A rapid and selective assay for measuring cell surface hydrophobicity of brewer's yeast cells

A rapid and selective assay was developed to measure cell surface hydrophobicity of brewer's yeast cells. During this so-called magnobead assay, bottom-fermenting yeast cells adhere to paramagnetic, polystyrene-coated latex beads which can easily be removed from the cell suspension by using a (samarium-cobalt) magnet. At pH 4·5, electrostatic repulsion between yeast cells and latex beads was found to be minimal and yeast cell adhesion was predominantly based on hydrophobic interactions. The percentage of cells adhering to the beads could be calculated and provided a measure for cell surface hydrophobicity. Cell surface hydrophobicity measured by the magnobead assay was found to yield similar results, as did determination of contact angles of water droplets on a layer of yeast cells, a standard method for measuring surface hydrophobicity. However, the magnobead assay has the following advantages: (i) it is a quick and simple method, and, more significantly, (ii) hydrophobicity can be measured under physiological conditions. Use of the magnobead assay confirmed that a higher level of cell surface hydrophobicity is correlated with stronger flocculence of brewer's lager yeast cells.     

Use of spent brewer's yeast in L‐(+) lactic acid fermentation

The application of by‐products from the brewing industry in lactic acid (LA) production was investigated in order to replace expensive nitrogen sources (such as yeast extract) with cheaper and renewable nitrogenous materials such as brewer's yeast (BY). In this study, brewer's spent grain (BSG) hydrolysate was used for L‐(+)‐LA fermentation by Lactobacillus rhamnosus ATCC 7469. The effect of pH control during the fermentation and the addition of various BY contents (5–50 g/L) in BSG hydrolysate on fermentation parameters was evaluated. BY addition significantly increased free amino nitrogen (FAN) concentration (by 25.2% at 5 g/L to 616% at 50 g/L). A strong positive correlation between FAN concentration in the hydrolysate and concentration of L‐(+)‐LA produced was observed (correlation coefficient of 0.913). A high cell viability of L. rhamnosus ATCC 7469 (1.95–3.32 × 10⁹ CFU/mL at the end of fermentation) was achieved in all fermentations with the addition of brewer's yeast. The addition of BY increased L‐(+)‐lactic acid yield and volumetric productivity up to 8.4% (5 g/L) and 48.3% (50 g/L). The highest L‐(+)‐LA yield (89%) and volumetric productivity (0.89 g/L h^?1) were achieved in fermentation of BSG hydrolysate with 50 g/L of BY. © 2019 The Institute of Brewing & Distilling     

Isolation and partial purification of mannose-specific agglutinin from brewer's yeast involved in flocculation

Yeast cell-agglutinating activity, designated agglutinin (possible lectin), was isolated from cell walls of both non-flocculent and flocculent brewer's yeast cells. Agglutinin-mediated aggregation of yeast cells in a manner similar to flocculation with respect to specific mannose-sensitivity, pH-dependence and calcium-dependence. Agglutinating activity was found to be heat-stable and protease-insensitive. Furthermore, addition of agglutinin to flocculent cells strongly stimulated the flocculation ability of the cells, whereas addition to non-flocculent cells rendered these cells weakly flocculent. Agglutinin was found to be released from flocculent cells during the course of a flocculation assay, but not from non-flocculent cells. Presence of mannose during the assay inhibited release of agglutinin. Our results suggest that (i) mannose-specific agglutinin is continuously synthesized during growth of brewer's yeast cells, (ii) agglutinin is present in cell walls of non-flocculent cells but is unable to bind its ligand on other cells, and (iii) the ability of yeast cells to flocculate in a flocculation assay depends, among other factors, on release of agglutinin from the cells. A 10-kDa polypeptide might represent one form of agglutinin.     

Lactic acid fermentation of brewer's spent grain hydrolysate by Lactobacillus rhamnosus with yeast extract addition and pH control

Lactic acid (LA) is a versatile chemical with a wide range of applications in food, pharmaceutical, cosmetic, textile and polymer industries. Brewer's spent grain (BSG) is the most abundant brewing by‐product. In this study BSG hydrolysates were used for LA fermentation by Lactobacillus rhamnosus ATCC 7469. The aim of this study was to evaluate the effects of pH control during fermentation, reducing sugar content and yeast extract content in BSG hydrolysate on LA fermentation parameters. The pH control greatly increased reducing sugar utilization, l ‐(+)‐LA content, yield and volumetric productivity. The highest l ‐(+)‐LA yield and volumetric productivity were achieved with the reducing sugar content of 54 g/L. Yeast extract addition significantly increased reducing sugar utilization, l ‐(+)‐LA content, L. rhamnosus cell viability, l ‐(+)‐LA yield and volumetric productivity. The highest l ‐(+)‐LA content (39.38 g/L), L. rhamnosus cell viability (9.67 log CFU/mL), l ‐(+)‐LA yield (91.29%) and volumetric productivity (1.69 g/L/h) were achieved with the reducing sugar content of 54 g/L and yeast extract content of 50 g/L. Copyright © 2017 The Institute of Brewing & Distilling     

Identification of regulatory elements in the AGT1 promoter of ale and lager strains of brewer's yeast

Agt1 is an interesting α-glucoside transporter for the brewing industry, as it efficiently transports maltotriose, a sugar often remaining partly unused during beer fermentation. It has been shown that on maltose the expression level of AGT1 is much higher in ale strains than in lager strains, and that glucose represses the expression, particularly in the ale strains. In the present study the regulatory elements of the AGT1 promoter of one ale and two lager strains were identified by computational methods. Promoter regions up to 1.9 kbp upstream of the AGT1 gene were sequenced from the three brewer's yeast strains and the laboratory yeast strain CEN.PK-1D. The promoter sequence of the laboratory strain was identical to the AGT1 promoter of strain S288c of the Saccharomyces Genome Database, whereas the promoter sequences of the industrial strains diverged markedly from the S288c strain. The AGT1 promoter regions of the ale and lager strains were for the most part identical to each other, except for one 22 bp deletion and two 94 and 95 bp insertions in the ale strain. Computational analyses of promoter elements revealed that the promoter sequences contained several Mig1- and MAL-activator binding sites, as was expected. However, some of the Mig1 and MAL-activator binding sites were located on the two insertions of the ale strain, and thus offered a plausible explanation for the different expression pattern of the AGT1 gene in the ale strains. Accordingly, functional analysis of A60 ale and A15 lager strain AGT1 promoters fused to GFP (encoding the green fluorescent protein) showed a significant difference in the ability of these two promoters to drive GFP expression. Under the control of the AGT1 promoter of the ale strain the emergence of GFP was strongly induced by maltose, whereas only a low level of GFP was detected with the construct carrying the AGT1 promoter of the lager strain. Thus, the extra MAL-activator binding element, present in the AGT1 promoter of the ale strain, appears to be necessary to reach a high level of induction by maltose. Both AGT1 promoters were repressed by glucose but their derepression was different, possibly due to a distinct distribution of Mig1 elements in these two promoters.     

Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus‐like strains. Lager yeasts are particularly adapted to low‐temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make‐up of lager yeast spore clones, we introduced molecular markers to analyse mating‐type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18°Plato at 18–25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd.     

Application of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry as a monitoring tool for in‐house brewer's yeast contamination: a proof of concept

Contamination of brewer's pitching yeast cultures with wild‐type yeasts or bacteria is unwanted as it can corrupt the fermentation outcome and causes huge economic losses for the brewing industry. The applicability of matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) as a fast tool to monitor the purity of brewer's yeast cultures was investigated. This proof of concept was examined for a brewer's yeast strain contaminated with wild‐type yeast and for bottled beer produced by fermentation with that particular contaminated brewer's yeast strain. The data demonstrated that MALDI‐TOF MS is very suitable to discriminate between brewing and non‐brewing yeast strains. Copyright © 2014 The Institute of Brewing & Distilling     

Characterization and determination of brewer's solid wastes composition

Agro‐industrial wastes are produced in large quantities around the world from the processing and manufacturing of food and beverages. The disposal of these wastes into the environment leads to damage to ecosystems owing to their composition rich in organic matter. In this context it may be noted that the brewing industry, whose production process includes processing steps and fermentation of vegetable raw materials such as barley and/or other grains used as adjuncts and hops, generates various byproducts. The worldwide consumption of these beverages and the current model of breweries, which includes production on a large scale, lead to the generation of large amounts of brewery waste, namely spent grain, hot trub and residual yeast. Owing to its composition, these residues exhibit significant potential for application in bioprocess technologies. In this study the three residues mentioned had their composition determined as a function of moisture, ash, total organic carbon (TOC), total and soluble nitrogen, reducing sugar and soluble free amino nitrogen. Moreover, the residues were characterized for total acidity, pH and chemical oxygen demand (COD) of total and soluble fractions. The three residues evaluated had high moisture content (>80%) and high organic matter content (TOC and COD, ~50% and >1000 mg/g, respectively), which can highlights the significant protein fraction (almost 50% for hot trub and residual yeast), suggesting the possibility of using these wastes for recovery. Copyright © 2015 The Institute of Brewing & Distilling     

Effects of soy protein hydrolysates on the growth and fermentation performances of brewer's yeast

Soy protein isolate was hydrolysed with Alcalase, Papain, Flavorzyme and Protemax, respectively, and further fractioned by ultrafiltration. The resulting soy protein hydrolysates (SPH) and their ultrafiltration fractions were used to examine their effects on the growth and fermentation performances of brewer's yeast. Results showed that degree of hydrolysis, molecular weight distribution and amino acid composition of SPH significantly affected the growth, viability and fermentation performance of brewer's yeast. The SPH prepared from different proteolytic enzymes exhibited distinct growth‐ and fermentation‐promoting activity for brewer's yeast. The SPH treated with Protemax for 9 h and with the molecular weight below 3 kDa showed the highest growth‐promoting activity and induced more rapidly reducing sugar consumption and higher ethanol production. The relatively lower molecular weight and the hydrophilic and electropositive amino acid residues (Lys, His, Arg and Ile) in SPH might be responsible for its functionality, promoting the growth and fermentation of brewer's yeast.     

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     

Acid hydrolysis and fermentation of brewer's spent grain to produce xylitol

The hemicellulosic fraction of brewer's spent grain (BSG) was hydrolysed with diluted acid under different conditions of liquid/solid ratio (8–12 g g⁻¹), sulfuric acid concentration (100–140 mg g⁻¹ dry matter) and reaction time (17–37 min) in order to produce a liquor with a large amount of xylose and good fermentability to produce xylitol. Results showed that all the evaluated reaction conditions were able to hydrolyse xylan and arabinan with efficiencies higher than 85.8 and 95.7% respectively, and even under the mildest reaction condition a considerable amount (92.7%) of the hemicellulosic fraction could be extracted. The hydrolysates presented different fermentabilities when used as fermentation media for xylitol production by Candida guilliermondii yeast, owing to the differences in their composition. Based on statistical analysis, the best condition for BSG acid hydrolysis was the use of a liquid/solid ratio of 8 g g⁻¹, 100 mg H₂SO₄ g⁻¹ dry matter and a reaction time of 17 min. Under this condition a high extraction efficiency of hemicellulosic sugars (92.7%) and good fermentation results (Y_P/S = 0.70 g g⁻¹ and Q_P = 0.45 g dm⁻³ h⁻¹) were attained. Copyright © 2005 Society of Chemical Industry     

Isolation of a lager yeast with an increased copy number of the YCK1 gene and high fermentation performance

The selection of yeast with good fermentation characteristics is critical for producing beer with desirable qualities. A yeast population was selected with an enhanced fermentation rate, referred to as high‐fermentation yeast (HFY), which was derived from the wild‐type Sacchromyces pastorianus yeast population (WTY). To identify genes that contribute to the fermentation performance, we compared the genetic profiles of the WTY and HFY populations by next‐generation sequencing. Several chromosomal regions were found to exhibit markedly different sequence coverage, suggesting chromosomal duplications and deletions, which might have occurred during selection of the HFY population. Among the genes with altered coverage, the copy number of the Saccharomyces eubayanus‐type YCK1 (SeYCK1) gene was almost two times higher in the HFY population than in the WTY population. The gene which is involved in glucose sensing in Saccharomyces cerevisiae was at a higher level in the HFY population throughout fermentation. These findings suggest that the chromosomal duplication of a region including the SeYCK1 gene locus of the HFY population is at least partially responsible for the differences in the fermentation properties between the WTY and HFY populations. © 2018 The Institute of Brewing & Distilling     

Effect of the respiro-fermentative balance during yeast propagation on fermentation and wort attenuation

Breweries use different yeast strains to create beers with different flavours and aromas. Yeast propagation must produce yeast that performs consistently from the first fermentation to harvesting and re-pitching in subsequent fermentations. Breweries propagate yeast in wort leading to low efficiency fermentative growth in Crabtree-positive yeast. There is limited knowledge on the impact on beer production when fermenting with yeast propagated in sugar limited and nutrient supplemented wort. It was hypothesised that propagating yeast in this way would have a positive impact on subsequent fermentation performance. Saccharomyces cerevisiae was propagated at the laboratory scale in standard wort with a high carbon to nitrogen (C:N) ratio (850) or in modified wort supplemented with yeast extract to achieve a low C:N ratio (100) and at varying sugar concentrations. Propagation in low C:N wort with 2°P sugar yielded a 27% decrease in fermentation efficiency and a 46% increase in cell production compared to 2°P high C:N wort. This suggests nitrogen is critical to the respiro-fermentative balance during growth. Yeast propagated in standard wort resulted in slower fermentations and significant under-attenuation compared to yeast grown in the modified wort with low sugar and high nitrogen. The results of this study suggest the nitrogen and sugar content drive the respiro-fermentative balance during yeast propagation. The metabolism of yeast during propagation induces significant downstream impacts on the subsequent fermentation performance and wort attenuation. © 2020 The Institute of Brewing & Distilling     

Effects of wheat gluten hydrolysates fractionated by different methods on the growth and fermentation performances of brewer's yeast under high gravity fermentation

Wheat gluten hydrolysates (WGH) were fractionated through ultrafiltration membrane with molecular weight (Mw) cut‐off of 3 kDa and ethanol precipitation, respectively. WGH and their fractions were used to examine their effects on the growth and fermentation performances of brewer's yeast under high gravity fermentation. Results showed that WGH and their fractions exhibited significant differences in biomass accumulation, viability, ethanol yield, free amino nitrogen and sugar consumptions under high gravity fermentation. Compared to WGH, the fractions with Mw < 3 kDa and the supernatant of WGH treated with ethanol precipitation showed better fermentation performance for brewer's yeast. The relatively lower molecular weight and the higher levels of Leu, Lys, His and Arg in these two fractions might be responsible for their bioactivity for brewer's yeast. Thus, both ultrafiltration and ethanol precipitation could be used as efficient methods for enriching peptides with significant growth‐ and fermentation‐promoting activity for brewer's yeast under high gravity fermentation.     

Fed‐batch l‐(+)‐lactic acid fermentation of brewer's spent grain hydrolysate

Brewer's spent grain (BSG) hydrolysates were used for l ‐(+)‐lactic acid (LA) fermentation by Lactobacillus rhamnosus ATCC 7469. The aim of this study was to evaluate fed‐batch LA fermentation of BSG hydrolysate with the addition of glucose, glucose and yeast extract, and wort during LA fermentation and its effect on fermentation parameters such as LA concentration, its volumetric productivity and yield, and L. rhamnosus cell viability. The highest LA yield, volumetric productivity and concentration of 93.3%, 2.0 g/L/h, and 116.1 g/L, respectively, were achieved with glucose and yeast extract addition during fermentation. In fed‐batch fermentation with glucose and yeast extract addition significantly higher LA concentration, yield and volumetric productivity (by 194.8; 2.2, and 20.7%, respectively) were achieved compared with batch fermentation. The results indicated that fed‐batch fermentation could be used to increase LA fermentation efficiency. Copyright © 2017 The Institute of Brewing & Distilling     

酿酒废酵母吸附共存重金属离子的研究

为研究酿酒废酵母吸附共存重金属离子的可行性,在单因素试验的基础上,采用响应曲面法探讨酿酒废酵母吸附共存离子Pb2+、Ni 2+、Cu2+的最佳工艺条件。结果表明:酿酒废酵母对共存重金属离子Pb2+、Ni 2+、Cu2+吸附的最佳条件为pH值3.15,吸附温度30℃,吸附时间60min,酵母浓度2.04g/L,离子浓度61.34mg/L,该条件下酿酒废酵母对共存离子的吸附率为62.78%。     

小麦面筋蛋白水解物对酿酒酵母增殖和发酵性能的影响

研究了小麦面筋蛋白水解物对酿酒酵母增殖和发酵性能的影响,结果表明,不同水解度的小麦面筋蛋白水解物对酿酒酵母的促增殖和发酵效果不同,其中水解度为13.96%的水解物具有最强的促酵母增殖和发酵效果,该水解物可使稳定期酵母生物量提高37.0%,表观发酵度提高8.8%,乙醇产量提高6.4%,氨基氮利用率提高13.0%,同时发酵时间缩短14.3%。      

外源海藻糖对啤酒酵母在热胁迫下的保护作用

为探究外源海藻糖对啤酒酵母在热胁迫下的保护作用,作者在3个热胁迫条件下检测外源海藻糖对啤酒酵母活力及活性的影响,并通过分析转录组数据对热胁迫下外源海藻糖对啤酒酵母的保护作用机理进行初步分析。结果表明,外源海藻糖可以提升啤酒酵母的耐热性,但具有一定限度,温度的升高和胁迫时间的延长导致不同浓度海藻糖的作用差别减小。另外,添加海藻糖可以增加酵母胞内海藻糖质量分数,添加海藻糖对啤酒酵母转录水平上的影响主要体现在核糖体的合成和相关功能上。结合本研究推测,外源海藻糖对啤酒酵母在热胁迫下的保护作用是通过增加胞内海藻糖质量分数实现的,上升的胞内海藻糖质量分数促进了核糖体合成及相关代谢,使核糖体功能增强,进而实现对啤酒酵母耐热性的增强。