Pilot‐scale brewing using self‐cloning bottom‐fermenting yeast with high SSU1 expression

A pilot‐scale fermentation was performed using SSU1‐overexpressing bottom‐fermenting yeast strains constructed by ‘self‐cloning’. In these strains, the gene SSU1, encoding a plasma membrane protein that excretes sulphite, was highly expressed. The rate of fermentation of the two SSU1‐overexpressing strains tested showed some reduction during the mid‐fermentation phase as compared with the parental strain. These differences, however, did not affect overall fermentation and the final apparent extracts had decreased to a level normally obtained during brewing. The concentration of hydrogen sulphide in the wort remained low during fermentation in the case of the two self‐cloning strains compared with the parent. The concentration of 2‐mercapto‐3‐methyl‐1‐butanol, a sulphur compound that causes an ‘onion‐like’ off‐flavour, was also reduced in the case of the self‐cloning strains, a result confirmed by sensory evaluation of the beer immediately after bottling. Furthermore, with these strains the anti‐oxidation potential of bottled beer, as measured by electron spin resonance, was improved and the concentration of trans‐2‐nonenal in bottled beer after 7 days of accelerated aging at 37°C was decreased. These observations, together with the lower stale flavour score determined by sensory evaluation of bottled beer after a month of aging at 25°C, indicated that the flavour stability of the beer had been successfully improved. Copyright © 2013 The Institute of Brewing & Distilling     

Processing of bottom‐fermented gluten‐free beer‐like beverages based on buckwheat and quinoa malt with chemical and sensory characterization

Typical beer contains significant amount of gluten, and being the third most popular beverage worldwide, the commercial production of its gluten‐free form is of rising interest. This research aimed to prepare bottom‐fermented beverages from buckwheat and quinoa and to explore their physical, chemical and sensory properties. Compared with barley, the analysis of brewing attributes of buckwheat and quinoa showed a lower malt extracts, longer saccharification times, higher total protein and fermentable amino nitrogen content and higher values of the iodine test and colour. Fermentability values, the wort pH and the soluble protein content were similar for barley and buckwheat, but different for quinoa, whereas only values of viscosity and beverage pH were similar between barley and quinoa. Both beverages, especially the quinoa beverage, contained a superior level of metal cations. The fermentable carbohydrate content in the buckwheat wort was comparable to barley but lower in quinoa; however, worts derived from both pseudocereals contained predominantly glucose. The amino acid content of the buckwheat wort was similar to barley, whereas the content in the quinoa beverage was almost twice as high. The content of volatile compounds commonly associated with beer aroma was comparable between the barley and buckwheat beverage but significantly lower in the quinoa; however, the latter contained some distinctive volatile substances not found in the other beverages. The organoleptic perception of the buckwheat beverage was better than that of the quinoa, although both showed a good general acceptance. In general, buckwheat appears quite similar to barley, whereas quinoa shows many unique properties. Copyright © 2014 The Institute of Brewing & Distilling     

Application of Plackett–Burman experimental design for investigating the effect of wort amino acids on flavour‐active compounds production during lager yeast fermentation

Aroma‐active higher alcohols and esters are produced intracellularly in the cytosol by fermenting lager yeast cells, which are of major industrial interest because they determine aroma and taste characteristics of the fermented beer. Wort amino acid composition and their utilization by yeast during brewer's wort fermentation influence both the yeast fermentation performance and the flavour profile of the finished product. To better understand the relationship between the yeast cell and wort amino acid composition, Plackett–Burman screening design was applied to measure the changes in nitrogen composition associated with yeast amino acids uptake and flavour formation during fermentation. Here, using an industrial lager brewing strain of Saccharomyces pastorianus , we investigated the effect of amino acid composition on the accumulation of higher alcohols and volatile esters. The objective of this study was to identify the significant amino acids involved in the flavour production during beer fermentation. Our results showed that even though different flavour substances were produced with different amino acid composition in the fermentation experiments, the discrepancies were not related to the total amount of amino acids in the synthetic medium. The most significant effect on higher alcohol production was exercised by the content of glutamic acid, aromatic amino acids and branch chain amino acids. Leucine, valine, glutamic acid, phenylalanine, serine and lysine were identified as important determinants for the formation of esters. The future applications of this information could drastically improve the current regime of selecting malt and adjunct or their formula with desired amino acids in wort. Copyright © 2017 The Institute of Brewing & Distilling     

Part III: the influence of serial repitching of Saccharomyces pastorianus on the production dynamics of some important aroma compounds during the fermentation of barley and gluten‐free buckwheat and quinoa wort

The present paper is the last report of a comprehensive study regarding the influence of the serial repitching of Saccharomyces pastorianus TUM 34/70 on the composition of a barley, buckwheat or quinoa fermentation medium. In particular, it focuses on the production dynamics of important volatile compounds typically associated with the aroma of beer. Samples were taken every 24 h after 11 serial repitchings of a single starter culture, analysed for the particular aroma compound content by distillation followed by gas chromatography with flame ionization detection. The term ‘serial repitching factor’ is used for the first time to support the visual evaluation of the influence of serial repitching. Results showed that the levels of methanol in the quinoa wort fermentation were only slightly higher than in barley and in practical terms independent of successive fermentation. The behaviour of acetaldehyde in quinoa was similar to that in barley. However, there was a final 2‐fold lower production of some important aroma compounds compared with barley and buckwheat and for this reason quinoa cannot be recommended as a gluten‐free substitute to produce a bottom‐fermented beer. Regarding the buckwheat wort fermentation, a 2‐ to 3‐times lower final acetaldehyde content than in barley is desirable, whereas a relatively high methanol content is not desirable. Barley and buckwheat showed comparable sum concentrations and similar overall profiles of some important aroma compounds. From this perspective, buckwheat appears to be a promising substitute for barley as a brewing raw material. The overall conclusions of our comprehensive study (Parts I–III) are that buckwheat shows adequate brewing properties to substitute for barley in the commercial preparation of a bottom‐fermented gluten‐free beer‐like beverage, and yeast can be repitched at least 11 times. In contrast, quinoa in practical terms shows no substitutional potential for barley in beer; however, it has many nutritious advantages, thus the commercial preparation of a unique, bottom‐fermented gluten‐free ‘non‐beer‐like’ beverage – where the yeast could be repitched six times at most – appears feasible. Copyright © 2015 The Institute of Brewing & Distilling     

Optimization of alcohol‐free beer production by lager and cachaça yeast strains using response surface methodology

Alcohol‐free beers (AFBs) are an attractive segment of the beer market both for the brewing industry and for consumers. While AFBs produced by arrested/limited fermentation often suffer from a lack of volatile compounds, beer flavour can be improved by yeast selection and optimization of fermentation conditions. The yeast selection strategy was demonstrated by comparing traditional lager yeast with selected cachaça yeast strains. Correspondingly, response surface methodology was used to enhance the formation of the flavour‐active volatile compounds by optimization of the fermentation conditions (original wort extract, fermentation temperature, pitching rate). Statistical analysis of the experimental data revealed the relative significance of process variables and their interactions. The developed quadratic model describing the responses of total esters and higher alcohols to changes in process variables was used to predict the ideal fermentation conditions in terms of flavour formation. The predicted conditions were experimentally verified and alternative strategies of AFB production are suggested. Copyright © 2016 The Institute of Brewing & Distilling     

Construction of self‐cloning industrial brewer's yeast with SOD1 gene insertion into PEP4 prosequence locus by homologous recombination

Superoxide dismutase (SOD, encoded by SOD1), which can scavenge active oxygen free radicals, is an ideal endogenous antioxidase in beer. In this study, the SOD1 expression cassette was constructed, and this cassette contained the PGK1 promoter, the PGK1 terminator and the SOD1 gene fused to the signal sequence of the yeast mating pheromone α‐factor (MFα1_s). One of the prosequences of the PEP4 gene (encoding proteinase A, PrA) in Saccharomyces cerevisiae strain S‐6 was replaced by the SOD1 expression cassette via homologous recombination and the self‐cloning strain S54PS, which could improve the antioxidant capability and foam stability of beer, was successfully obtained. Fermentation results showed that the SOD activity of the final beer brewed with S54PS was increased by 21.06%. Accordingly, the DPPH‐radical scavenging activity of S54PS increased by 30.6% compared with that yielded by the parental strain S‐6. Furthermore, the PrA activity of S54PS was always lower than that of the parental strain at all stages of beer fermentation. The head retention of the beer (255 ± 4 s) was better than that of the parental strain (224 ± 1 s). Hence, this research implies that S54PS exhibits good brewing performance and can be applied to improve the industrial brewing process. Copyright © 2016 The Institute of Brewing & Distilling     

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.     

Improving the performance of industrial ethanol‐producing yeast by expressing the aspartyl protease on the cell surface

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell‐surface anchored expressed in industrial ethanol‐producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild‐type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98–99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild‐type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Improved gap repair cloning in yeast: treatment of the gapped vector with Taq DNA polymerase avoids vector self‐ligation

Gap repair is a fast and efficient method for assembling recombinant DNA molecules in Saccharomyces cerevisiae. This method produces a circular DNA molecule by homologous recombination between two or more linear DNA fragments, one of which is typically a vector carrying replicative sequences and a selective marker. This technique avoids laborious and costly in vitro purification and ligation of DNA. The DNA repair machinery can also close and ligate the linear vector by mechanisms other than homologous recombination, resulting in an empty vector. The frequency of these unwanted events can be lowered by removing the 5′‐phosphate groups using phosphatase, which is the standard method used for in vitro ligation. However, phosphatase treatment is less effective for gap repair cloning than for in vitro ligation, presumably due to the ability of the S. cerevisiae DNA repair machinery to efficiently repair the missing phosphate group to allow religation. We have developed a more efficient method to prevent vector religation, based on treatment of the vector fragment with Taq DNA polymerase and dATP. This procedure prevents vector recircularization almost completely, facilitating the screening for true recombinant clones. Copyright © 2012 John Wiley & Sons, Ltd.     

Study on the self‐assembly property of type I collagen prepared from tilapia (Oreochromis niloticus) skin by different extraction methods

Type I collagen was prepared from tilapia (Oreochromis niloticus) skin by acetic acid and pepsin process at 4 °C, respectively (ASC and PSC), and hot‐water method separately at 25, 35 and 45 °C (C‐25, C‐35 and C‐45). Their structure and self‐assembly property were discussed. SDS‐PAGE patterns suggested that pepsin hydrolysis and the 35 and 45 °C extraction produced collagen with much reduced proportions of α‐ and β‐chains. Fourier transform infrared spectroscopy spectra revealed that pepsin hydrolysis did not change the conformation of collagen, but higher extraction temperature did. Self‐assembly curves and atomic force microscopy (AFM) observations showed that only ASC, PSC and C‐25 could self‐assemble into fibrils with D‐periodicity, but the reconstruction rate of C‐25 was lower. Besides, PSC had relatively higher resolution ratio compared with others. Overall, pepsin‐extracted collagen displayed higher solubility and better fibril‐forming capacity, having the potential of applying in biomaterials and food‐packaging materials.     

Development of a flavour fingerprint by GC‐MS and GC‐O combined with chemometric methods for the quality control of Korla pear (Pyrus serotina Reld)

Based on solid‐phase micro‐extraction/gas chromatography‐mass spectrometry (SPME/GC‐MS) and gas chromatography‐olfactometry (GC‐O), a new method was described for the first time to establish flavour fingerprint for the quality control of Korla pear. Twenty‐three batches of Korla pear collected from different regions in Xinjiang, China were analysed via GC‐MS, among which 21 were selected to establish the fingerprint. The results of the analysis were combined with GC‐O assessment to obtain 26 common odour‐active compounds as the characteristics of the Korla pear flavour fingerprint. The method for flavour fingerprint analysis was then validated on the basis of the relative retention time and relative peak area of the common peaks, sample stability and similarity analysis. The similarities in the 21 Korla pear samples were more than 0.80, thereby indicating that the samples from different batches were consistent to a particular extent. The chemometrics method (principal component analysis, PCA) was performed to develop a flavour fingerprint that is suitable for identifying and differentiating Korla pears and can be used for quality control.     

Scale‐up batch fermentation of bioethanol production from the dry powder of Jerusalem artichoke (Helianthus tuberosus L.) tubers by recombinant Saccharomyces cerevisiae

This study is a first exploration of industrial bioethanol production from the dry powder of Jerusalem artichoke (Helianthus tuberosus L.) tubers by recombinant S. cerevisiae 6525. Scale‐up fermentation of bioethanol production from the dry powder of Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae 6525 was carried out at the scale of 50 and 500 L agitating fermentors. For the 50 L fermentor, 85.67 g/L of ethanol was obtained within 72 h of fermentation, and the ethanol yield was 90.1%. For the 500 L fermentor, the highest ethanol concentration of 77.00 g/L was achieved at 84 h, and the ethanol yield was 81.01%. These results indicated that a relatively high yield of ethanol could also be obtained from a scaled‐up fermentation of Jerusalem artichoke powder. Thus, it may be feasible to use a Jerusalem artichoke tuber feedstock to carry out ethanol fermentations using the recombinant S. cerevisiae 6525 for industrial production. Copyright © 2016 The Institute of Brewing & Distilling