of average ethanol content with a high volatile flavour compound concentration. Two other strains could possibly be used for pre‐fermentation as a bio‐flavouring agent for beers that have been post‐fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavour‐forming properties. Copyright © 2015 John Wiley & Sons, Ltd.     

A novel approach for the production of a non‐alcohol beer (≤0.5% abv) by a combination of limited fermentation and vacuum distillation

Despite the increasing demand, the production of non‐alcohol beers is still limited by unsatisfactory or artificial flavour and taste. In this study, a novel approach to producing non‐alcohol beer is presented, in which the alcohol‐reducing techniques, limited fermentation and vacuum distillation were combined. Starting from barley and wheat malts, wort with a low level of fermentable sugars was prepared by infusion mashing and lautering. Limited fermentation was carried out by Saccharomycodes ludwigii at 18°C. When the level of fermentable sugar was reduced by 25%, the fermented wort was quickly cooled from 18 to 0°C and held at that temperature for two days. The young beer was obtained after degassing and removal of yeast and was then subjected to vacuum distillation at 0.06 MPa to remove the alcohol. The concentrated extract is suitable for storage and transportation. The final product of non‐alcohol beer was obtained by dilution with deoxygenated water and carbonation with 6.0 g/L CO₂, followed by addition of 8–12% of regular beer and equilibration for 2–3 days to develop normal beer aroma. The results showed that the non‐alcohol beer had several favourable properties, including the alcohol level of <0.5% (v /v), colour 7.0 (EBC), thiobarbituric acid value of 1.05 and ratio of alcohols to esters of 1.08. Compared with other methods for the production of non‐alcohol beer, this novel approach produced a favourable alternative to regular beers with similar flavour characteristics and satisfactory stability. Copyright © 2017 The Institute of Brewing & Distilling     

Evaluation of the fermentative potential of Candida zemplinina yeasts for craft beer fermentation

The fermentative potential of Candida zemplinina Y.01667 and Y.01670 was evaluated to explore the potential use of these yeasts for craft beer fermentations. Fermentation experiments were carried out at different temperatures and soluble solid concentrations, using synthetic media with glucose syrup as a sugar source and with a laboratory malt wort plus different adjuncts. Results showed that both strains fermented well at 14 °C and had improved fermentative activity at 20 °C. The fermentative kinetics of C. zemplinina Y.01667 and Y.01670 were not affected when experiments at higher concentrations of soluble solids were conducted. Furthermore, C. zemplinina strains had better growth, higher viable cells counts, less free amino nitrogen consumption, lower sedimentation rates and slighter changes in pH values, when compared with results of the lager beer yeast Saccharomyces cerevisiae S‐23 in the synthetic medium tested. Fermentations in a malt wort with different adjuncts indicated that C. zemplinina Y.01670 could possibly be used as a yeast in craft beer production. Copyright © 2016 The Institute of Brewing & Distilling     

West African sorghum beer fermented with Lactobacillus delbrueckii and Saccharomyces cerevisiae: fermentation by‐products

Physicochemical quality parameters and volatile fermentation by‐products were determined in West African sour sorghum beer (pito) fermented with pure cultures of Lactobacillus delbrueckii and Saccharomyces cerevisiae compared with pito prepared by traditional spontaneous fermentation. Levels of by‐products were also compared with those found in similar beer types. Similar levels of apparent extract, alcohol, pH, lactic acid and bitterness were obtained for pure culture and traditional fermentations, although differences were observed in colour and turbidity. Significant statistical differences were obtained for all of the volatile aroma compounds analysed. The pure culture approach resulted in a higher level of total volatile compounds (353 mg/L) of which higher alcohols accounted for 88%, predominately n‐propanol. The traditional approach had total volatiles of 229 mg/L with 86% higher alcohols but with iso‐amyl alcohol predominating. Ester levels were low in the pure culture beer but with a relatively high level of acetaldehyde. Fermenting pito with pure cultures yielded a product with similar physicochemical quality as traditional pito but with a suggestion of a more pronounced aroma whose impact on the overall product quality will require consumer acceptance and sensory evaluation. © 2019 The Institute of Brewing & Distilling     

Fed‐batch fermentation with glucose syrup as an adjunct for high‐ethanol beer brewing

To produce a beer with a high ethanol content, preliminary research on fed‐batch fermentation profiles with glucose syrup as an adjunct during the primary fermentation period was conducted. The ethanol concentration of the beer was elevated by feeding a glucose syrup into the fermentors at a later stage of primary fermentation. Fermentation trials were carried out using a typical lager strain, SC‐9, with a pitching rate at 7.0 × 10⁶ cells/mL. An all‐malt wort (12.5°P) was employed and the primary fermentation temperature was 14 °C. Glucose syrup was supplemented when the concentration of residual reducing sugars was decreased to ~10 g/L. Results showed that the supplemented glucose was consumed rapidly and that the ethanol concentration in the final beer was raised to 67.9 g/L. Additional growth of yeast was observed after feeding accompanied by a low yield of ethanol (~0.46 g/g). Formation of diacetyl was enhanced by yeast growth and two additional peaks were obtained after feeding. The peak value of the diacetyl concentration was 1.90 mg/L. The fed‐batch fermentation resulted in a beer with an overproduction of higher alcohols and esters, indicating that brewing under these experimental conditions led to an unbalanced flavour profile. Results of optimization demonstrated that the optimal conditions were found to be 15°P for initial wort extract, 10 °C for fermentation temperature and 20 × 10⁶ cells/mL for yeast pitching rate, leading to total higher alcohols of 173.8 mg/L, total esters of 22.8 mg/L and an acetaldehyde concentration of 40.5 mg/L. A 12 day maturation and fermentation temperature of 8 °C was needed to reduce the acetaldehyde to 14.3 mg/L. Copyright © 2014 The Institute of Brewing & Distilling     

High‐pressure homogenization: a non‐thermal process applied for inactivation of spoilage microorganisms in beer

The inactivation of spoilage microorganisms in beer using high‐pressure homogenization (HPH) was studied with the aim of evaluating the possibility of changing the conventional pasteurization process using this particular process. The homogenization pressure required for the inactivation of lactic acid bacteria, acetic bacteria and yeasts was investigated. For the most resistant microorganisms, the pressure inactivation kinetics and the effects of multiple process passes, initial temperature of the beer and the CO₂ concentration were studied. The results indicated that Lactobacillus delbrueckii was the most resistant microorganism tested, requiring 250 MPa to reach a six decimal reduction. Additionally, results showed that L. delbrueckii inactivation followed a second‐order kinetic process. A multi‐pass process and the use of a high initial beer temperature increased inactivation by HPH with L. delbrueckii, allowing the use of 150 MPa to achieve a five log cycle of inactivation. In contrast, a high CO₂ concentration reduced the efficacy of the HPH process. The results that were obtained are useful for high‐pressure homogenization applications in breweries and help to elucidate the effect of this new technology in a beverage that is both alcoholic and carbonated. Copyright © 2013 The Institute of Brewing & Distilling     

West African sorghum beer fermented with Lactobacillus delbrueckii and Saccharomyces cerevisiae: shelf‐life and consumer acceptance

The fermentation profiles, shelf‐life and consumer acceptance of traditional West African sour sorghum beer (pito) fermented with pure commercial starter cultures of lactic acid bacteria (L. delbrueckii) and yeast (Saccharomyces cerevisiae) were evaluated. The beers from this ‘pure culture’ approach were compared with the spontaneous fermentation of pito wort. Lactic acid formation, pH change and extract utilisation were monitored during fermentation. Lactic acid content was used as a measure of sourness to establish the spoilage level of over‐sourness. Further, regression models relating sourness to the time the drink was kept were used to predict the shelf‐life. Consumer acceptance of the product was evaluated using a novel nine‐point hedonic scale. The pure culture and traditionally fermented beers followed similar lactic acid and fermentation profiles but strain‐specific differences were observed. Similar levels of pH, lactic acid level and extract utilisation were achieved. An improvement in shelf‐life of two days was found over traditionally fermented pito. There was no statistical difference between the two pito products for overall liking and taste. However, there was a preference for the aroma of the pure culture pito. It is suggested that the use of pure cultures will facilitate the scale‐up of pito production. © 2019 The Institute of Brewing & Distilling     

Optimal control of beer fermentation processes with Lipschitz‐constraint on the control

Nowadays, one can find in almost all industrial products a trail of mathematical optimization. In particular, the theory and algorithms of optimal control have helped in various fields to reduce the production time and to improve the quality of the considered products. As a special class of applications, two optimal control formulations for the fermentation process of beer are presented. The reactions of the fermentation processes are modelled by a system of ordinary differential equations that are steered by the heating and cooling of the involved substrates. The occurring physical limitations, such as temperature limits and the requirement to avoid scenarios with unrealistic bang‐bang controls, give rise to optimal control problems with general control constraints. Therefore, this paper has reviewed a forward–backward sweeping method for solving these kinds of optimal control problems and presents encouraging numerical results that were obtained by this approach. Copyright © 2014 The Institute of Brewing & Distilling     

A rapid and sensitive non‐radioactive method applicable for genome‐wide analysis of Saccharomyces cerevisiae genes involved in small RNA biology

Conventional isolation and detection methods for small RNAs from yeast cells have been designed for a limited number of samples. In order to be able to conduct a genome‐wide assessment of how each gene product impacts upon small RNAs, we developed a rapid method for analysing small RNAs from Saccharomyces cerevisiae wild‐type (wt) and mutants cells in the deletion and temperature‐sensitive (ts) collections. Our method implements three optimized techniques: a procedure for growing small yeast cultures in 96‐deepwell plates, a fast procedure for small RNA isolation from the plates, and a sensitive non‐radioactive northern method for RNA detection. The RNA isolation procedure requires only 4 h for processing 96 samples, is highly reproducible and yields RNA of good quality and quantity. The non‐radioactive northern method employs digoxigenin (DIG)‐labelled DNA probes and chemiluminescence. It detects femtomole levels of small RNAs within 1 min exposure time. We minimized the processing time for large‐scale analysis and optimized the stripping and reprobing procedures for analyses of multiple RNAs from a single membrane. The method described is rapid, sensitive, safe and cost‐effective for genome‐wide screens of novel genes involved in the biogenesis, subcellular trafficking and stability of small RNAs. Moreover, it will be useful to educational laboratory class venues and to research institutions with limited access to radioisotopes or robots. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparative analysis of the fermentation performance of high‐quality milk beer strains (Kluyveromyces marxianus) and optimisation of medium formula for high‐density fermentation

Milk beer is a new type of dairy beverage in China and increasingly popular with consumers. The strain FXJ1 isolated in our laboratory was an excellent strain for the production of milk beer with alcohol content of 0.48%. Our research verified that the strain FXJ1 was superior to the commercial strain MJ for milk beer production and got the optimal medium formula (67.37 g/L sucrose, 29.7 g/L yeast extract, 15.61 g/L corn steep liquor, 4.13 g/L KH₂PO₄ and 0.3 g/L MgSO₄) which allowed the biomass production of strain FXJ1 to achieve 8.88 ± 0.08 g/L and increase 2.94‐fold as compared with yeast extract peptone dextrose base medium.     

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non‐homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR‐amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour‐intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ‐mediated integrative transformation with PCR‐amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.